CA1256368A - Process for the preparation of an immune-reactive porous carrier material - Google Patents
Process for the preparation of an immune-reactive porous carrier materialInfo
- Publication number
- CA1256368A CA1256368A CA000497115A CA497115A CA1256368A CA 1256368 A CA1256368 A CA 1256368A CA 000497115 A CA000497115 A CA 000497115A CA 497115 A CA497115 A CA 497115A CA 1256368 A CA1256368 A CA 1256368A
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- CA
- Canada
- Prior art keywords
- immuno
- process according
- reaction
- inhibitor
- carrier material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/824—Immunological separation techniques
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/826—Additives, e.g. buffers, diluents, preservatives
Abstract
Abstract Process for the preparation of an immuno-reactive porous carrier material The present invention provides a process for the preparation of an immuno-reactive, porous carrier material by application of a solution of a first reaction component of an immuno-reaction and of a solution of a second component of an immuno-reaction coprecipitating therewith, incubation of the carrier material impregnated with the solutions for the immuno-precipitation, optional washing and subsequent drying of the impregnated carrier material, wherein a solution of both components of the immuno-reaction is prepared, which solution contains an inhibitor for the immuno precipitation, the carrier material is impregnated with this solution and then the immuno-precipitation is initiated by removal of the inhibitor or by removal of the inhibiting action.
Description
~ ~;2si636al lhe present invention is concerned with a process for the production of an immun~-reactive porous carrier materialq Immuno-reactive porous carrier materials play an important part in various branches of technology, For example, such materials are required for analgtical and preparative processes in which one component of an immune reactio,n is used Pixed to an insoluble matri~.
The fixing of the component of the immune reaction o to the insoluble carrier can take place by chemical or physical forces. ~hus, methods for the production of covalent bonds between a solid carrier material and a chemical substance to be bound thereon have been known for a ~ong time.
In general, however, a disadvantage of these methods is the fact that they bring about a chernical change of the biologically-active material which, in many cases, also results in a change of the biological activity.
Another knolm process is the inclu~ion polymerisation of such biologicallg-active substances~ Here, as a rule, the molecule as such remains unchanged and, therefore, also retains its biological effectiveness unchanged.
However, its accessibility to the other component of the immune reaction present in a liquid phase is drastically limited.
Therefore~ in mar1g cases, use has been made of a third known process which avoids the di~advantages of ~25636~3 covalent binding and of inclusion pol~merisation, namely, the adsorpti~e fixin~ to an appropriate carrier material. However, the method has, in turn, the disad-vantage that binding to the solid carrier is su~-stantially l~eaker than in the case of the two first-mentioned methods and, as a rule, an acceptable binding stability could o~ly be achieved on particular s~nthetic resin surfaces.
Special problems arose with regard to the adhesive lo strength in the case of an adsorptive binding, i.e. when not onlg a covalent fixing but also an inclusion pol~mer-isation is to be avoided, ~hen porous carrier materials are employed. I
From UjS. Patent Specification No. 3,888,629, a method i~ known which solves this problem of fixing adhesion b~ allowing a reaction to take place between the two components of an immuno-reaction, i.e. between antibody and antigen, in the porous carrier material. For this purpose, the porous carrier material is impregnated either ~ith a solution of the first component of the immuno-reaction and thereafter with a solution of the second component of the immune reaction, so that the immuno--reaction itself can take place in the porous carrier material with permanent fixing of the ~esired immuno-- reactive material without chemical change or the disadvantages regarding its accessibility for other reaction components (see P~ 0 82/02601), or, in a second known method, the two solutions of the particular cornponents ~25~3~13 of the immune reaction are firs-t rapidly mixed to~ether and then the porous material is. immediately impre~nated therewith (see U.SO Patent Specification No. 3,888,629).
~ his method of binding an immune~reac~ive material on to a porous carrier material admittedl~ solves the above-described problems but, nevertheless, displays another disadvahtage, namely, a non~uniform distribution of the fixed immuno-reactive substance on the carrier materialO
~his disadvanta~e is especially s~rious when this immuno~
-reactive porous carrier material is to be used, for example~
for the purpose of the quantitative analysis of haptens or proteins in low concentrations. ~he requirement for stability of the antigen-antibod~ meshwork, as well as for low equilibrium-dissociation concentrations of the immuno--reactive component from the immuno~complexes relative to the concentration of the haptens or proteins to be deter-mined,can onlY be achieved when the antibodg used has a hi~h affinity for the antigen in question. As is known from experience, the precipitate formation ln the case of mixing such immuno-reactive cornponents takes place spon-taneously, In the case of the production of technical amounts of an immuno-adsorber by impre~nation of a porous carrier material with solutions of the immuno-reactive components which have been mixed together shortly before, as is to be expected, a chronologically and spatially uncontrollable precipitate formation occurs on the carrier material.
. ~ , .
~;~5~3~;~
~he quantitative dosa~ing of the immunosorbent pre ferably take~ pIace bg cutting out a particular area of paper in the case of an immuno-reactive paper, by counting out a particular number of spheroids in the case of a spheroidal porous carrier material or by wei~hing out a definite amount of the immuno-reactive material..However, in the case of non-uniform distribution of the fixed immuno-rsact~ve material, such simple methods of dosaging cannot be employed.
A further di~advantage of the known immune precipit-ation techniques is that very highly purified antigens are needed whicn requires a pre-purification by immunosorption~ and is thus!~laborious method. of production.
~herefore, it is an object of the present invention lS to provide a simple method for the production of a porous immuno-reactive carrier ma-terial which has a superlor homogeneity with regard to the distribution of the immune-reactive component and whick can be produced easily and simply and which, in -~articular~ does not requiFe an~
laborious pre-purification for the production~ .
Thus, according to the present invention, there i5 provided a process for the production of an immuno--reactive porous carrier material by application of a solution of a first component of an immuno-reaction and of a solution of a second component of an immuno~reaction coprecipitating therewith, incubation of the carrier -material impregnated with these solution~ for immuno--precipitation, optional washin~ and subsequent drying ~25~
of the impregnated carrier material, wherein a solution of both components of the immuno-reaction is prepared, which solution contains an inhibitor for the immune pre~
cipitation, the carrier material is impregnated with this solution and then the immuno~precipitation is initiated by removal of the inhibitor or by removal of the inhibitin~
action.
An important feature of the present invention is the use of an immuno-precipitation inhibitor which is reversibly active and whlch loses its inhibiting action by simple removal or by chemical chan~e. In the presence of the inhibitor, the components of the immuno-reaction leadin~ to the precipitation can be completely homo~
geneously distributed and, because of precipitation which only ta~es place very slowly, there is obtained a cornpletely uniform co~ering of the carrier material with the partic-ularly desired immuno-reactive substance which, even in the case of a large-scale batch, ~ives completel~ uniform and reproducible results.
In contradistinction to the known process of sequential application of antigen and antibody (hetero~enous process), such as is described hereinafter in comparative Example 4, in the case of the "homogeneous" process according to the present invention, impregnation is carried out ~Jith a homogeneous solution of anti~en and antibody A differ-entiation is thereby to be made between two possible embodiments of the process according to the present invention: in the case of a "l step proces~", the ~;25~3~i~
inhibitor which i~ contained in the impregnation solution ~5 removed from the solution or is made inactive durin~
the production process and, in the case of the "2-step process"~ the porous carrier is firs-t pre-impregnated with an active material which removes the action of the inhibitor in the case of the subsequent impregna~ion with the immunQ-reacti~re components.
One possibility for the removal of the inhibitor is a ~JashinF step before the drying and also, by means of additives to the wash solution, the action of the inhibitor in the porous carrier material can be removed.
~owever, the washin~ ~tep i~ not an essential step of the processO Thus, for example, the precipitation can be initiated by evaporation of a volatile inhibitor.
~s inhibitors of the immuno-precipitation, within the scope of the pre~ent invention there are preferably used those substances which are used as desorption a~ents in the case of immunosorptive purifications~ For this purpose, there are especiallg preferred acids, base~ and chaotropic . ions of the ~-Iofmeister series (lgotropic series) such as are described, for example, in "Structure and Stabil1ty of Biological l~lacromolecules"~ 1969, pub Marcel Dekker Inc.
New York, page L~27, as well as certain or~anic compounds or solvents, such as acetonitrile~ urea or glycerol.
~s appropriate acids which can be used in the scope o~
the present invention, there can be employed not only volatile but also non-volatile acids. l~ter impre~nation ~25~3~3 of the carrier material, volatile acids can easily be removed for the removal of the inhibiting action, for example by heating, the application of a vacuum or the like. In the case of non-volatile acids, an analogous effect can be achieved by the addition of a salt of a volatile acid which is decomposed by the non-volatile acid, with the liberation of the volatile acid. Preferred examples include acetic acid, propionic acid and hydro-chloric acid for the volatile acids. In the same way, volatile and non-volatile bases, for exarnple ammonia and t-phosphate, can be used.
~urthermore, as inhibitors, there can also be used or~anic compounds whicn can reversibly influence not only the protein but also the water structure and which are described, for example, in J.~. ~randts "Conformational ~ransitions ~f Proteins in l~ater" contained in "Structure and Stability of Biological Macromolecules"~ 1969, pub.
Marcel Dekker Inc.~ ew York, pp. 213 - 290. Glycerol and urea are hereby especially preferred.
As inhibitors, there can also be used chaotropic ions, such as thio~yanates and iodides. Examples of other appropriate ions include fluorides, bromides, perchlorates, guanidine and sulphates.Their abstraction for the purpose of removing the precipitation inhibition can take place by extraction with, for example, organic solvents or mixtures of organic solvents, for exaMple esters, or mixtures of org,anic solvents and water, fo~ example water/alcohol mixtures, possibl~ with the addition of ionophores or ~2S63~3 the li~e~ As a rule, it i~ hereb~ already suf~icient to chan~e the ionic stren~th in order to achieve the desired effect but a complete removal of the inhibitor can also tale place. ~;n addition of a complex former, such as eth~lenediamine-tetraacetic acid (~Dq'~), can also be considered, for example for the removal of inhibiting metal salts, such as ma~nesium chloride~
The molar concentrations of the immunc-reactive components leading to the precipitation can be varied ~ithin wide limits. In the case o~ the present invention, they are preferably used in such a manner that that component, the immuno-reac~ivity of which is to be utilisea in the finished carrier material, i~ not present in excess The component leadin~ to the precipitation is especiallg preferably used in the ratio of the ~eidelber~er maximum. This means that the two reaction components are used in the ratio which i~ most favourable for the pre-cipitation ThiS ratio can easily be previously ascertained by turbidity tests. '~n appropriate turbidity curve is to be found, for example9 in "r~ethods in Immunology and Immunochemistr~", Volume III9 pub. Academic Press, Chapter 13, page lO. In the case of the production of such a curve, the turbidit~ is plotted which is obtained in the case of constant amount of one of the reaction components with increasin~ amounts of the other reaction component q1he turbidity maximum is the ~Ieidelber~er maximum.
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In or~er -to achieve maximum stability of the carrier-fixed immuno-reactive body, one of the components of the immuno~-precipitation should preferabl~ be a precipitating antibody ~Jith an affinity for the antigen which is as high as possible.
The compol1ents of the immuno-precip~tation in the scope of the present invention are~ on the one hand, an anti~en~
for example a hapten or a protein, and, on the other hand~
an antibod~ ~'he antigen can, of course, itself also be an antibody. In such a case, i.e. when the immuno-reactive component to be precipitated is itself an antibody, this can also be used as a ~ab, Fab' or (Fab')2 fra~ment, namely, not only of a monoclonal but also of a polyclonal antibod~ The precip~tating anti-antibody which is then used for the precipitation MUSt be correspondingly chosen~
The anti-antibody itself can then be polyclonal or mono-clonal or a (~ab')2 fra~ment thereof. If the anti-antibody is a monoclonal antibody or a lragment thereof, then this should preferably reco;~ni5e two different epitopes on the antigen. However, monoclonal antibodies or (Fab')2 fra~ments thereof can also be used which onl~ recognise one~epitope, which is more usual, lif this epitope is present at least twice on the antigen. ~ixtures of monoclonal antibodies can also be used as anti-antibody fraction for the immuno--precipitation~
~s imMuno~reactive material to be precip2tated, there can preferably also be used a pro~ein to which a hapten or antiuen is coupled In this case, a precipitatin~r antibod~
~2563~
is preferably used as ~e~nd Gomponent of the immune reaction which is directed against the protein. In a further embodiment, a hapten or antigen can be coupled to the precipitating antibody. q1he other component of the immune reaction can thereby be unlabelled or be coupled with the same or another hapten or antibod~7.
~lternatively, the protein to be precipitated is itself a specific antibody which is precipitated bg an anti-antibod~.
lo As already mentioned above, it is a special advantage of the process according to the present invention that at least one component of the immune reaction can be used in non-purified form. In contradistinction to the known immuno-precipitation processes, in which the precipitation takes place immediately after combination of the immune reaction components, in the case of the process according to the present invention, the speed of the precipitation in suspension or in the porous carrier material can take place controlled in such a manner that no entrainment effects ocçur in the case of foreign substances and the latter do not have to be previously removed but rather can simplv be washed out after the immuno-precipitation.
Such a washing step is preferrcd since no precipitate particles, less stable immun~-complexes and adsorptively bound antigens are herebg removed. ~herefore, for the washing, there can be used buffers of high ionic strength, deter~ents, organic solvents and the like, as well as additives ~tabilising the precipitates, such a~ carbo-hydrates and proteins.
~L~2,S~ii3~3 If a ~revious purirication of the components of the im~une precipitation purification i~ to be omitted, it is preferable Pirst to mix the particular antigen and the particular antibody in the optimum (Heidelberger) ratio, to wash the precipitate obtained and a~ain to dissolve by the addition of the inhibitor of the immuno~precip-itation, preferably by the addition of acid. ~here is thus obtained a mixture of the components of the immuno~
-precipitation reaction which can be applied direc~ly to the porous carrier material.
lithin the scope of the present invention, it is, of course, also possible previously separately to purify the components oi the immuno--reaction in the usual wa~, via immuno-adsorbers, then to elute with an inhibitor and either to use the eluates directly for the process accordin~ to the present invention or first to produce a storage-stable form of the reaction cornponents by~ lyo-philisation. 'rhe inhibition of the immuno-precipitation, as well as the slowin~ down of the precipitation in the carrier material after removal of the inhibitin~ action can be assis-ted by physical methods, for example lowering the temperature or increasing the viscosity.
~s carriers within the scope of the present invention, there can be used carriers which are conventional for immu~o-reactive substances. ~uch a solid c~rrier material, which is frequentl~ also referred to as a matri}~, can be made, for example, of glass, synthetic resin, oaper, porous ~letal and the Iike, provided that the carrier _12-~.25~
material is sufficiently permeated by interconnected, liquid-permeable hollow ~paces I~atural, synthetic, organic or inorganic poIymers can be used. Furthermore, fibrous, spongy or sintered substances can be used.
As carrier materials~ there can be used planar, partic-ulate or other three-dimensional porous bodies. However, it is preferred to use planar 7 porous carriers, such as paper, films of foamed material, glass fibre mat~ and the like. Since the process according to the present invention gives a completely homogeneous distribution of the immuno-reactive substance in tbis planar matrix, a dosaging can take place extremely simply on the basis of unit surface areas~ Such immuno-reactive, planar carrier materials are especially suitable for use as solid phases in heterogeneous immunoassays.
~he use of an immunQ-reackive carrier material produced accordingr to the presen-t invention in the scope of a hetero~enous enzyme immunoassay can, for example, take place in such a manner that hap~en (H) or protein ~P)~
which is contained in a sample, such as buffer solution, serum, plasma, urine culture supernatant or the like, is mixed with a labelled binder (B). ~s binders, there can be used, inter alia, antibodles, Fab fra~ments or Fab fragments, as well as li~ands, which react specific-ally with the hapten or protein ~s labellin~ for the binder, there can be used, ~or example, an enzyme, a fluorescent label or radioisotope and, in the following Examples~ ~ galactosidase is used. ~he molar amount of ~256~8 the added binder can be not only in excess but al~o in in~ufficiency with regard to the hapten or protein present in the sample, T~is mixture is incubated for a constant time during which the comple~es H-B or P-~ are formed. After the expiry of this period of time, three species are present in the reaction mixture, namely, the complex consisting of hapten/protein, and binder (E-B, P-B), residual free hapten/protein (EI/P) and residual free binder (B).
The separation of these species takes place in a second step by means of immunosorption For this purpose, the mixture is applied to immuno-precipitation solid phase pro~uced accordin6 to the present invention which contains bound the hapten to be detected or the antibody to the protein to be determined.
In the case of the hapten test, the free binder but not the binder saturated with hapten binds to the solid phase~
Conse~uently~ the supernatant or the eluate of the solid phase contains the binder saturated with the hapten of the sample. The quantl~ati~e determination of the hapten now ta~es place via the labelling of the binder and., in the following ~xamples, via the determination of ~-galactr-osidase by m~ans of o-nitrophenyl-~-D-galactoside or chlorophenol red ~-D-galactoside.
In the case of the protein test, the complex of pro-tein and binder but not the free binder binds to the solid phase, residues of the free binder are removed by washing from the immunosorbent produced according to the present -lL~_ ,~, .
c invention The quantitative detection of the protein takes place via the labelled binder and, in the following Examples, via the determination of the ~-galactosidase by means of Q-nitrophenyl-~-D-galactoside or chlorophenol red ~~D-galactoside Another use is in competitive imm~no-tests. ~he use of the immuno-reactive carrier material prcduced accordin~ to the present invention can take place in such a manner that the analyte (hapten or protein), which is contained in the sample, is mixed with a constant amount of labelled analyte. '~he labelling can be, for example, an enzyme, a fluorescent label, radioisotope or the like. ~his mixture is applied to the matrix. On the matrix, there is immobil-ised an antlbody which i5 directed against the analytesc The mixture is incubated on the matrix for a definite time.
During this time, not only unchan~ed analyte but also labelled analyte compete for the binding places on the matrix. The more analyte is present in the sample, the le~s labelled analyte is bound by the matrix and vice versa. ~t the end of the incubation phase, the liquid is removed from the porous matrix, for example by centri-fuging. ~he amount of labelled analyte is then determined either in the ~ree phase or bound to the matrix.
Ano~her use of the carrier material produced according to the present inventlon can take place in such a manner that the analyte (hapten or protein) is mixed ~lith a constan-t amount of labelled antibody which is directed against the analyte. Possible kinds of labelling o~ the antibody have been describe~ hereinbeforeO ~hi~ mixture ~,z56368 is then either incubated for a definite time and then applied to the porous carrier material or, alternatively, applied to the porous carrier material immediatel~ after mi~ing. If the analyte is a hapten, the carrier material contains the hapten to be detected or a derivative thereof in fixed form and if the analyte is a protein, the carrier material contains -the protein to be detected or a derivative thereof also in fixed form. If the first rni.~ture is incubated be:Eore application to the carrier material, then labelled antibody bmds wlth remainLng free binding places ~o the porous carrier material. If the mixture i~ applied immediatel~ to the carrier material, then the anal~te from the sample and the analyte fi~ed on th~ carrier material compete for the bindin~ places of the labelled antibody. ~t the end of the incubation phase, the liquid is reMoved from the porou~ carrier material and the amount of labelled antibod~ is deter-mined either in the li~uid phase or on the porous carrier material.
The inven-tion is further described with reference to -the accompanying drawings and Examples, in which:
Fig.l shows a calibration curve for the digoxin immune precipitate fleeces according to Example 3;
Fig. 2 shows a calibration curve analogously to Eig. 1 for digoxin immune precipi-tate fleeces accord-ng to Example ~ (not an example according to the nvention);
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Fig. 3 shows a calibration curve analogously to Fig. 1 for digoxin immune precipitate fleeces accord-ing to Example 5;
Fig. 4 shows a calibration curve for a DPH immune precipitate fleece according to Example 6;
Fig. 5 shows a calibration curve for the TSH
immune precipitate fleece according to Example 7;
Fig. 6 shows a calibra-tion curve for the TSH
immune precipitate fleece according to Example 8, and Fig. 7 shows in -the form of a graph the formation of precipitate dependent on the pH according to Example 9.
'rhe following ~xample~ are given for the purpo~e of illustrating the pre~ent invention.
Obtaining the materials u~ed for the test5 and carrying out of the test:
a) Produr~tion of pol~ha~ten~ (PH):
'~he production of P~ kno~m in the art. '~hu~ for example, a di~oxin P~ or a diphenylhydantoin PH can be obtained via reacti~e asymmetrical dicarboxylic acid ester/activated hapten ester and binding thereof to a carrier protein.
-16a-., .... , . , . . , . . . , . , . ~ .. .. .. . . . .. . ..
~ ~;25Ei3~
The preparation of T3- and ~4-PH can take place 7 for example, by direct coupling of the N~2 group~ of horMone and protein by means of bis-imidates (see European Patent Spccification A 0078952) or by the carbodiimide reaction (see Aherne et aI., Brit. J. Clin~ PharmO, 39 56/1976) or b~ the mixed anhydride reaction (see ~rlanger et al , Methods in Immunolo~y and Immunochemi~try, ed. Williams and Chase, pub. Academic Press, New York, 1967, pp. 149 et ~ lternative~y, in a first ~tep, the ~2 function of ~3 or T4 can be protected with an acetgl, trifluoro-acetyl, tert.-butoxycarbonyl or benzJloxycarbonyl group.
Subse~uently~ the carboxyl function is converted into an activated ester, for example N-h~droxy~uccinimide e~ter, N_hydroxyphthalimide ester, N--hydroxybenztriazole esterO
An example of such an activated T'~ is described in ~xample 3 of European Patent Specmfication A 0108400. Reaction with the carrier protein grives the PH.
Ihe choice of the carrier proteins is not subjected to any limitation~ in~ofar as a corresponding "precip-itating" antibody is available or can be produced.
Insofar as a immunosorptive purification of antigen and an-tibody has taken place, a~ carrier there was used the "affinity adsorber, glutardialdehyde activated" of Boehrin~er Mannheim G~bH (order No~ 665525) ~'or t he ~xamples described hereinafter, accordin~ to the instrllc-tions of the manufac-turer, for the antigen puriEication a sheep antibod~ against rabbit IgG or a ~pecific anti-body a~ainst the hapten was bound to the carrier and for ~ 2X~3~3 - "
the antibody purification rabbit I~G was bound to the carrier, The immunosorption took place as described in the worlcinv instructions for the affinity ad~orbent.
b) Production of the binder (di~oY~in as examPle~:
Obtaining the antiserum directed a~ainst dig~oxin, The production of the immunogen, namely, human serum albumin conjugated with digoxin, took place in the manner described in detail by V.P. 3utler jr, and J.E. Chen., Proc. Nat. Acad, Sci, US., 51~ 71-78/1967~ as well as by T.',l. Smith, V.P. Butler and ~. ~aber, Biochemistry~ 9, 331-337/1970~ Sheep were immunised with this immuno~en and the corresponding antiserum obtained.
Production of the binder.
Antiserum directed against digoxin was purified to the IgG fraction via ammonium sulphate precipitation and passage over D~E-cellulose. Papain splittin~ was carried out according to the method of R~ ~o Porter (Biochem. J~, 73~ 119-126/1959)~
The ~ab fragments were separated off from the non-digested IgG molecules and the Fc fra~ments by means of gel filtration over Sephadex~G 100 and ion exchan~er chromatography over DE~E-cellulose according to the methods described in the literature (see K. rlalinowski and ~iO
Manski in "llethods in Enzymology"; J,J. ~angone and H, van Vunakis ed~., pub. Academic Press, Vol. 73, 418-459/19~
The resulting ~ab fraction ~as purified chromatographically and coupled to ~-galactosidase according to the method of T. Yitiwaga in 'l~nzyme Immunoassay , I~hikawa, T. Kawai and h, ~'iyai eds., I~aku 15hoin, Tokyo-i~lew York, pp~81-89/1981 ~ Trade Mark -18-. . . , . . . ~
~;~56~
c) Pro_uction of sheep antlbod~l a~ainst rabbit or mouse I~G/FcY.
Rabbit slau~rhter serum or mouse serum was subjected to an ammonium sulphate precipitation. A~ter passage over DEAE-cellulose and papain splittin~, gel and ion exchange chromatography (see b)), there are obtained the Fc fra~ments of the rabbit and mou~ IgG's as immuno~ens.
'rhe working up of the sheep antisera took place as described under b) (immunosorption see a))O
d) Carr~,~in~ out of the test (di,roxin as example).
Digoxin standards in human serum (taken from the 3~I~A
kit of Boehringer Mannheim GmbH; order No. 199656) were diluted 1:7~5 with 0.9,o aqueous sodium chloride solution.
200 ~ 1~ diluted standard and 200 Jll. binder (20 mU/mlO, ~etermined with o-nitrophenyl-~-D-galactoside), produced as described in b), were incubated in phos~hate-buffered ~aline (PBS)(pH 7.2) at 37C~
Thereafter, 50 JLl. of the mixture were applied to 1 cm2 of the planar, immuno-reactive carrier material, Incubation is carried out for a further 5 minutes at 37C., followed by centrifugation for 1 minute in an Eppendorf centrifu~e, '~he enzyme activitY of the dit~oxin-binder complex can now be determined iin the free pha~e kinetically by ~he addition of chlorophenol red ~D-t~ralactoside (prepared according to Federal Republic of Germany Patent Specification Mo. 33 45 74~) and measured at 578 nm.
~;Ei3E;8 In the Examples, ther0 is, in each case, shown the course of the "rising" calibration curve, the digoxin concentration o~ the undiluted serum (X axis) being pIotted against the measured optical density at 578 nm (Y axis).
Alternatively, the excess amount o~ binder, i.e. the amount bound to the solid phase, can be determined as above and plotted against the particular di~oxin con-centration, a "decreasing" calibration curve being lo obtained.
Example l.
Preparation of ~3 (triiodothyronine) immune Precipitate fleece.
As anti~en, there was used polyhapten consisting of rabbit I~G and T3 bound thereto (mole ratio IgG:T3 = 1 3) ~he antigen was purified immunosorptively over an anti-~3 column (see a)).
As antibody, there was used polyclonal antibody directed a~ainst the ~c part o~ the rabbit I~G~ ~he antibody was purified immunosorptively over a rabbit I~G
column (see a)).
As ~leece, -there was used a mixed fleece of cellulose and polyester (obtained from I~alff, Euskirchen, Germany) or o~ lint, cellulose and polyamide (obtained fr~ Binzer3 Hatzfeld, Eder~ Germany).
~ntigen and antibody were taken up separatel~ in l mM
acetic acid (in each case c = l mg /ml.) and left to stand for l hour at ambient temperature. ~he solutions ~2563~
were then mixed in a ratio of 1:3 (the antigen/antibody ratio t~at corre~ponded to the Heidelberger maximum) and diluted with a nlnefold volume of a solution of 250 mM
acetic acid and 5 mM sodium acetate~ , The fleece was drawn through the impre~nation solution, squeezed out and dried for 60 minutes at 30C. in a circulating-air drging cabinet. It was then wa~hed by descending chromatography for 1 hour with PBS buffer ~(pI~ 6.0) and dried for 30 minutes at 30C. in a circulating air dr~ing cabinet, ~ r'he ratio of measurement range (standard 5 ng. ~3/ml, -standard 0) to standard 0 was llo 8 ( see ~able 1)~
' ~able 1 Comparison_of ~3 immun_,~recitate fleece ~e~ared b~
different processes . . . _ proce~ ~xample measurement range/standard 0 _ ~
hom. 2-step 2 12.0 hom. l-step 1 11.8 heterog. 2-step analogue 4 1'.6 suspension analo~ue 5 0.19 _ __ . ~
Example 2.
~reParation of ~ immune precipi,tate fleece (homo~eneous
The fixing of the component of the immune reaction o to the insoluble carrier can take place by chemical or physical forces. ~hus, methods for the production of covalent bonds between a solid carrier material and a chemical substance to be bound thereon have been known for a ~ong time.
In general, however, a disadvantage of these methods is the fact that they bring about a chernical change of the biologically-active material which, in many cases, also results in a change of the biological activity.
Another knolm process is the inclu~ion polymerisation of such biologicallg-active substances~ Here, as a rule, the molecule as such remains unchanged and, therefore, also retains its biological effectiveness unchanged.
However, its accessibility to the other component of the immune reaction present in a liquid phase is drastically limited.
Therefore~ in mar1g cases, use has been made of a third known process which avoids the di~advantages of ~25636~3 covalent binding and of inclusion pol~merisation, namely, the adsorpti~e fixin~ to an appropriate carrier material. However, the method has, in turn, the disad-vantage that binding to the solid carrier is su~-stantially l~eaker than in the case of the two first-mentioned methods and, as a rule, an acceptable binding stability could o~ly be achieved on particular s~nthetic resin surfaces.
Special problems arose with regard to the adhesive lo strength in the case of an adsorptive binding, i.e. when not onlg a covalent fixing but also an inclusion pol~mer-isation is to be avoided, ~hen porous carrier materials are employed. I
From UjS. Patent Specification No. 3,888,629, a method i~ known which solves this problem of fixing adhesion b~ allowing a reaction to take place between the two components of an immuno-reaction, i.e. between antibody and antigen, in the porous carrier material. For this purpose, the porous carrier material is impregnated either ~ith a solution of the first component of the immuno-reaction and thereafter with a solution of the second component of the immune reaction, so that the immuno--reaction itself can take place in the porous carrier material with permanent fixing of the ~esired immuno-- reactive material without chemical change or the disadvantages regarding its accessibility for other reaction components (see P~ 0 82/02601), or, in a second known method, the two solutions of the particular cornponents ~25~3~13 of the immune reaction are firs-t rapidly mixed to~ether and then the porous material is. immediately impre~nated therewith (see U.SO Patent Specification No. 3,888,629).
~ his method of binding an immune~reac~ive material on to a porous carrier material admittedl~ solves the above-described problems but, nevertheless, displays another disadvahtage, namely, a non~uniform distribution of the fixed immuno-reactive substance on the carrier materialO
~his disadvanta~e is especially s~rious when this immuno~
-reactive porous carrier material is to be used, for example~
for the purpose of the quantitative analysis of haptens or proteins in low concentrations. ~he requirement for stability of the antigen-antibod~ meshwork, as well as for low equilibrium-dissociation concentrations of the immuno--reactive component from the immuno~complexes relative to the concentration of the haptens or proteins to be deter-mined,can onlY be achieved when the antibodg used has a hi~h affinity for the antigen in question. As is known from experience, the precipitate formation ln the case of mixing such immuno-reactive cornponents takes place spon-taneously, In the case of the production of technical amounts of an immuno-adsorber by impre~nation of a porous carrier material with solutions of the immuno-reactive components which have been mixed together shortly before, as is to be expected, a chronologically and spatially uncontrollable precipitate formation occurs on the carrier material.
. ~ , .
~;~5~3~;~
~he quantitative dosa~ing of the immunosorbent pre ferably take~ pIace bg cutting out a particular area of paper in the case of an immuno-reactive paper, by counting out a particular number of spheroids in the case of a spheroidal porous carrier material or by wei~hing out a definite amount of the immuno-reactive material..However, in the case of non-uniform distribution of the fixed immuno-rsact~ve material, such simple methods of dosaging cannot be employed.
A further di~advantage of the known immune precipit-ation techniques is that very highly purified antigens are needed whicn requires a pre-purification by immunosorption~ and is thus!~laborious method. of production.
~herefore, it is an object of the present invention lS to provide a simple method for the production of a porous immuno-reactive carrier ma-terial which has a superlor homogeneity with regard to the distribution of the immune-reactive component and whick can be produced easily and simply and which, in -~articular~ does not requiFe an~
laborious pre-purification for the production~ .
Thus, according to the present invention, there i5 provided a process for the production of an immuno--reactive porous carrier material by application of a solution of a first component of an immuno-reaction and of a solution of a second component of an immuno~reaction coprecipitating therewith, incubation of the carrier -material impregnated with these solution~ for immuno--precipitation, optional washin~ and subsequent drying ~25~
of the impregnated carrier material, wherein a solution of both components of the immuno-reaction is prepared, which solution contains an inhibitor for the immune pre~
cipitation, the carrier material is impregnated with this solution and then the immuno~precipitation is initiated by removal of the inhibitor or by removal of the inhibitin~
action.
An important feature of the present invention is the use of an immuno-precipitation inhibitor which is reversibly active and whlch loses its inhibiting action by simple removal or by chemical chan~e. In the presence of the inhibitor, the components of the immuno-reaction leadin~ to the precipitation can be completely homo~
geneously distributed and, because of precipitation which only ta~es place very slowly, there is obtained a cornpletely uniform co~ering of the carrier material with the partic-ularly desired immuno-reactive substance which, even in the case of a large-scale batch, ~ives completel~ uniform and reproducible results.
In contradistinction to the known process of sequential application of antigen and antibody (hetero~enous process), such as is described hereinafter in comparative Example 4, in the case of the "homogeneous" process according to the present invention, impregnation is carried out ~Jith a homogeneous solution of anti~en and antibody A differ-entiation is thereby to be made between two possible embodiments of the process according to the present invention: in the case of a "l step proces~", the ~;25~3~i~
inhibitor which i~ contained in the impregnation solution ~5 removed from the solution or is made inactive durin~
the production process and, in the case of the "2-step process"~ the porous carrier is firs-t pre-impregnated with an active material which removes the action of the inhibitor in the case of the subsequent impregna~ion with the immunQ-reacti~re components.
One possibility for the removal of the inhibitor is a ~JashinF step before the drying and also, by means of additives to the wash solution, the action of the inhibitor in the porous carrier material can be removed.
~owever, the washin~ ~tep i~ not an essential step of the processO Thus, for example, the precipitation can be initiated by evaporation of a volatile inhibitor.
~s inhibitors of the immuno-precipitation, within the scope of the pre~ent invention there are preferably used those substances which are used as desorption a~ents in the case of immunosorptive purifications~ For this purpose, there are especiallg preferred acids, base~ and chaotropic . ions of the ~-Iofmeister series (lgotropic series) such as are described, for example, in "Structure and Stabil1ty of Biological l~lacromolecules"~ 1969, pub Marcel Dekker Inc.
New York, page L~27, as well as certain or~anic compounds or solvents, such as acetonitrile~ urea or glycerol.
~s appropriate acids which can be used in the scope o~
the present invention, there can be employed not only volatile but also non-volatile acids. l~ter impre~nation ~25~3~3 of the carrier material, volatile acids can easily be removed for the removal of the inhibiting action, for example by heating, the application of a vacuum or the like. In the case of non-volatile acids, an analogous effect can be achieved by the addition of a salt of a volatile acid which is decomposed by the non-volatile acid, with the liberation of the volatile acid. Preferred examples include acetic acid, propionic acid and hydro-chloric acid for the volatile acids. In the same way, volatile and non-volatile bases, for exarnple ammonia and t-phosphate, can be used.
~urthermore, as inhibitors, there can also be used or~anic compounds whicn can reversibly influence not only the protein but also the water structure and which are described, for example, in J.~. ~randts "Conformational ~ransitions ~f Proteins in l~ater" contained in "Structure and Stability of Biological Macromolecules"~ 1969, pub.
Marcel Dekker Inc.~ ew York, pp. 213 - 290. Glycerol and urea are hereby especially preferred.
As inhibitors, there can also be used chaotropic ions, such as thio~yanates and iodides. Examples of other appropriate ions include fluorides, bromides, perchlorates, guanidine and sulphates.Their abstraction for the purpose of removing the precipitation inhibition can take place by extraction with, for example, organic solvents or mixtures of organic solvents, for exaMple esters, or mixtures of org,anic solvents and water, fo~ example water/alcohol mixtures, possibl~ with the addition of ionophores or ~2S63~3 the li~e~ As a rule, it i~ hereb~ already suf~icient to chan~e the ionic stren~th in order to achieve the desired effect but a complete removal of the inhibitor can also tale place. ~;n addition of a complex former, such as eth~lenediamine-tetraacetic acid (~Dq'~), can also be considered, for example for the removal of inhibiting metal salts, such as ma~nesium chloride~
The molar concentrations of the immunc-reactive components leading to the precipitation can be varied ~ithin wide limits. In the case o~ the present invention, they are preferably used in such a manner that that component, the immuno-reac~ivity of which is to be utilisea in the finished carrier material, i~ not present in excess The component leadin~ to the precipitation is especiallg preferably used in the ratio of the ~eidelber~er maximum. This means that the two reaction components are used in the ratio which i~ most favourable for the pre-cipitation ThiS ratio can easily be previously ascertained by turbidity tests. '~n appropriate turbidity curve is to be found, for example9 in "r~ethods in Immunology and Immunochemistr~", Volume III9 pub. Academic Press, Chapter 13, page lO. In the case of the production of such a curve, the turbidit~ is plotted which is obtained in the case of constant amount of one of the reaction components with increasin~ amounts of the other reaction component q1he turbidity maximum is the ~Ieidelber~er maximum.
_9_ 63~
In or~er -to achieve maximum stability of the carrier-fixed immuno-reactive body, one of the components of the immuno~-precipitation should preferabl~ be a precipitating antibody ~Jith an affinity for the antigen which is as high as possible.
The compol1ents of the immuno-precip~tation in the scope of the present invention are~ on the one hand, an anti~en~
for example a hapten or a protein, and, on the other hand~
an antibod~ ~'he antigen can, of course, itself also be an antibody. In such a case, i.e. when the immuno-reactive component to be precipitated is itself an antibody, this can also be used as a ~ab, Fab' or (Fab')2 fra~ment, namely, not only of a monoclonal but also of a polyclonal antibod~ The precip~tating anti-antibody which is then used for the precipitation MUSt be correspondingly chosen~
The anti-antibody itself can then be polyclonal or mono-clonal or a (~ab')2 fra~ment thereof. If the anti-antibody is a monoclonal antibody or a lragment thereof, then this should preferably reco;~ni5e two different epitopes on the antigen. However, monoclonal antibodies or (Fab')2 fra~ments thereof can also be used which onl~ recognise one~epitope, which is more usual, lif this epitope is present at least twice on the antigen. ~ixtures of monoclonal antibodies can also be used as anti-antibody fraction for the immuno--precipitation~
~s imMuno~reactive material to be precip2tated, there can preferably also be used a pro~ein to which a hapten or antiuen is coupled In this case, a precipitatin~r antibod~
~2563~
is preferably used as ~e~nd Gomponent of the immune reaction which is directed against the protein. In a further embodiment, a hapten or antigen can be coupled to the precipitating antibody. q1he other component of the immune reaction can thereby be unlabelled or be coupled with the same or another hapten or antibod~7.
~lternatively, the protein to be precipitated is itself a specific antibody which is precipitated bg an anti-antibod~.
lo As already mentioned above, it is a special advantage of the process according to the present invention that at least one component of the immune reaction can be used in non-purified form. In contradistinction to the known immuno-precipitation processes, in which the precipitation takes place immediately after combination of the immune reaction components, in the case of the process according to the present invention, the speed of the precipitation in suspension or in the porous carrier material can take place controlled in such a manner that no entrainment effects ocçur in the case of foreign substances and the latter do not have to be previously removed but rather can simplv be washed out after the immuno-precipitation.
Such a washing step is preferrcd since no precipitate particles, less stable immun~-complexes and adsorptively bound antigens are herebg removed. ~herefore, for the washing, there can be used buffers of high ionic strength, deter~ents, organic solvents and the like, as well as additives ~tabilising the precipitates, such a~ carbo-hydrates and proteins.
~L~2,S~ii3~3 If a ~revious purirication of the components of the im~une precipitation purification i~ to be omitted, it is preferable Pirst to mix the particular antigen and the particular antibody in the optimum (Heidelberger) ratio, to wash the precipitate obtained and a~ain to dissolve by the addition of the inhibitor of the immuno~precip-itation, preferably by the addition of acid. ~here is thus obtained a mixture of the components of the immuno~
-precipitation reaction which can be applied direc~ly to the porous carrier material.
lithin the scope of the present invention, it is, of course, also possible previously separately to purify the components oi the immuno--reaction in the usual wa~, via immuno-adsorbers, then to elute with an inhibitor and either to use the eluates directly for the process accordin~ to the present invention or first to produce a storage-stable form of the reaction cornponents by~ lyo-philisation. 'rhe inhibition of the immuno-precipitation, as well as the slowin~ down of the precipitation in the carrier material after removal of the inhibitin~ action can be assis-ted by physical methods, for example lowering the temperature or increasing the viscosity.
~s carriers within the scope of the present invention, there can be used carriers which are conventional for immu~o-reactive substances. ~uch a solid c~rrier material, which is frequentl~ also referred to as a matri}~, can be made, for example, of glass, synthetic resin, oaper, porous ~letal and the Iike, provided that the carrier _12-~.25~
material is sufficiently permeated by interconnected, liquid-permeable hollow ~paces I~atural, synthetic, organic or inorganic poIymers can be used. Furthermore, fibrous, spongy or sintered substances can be used.
As carrier materials~ there can be used planar, partic-ulate or other three-dimensional porous bodies. However, it is preferred to use planar 7 porous carriers, such as paper, films of foamed material, glass fibre mat~ and the like. Since the process according to the present invention gives a completely homogeneous distribution of the immuno-reactive substance in tbis planar matrix, a dosaging can take place extremely simply on the basis of unit surface areas~ Such immuno-reactive, planar carrier materials are especially suitable for use as solid phases in heterogeneous immunoassays.
~he use of an immunQ-reackive carrier material produced accordingr to the presen-t invention in the scope of a hetero~enous enzyme immunoassay can, for example, take place in such a manner that hap~en (H) or protein ~P)~
which is contained in a sample, such as buffer solution, serum, plasma, urine culture supernatant or the like, is mixed with a labelled binder (B). ~s binders, there can be used, inter alia, antibodles, Fab fra~ments or Fab fragments, as well as li~ands, which react specific-ally with the hapten or protein ~s labellin~ for the binder, there can be used, ~or example, an enzyme, a fluorescent label or radioisotope and, in the following Examples~ ~ galactosidase is used. ~he molar amount of ~256~8 the added binder can be not only in excess but al~o in in~ufficiency with regard to the hapten or protein present in the sample, T~is mixture is incubated for a constant time during which the comple~es H-B or P-~ are formed. After the expiry of this period of time, three species are present in the reaction mixture, namely, the complex consisting of hapten/protein, and binder (E-B, P-B), residual free hapten/protein (EI/P) and residual free binder (B).
The separation of these species takes place in a second step by means of immunosorption For this purpose, the mixture is applied to immuno-precipitation solid phase pro~uced accordin6 to the present invention which contains bound the hapten to be detected or the antibody to the protein to be determined.
In the case of the hapten test, the free binder but not the binder saturated with hapten binds to the solid phase~
Conse~uently~ the supernatant or the eluate of the solid phase contains the binder saturated with the hapten of the sample. The quantl~ati~e determination of the hapten now ta~es place via the labelling of the binder and., in the following ~xamples, via the determination of ~-galactr-osidase by m~ans of o-nitrophenyl-~-D-galactoside or chlorophenol red ~-D-galactoside.
In the case of the protein test, the complex of pro-tein and binder but not the free binder binds to the solid phase, residues of the free binder are removed by washing from the immunosorbent produced according to the present -lL~_ ,~, .
c invention The quantitative detection of the protein takes place via the labelled binder and, in the following Examples, via the determination of the ~-galactosidase by means of Q-nitrophenyl-~-D-galactoside or chlorophenol red ~~D-galactoside Another use is in competitive imm~no-tests. ~he use of the immuno-reactive carrier material prcduced accordin~ to the present invention can take place in such a manner that the analyte (hapten or protein), which is contained in the sample, is mixed with a constant amount of labelled analyte. '~he labelling can be, for example, an enzyme, a fluorescent label, radioisotope or the like. ~his mixture is applied to the matrix. On the matrix, there is immobil-ised an antlbody which i5 directed against the analytesc The mixture is incubated on the matrix for a definite time.
During this time, not only unchan~ed analyte but also labelled analyte compete for the binding places on the matrix. The more analyte is present in the sample, the le~s labelled analyte is bound by the matrix and vice versa. ~t the end of the incubation phase, the liquid is removed from the porous matrix, for example by centri-fuging. ~he amount of labelled analyte is then determined either in the ~ree phase or bound to the matrix.
Ano~her use of the carrier material produced according to the present inventlon can take place in such a manner that the analyte (hapten or protein) is mixed ~lith a constan-t amount of labelled antibody which is directed against the analyte. Possible kinds of labelling o~ the antibody have been describe~ hereinbeforeO ~hi~ mixture ~,z56368 is then either incubated for a definite time and then applied to the porous carrier material or, alternatively, applied to the porous carrier material immediatel~ after mi~ing. If the analyte is a hapten, the carrier material contains the hapten to be detected or a derivative thereof in fixed form and if the analyte is a protein, the carrier material contains -the protein to be detected or a derivative thereof also in fixed form. If the first rni.~ture is incubated be:Eore application to the carrier material, then labelled antibody bmds wlth remainLng free binding places ~o the porous carrier material. If the mixture i~ applied immediatel~ to the carrier material, then the anal~te from the sample and the analyte fi~ed on th~ carrier material compete for the bindin~ places of the labelled antibody. ~t the end of the incubation phase, the liquid is reMoved from the porou~ carrier material and the amount of labelled antibod~ is deter-mined either in the li~uid phase or on the porous carrier material.
The inven-tion is further described with reference to -the accompanying drawings and Examples, in which:
Fig.l shows a calibration curve for the digoxin immune precipitate fleeces according to Example 3;
Fig. 2 shows a calibration curve analogously to Eig. 1 for digoxin immune precipi-tate fleeces accord-ng to Example ~ (not an example according to the nvention);
2563~
Fig. 3 shows a calibration curve analogously to Fig. 1 for digoxin immune precipitate fleeces accord-ing to Example 5;
Fig. 4 shows a calibration curve for a DPH immune precipitate fleece according to Example 6;
Fig. 5 shows a calibration curve for the TSH
immune precipitate fleece according to Example 7;
Fig. 6 shows a calibra-tion curve for the TSH
immune precipitate fleece according to Example 8, and Fig. 7 shows in -the form of a graph the formation of precipitate dependent on the pH according to Example 9.
'rhe following ~xample~ are given for the purpo~e of illustrating the pre~ent invention.
Obtaining the materials u~ed for the test5 and carrying out of the test:
a) Produr~tion of pol~ha~ten~ (PH):
'~he production of P~ kno~m in the art. '~hu~ for example, a di~oxin P~ or a diphenylhydantoin PH can be obtained via reacti~e asymmetrical dicarboxylic acid ester/activated hapten ester and binding thereof to a carrier protein.
-16a-., .... , . , . . , . . . , . , . ~ .. .. .. . . . .. . ..
~ ~;25Ei3~
The preparation of T3- and ~4-PH can take place 7 for example, by direct coupling of the N~2 group~ of horMone and protein by means of bis-imidates (see European Patent Spccification A 0078952) or by the carbodiimide reaction (see Aherne et aI., Brit. J. Clin~ PharmO, 39 56/1976) or b~ the mixed anhydride reaction (see ~rlanger et al , Methods in Immunolo~y and Immunochemi~try, ed. Williams and Chase, pub. Academic Press, New York, 1967, pp. 149 et ~ lternative~y, in a first ~tep, the ~2 function of ~3 or T4 can be protected with an acetgl, trifluoro-acetyl, tert.-butoxycarbonyl or benzJloxycarbonyl group.
Subse~uently~ the carboxyl function is converted into an activated ester, for example N-h~droxy~uccinimide e~ter, N_hydroxyphthalimide ester, N--hydroxybenztriazole esterO
An example of such an activated T'~ is described in ~xample 3 of European Patent Specmfication A 0108400. Reaction with the carrier protein grives the PH.
Ihe choice of the carrier proteins is not subjected to any limitation~ in~ofar as a corresponding "precip-itating" antibody is available or can be produced.
Insofar as a immunosorptive purification of antigen and an-tibody has taken place, a~ carrier there was used the "affinity adsorber, glutardialdehyde activated" of Boehrin~er Mannheim G~bH (order No~ 665525) ~'or t he ~xamples described hereinafter, accordin~ to the instrllc-tions of the manufac-turer, for the antigen puriEication a sheep antibod~ against rabbit IgG or a ~pecific anti-body a~ainst the hapten was bound to the carrier and for ~ 2X~3~3 - "
the antibody purification rabbit I~G was bound to the carrier, The immunosorption took place as described in the worlcinv instructions for the affinity ad~orbent.
b) Production of the binder (di~oY~in as examPle~:
Obtaining the antiserum directed a~ainst dig~oxin, The production of the immunogen, namely, human serum albumin conjugated with digoxin, took place in the manner described in detail by V.P. 3utler jr, and J.E. Chen., Proc. Nat. Acad, Sci, US., 51~ 71-78/1967~ as well as by T.',l. Smith, V.P. Butler and ~. ~aber, Biochemistry~ 9, 331-337/1970~ Sheep were immunised with this immuno~en and the corresponding antiserum obtained.
Production of the binder.
Antiserum directed against digoxin was purified to the IgG fraction via ammonium sulphate precipitation and passage over D~E-cellulose. Papain splittin~ was carried out according to the method of R~ ~o Porter (Biochem. J~, 73~ 119-126/1959)~
The ~ab fragments were separated off from the non-digested IgG molecules and the Fc fra~ments by means of gel filtration over Sephadex~G 100 and ion exchan~er chromatography over DE~E-cellulose according to the methods described in the literature (see K. rlalinowski and ~iO
Manski in "llethods in Enzymology"; J,J. ~angone and H, van Vunakis ed~., pub. Academic Press, Vol. 73, 418-459/19~
The resulting ~ab fraction ~as purified chromatographically and coupled to ~-galactosidase according to the method of T. Yitiwaga in 'l~nzyme Immunoassay , I~hikawa, T. Kawai and h, ~'iyai eds., I~aku 15hoin, Tokyo-i~lew York, pp~81-89/1981 ~ Trade Mark -18-. . . , . . . ~
~;~56~
c) Pro_uction of sheep antlbod~l a~ainst rabbit or mouse I~G/FcY.
Rabbit slau~rhter serum or mouse serum was subjected to an ammonium sulphate precipitation. A~ter passage over DEAE-cellulose and papain splittin~, gel and ion exchange chromatography (see b)), there are obtained the Fc fra~ments of the rabbit and mou~ IgG's as immuno~ens.
'rhe working up of the sheep antisera took place as described under b) (immunosorption see a))O
d) Carr~,~in~ out of the test (di,roxin as example).
Digoxin standards in human serum (taken from the 3~I~A
kit of Boehringer Mannheim GmbH; order No. 199656) were diluted 1:7~5 with 0.9,o aqueous sodium chloride solution.
200 ~ 1~ diluted standard and 200 Jll. binder (20 mU/mlO, ~etermined with o-nitrophenyl-~-D-galactoside), produced as described in b), were incubated in phos~hate-buffered ~aline (PBS)(pH 7.2) at 37C~
Thereafter, 50 JLl. of the mixture were applied to 1 cm2 of the planar, immuno-reactive carrier material, Incubation is carried out for a further 5 minutes at 37C., followed by centrifugation for 1 minute in an Eppendorf centrifu~e, '~he enzyme activitY of the dit~oxin-binder complex can now be determined iin the free pha~e kinetically by ~he addition of chlorophenol red ~D-t~ralactoside (prepared according to Federal Republic of Germany Patent Specification Mo. 33 45 74~) and measured at 578 nm.
~;Ei3E;8 In the Examples, ther0 is, in each case, shown the course of the "rising" calibration curve, the digoxin concentration o~ the undiluted serum (X axis) being pIotted against the measured optical density at 578 nm (Y axis).
Alternatively, the excess amount o~ binder, i.e. the amount bound to the solid phase, can be determined as above and plotted against the particular di~oxin con-centration, a "decreasing" calibration curve being lo obtained.
Example l.
Preparation of ~3 (triiodothyronine) immune Precipitate fleece.
As anti~en, there was used polyhapten consisting of rabbit I~G and T3 bound thereto (mole ratio IgG:T3 = 1 3) ~he antigen was purified immunosorptively over an anti-~3 column (see a)).
As antibody, there was used polyclonal antibody directed a~ainst the ~c part o~ the rabbit I~G~ ~he antibody was purified immunosorptively over a rabbit I~G
column (see a)).
As ~leece, -there was used a mixed fleece of cellulose and polyester (obtained from I~alff, Euskirchen, Germany) or o~ lint, cellulose and polyamide (obtained fr~ Binzer3 Hatzfeld, Eder~ Germany).
~ntigen and antibody were taken up separatel~ in l mM
acetic acid (in each case c = l mg /ml.) and left to stand for l hour at ambient temperature. ~he solutions ~2563~
were then mixed in a ratio of 1:3 (the antigen/antibody ratio t~at corre~ponded to the Heidelberger maximum) and diluted with a nlnefold volume of a solution of 250 mM
acetic acid and 5 mM sodium acetate~ , The fleece was drawn through the impre~nation solution, squeezed out and dried for 60 minutes at 30C. in a circulating-air drging cabinet. It was then wa~hed by descending chromatography for 1 hour with PBS buffer ~(pI~ 6.0) and dried for 30 minutes at 30C. in a circulating air dr~ing cabinet, ~ r'he ratio of measurement range (standard 5 ng. ~3/ml, -standard 0) to standard 0 was llo 8 ( see ~able 1)~
' ~able 1 Comparison_of ~3 immun_,~recitate fleece ~e~ared b~
different processes . . . _ proce~ ~xample measurement range/standard 0 _ ~
hom. 2-step 2 12.0 hom. l-step 1 11.8 heterog. 2-step analogue 4 1'.6 suspension analo~ue 5 0.19 _ __ . ~
Example 2.
~reParation of ~ immune precipi,tate fleece (homo~eneous
2-step process),, ~ ntigen, antibody and fleece are the same as in Example lo ~he fleece is impregnated wit~ 50 mM aqueou~ sodium chloride solution and subsequently dried ~or 3G minutes at 50C, in a circulating-air drying cabinet.
, -21-., --; :~ .
~ntif3en and antibody are taken up separately in l acetic acid (in each case c - l mg./ml.) and left to stand for l hour at ambient temperature. The two solutions are mixed in a ratio of 1:3 and diluted with a ninefold volume of 50 m~l acetic acid.
The soIution is applied to the fleece (liquid satur-ation of the fleece) and the impregnated fleece is dried for 6~minutes at 30C. in a circulating~air dr~ying cabinet.
It is then ~ashed by descending chromatofrraphy ~ith PBS
buffer (pH 6.0) and dried for 30 minutes at 30C. in a circulating-air dryin~ cabinet.
'~he ratio of measurement range (standard 5 n,,, 'lt3/ml.
sta-ndard 0) to standard 0 in 12.0 (see ~able 1).
Example ~.
Preparation of difroxin immune precipitate fleeces homofreneous l-step process).
As antigen, there was used polyhapten~ consisting o`
rabbiit I3G ahd di~oxin bound thereon (IgG:di30xin 1:3 to 1:4). qthe antigen was purified immunosorptively via an anti~ Fc' colur,~n (see a)), As antibody there was used the same polyclonal antibody as in 3xample l.
As fleece, there ~s used a mixed fleece of cellulose and polyester (obtained from Kalff, 3uskirchen, Germany).
,~ntigen and antibody were taken up se?arately in lO0 mM
acetic acid in concentrations of l m~,/ml. and ~.5 mæ,/ml., respectively~and the two solutions mixed in a ratio of 1:1 '~he irpref~nation solution was then diluted with the four-;6368 $
fold volume of 2C0 mM sodium citrate buf~er (pH 3~0), ~he ~leece was drawn through -the impregnation solution, squeezed out, dried for 10 minutes at 75C with circul a-tin~ air and subsequentlg washea as in Example 1 and dried as before.
~he calibration curve sho~m in Fig. 1 of the accompany~
ing drawings was obtained with the so-produced digoxin immune precipitate fleeces, The standards 0~ 0.3, 0.75, 1.75, 3 and 5 ng. digoxin/ml. were used. ~he ratio of measurement range (standard 5 ngO digoxin~ml. - standard 0) to standard 0 was 4.9 ~xample ~a~
Pre~aration of_digoxi mune precipitate fleece (without washin~ step).
l~ntigen (200 mg,/10) and antibody (700 m~./l.) are taken up separately in 50 rnM acetic acid, After 15 to 30 minutes at ambient temperature, the two solutions are mixed in the ratio of 1:1. In a subsequent ~tep, this soIution is diluted with the same volume of 50 mM acetic, acid plus 40 mM sodium chloride. Fleece are impregnated t'nerewith and then dried for 10 minutes at 75G~
~he fleece is used directly in the test, ~he ca~ibration curve obtained therewith corresponds to ~'ig. 1 of the accompanying drawings~
Example 4 (comparison)~
Prel~aration of di~oxin immune preci~itate fleece (he-tero~enous 2-step process).
~2563~
Antig2n, antibody and fleece are the same as in ~xample 3.
Anti~en and antibod~ are taken up separa-tel~ in 1 ~rl acetic acid (c = 5 mg /ml. and 17.5 mg,/mlO, respectively) ~hey are lef-t to stand for 30 minutes at ambient temper-ature, then the antigen solution is diluted with a 50 fold volume of PBS buffer (pH 600) and impregnated on to the fleece which is dried for 10 minutes at 75C. in a circul-ating-air dr~ing cabinet. ~he dried ~leece is subsequently impregnated ~rith the antibody solution which have been di~uted 50 fold with the above buffer, followed by drying for 10 minutes at 75Co ~fter washing the fleece with the same buffer, it is dried for 10 minutes at 75C.
The curve obtained from the calibration ~raph is shown in ~ig, 2 of the accompan~ing drawings. ~he ratio of measure-ment range (s-tandard 5 ng. digoxin/ml. - standard 0) to standard 0 is 1.6.
As an alternative to the above procedure, the antibody can be impregnated first of all, followed by the antigen.
Example 5 (com~arison)~
.
Pre~aration o~ di~oxin immune precipitate fleece (suspension process), Antigen, antibody and fleece are the same as in ~xample ~0 Antigen and antibody are each taken up in 100 ~ acetic acid (c = 1 mg~/ml, and 3.5 mg~/ml., respectively) and left to stahd for 15 minutes at ambient temperature. 1 part of the anti~en solution, 1 part of the antibod~ solution and 1~5 part~ of 100 r~M potassium phosphate buffer (pH 7.6) ~re o mixed and then left to stand for l hour at a~bient temperature. ~he suspension formed is centrifuged off, the supernatant is discarded and the precipitate is resuspended (homogenised) in a 9 fold volume of PBS buffer ~pH 6.0). lThe suspension is impregnated on to the fleece in such a manner that the fleece is saturated with liquid. ~hereafter, the fleece is dried for lO minutes at 75C. in a circurating-air drying cabinet. After washing for l hour with PBS buffer (p~ 6.0), the fleece is again dried for lO minutes at 75C.
The curve obtained from the calibration~raph is shown in Fi~, 3 of the accompanying drawinOsO The ratio of mea~ure-ment range (standard 5 ng. digoxin/ml~ - standard 0) to standard 0 is 0.19.
Example 6.
Preparation of di~hen~lh~dantoin (DP~) immune ~recipitate fleece (homo~eneous l-step process with, precipitate washin~s).
A~ antigen, there was used polyhapten consisting of rabbit IgG and diphen~lhgdantoin bound thereon. --As antibody, there was used polyclonal antibody directed against the ~c part of the rabbit IgG.
~s fleece, there was used a mixed fleece of cellulose and polyester (obtained from ~alff, ~uskirchen, Germany), ~;ntigen and antibody (each purified non-immunosorptivel~) are~ch taken up in lO0 mM acetic acid (c = 1 mg./ml, and 20 mg,~ml. 9 respectively) and left to stand for 15 minutes at ambient temperature, ~:~5~316~
l part of antigen solution, 2 parts of antibody solution and 2 parts of lO0 mM potassium phospha~e buffer (pH 7.6) are mixed and the su~ ension left to stand for l hour at ambient temperature. The precipitate is centrifuged down the supernatant is discarded and the precipitate is resuspended in a ninefold volume of 100 m~ potassium phosphate buffer. This centrifugation /washing step is repeatel three times and th~n the precipitate is washed twi~e in the same manner with distilled water to remove lo the salt.
~he precipitate is taken up in 100 mM acetic acid (c = 5 mg./ml.). ~he clear antigen/antibody solution is left to stand for 15 minutes at ambient temperature and then diluted with a fivefold volume of 200 mM sodium citrate buffer (pH ~.0), ~he fleece is drawn through this impregnation solution~
squeezed out, dried for lO minutes at 75C, and subsequently washed as in Example l and dried as before.
~he calibration curve shown in Fig. 4 of the accomp-a ~, ~ drawings is obtained with the so-produced DPH immune precipitate fleece. ~he standards 0. 2.5, 57 lO~ 20 and 30 ~ g. DPH/ml. are used. ~he ratio of m~asurement range (standard 30 ~ g. DPH/ml. - standard 0) to standard 0 is 18.2.
~xample 7.
Preparation of ~H immune precipitate flesce (homogeneous l~step process with ~recipitate washing~).
~ ~5636~3 ,`
As antigen there was used monoclonal antibody directed a~ainst human TSH. The antibody (from ascites) was purified by ammonium sulphate precipitation and chromato~raph~ on D~A~-cellulose.
As antibod~ there was used polyclonal antibody directed against the Fc part of mouse IgG. The antibody was purified by ammonium sulphate precipitation and chromatography on D~AE-cellulose.
As fleece there was used a mixed fleece of cellulose and lo polgester (obtained from ~alff, ~uskirchen, German~).
Antigen and antibod~t we~e each taken up in 100 mM
potassium phosphate buffer (pH 6.0) and 0~9,v sodium chloride (c = 1 m~,~/ml. and 17 mg~/ml., respectively). The two solutions were mixed and the suspension then left to stand for 30 minutes at ambient temperature. The precipitate was centrifuged down, the supernatant was discarded and the precipitate was resuspended in a ninefold volume of the above buffer. Ihis centrifugation ywashing procedure was repea-~ted five times and then the precipitate was washed twice in the same manner with distilled water to remove the salt.
The precipitate was taken up in 5G mM acetic acid (c = 5 mg./ml.), the clear anti~en/antibody solution then left to stand for 15 minutes at ambient temperature and subse~uentl~ diluted ~ith a fourfold volume of 20 rnM sodium acetate ~olution.
The fleece is drawn throu~h this impregnation solution, squeezed out and then dried for 30 minutes at 37C. in a ~25636~3 circulating-air drying cabinet. It is then washed for 1 hour by descending chromatography with phosphate buffer (pH 6.5) and dried for 30 minutes at 37C.
The calibration eurve shown in Fig. 5 of the accompanying drawings was obtained with the so-produced TSH immune precipitate fleece. The standards 0, 2, 6, 12, 24 and 48 ~U TSH/ml. were used.
Example 8 Preparation of TSH immune preeipitate fleeee (homo-geneous 2-step proeess with precipitate washing) The washed precipitate of Example 7 was used.
Use was also made of the same fleeee as in Example 7.
The dried fleece was subsequently impregnated with 50 mM aqueous sodium ehloride solution and dried for 10 minutes at 75C.
The preeipitate was taken up in an amount of 500 mM aeetie aeid whieh was just suffieient to dis-solve the preeipitate. It was then diluted with 5 mM
aeetie aeid to c = 1 mg./ml. The solution was applied to the fleece in an amount suffieient to saturate the fleeee with the liquid. The fleece was then dried for 30 minutes at 30C. in a eireulating air drying eabinet. The fleece was then washed chromato-graphically with sodium acetate solution and sub-sequently with phosphate buffer (pH 6.5) and dried at 37C.
The calibration eurve shown in Fig. 6 of the aceompanying drawings was obtained with the so-produeed TSH immune precipitate fleeee. The standards 0, 2, 6, 12, 24 and 48 IU TSH/ml. were used.
i~
5~ ;8 ~ample 9.
Comparison of the ~tabilities of _,the impregnation solutions.
Antigen and antibo~y are the same as in ~xample 1.
Antigen and antibody are, in each case, separately dissolved in a solution of inhibitor, O~lM aqueous sodium chloride solution and 0.5~ bovine serum albumin. ~he t~o solutions are adjustcd ~lth 0.2~ h~drochlori~ acid (acetic acid and propionic acid give comparable results) to pEI 6.0, p~I 4.0 and pI-I 2Ø 'rhe solutions of anti~en and antibody of the same ~ are then mixed and the chronolosical course of the precipitate formation is monitoredr Fig. 7 of the accomp-anying drawings shows that at pH 6.0, immediately after mixing of antigen and antibody, immuno-precipitation takes place. A~ pII 4.0 there is a poorer precipitation which is not completed after 1 hour and at pH 2.0 precip-itation no longer occurs.
Example 10.
Preparation of ~r4 ~tetraiodothyronine) iMmune ~recipitate fleece~(without wash ~
As antigen there was used polyhapten consisting of rabbit I~G and ~4 bound thereon (mole ratio of IgG:'~4,=
1:3). The antigen was purified immunosorptively over an anti-T4 colu~nn (see a)).
As antibody there wa~ u~ed polyclonal antibody directed against the ~'c part of the rabbit IgGo The antibody was purified immunosorptively over a ra~bit IgG column (see a)).
. ~ .
~L2S~8 i~s fleece there was used a mix-ture of cellulose and polyester (obtainable from Kalff, Eu~kirchen~ Germany) or of lint, cellulose, polyamide (obtainable from Binzer, ~atzfeld, Eder, Germany).
Pre~aration of the m trix:
~ ntigen (400 mg./ 1 ) and antibody (1400 m~,/l ) were taken up separately in 50 mM acetic acid plus Oo5,c ~dium chlorideD .~*ter 15 to 30 minutes, both solution~ were mixed in a ratio of 1/1 and fleece impregnated therewith.
~fter dr~ing for 10 minutes at 75C., the fleece was impregnated ~ith PBS and dried for 10 minutes at 75~0 Example lOa, Presaration of Tl~ immune precipitate fleece ~with ~Jashing ~tep)~ -Antic~en (2 mg./ml.) and antibody (7 mg./ml.) aredissolved separately in distilled water. Both solutions are diluted with 50 mM aqueous trisodium phosphate ~olution in the ratio of 1 + ~, ~qual volumes of the diluted solutions are mixed and fleece impre~na-ted therewith, The fleece are dried for 10 minutes at 75C. in a circulating-air drying cabinet. Subsequently, the fleece are wa hed by descending chromatography ~/ith 10 mM phosphate buffer~until the eluate has a p~I value of 5Ø lifter washing, the fleece are dried, ~xam~le lOb ~repar~tion of T~ immune precipitate flcece (without as,lin~r step),
, -21-., --; :~ .
~ntif3en and antibody are taken up separately in l acetic acid (in each case c - l mg./ml.) and left to stand for l hour at ambient temperature. The two solutions are mixed in a ratio of 1:3 and diluted with a ninefold volume of 50 m~l acetic acid.
The soIution is applied to the fleece (liquid satur-ation of the fleece) and the impregnated fleece is dried for 6~minutes at 30C. in a circulating~air dr~ying cabinet.
It is then ~ashed by descending chromatofrraphy ~ith PBS
buffer (pH 6.0) and dried for 30 minutes at 30C. in a circulating-air dryin~ cabinet.
'~he ratio of measurement range (standard 5 n,,, 'lt3/ml.
sta-ndard 0) to standard 0 in 12.0 (see ~able 1).
Example ~.
Preparation of difroxin immune precipitate fleeces homofreneous l-step process).
As antigen, there was used polyhapten~ consisting o`
rabbiit I3G ahd di~oxin bound thereon (IgG:di30xin 1:3 to 1:4). qthe antigen was purified immunosorptively via an anti~ Fc' colur,~n (see a)), As antibody there was used the same polyclonal antibody as in 3xample l.
As fleece, there ~s used a mixed fleece of cellulose and polyester (obtained from Kalff, 3uskirchen, Germany).
,~ntigen and antibody were taken up se?arately in lO0 mM
acetic acid in concentrations of l m~,/ml. and ~.5 mæ,/ml., respectively~and the two solutions mixed in a ratio of 1:1 '~he irpref~nation solution was then diluted with the four-;6368 $
fold volume of 2C0 mM sodium citrate buf~er (pH 3~0), ~he ~leece was drawn through -the impregnation solution, squeezed out, dried for 10 minutes at 75C with circul a-tin~ air and subsequentlg washea as in Example 1 and dried as before.
~he calibration curve sho~m in Fig. 1 of the accompany~
ing drawings was obtained with the so-produced digoxin immune precipitate fleeces, The standards 0~ 0.3, 0.75, 1.75, 3 and 5 ng. digoxin/ml. were used. ~he ratio of measurement range (standard 5 ngO digoxin~ml. - standard 0) to standard 0 was 4.9 ~xample ~a~
Pre~aration of_digoxi mune precipitate fleece (without washin~ step).
l~ntigen (200 mg,/10) and antibody (700 m~./l.) are taken up separately in 50 rnM acetic acid, After 15 to 30 minutes at ambient temperature, the two solutions are mixed in the ratio of 1:1. In a subsequent ~tep, this soIution is diluted with the same volume of 50 mM acetic, acid plus 40 mM sodium chloride. Fleece are impregnated t'nerewith and then dried for 10 minutes at 75G~
~he fleece is used directly in the test, ~he ca~ibration curve obtained therewith corresponds to ~'ig. 1 of the accompanying drawings~
Example 4 (comparison)~
Prel~aration of di~oxin immune preci~itate fleece (he-tero~enous 2-step process).
~2563~
Antig2n, antibody and fleece are the same as in ~xample 3.
Anti~en and antibod~ are taken up separa-tel~ in 1 ~rl acetic acid (c = 5 mg /ml. and 17.5 mg,/mlO, respectively) ~hey are lef-t to stand for 30 minutes at ambient temper-ature, then the antigen solution is diluted with a 50 fold volume of PBS buffer (pH 600) and impregnated on to the fleece which is dried for 10 minutes at 75C. in a circul-ating-air dr~ing cabinet. ~he dried ~leece is subsequently impregnated ~rith the antibody solution which have been di~uted 50 fold with the above buffer, followed by drying for 10 minutes at 75Co ~fter washing the fleece with the same buffer, it is dried for 10 minutes at 75C.
The curve obtained from the calibration ~raph is shown in ~ig, 2 of the accompan~ing drawings. ~he ratio of measure-ment range (s-tandard 5 ng. digoxin/ml. - standard 0) to standard 0 is 1.6.
As an alternative to the above procedure, the antibody can be impregnated first of all, followed by the antigen.
Example 5 (com~arison)~
.
Pre~aration o~ di~oxin immune precipitate fleece (suspension process), Antigen, antibody and fleece are the same as in ~xample ~0 Antigen and antibody are each taken up in 100 ~ acetic acid (c = 1 mg~/ml, and 3.5 mg~/ml., respectively) and left to stahd for 15 minutes at ambient temperature. 1 part of the anti~en solution, 1 part of the antibod~ solution and 1~5 part~ of 100 r~M potassium phosphate buffer (pH 7.6) ~re o mixed and then left to stand for l hour at a~bient temperature. ~he suspension formed is centrifuged off, the supernatant is discarded and the precipitate is resuspended (homogenised) in a 9 fold volume of PBS buffer ~pH 6.0). lThe suspension is impregnated on to the fleece in such a manner that the fleece is saturated with liquid. ~hereafter, the fleece is dried for lO minutes at 75C. in a circurating-air drying cabinet. After washing for l hour with PBS buffer (p~ 6.0), the fleece is again dried for lO minutes at 75C.
The curve obtained from the calibration~raph is shown in Fi~, 3 of the accompanying drawinOsO The ratio of mea~ure-ment range (standard 5 ng. digoxin/ml~ - standard 0) to standard 0 is 0.19.
Example 6.
Preparation of di~hen~lh~dantoin (DP~) immune ~recipitate fleece (homo~eneous l-step process with, precipitate washin~s).
A~ antigen, there was used polyhapten consisting of rabbit IgG and diphen~lhgdantoin bound thereon. --As antibody, there was used polyclonal antibody directed against the ~c part of the rabbit IgG.
~s fleece, there was used a mixed fleece of cellulose and polyester (obtained from ~alff, ~uskirchen, Germany), ~;ntigen and antibody (each purified non-immunosorptivel~) are~ch taken up in lO0 mM acetic acid (c = 1 mg./ml, and 20 mg,~ml. 9 respectively) and left to stand for 15 minutes at ambient temperature, ~:~5~316~
l part of antigen solution, 2 parts of antibody solution and 2 parts of lO0 mM potassium phospha~e buffer (pH 7.6) are mixed and the su~ ension left to stand for l hour at ambient temperature. The precipitate is centrifuged down the supernatant is discarded and the precipitate is resuspended in a ninefold volume of 100 m~ potassium phosphate buffer. This centrifugation /washing step is repeatel three times and th~n the precipitate is washed twi~e in the same manner with distilled water to remove lo the salt.
~he precipitate is taken up in 100 mM acetic acid (c = 5 mg./ml.). ~he clear antigen/antibody solution is left to stand for 15 minutes at ambient temperature and then diluted with a fivefold volume of 200 mM sodium citrate buffer (pH ~.0), ~he fleece is drawn through this impregnation solution~
squeezed out, dried for lO minutes at 75C, and subsequently washed as in Example l and dried as before.
~he calibration curve shown in Fig. 4 of the accomp-a ~, ~ drawings is obtained with the so-produced DPH immune precipitate fleece. ~he standards 0. 2.5, 57 lO~ 20 and 30 ~ g. DPH/ml. are used. ~he ratio of m~asurement range (standard 30 ~ g. DPH/ml. - standard 0) to standard 0 is 18.2.
~xample 7.
Preparation of ~H immune precipitate flesce (homogeneous l~step process with ~recipitate washing~).
~ ~5636~3 ,`
As antigen there was used monoclonal antibody directed a~ainst human TSH. The antibody (from ascites) was purified by ammonium sulphate precipitation and chromato~raph~ on D~A~-cellulose.
As antibod~ there was used polyclonal antibody directed against the Fc part of mouse IgG. The antibody was purified by ammonium sulphate precipitation and chromatography on D~AE-cellulose.
As fleece there was used a mixed fleece of cellulose and lo polgester (obtained from ~alff, ~uskirchen, German~).
Antigen and antibod~t we~e each taken up in 100 mM
potassium phosphate buffer (pH 6.0) and 0~9,v sodium chloride (c = 1 m~,~/ml. and 17 mg~/ml., respectively). The two solutions were mixed and the suspension then left to stand for 30 minutes at ambient temperature. The precipitate was centrifuged down, the supernatant was discarded and the precipitate was resuspended in a ninefold volume of the above buffer. Ihis centrifugation ywashing procedure was repea-~ted five times and then the precipitate was washed twice in the same manner with distilled water to remove the salt.
The precipitate was taken up in 5G mM acetic acid (c = 5 mg./ml.), the clear anti~en/antibody solution then left to stand for 15 minutes at ambient temperature and subse~uentl~ diluted ~ith a fourfold volume of 20 rnM sodium acetate ~olution.
The fleece is drawn throu~h this impregnation solution, squeezed out and then dried for 30 minutes at 37C. in a ~25636~3 circulating-air drying cabinet. It is then washed for 1 hour by descending chromatography with phosphate buffer (pH 6.5) and dried for 30 minutes at 37C.
The calibration eurve shown in Fig. 5 of the accompanying drawings was obtained with the so-produced TSH immune precipitate fleece. The standards 0, 2, 6, 12, 24 and 48 ~U TSH/ml. were used.
Example 8 Preparation of TSH immune preeipitate fleeee (homo-geneous 2-step proeess with precipitate washing) The washed precipitate of Example 7 was used.
Use was also made of the same fleeee as in Example 7.
The dried fleece was subsequently impregnated with 50 mM aqueous sodium ehloride solution and dried for 10 minutes at 75C.
The preeipitate was taken up in an amount of 500 mM aeetie aeid whieh was just suffieient to dis-solve the preeipitate. It was then diluted with 5 mM
aeetie aeid to c = 1 mg./ml. The solution was applied to the fleece in an amount suffieient to saturate the fleeee with the liquid. The fleece was then dried for 30 minutes at 30C. in a eireulating air drying eabinet. The fleece was then washed chromato-graphically with sodium acetate solution and sub-sequently with phosphate buffer (pH 6.5) and dried at 37C.
The calibration eurve shown in Fig. 6 of the aceompanying drawings was obtained with the so-produeed TSH immune precipitate fleeee. The standards 0, 2, 6, 12, 24 and 48 IU TSH/ml. were used.
i~
5~ ;8 ~ample 9.
Comparison of the ~tabilities of _,the impregnation solutions.
Antigen and antibo~y are the same as in ~xample 1.
Antigen and antibody are, in each case, separately dissolved in a solution of inhibitor, O~lM aqueous sodium chloride solution and 0.5~ bovine serum albumin. ~he t~o solutions are adjustcd ~lth 0.2~ h~drochlori~ acid (acetic acid and propionic acid give comparable results) to pEI 6.0, p~I 4.0 and pI-I 2Ø 'rhe solutions of anti~en and antibody of the same ~ are then mixed and the chronolosical course of the precipitate formation is monitoredr Fig. 7 of the accomp-anying drawings shows that at pH 6.0, immediately after mixing of antigen and antibody, immuno-precipitation takes place. A~ pII 4.0 there is a poorer precipitation which is not completed after 1 hour and at pH 2.0 precip-itation no longer occurs.
Example 10.
Preparation of ~r4 ~tetraiodothyronine) iMmune ~recipitate fleece~(without wash ~
As antigen there was used polyhapten consisting of rabbit I~G and ~4 bound thereon (mole ratio of IgG:'~4,=
1:3). The antigen was purified immunosorptively over an anti-T4 colu~nn (see a)).
As antibody there wa~ u~ed polyclonal antibody directed against the ~'c part of the rabbit IgGo The antibody was purified immunosorptively over a ra~bit IgG column (see a)).
. ~ .
~L2S~8 i~s fleece there was used a mix-ture of cellulose and polyester (obtainable from Kalff, Eu~kirchen~ Germany) or of lint, cellulose, polyamide (obtainable from Binzer, ~atzfeld, Eder, Germany).
Pre~aration of the m trix:
~ ntigen (400 mg./ 1 ) and antibody (1400 m~,/l ) were taken up separately in 50 mM acetic acid plus Oo5,c ~dium chlorideD .~*ter 15 to 30 minutes, both solution~ were mixed in a ratio of 1/1 and fleece impregnated therewith.
~fter dr~ing for 10 minutes at 75C., the fleece was impregnated ~ith PBS and dried for 10 minutes at 75~0 Example lOa, Presaration of Tl~ immune precipitate fleece ~with ~Jashing ~tep)~ -Antic~en (2 mg./ml.) and antibody (7 mg./ml.) aredissolved separately in distilled water. Both solutions are diluted with 50 mM aqueous trisodium phosphate ~olution in the ratio of 1 + ~, ~qual volumes of the diluted solutions are mixed and fleece impre~na-ted therewith, The fleece are dried for 10 minutes at 75C. in a circulating-air drying cabinet. Subsequently, the fleece are wa hed by descending chromatography ~/ith 10 mM phosphate buffer~until the eluate has a p~I value of 5Ø lifter washing, the fleece are dried, ~xam~le lOb ~repar~tion of T~ immune precipitate flcece (without as,lin~r step),
-3~~
~:25~i36,~
`;
A-~ntiven (2 mg.~ml.) and antibod~ (7 m,s.~ml.) are dissolved ~eparately in distilled water. Both solutions are diluted with 5G mM aqueous ammonia solution in the ratio of 1 ~ 4~
~qual volumes of the diluted solutions are mixed and fleece impregnated therewith~ The fleece are dried for 10 minutes at 75C, and used directl~ in the test.
The calibration curves obtained with the fleece of ~xamples 10, lOa and lOb correspond to the calibration curves of the preceding ~xamples.
~:25~i36,~
`;
A-~ntiven (2 mg.~ml.) and antibod~ (7 m,s.~ml.) are dissolved ~eparately in distilled water. Both solutions are diluted with 5G mM aqueous ammonia solution in the ratio of 1 ~ 4~
~qual volumes of the diluted solutions are mixed and fleece impregnated therewith~ The fleece are dried for 10 minutes at 75C, and used directl~ in the test.
The calibration curves obtained with the fleece of ~xamples 10, lOa and lOb correspond to the calibration curves of the preceding ~xamples.
Claims (27)
1. In a process for the preparation of an immuno-reactive, porous carrier material by appli-cation to a porous carrier of a solution of a first reaction component of an immuno-reaction and of a solution of a second component of the immuno-reaction precipitating therewith and incubation of the carrier material impregnated with the solutions for the immuno-precipitation, the steps of:
i) preparing a solution of said first and second reaction components of the immuno-reaction precipitation including an inhibitor for the immune-reaction pre-cipitation, ii) impregnating the carrier material with the solution from i), and iii) initiating the immuno-precipitation reaction by removal of the inhibitor or by neutralization of the inhibiting action of the inhibitor.
i) preparing a solution of said first and second reaction components of the immuno-reaction precipitation including an inhibitor for the immune-reaction pre-cipitation, ii) impregnating the carrier material with the solution from i), and iii) initiating the immuno-precipitation reaction by removal of the inhibitor or by neutralization of the inhibiting action of the inhibitor.
2. A process according to claim 1, wherein the components of the immuno-reaction are a protein and an antibody of a fragment thereof directed against this protein.
3. A process according to claim 2, wherein said protein comprises a protein to which a hapten or antigen is coupled.
4. A process according to claim 2, wherein said antibody or fragment thereof directed against the protein comprises an antibody or fragment thereof to which is coupled a hapten or antigen.
5. A process according to claim 3, wherein said antibody or fragment thereof directed against the protein comprises an antibody or fragment thereof to which is coupled a hapten or antigen.
6. A process according to claim 2, 3 or 4, wherein said protein comprises a specific anti-body.
7. A process according to claim 1, 2 or 3, wherein following said incubation the impregnated carrier material is washed and subsequently dried.
8. A process according to claim 1, wherein said inhibitor is an acid or a base.
9. A process according to claim 8, wherein said inhibitor is a volatile acid or base which is evaporated off after impregnation of the carrier mate-rial.
10. A process according to claim 8, wherein said inhibitor is a non-volatile acid or base and a carrier material is employed which is impregnated with a salt of a volatile acid or base.
11. A process according to claim 1, 2 or 3, wherein said inhibitor is glycerol or urea and, after impregnation of the carrier material, this is extracted with a mixture of at least one organic solvent and water.
12. A process according to claim 1, 2 or 3, wherein said inhibitor is a metal salt, the inhibiting action of which in the carrier material is removed by means of a complex former.
13. A process according to claim 1, wherein said inhibitor is a chaotropic ion of the Hofmeister series.
14. A process according to claim 13, wherein said inhibitor is a thiocyanate.
15, A process according to claim 13, wherein said inhibitor is an alkali metal iodide.
16. A process according to claim 1, 2 or 3, wherein the components of the immuno-reaction are used in the ratio of the Heidelberger maximum.
17. A process according to claim 4, 5 or 8, wherein the components of the immuno-reaction are used in the ratio of the Heidelberger maximum.
18. A process according to claim 13, 14 or 15, wherein the components of the immuno-reaction are used in the ratio of the Heidelberger maximum.
19. A process according to claim 1, 2 or 3, wherein at least one component of the immuno-reaction is used in non-purified form.
20. A process according to claim 4, 5 or 8, wherein at least one component of the immuno-reaction is used in non-purified form.
21. A process according to claim 13, 14 or 15, wherein at least one component of the immuno-reaction is used in non-purified form.
22. A process according to claim 1, 2 or 3, wherein from the components of the immuno-reaction there is first prepared a suspension precipitate which, after washing, is dissolved by the addition of an inhibitor and subsequently used for the impregnation of the porous carrier.
23. A process according to claim 4, 5 or 8, wherein from the components of the immuno-reaction there is first prepared a suspension precipitate which, after washing, is dissolved by the addition of an inhibitor and subsequently used for the impregnation of the porous carrier.
24. A process according to claim 13, 14 or 15, wherein from the components of the immuno-reaction there is first prepared a suspension precipitate which, after washing, is dissolved by the addition of an inhibitor and subsequently used for the impregnation of the porous carrier.
25. A process for the preparation of an immuno-reactive, porous carrier material comprising:
i) providing a solution containing a first component of an immuno-reaction, a second component of the immuno-reaction, the first and second components having a pro-perty of reacting to form an immuno-precipitate, and an inhibitor effective to suppress the formation of the pre-cipitate from the first and second components, ii) impregnating a porous carrier material with said solution, and iii) initiating the immuno-precipitation reaction by removal of the inhibitor or neutralization of the inhibiting action of the inhibitor.
i) providing a solution containing a first component of an immuno-reaction, a second component of the immuno-reaction, the first and second components having a pro-perty of reacting to form an immuno-precipitate, and an inhibitor effective to suppress the formation of the pre-cipitate from the first and second components, ii) impregnating a porous carrier material with said solution, and iii) initiating the immuno-precipitation reaction by removal of the inhibitor or neutralization of the inhibiting action of the inhibitor.
26. A process according to claim 25, wherein step iii) comprises removal of the inhibitor.
27. A process according to claim 25, wherein step iii) comprises neutralization of the inhibiting action of the inhibitor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3446636.3 | 1984-12-20 | ||
| DE19843446636 DE3446636A1 (en) | 1984-12-20 | 1984-12-20 | METHOD FOR PRODUCING AN IMMUNREACTIVE POROUS CARRIER MATERIAL |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1256368A true CA1256368A (en) | 1989-06-27 |
Family
ID=6253391
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000497115A Expired CA1256368A (en) | 1984-12-20 | 1985-12-06 | Process for the preparation of an immune-reactive porous carrier material |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US4820644A (en) |
| EP (1) | EP0185372B1 (en) |
| JP (1) | JPS61151463A (en) |
| AT (1) | ATE41060T1 (en) |
| AU (1) | AU566478B2 (en) |
| CA (1) | CA1256368A (en) |
| DD (1) | DD244418A5 (en) |
| DE (2) | DE3446636A1 (en) |
| DK (1) | DK161992C (en) |
| ES (1) | ES8704269A1 (en) |
| IE (1) | IE58809B1 (en) |
| NO (1) | NO165696C (en) |
| ZA (1) | ZA859674B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK289686A (en) * | 1986-06-19 | 1987-12-20 | Immuntech As | PROCEDURE FOR PREPARING SURFACE BASED IMMUNAL ANALYSIS |
| DE3620653A1 (en) * | 1986-06-20 | 1987-12-23 | Boehringer Mannheim Gmbh | METHOD FOR PRODUCING AN IMMUNREACTIVE SUPPORT MATERIAL FOR HETEROGENEOUS IMMUNOLOGICAL ANALYSIS |
| DE3735684A1 (en) * | 1987-10-22 | 1989-05-03 | Boehringer Mannheim Gmbh | IMMUNREACTIVE CARRIER MATERIAL AND METHOD FOR THE PRODUCTION THEREOF |
| DE3802366A1 (en) * | 1988-01-27 | 1989-08-10 | Boehringer Mannheim Gmbh | CARRIER TABLES FOR REMOVABLE IMPREGNATED REAGENTS |
| US5268306A (en) * | 1988-02-29 | 1993-12-07 | Boehringer Mannheim Gmbh | Preparation of a solid phase matrix containing a bound specific binding pair |
| DE3806431A1 (en) * | 1988-02-29 | 1989-09-07 | Boehringer Mannheim Gmbh | METHOD FOR PRODUCING A SOLID PHASE MATRIX |
| DE3826057A1 (en) * | 1988-07-30 | 1990-02-01 | Boehringer Mannheim Gmbh | TEST TESTER FOR THE ANALYTICAL DETERMINATION OF AN INGREDIENT OF A LIQUID SAMPLE |
| US5338513A (en) * | 1988-07-30 | 1994-08-16 | Boehringer Mannheim Gmbh | Test carrier for the analytical determination of a component of a liquid sample |
| US5888728A (en) * | 1988-10-17 | 1999-03-30 | Molecular Devices Corporation | Hapten derivatized capture membrane and diagnostic assays using such membrane |
| JPH0820448B2 (en) * | 1989-01-18 | 1996-03-04 | 帝人株式会社 | Method for producing fixed antibody |
| US5064541A (en) * | 1989-04-07 | 1991-11-12 | Abbott Laboratories | Devices and methods for the collection of a predetermined volume of plasma or serum |
| US4933092A (en) * | 1989-04-07 | 1990-06-12 | Abbott Laboratories | Methods and devices for the separation of plasma or serum from whole blood |
| AU6870091A (en) * | 1989-11-08 | 1991-06-13 | Fmc Corporation | Combined centrifuge tube and porous selection means for separation and recovery of biological materials |
| US5424219A (en) * | 1991-10-25 | 1995-06-13 | Cytech Biomedical, Inc. | Method of performing assays for biomolecules and solid supports for use in such methods |
| US6927062B2 (en) * | 2002-11-25 | 2005-08-09 | Agdia, Inc. | Controls and standards for assays and method for manufacture thereof |
| US7393453B2 (en) * | 2004-06-23 | 2008-07-01 | Kham Lin | Method for analysis of perchlorate |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE13156C (en) * | Dr. C. OTTO & CO. in Dahlhausen a. d. Ruhr | Equipment on coke ovens, consisting in preheating the coke oven gases after the tar and ammonia content has been removed from them, before they are burned | ||
| CA983358A (en) * | 1971-09-08 | 1976-02-10 | Kenneth D. Bagshawe | Performance of chemical or biological reactions |
| US3966897A (en) * | 1973-04-02 | 1976-06-29 | Marine Colloids, Inc. | Medium for use in bioassay and method of using same |
| US4205954A (en) * | 1978-05-26 | 1980-06-03 | Warner-Lambert Company | Kinetic latex agglutinometry |
| JPS587332Y2 (en) * | 1978-06-06 | 1983-02-08 | 富士写真フイルム株式会社 | Multilayer blood chemistry analysis material |
| IL55564A (en) * | 1978-09-13 | 1981-12-31 | Ames Yissum Ltd | Method for the quantitative determination of antigens in aqueous solutions |
| US4495296A (en) * | 1979-05-21 | 1985-01-22 | New York Blood Center, Inc. | Labeled anti-hapten antibodies and their use as a universal reagent for solid phase radio- and/or enzyme-immunoassays |
| JPS5670460A (en) * | 1979-11-13 | 1981-06-12 | Fuji Photo Film Co Ltd | Multilayer chemical analysis material for analysis of water liquid |
| JPS57200862A (en) * | 1981-06-05 | 1982-12-09 | Fuji Photo Film Co Ltd | Mutilayer analysis element utilizing unique binding reaction |
| FR2523311B1 (en) * | 1982-03-12 | 1985-12-27 | Pasteur Institut | COUPLING PRODUCT BETWEEN AN ALBUMIN AND A SPECIFIC LIGAND, OBTAINING AND APPLICATIONS IN THE BIOLOGICAL FIELD |
| EP0121303B1 (en) * | 1983-03-04 | 1989-07-26 | Noctech Limited | Diagnostic agent, a process for its preparation and its use in diagnostic methods |
| US4687734A (en) * | 1984-06-07 | 1987-08-18 | Chester Samuel J | Immunoassay for the detection of human colon cancer using a polyclonal antibody derived from a capillary culture of tumor cells |
-
1984
- 1984-12-20 DE DE19843446636 patent/DE3446636A1/en not_active Withdrawn
-
1985
- 1985-12-06 CA CA000497115A patent/CA1256368A/en not_active Expired
- 1985-12-06 IE IE308985A patent/IE58809B1/en not_active IP Right Cessation
- 1985-12-10 AU AU51064/85A patent/AU566478B2/en not_active Ceased
- 1985-12-10 US US06/807,454 patent/US4820644A/en not_active Expired - Lifetime
- 1985-12-19 AT AT85116212T patent/ATE41060T1/en not_active IP Right Cessation
- 1985-12-19 EP EP85116212A patent/EP0185372B1/en not_active Expired
- 1985-12-19 ZA ZA859674A patent/ZA859674B/en unknown
- 1985-12-19 NO NO855160A patent/NO165696C/en unknown
- 1985-12-19 DD DD85284778A patent/DD244418A5/en not_active IP Right Cessation
- 1985-12-19 DK DK594485A patent/DK161992C/en not_active IP Right Cessation
- 1985-12-19 DE DE8585116212T patent/DE3568490D1/en not_active Expired
- 1985-12-20 ES ES550286A patent/ES8704269A1/en not_active Expired
- 1985-12-20 JP JP60285930A patent/JPS61151463A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| DK161992C (en) | 1992-02-03 |
| DK594485A (en) | 1986-06-21 |
| IE58809B1 (en) | 1993-11-17 |
| ZA859674B (en) | 1986-09-24 |
| NO165696B (en) | 1990-12-10 |
| JPH0521510B2 (en) | 1993-03-24 |
| DD244418A5 (en) | 1987-04-01 |
| NO855160L (en) | 1986-06-23 |
| ES8704269A1 (en) | 1987-04-01 |
| DE3446636A1 (en) | 1986-06-26 |
| AU5106485A (en) | 1986-06-26 |
| JPS61151463A (en) | 1986-07-10 |
| IE853089L (en) | 1986-06-20 |
| DK594485D0 (en) | 1985-12-19 |
| ATE41060T1 (en) | 1989-03-15 |
| EP0185372A1 (en) | 1986-06-25 |
| DE3568490D1 (en) | 1989-04-06 |
| AU566478B2 (en) | 1987-10-22 |
| DK161992B (en) | 1991-09-02 |
| EP0185372B1 (en) | 1989-03-01 |
| US4820644A (en) | 1989-04-11 |
| ES550286A0 (en) | 1987-04-01 |
| NO165696C (en) | 1991-03-20 |
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