CA1124643A - Carrier tagged with releasable fluorophores - Google Patents
Carrier tagged with releasable fluorophoresInfo
- Publication number
- CA1124643A CA1124643A CA333,870A CA333870A CA1124643A CA 1124643 A CA1124643 A CA 1124643A CA 333870 A CA333870 A CA 333870A CA 1124643 A CA1124643 A CA 1124643A
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- CA
- Canada
- Prior art keywords
- reactant
- carrier
- fluorophores
- immunological
- reagent
- 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.)
- Expired
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Abstract
ABSTRACT OF THE DISCLOSURE
A method of composition is featured for performing a fluoroimmunoassay of a biological fluid sample for an immunological reactant. A conjugate of a carrier labelled with fluorophores and coupled to an immunological reactant is mixed with the sample and a known quantity of binding agent. After equilibrium is accomplished, the bound and unbound portions are separated. The carrier in a selected portion is chemically treated to liberate the fluorophores.
The fluorescent level is then measured and compared with a standard.
Docket 2135-A
A method of composition is featured for performing a fluoroimmunoassay of a biological fluid sample for an immunological reactant. A conjugate of a carrier labelled with fluorophores and coupled to an immunological reactant is mixed with the sample and a known quantity of binding agent. After equilibrium is accomplished, the bound and unbound portions are separated. The carrier in a selected portion is chemically treated to liberate the fluorophores.
The fluorescent level is then measured and compared with a standard.
Docket 2135-A
Description
~46~3 Field of the Invention _ e The invention relates to a method and composition for performing a fluoroimmunoassay of a biological fluid sample and, more particularly, for performing a more sensitive fluoroimmunoassay using a conjugate formed by heavily fluorescently tagging a carrier, to the extent that self-quenching between the bound fluorophores may take place, which is subsequently chemically treated to liberate the fluorophores for measurement.
Background of the Invention It is known that a greater sensitivity can be achieved in a fluoroimmunoassay, i~ the competitive reactant is heavily labelled. However, as the number of fluorophores bound to such reactant is increased, the spacing between adjacent fluorophores decreases until a critical distance of 60Ao to lOOAo is reached. At this critical distance, or less, the fluorophores are self-quenching, i.e., the fluorescent level becomes less than an equivalent number of fluorophore molecules distanced greater than lOOAo. Quenching also occurs in certain assays, e.g., for thyroid hormones, because of the iodine present in such materials. In the past, fluorometric assays for these reactants have been extremely difficult.
~ he invention heavily labels a competing reactant ~ith fluorophores, which may be subjected to self-quenching, and then the fluorophores are separated from the reagent after competitive binding has occurred. Following separation, the fluorophores are spaced beyond the critical ~;! ~
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distance, whereby self-quenchin~ ceases and the available fluorescent level is restored. Accordingly, a more sensitive fluoroimmunoassay can be achieved.
SUMMARY OF THE INVENTION
This invention pertains to a method and reagent for performing a fluoroimmunoassay using a heavily labelled reagent which competes with the immunological reactant in the sample for a known quantity of a comp~ementing binding agent. The reagent comprises a conjugate formed by tagging a plurality of fluorophores to a ~arrier capable of being chemically treated, so as to rel~ase the fluorophor~s.
The rea~ent is coupled to a competitive r(eactant ~o form a conjugate. The sample and the conjugat~e are mixed with a binding agent. After the competitive-b~nding reaction, the bound immunological reactants and unb~und immunological reactants are separated. The carrier, i~ either or both separated portions, is then chemically tr~ated, or lysed, to liberate the otherwise quenched, close~y-packed fluorophores to greatly enhance the fluorescent level. The fluorescent level of the liberated fluorophores is th~ compared to a standard level of fluorescence to deter~ine the amount of immunological reactant in the sample.
In another embodiment of the in~entive method, the fluorophores are directly labelled to a competing reactant without the need for a carrier. In this case, the competing reactant must be chemically tre.atable to liberate the fluorophores.
Preferably, carriers are select~d to have many wc/
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fluorophore-binding sites whereby a large number of fluorophores can be attached t~ereto. As the problem of se~f-quenching is avoided by this invention, a large number of binding sites are preferred. For example, such carriers may be very long chain molecules such as proteins, polymers, or polysaccharides.
The reactant in the biological fluid sample may be either an antigen or an antibody. The competing immunological reactant conjugated with the carrier will be a corresponding antigen or antibody or, in some cases, a hapten.
It is an object of this invention to provide an improved method and reagent for performing a fluoroimmunoassay.
It is another object of the invention to provide a more sensitive fluoroimmunoassay.
These and other objects of the invention are achieved by: conjugating one reactant of said fluoroimmuno-assay with a carrier tagged with a plurality of fluorophores to provide a quenched level of fluorescence; and liberating the fluorophores from said carrier following reaction with its complement.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Additional objects and features will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:
Fig. 1 depicts the formation of a mixture ` comprising the fluid sample, the competing conjugate~ and a binding a~ent;
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Fig. 2 shows the equilibrated products of the mixture of Fig l;
Fig. 3 illustrates a separa-ted portion of the equilibrated products of Fig. 2;
Fig. ~ depicts the products of Fig. 3; following liberation of the fluorophores previously bound to the carrier.
Fig. 5 shows the liberated fluorophores after separation and wash from the products of Fig. 4; and Fig. 6 illustrates a graph depicting the increase in fluorescent level prior to and following liberation of the fluorophores from the carrier.
DETAILED DESCRIPTION
Generally speaking, the inventive method for assaying a f~uid sample for a reactant comprises the steps of: (a) using a reagent comprising a tagged carrier capable of being coupled to a competing reactant to form a conjugate. The tagged carrier has a plurality of fluoro-phores. When the carrier is treated chemically, the fluorophores release from the carrier. After a conjugatP
is formed by coupling a competing reactant to the tagged carrier reagent; (b) a mixture is then formed of the sample, a known amount of the conjugate and a binding agent;
(c) after equilibration, the mixture is separated into a first bound and a second unbound portion, i.e., unbound conjugate reactant and unbound sample reactant are removed from the bound conjugate reactant and bound sample reactant;
(d) to either the first bound portion or the second unbound portion of the mixture, an enzyme, lysing agent or other chemical reagent is added to liberate the tagged w~
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fluorophores; (e) the fluorescent level of the liherated fluorophores is measured and cor~lpared ~ith a standard fluorescent level.
Now referring to Fig. 1, the conjugate 10 of method step (a) is shown. me conjugate 10 is formed by fluorescently tagging (labeling) a carrier molecule 11 with as n~ny fluorophores 12 as possible. The carrier molecule 11 is a long chain molecule having many fluorophore binding sites. Such a molecule can be a protein, cellulose, a cellulose derivative, a polysaccharide, a polymer, etc.
The carrier 10 must be capable of either being lysed by an enzyme or other lysing agent 13 (Fig. 3), or treated chemically to free the fluoro-phores. m e tagged carrier is the inventive reagent which will enhance the fluorescent level of the immunoreaction, whereby the sensitivity of the assay will be greatly improved.
To this reagent, a reactant such as an antigen (Ag) a hapten, or an antibody (~b) is coupled to form the conjugate 10. In the illustration of Fig. 1, antigens (~g) 14 have been coupled to the OE rier 11. The antigens (Ag) 14 compete with antigens (Ag) 15 in the fluid sa~ple.
Antibodies 16 will bind with either antigens 14 or antigens 15. As described, antibodies 16 are bound to a solid phase 17, to form a binding agent, as is well known in the art. The solid phase 17 can be magnetic particulate cellulose, glass, latexes, plastics, agaroses, Sepharoses,~ and Sephadexes,* etc. The solid phase particulate may be shaped as spheres, rods, tubes or some other generally uniformly-shaped particles, etc.
As sh~n in Fig. 1, a mixture is made of the sample containing an unknown amount of antigens 15, a known amount of the conjugate 10 and a kncwn amount of binding agent. The * Trademark jb/ - 6 -: ~ : ., -.
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. :. ' :' binding agent will equilibrate with the antigens 14 and 15, respectively, as aforementioned, to form the reaction products shown in Fig. 2. The mixture now contains bound and unbound, or free, fractions, i.e., bound molecules comprising antigen-antibody linkages, and unbound molecules containing free antigens 14 and free antigens 15.
Fig. 3 illustrates the bound product portion of the product mixture of Fig. 2 after separation and washing.
The bound product portion is treated with an appropriate enzyme or lysing agent 13, that will digest or lyse the long chain carrier molecule 11.
In one embodiment of this invention, the carrier 11 may be treated chemically to release the fluorophores 12 which are bound to it, by breaking-up the carrier molecule.
By way of example, a protein molecule carrier may be lysed by enzymes such as: pepsin (3.4.23.1), bromelin (3.4.22.5), trypsin (3.4.21.4), chymotrypsin (3.4.21.1), papain (3.4.22.2), pronase (3.4.21.4 and 3.4.24.4) etc. Where a cellulose mole-cule is used as the carrier, a cellulose (3.2.1.4) enzyme such as emulsin may be used. Where the carrier is a polysaccharide such as dextran, a dextranase (3.2.1.11) enzyme can be employed.
The results of the lysing of carrier 11 by the enzyme 13 are shown in Fig. 4. The carrier 11 will be lysed into many fragments 20, to liberate the fluorophores 12. The liberating of the fluorophores 12 allows them to become spaced apart in solution, thus freeing their fluorescent characteristics and avoiding any self-quenching effects. The fluorescent level will be proportional to the number of antigen-antibody reactions that have taken place. Therefore, the measured fluorescent level of the liberated fluorophores 12 can be compared to a standard level of fluorescence in order to determine the amount of antigens 15 in the sample.
'~
! As aorementioned, the unbound or free portion o~ Fig. 2, ~' may also be treated with an enzyme 13 to obt~in a luorescent 3 ', level proportional to the number of competing antigen-antibody reactions. The amount of antigens lS in the sample ~ay likewise 5 I be obtained from this data, because a known amount of reagent 6 I and binding agent are used in the reaction. The fluorescent level of the free portion, therefore, can similarly be compared 7 ~o a standard fluorescent le~el. Where the free or unbo~nd 8 i portion is treated, the additional separation and ~ashing 9 ¦ step of Figs. 4 and 5 will not be neces~ary.
10 j As is known in the art, the bound reaction products of ll ~ Fig. 4 are separated and washed, and appropriate adjustment in i pH or other conditions are made in the solution, as depicted l~ ¦ in Fig. 5. The fluorophores 12 are then subjected to ultra-13 I violet light, and the fluorescence is measured in a fluorometer. r 14 I The fluorophores 12 which may be used in the invention lS 'are those generally available for these procedures, su~h as:
l6 !fluorescein, dansyl, rhodamine, ~luorescamine, pyrene, 17 lacridine and 2-methoxy-2~4-diphenyl-3(2H)-furanonQ (M~P~.
li~ IIt may be found that certain ones of these fluorophcres will ¦~provide better binding to certain ones of the carri~rs ll, ¦~r will provide a higher level of fluorescence as will ke ¦ obvious to those skilled in the art.
21l~ Referring to Fig. 6, a curve 25 illustrates ~he releaced `fluorescence vs. time, of fluorescein tagge~l molecules o~
~ j Human Serum Albumin (HSA), which have been di~ested ~l t:~e
Background of the Invention It is known that a greater sensitivity can be achieved in a fluoroimmunoassay, i~ the competitive reactant is heavily labelled. However, as the number of fluorophores bound to such reactant is increased, the spacing between adjacent fluorophores decreases until a critical distance of 60Ao to lOOAo is reached. At this critical distance, or less, the fluorophores are self-quenching, i.e., the fluorescent level becomes less than an equivalent number of fluorophore molecules distanced greater than lOOAo. Quenching also occurs in certain assays, e.g., for thyroid hormones, because of the iodine present in such materials. In the past, fluorometric assays for these reactants have been extremely difficult.
~ he invention heavily labels a competing reactant ~ith fluorophores, which may be subjected to self-quenching, and then the fluorophores are separated from the reagent after competitive binding has occurred. Following separation, the fluorophores are spaced beyond the critical ~;! ~
WC~
.;, ~
distance, whereby self-quenchin~ ceases and the available fluorescent level is restored. Accordingly, a more sensitive fluoroimmunoassay can be achieved.
SUMMARY OF THE INVENTION
This invention pertains to a method and reagent for performing a fluoroimmunoassay using a heavily labelled reagent which competes with the immunological reactant in the sample for a known quantity of a comp~ementing binding agent. The reagent comprises a conjugate formed by tagging a plurality of fluorophores to a ~arrier capable of being chemically treated, so as to rel~ase the fluorophor~s.
The rea~ent is coupled to a competitive r(eactant ~o form a conjugate. The sample and the conjugat~e are mixed with a binding agent. After the competitive-b~nding reaction, the bound immunological reactants and unb~und immunological reactants are separated. The carrier, i~ either or both separated portions, is then chemically tr~ated, or lysed, to liberate the otherwise quenched, close~y-packed fluorophores to greatly enhance the fluorescent level. The fluorescent level of the liberated fluorophores is th~ compared to a standard level of fluorescence to deter~ine the amount of immunological reactant in the sample.
In another embodiment of the in~entive method, the fluorophores are directly labelled to a competing reactant without the need for a carrier. In this case, the competing reactant must be chemically tre.atable to liberate the fluorophores.
Preferably, carriers are select~d to have many wc/
- . ~
fluorophore-binding sites whereby a large number of fluorophores can be attached t~ereto. As the problem of se~f-quenching is avoided by this invention, a large number of binding sites are preferred. For example, such carriers may be very long chain molecules such as proteins, polymers, or polysaccharides.
The reactant in the biological fluid sample may be either an antigen or an antibody. The competing immunological reactant conjugated with the carrier will be a corresponding antigen or antibody or, in some cases, a hapten.
It is an object of this invention to provide an improved method and reagent for performing a fluoroimmunoassay.
It is another object of the invention to provide a more sensitive fluoroimmunoassay.
These and other objects of the invention are achieved by: conjugating one reactant of said fluoroimmuno-assay with a carrier tagged with a plurality of fluorophores to provide a quenched level of fluorescence; and liberating the fluorophores from said carrier following reaction with its complement.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Additional objects and features will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:
Fig. 1 depicts the formation of a mixture ` comprising the fluid sample, the competing conjugate~ and a binding a~ent;
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: ' `
Fig. 2 shows the equilibrated products of the mixture of Fig l;
Fig. 3 illustrates a separa-ted portion of the equilibrated products of Fig. 2;
Fig. ~ depicts the products of Fig. 3; following liberation of the fluorophores previously bound to the carrier.
Fig. 5 shows the liberated fluorophores after separation and wash from the products of Fig. 4; and Fig. 6 illustrates a graph depicting the increase in fluorescent level prior to and following liberation of the fluorophores from the carrier.
DETAILED DESCRIPTION
Generally speaking, the inventive method for assaying a f~uid sample for a reactant comprises the steps of: (a) using a reagent comprising a tagged carrier capable of being coupled to a competing reactant to form a conjugate. The tagged carrier has a plurality of fluoro-phores. When the carrier is treated chemically, the fluorophores release from the carrier. After a conjugatP
is formed by coupling a competing reactant to the tagged carrier reagent; (b) a mixture is then formed of the sample, a known amount of the conjugate and a binding agent;
(c) after equilibration, the mixture is separated into a first bound and a second unbound portion, i.e., unbound conjugate reactant and unbound sample reactant are removed from the bound conjugate reactant and bound sample reactant;
(d) to either the first bound portion or the second unbound portion of the mixture, an enzyme, lysing agent or other chemical reagent is added to liberate the tagged w~
.~, . , ~ , . .
fluorophores; (e) the fluorescent level of the liherated fluorophores is measured and cor~lpared ~ith a standard fluorescent level.
Now referring to Fig. 1, the conjugate 10 of method step (a) is shown. me conjugate 10 is formed by fluorescently tagging (labeling) a carrier molecule 11 with as n~ny fluorophores 12 as possible. The carrier molecule 11 is a long chain molecule having many fluorophore binding sites. Such a molecule can be a protein, cellulose, a cellulose derivative, a polysaccharide, a polymer, etc.
The carrier 10 must be capable of either being lysed by an enzyme or other lysing agent 13 (Fig. 3), or treated chemically to free the fluoro-phores. m e tagged carrier is the inventive reagent which will enhance the fluorescent level of the immunoreaction, whereby the sensitivity of the assay will be greatly improved.
To this reagent, a reactant such as an antigen (Ag) a hapten, or an antibody (~b) is coupled to form the conjugate 10. In the illustration of Fig. 1, antigens (~g) 14 have been coupled to the OE rier 11. The antigens (Ag) 14 compete with antigens (Ag) 15 in the fluid sa~ple.
Antibodies 16 will bind with either antigens 14 or antigens 15. As described, antibodies 16 are bound to a solid phase 17, to form a binding agent, as is well known in the art. The solid phase 17 can be magnetic particulate cellulose, glass, latexes, plastics, agaroses, Sepharoses,~ and Sephadexes,* etc. The solid phase particulate may be shaped as spheres, rods, tubes or some other generally uniformly-shaped particles, etc.
As sh~n in Fig. 1, a mixture is made of the sample containing an unknown amount of antigens 15, a known amount of the conjugate 10 and a kncwn amount of binding agent. The * Trademark jb/ - 6 -: ~ : ., -.
..
: . . .
. .. .. .
. :. ' :' binding agent will equilibrate with the antigens 14 and 15, respectively, as aforementioned, to form the reaction products shown in Fig. 2. The mixture now contains bound and unbound, or free, fractions, i.e., bound molecules comprising antigen-antibody linkages, and unbound molecules containing free antigens 14 and free antigens 15.
Fig. 3 illustrates the bound product portion of the product mixture of Fig. 2 after separation and washing.
The bound product portion is treated with an appropriate enzyme or lysing agent 13, that will digest or lyse the long chain carrier molecule 11.
In one embodiment of this invention, the carrier 11 may be treated chemically to release the fluorophores 12 which are bound to it, by breaking-up the carrier molecule.
By way of example, a protein molecule carrier may be lysed by enzymes such as: pepsin (3.4.23.1), bromelin (3.4.22.5), trypsin (3.4.21.4), chymotrypsin (3.4.21.1), papain (3.4.22.2), pronase (3.4.21.4 and 3.4.24.4) etc. Where a cellulose mole-cule is used as the carrier, a cellulose (3.2.1.4) enzyme such as emulsin may be used. Where the carrier is a polysaccharide such as dextran, a dextranase (3.2.1.11) enzyme can be employed.
The results of the lysing of carrier 11 by the enzyme 13 are shown in Fig. 4. The carrier 11 will be lysed into many fragments 20, to liberate the fluorophores 12. The liberating of the fluorophores 12 allows them to become spaced apart in solution, thus freeing their fluorescent characteristics and avoiding any self-quenching effects. The fluorescent level will be proportional to the number of antigen-antibody reactions that have taken place. Therefore, the measured fluorescent level of the liberated fluorophores 12 can be compared to a standard level of fluorescence in order to determine the amount of antigens 15 in the sample.
'~
! As aorementioned, the unbound or free portion o~ Fig. 2, ~' may also be treated with an enzyme 13 to obt~in a luorescent 3 ', level proportional to the number of competing antigen-antibody reactions. The amount of antigens lS in the sample ~ay likewise 5 I be obtained from this data, because a known amount of reagent 6 I and binding agent are used in the reaction. The fluorescent level of the free portion, therefore, can similarly be compared 7 ~o a standard fluorescent le~el. Where the free or unbo~nd 8 i portion is treated, the additional separation and ~ashing 9 ¦ step of Figs. 4 and 5 will not be neces~ary.
10 j As is known in the art, the bound reaction products of ll ~ Fig. 4 are separated and washed, and appropriate adjustment in i pH or other conditions are made in the solution, as depicted l~ ¦ in Fig. 5. The fluorophores 12 are then subjected to ultra-13 I violet light, and the fluorescence is measured in a fluorometer. r 14 I The fluorophores 12 which may be used in the invention lS 'are those generally available for these procedures, su~h as:
l6 !fluorescein, dansyl, rhodamine, ~luorescamine, pyrene, 17 lacridine and 2-methoxy-2~4-diphenyl-3(2H)-furanonQ (M~P~.
li~ IIt may be found that certain ones of these fluorophcres will ¦~provide better binding to certain ones of the carri~rs ll, ¦~r will provide a higher level of fluorescence as will ke ¦ obvious to those skilled in the art.
21l~ Referring to Fig. 6, a curve 25 illustrates ~he releaced `fluorescence vs. time, of fluorescein tagge~l molecules o~
~ j Human Serum Albumin (HSA), which have been di~ested ~l t:~e
2~l proteolytic enzyme Bromelin (3~4~22~5)~ Point "~" depic~s , ¦~the initial Docket 2135-A
~ ,-:17 i, ~ - 8 -:-level of fluorescence prior to the digestion of the HSAmolecule; about 25~ of the maximum expected level o~ fluor-escence for a free amount of unbound fluorescein.
As the HSA molecule is digested by -the Bromelin, an increasing amount of fluorophores are released, and the fluorescent level is increased. As the HSA molecule is completely digested by the Bromelin, i.e., after a reaction time of approximately 150 minutes, almost all of the fluoro-phores will be released to give a fluorescent level approach-ing 90% of maximum, as shown by point "B".
Fig. 6, therefore, shows that a carrier molecule 11,such as fluorescein-labeled HSA, can function to enhance a fluoroimmunoassay of an antigen or antibody, according to the outlined method.
Where the molecule can be lysed by more than one enzyme, several enzymes may be simultanepusly used to break up the carrier molecule into finer se~ments. This will give rise to a greater fluorescence. For example, in a protein molecule carrier, trypsin (3.4.21.4) will cleave the protei~
molecule at amino acid linkages arginine and lysine. If the en2yme chymotrypsin t3~4~2l~l) is also used to digest the protein, the additional amino acid linkages of phenylalanine, leucine, and tyrosine will also be cleaved. Thus, the protein molecule will be broken into smaller segments, and hence, the attached fluorophores will enjoy a greater release into the solution. Consequently, a greater fluorescent level should be obtained.
In another embodiment of the inventive method, it is contemplated to liberate the fluorophores from a protein or polymer molecule without the necessity of having to lyse or digest the B
carrier molecule. In an article to Martin J. Lee, Ph.D., et al entitled: "The Inhibition of Mitochondrial Energized Processes by Fluorescein Mercuric Acetate"; Bioencrgetics (1971) 2, pp.l3-31; it is taught that fluorescent molecules such as Fluorescein Mercuric Acetate can attach reversibly to sulfhydryl groups (SH) of proteins and other po]~mers.
These fluorophores can be released by agents such as dithiothreitol.
The invention envisions using this technique to enhance the fluorescent level in a fluoroimmunoassay as previously described herein. The liberation of the fluorophores from the reactant carrier conjugate without the need for lysing suggests an alternate embodiment to the invention which may provide certain advantages such as quicker release time for the fluorophores. This procedure will be particularly useful where the carrier con-tains many sulfhydryl groupings, such that the carrier can be heavily labeled.
Also, where reactants themselves can be tagged fluorescently, it may be advantageous to bind and then liber`ate the fluorophores directly to and from the reactant without need for a carrier molecule. This alternate embodiment to the inventive method will be useful where the reactant is a long chain molecule that can be heavily tagged, or where the fluorescence is completely quenched as in the case of the thyroid hormone assay.
Having described the invention, what is ~esired to be protected by Letters Patent i5 presented in the ~ollowing appended claims.
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~ ,-:17 i, ~ - 8 -:-level of fluorescence prior to the digestion of the HSAmolecule; about 25~ of the maximum expected level o~ fluor-escence for a free amount of unbound fluorescein.
As the HSA molecule is digested by -the Bromelin, an increasing amount of fluorophores are released, and the fluorescent level is increased. As the HSA molecule is completely digested by the Bromelin, i.e., after a reaction time of approximately 150 minutes, almost all of the fluoro-phores will be released to give a fluorescent level approach-ing 90% of maximum, as shown by point "B".
Fig. 6, therefore, shows that a carrier molecule 11,such as fluorescein-labeled HSA, can function to enhance a fluoroimmunoassay of an antigen or antibody, according to the outlined method.
Where the molecule can be lysed by more than one enzyme, several enzymes may be simultanepusly used to break up the carrier molecule into finer se~ments. This will give rise to a greater fluorescence. For example, in a protein molecule carrier, trypsin (3.4.21.4) will cleave the protei~
molecule at amino acid linkages arginine and lysine. If the en2yme chymotrypsin t3~4~2l~l) is also used to digest the protein, the additional amino acid linkages of phenylalanine, leucine, and tyrosine will also be cleaved. Thus, the protein molecule will be broken into smaller segments, and hence, the attached fluorophores will enjoy a greater release into the solution. Consequently, a greater fluorescent level should be obtained.
In another embodiment of the inventive method, it is contemplated to liberate the fluorophores from a protein or polymer molecule without the necessity of having to lyse or digest the B
carrier molecule. In an article to Martin J. Lee, Ph.D., et al entitled: "The Inhibition of Mitochondrial Energized Processes by Fluorescein Mercuric Acetate"; Bioencrgetics (1971) 2, pp.l3-31; it is taught that fluorescent molecules such as Fluorescein Mercuric Acetate can attach reversibly to sulfhydryl groups (SH) of proteins and other po]~mers.
These fluorophores can be released by agents such as dithiothreitol.
The invention envisions using this technique to enhance the fluorescent level in a fluoroimmunoassay as previously described herein. The liberation of the fluorophores from the reactant carrier conjugate without the need for lysing suggests an alternate embodiment to the invention which may provide certain advantages such as quicker release time for the fluorophores. This procedure will be particularly useful where the carrier con-tains many sulfhydryl groupings, such that the carrier can be heavily labeled.
Also, where reactants themselves can be tagged fluorescently, it may be advantageous to bind and then liber`ate the fluorophores directly to and from the reactant without need for a carrier molecule. This alternate embodiment to the inventive method will be useful where the reactant is a long chain molecule that can be heavily tagged, or where the fluorescence is completely quenched as in the case of the thyroid hormone assay.
Having described the invention, what is ~esired to be protected by Letters Patent i5 presented in the ~ollowing appended claims.
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Claims (26)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of enhancing the fluorescent level of a fluoroimmunoassay, comprising the steps of:
(a) conjugating one reactant of said fluoro-immunoassay with a carrier tagged with a plurality of fluoro-phores to induce self-quenching therebetween; and (b) liberating the fluorophores from said carrier following reaction with its complement.
(a) conjugating one reactant of said fluoro-immunoassay with a carrier tagged with a plurality of fluoro-phores to induce self-quenching therebetween; and (b) liberating the fluorophores from said carrier following reaction with its complement.
2. The method of claim 1, wherein said carrier is selected from a group consisting of: a protein; cellulose;
a cellulose derivative; a polymer; and a polysaccharide.
a cellulose derivative; a polymer; and a polysaccharide.
3. The method of claim 1, wherein the step (b) further comprises the step (c) of: lysing said carrier by at least one enzyme.
4. The method of claim 1, wherein said reactant is an antigen.
5. The method of claim 1, wherein said reactant is an antibody.
6. The method of claim 1, wherein said reactant is a hapten.
7. The method of claim 1, wherein said fluoro-phores are selected from a group consisting of: fluorescein, dansyl, rhodamine, fluorescamine, pyrene, acradine, 2-methoxy-2,4-diphenyl-3(2H)-furanone, and fluorescein mercuric acetate.
8. A reagent for use in fluoroimmunoassay of a fluid sample for an immunological analyte, comprising:
a carrier that can be fluorescently tagged and then treated to release fluorescence, said carrier being heavily tagged with fluorophores to induce self-quenching therebetween, and being adapted to be conjugated with an immunological reactant, said carrier being further adapted to liberate said fluorophores.
a carrier that can be fluorescently tagged and then treated to release fluorescence, said carrier being heavily tagged with fluorophores to induce self-quenching therebetween, and being adapted to be conjugated with an immunological reactant, said carrier being further adapted to liberate said fluorophores.
9. The reagent of claim 8, wherein said carrier is selected from a group consisting of: a protein, cellulose, a cellulose derivative, a polysaccharide, and a polymer.
10. The reagent of claim 8, wherein said fluoro-phores are selected from a group of materials consisting of: fluorescein, dansyl, rhodamine, fluorescamine, pyrene, acradine, 2-methoxy-2,4-diphenyl-3(2H)-furanone, and fluorescein mercuric acetate.
11. The reagent of claim 8, wherein said reactant is an antigen.
12. The reagent of claim 8, wherein said reactant is an antibody.
13. The reagent of claim 8, wherein said reactant is a hapten.
14. The reagent of claim 8, wherein said carrier is adapted to be lysed by at least one enzyme.
15. A method of fluoroimmunoassaying a fluid sample for an immunological reactant, comprising the steps of:
(a) forming a mixture of a sample containing an immunological reactant to be assayed with a known amount of a tagged competing immunological reactant, said competing immunological reactant being tagged with a plurality of fluorophores to induce self-quenching therebetween, and a binding agent that equilibrates with both said sample reactant and said competing reactant;
(b) equilibrating said mixture to form a first portion comprising reactants bound to said binding agent, and a second portion comprising reactants which are not bound to said binding agent;
(c) separating the mixture into said first and second portions, respectively;
(d) liberating fluorophores from said tagged immunological reactant in either one of said first or second portions; and (e) measuring the fluoroescent level of said liberated fluorophores.
(a) forming a mixture of a sample containing an immunological reactant to be assayed with a known amount of a tagged competing immunological reactant, said competing immunological reactant being tagged with a plurality of fluorophores to induce self-quenching therebetween, and a binding agent that equilibrates with both said sample reactant and said competing reactant;
(b) equilibrating said mixture to form a first portion comprising reactants bound to said binding agent, and a second portion comprising reactants which are not bound to said binding agent;
(c) separating the mixture into said first and second portions, respectively;
(d) liberating fluorophores from said tagged immunological reactant in either one of said first or second portions; and (e) measuring the fluoroescent level of said liberated fluorophores.
16. The method of claim 15, wherein said immunological reactant is an antigen and said binding agent is an antibody which specifically binds to said antigen.
17. The method of claim 15, wherein said immunological reactant is an antibody and said binding agent is an antigen which specifically binds to said antibody.
18. The method of claim 15, further comprising the step of:
(f) coupling said binding agent to a solid phase prior to step (b).
(f) coupling said binding agent to a solid phase prior to step (b).
19. The method of claim 18, wherein said solid phase is selected from a group of materials consisting of: magnetic particulate, cellulose, glass, latexes, plastics, agaroses, Sepharoses?, and Sephadexes?.
20. The method of claim 18, wherein said solid phase may be in the form of tubes.
21. The method of claim 18, wherein said solid phase may be in the nt form of rods.
22. The method of claim 18, wherein said solid phase may be in the form of spheres.
Docket 2135-A
Docket 2135-A
23. The method of claim 18, wherein said solid phase may be in the form of particles.
24. The method of claim 15, wherein said fluorophores are selected from a group consisting of:
fluorescein, dansyl, rhodamine, fluorescamine, pyrene, 2-methoxy-2,4-diphenyl-3(2H)-furanone, acradine and fluorescein mercuric acetate.
fluorescein, dansyl, rhodamine, fluorescamine, pyrene, 2-methoxy-2,4-diphenyl-3(2H)-furanone, acradine and fluorescein mercuric acetate.
25. The method of claim 15, further comprising the step of:
(f) comparing the measured fluorescent level with a standard fluorescent level.
(f) comparing the measured fluorescent level with a standard fluorescent level.
26. The method of claim 15, wherein the fluoro-phores liberated in step (d) comprise chemically treating said first or second portion.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US96511878A | 1978-11-30 | 1978-11-30 | |
| US965,118 | 1978-11-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1124643A true CA1124643A (en) | 1982-06-01 |
Family
ID=25509477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA333,870A Expired CA1124643A (en) | 1978-11-30 | 1979-08-16 | Carrier tagged with releasable fluorophores |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1124643A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5132242A (en) * | 1987-07-15 | 1992-07-21 | Cheung Sau W | Fluorescent microspheres and methods of using them |
| US5194300A (en) * | 1987-07-15 | 1993-03-16 | Cheung Sau W | Methods of making fluorescent microspheres |
| US5384241A (en) * | 1987-09-11 | 1995-01-24 | Enzo Diagnostics, Inc. | Specific binding assay compound with inhibitive self-quenching characteristics |
-
1979
- 1979-08-16 CA CA333,870A patent/CA1124643A/en not_active Expired
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5132242A (en) * | 1987-07-15 | 1992-07-21 | Cheung Sau W | Fluorescent microspheres and methods of using them |
| US5194300A (en) * | 1987-07-15 | 1993-03-16 | Cheung Sau W | Methods of making fluorescent microspheres |
| US5384241A (en) * | 1987-09-11 | 1995-01-24 | Enzo Diagnostics, Inc. | Specific binding assay compound with inhibitive self-quenching characteristics |
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