CA2015367A1 - Radial immunodiffusion and like techniques - Google Patents

Abstract

ABSTRACT
RADIAL IMMUNODIFFUSION AND LIKE TECHNIQUES

In a known method of immunodiffusion, a sample containing a first agent such as an antigen is introduced into a well in a lamina of agarose gel containing a second agent such as a complementary antibody. The first agent diffuses through the gel and becomes releasably bound to the second agent and, when the concentrations of agents are optimal, the agents form an extended matrix incorporating light-scattering aggregates. The size of the visible matrix enables the concentration of one of the agents to be assessed when that of the other agent is known. Each aggregate comprises very large numbers of the molecules of the agents. The invention provides an improved method in which the second agent is attached to carrier means in the gel (3) so that the carrier means constitutes part of the visible aggregations. The carrier means may constitute the gel itself and/or it may constitute particulate material such as polystyrene particles.

Description

2;~fl~ '91~ 5~021 ~56 1368 BARI~ER BRETTELL ~) NACRAE ~)o3/~Jls i7 T~is invention is conce~ned wi~h improvements in and rel~ting to radi~l ~nunodi~fusion a~d like techniques.

Several ~e~hods of measuring the co~centration o~
a pro~einaceous anti~en in a sample a~e already know~.
A fixst of these methods is well-known ~nd ~idel~ ~sed 10 and is normally referred to as im~u~odi~fusion. In that method a sampl.e of the material at a predetermined dilution is i~ rod~ced into a well in a plate of agarose gel cohtaining an antibod~ specifi~ to that antigen, the ahti~ody being present a~ a predeterm~ed concen~ration in ~he gel. The sample diffuses radially ~rom ~he well into t~e gel, where t~e anti~en becomes releasably bound to the a~tibody. ~his results in the formation of a matrix which is ~isible as a ring, o~
someti~es as ~ di~c, concen~ric with the well. With the passage of ti~e the diameter of the ~ing or ~isc increases ~ntil a maximum diameter is ~eached, and it is usu~l for the inner p~rt of t~e rin~ or disc to beco~e less distinct or ~o disappeal as t~e m~ximum outer diameter is ~eached. Experimen~ has shown that t~e concentration o~ antigen in ~he original sample is directly proportional to the maximum diame~er of t~e ring or disc The ~ethod is des~ri~ed in the ~ollowing paper: Mancini G., Carbonara A.O. and ~eremans l~w~~ ~ , 2, 235 (1~5).
In a con~erse of that first method, whic~ is of use in assessing the concen~r~tioh o~ an~i~ody in s~ple, t~e agarose ~el con~aLns a predetermined concentration of the antigen; w~ile ~le s~mple ~ontains an unknown concent~atlon o~ the complementary an~ibod~.

:'^1' :;~;' ~ - -~.~-, . . . ..

'Jll 1~ 'U~l ~lo~ 13~ lLl~LL ~ .UA~ A~ J~ JlJ
2~;3~

The visibLe ~atrix is most c~early formed ~hen the ~oncen~ra~ions of the p~o~ein and the antibody are optimal. At those opt~m~l co~centrations most o~ the antibodies become bo~nd ~o t~o separate molecules of antigen while most of the an~igen moleoules ~ecome bo~na to two or more separa~e a~tibodies so that extende~ aggregations of molecules are fo~ed, those aggreg~tions constituting the visible basis of the matrix. Where there is an excess of an~i~odies fe~ ox 1~ none of t~e antibodies are a~le to link together separate antigen molecules. Likewise ~here t~ere is an excess of antigen molecules t so~e pairs of antigen molecules may eac~ be linked together ~y ~n antibod~
~oleeule but those linked pai~s are not linked to fonm large visible complexes as insu~icient antibody molecules are presen~.

For a ~atri~ to inter~ere with t~e direct passage of light through a ~el, it must comprise indivi~al aggregations o~ molecules eao~ of which is of an extent w~ich is at least abo~t one ~wentieth of the wa~eleng~h of the lig~t~ In practi~e, noticeable scattering occu~
wheh ~here are aggrega~ions each of whi~h is so~e 20 to 400 nm in diameter As an individual antibod~
~5 ~olecule or a typical indi~id~al antigen ~olecule is only a ~ew nm a~ross r e.g. a~out S to 8 nm across, it means that no scattering of ligh~ ~hat is visible ~o ~he e~e ocCurs ~ntil there are formed aggregations of mole~ules each consisting of a ~ery la~e n~mber of molecules~ typi~ally at least a few million molecules.
~t also means ~at ~he con~entration of an~ibody and antigen molecules in the ~ters~ices 0~ the gel is ~el~tively hi~h. This in t~rn p~es~pposes an zdequ~t~
concen~ation o~ the antibody (or conv~rsely o~ the ~5 antigen) in ~he gel initially.

i~,' . ., ~,. ~.
'~;~ " .-; ' ' ' 2~5367 While that type of me~hod of radial imm~nodiffusion is ex~remely valuable for some purposes it does nevertheless suffer from ~he draw~ack that its sensi~ivity is limited owin~ to the need to enable S relatively larqe aggre~ations of ~olecules to be formed. Moreo~er, the ~ethoa i~ slow at low concentrations.

A second known method of measuring the concentration of proteinaceo~s antigen in a sample makes ~se of a liquid reagent comprising a suspension o~ latex particles coated with ~ntibodies. ~he particles are suffioiently small that their presence does not greatly xeduce ~he clarity of the reagent. In lS use, however, when a pro~einaceous antigen is ~dd~d ~o the xeagent, ~he an~igen links some of the ooated particles together so that aggregations are fonmed ~hi~h noti~eab~y reduce the clarity. The conoentratio~
of the anti~en can be determined from meaqurements of ~he xesul~ant opaci~y of the reag~nt. While this method ca~ be oarxied out r~lativel~ rapidly and can be made more sensiti~e than the ~irst method described abo~e, it requires t~e ~se of relatively co~plex and expensive apparatus to provide a~urate meas~ements of opaci~y.

~ he present in~ention provides~ ~mong other ~hings, a modification. of the existing methoas whi~h can ~void or reduce at least so~e of the difficultie~
associ~ted with those me~hods~

From a first aspect the pxese~t inventio~ con~ists in ~ method o~ de~eoting the presence of ~he first of a pair of complementary matrix-forming agents in a 3S sample, in w~ich method the sample is ~pplied to a test bo~y ~omprising ~ gel in which there is present ~he .: . , , . .~:, . ~ . . .

2.5 0~ ' ~0 11: 1;)9 ~r~21 ~51~ 136,~ BARRER BRET'rELL ~ IACRAE ~
2~S3~7 se~ond o~ said pai~ of co~plementary mat~ix-for~in~
agen~s, said second agent being a~tached to car~ier means, allowing said first a~e~t (if present) to di~f~se th~ough the ~el ~ntil it is incorporated in a S light-s~atterirlg m~trix, the light-soattering properties of the ma~xix differing fro~ those of the test body, and usin~ a change in t~e light-scattering resulting f~om ~he fonmation of ~he ~atrix to detect ~he p~esence of the matrix and Gonse~uently the presence o~ saîd ~irs~ agent in the sa~ple~

The matrix is referred to above as being light-sc~tering matrix, and it is this property tAat erlables ~he presence o~ the matrix to be sensed and ca~
also conven~en~y be used in determini~g its extent~
While t~e light-scat~ering effect ~ay ~e sen~ed visu~lly by si~ple hum~n observatlonr it may alternatively be se~sed ~y inanimate sensîng means such as by a device incorporating a light-sensitive detector~ ~oreover, although it is ehvîsaged that the light of w~ich the scattering is sensed would normally be in the visible spectru~ this is not necessari~y so, ana it is witbin ~e scope of the in~Ten~ion ~o tlse a me~hod in w~ich the li~ht o~ which the scattering is sensed i~ o~tside the ~isible spect~um, fo~ example, it ~-~ay be in the ultra~iole~ par~ of ~he spect~um. ~ :~

Fro~ a sec~nd ~spec~ the prQse~t invention consists in ~ test body fo~ ~se in carr~ing out a ~ :
method in a~cordan~e wit~ the fixst aspect of ~he pxese~t; inve~tion and comprising a gel in whic~ there is pre~ent said secand ma~rix-forming agent a~tached ~o ca~rier means.
:
~he caxrier means may co~prise t~e ~el itself, the - -second matrix-~orming agent ~ein~ attaçhed to t~e gel.

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: . . :, ~3 1)~ a~ `U21 ~56 13~8 BARKER BRETTELL ~ IACRAE ~JO~
2~

Alternatively or in ~d~ition the carrier means may comprise parti~late materialA

The test body p~e~erably resembles a test body o~
~he kind used i~ carrying owt the known ~ethod described above. T~at is, t~e tes~ body is conveniently in t~e form o~ a lamina. This is pre~erabl~ fornled with a hole or nwell" into which a sa~ple is or CAh ~e ~ trod~ced. The lami~a is preferably o~ orm ~hic~ness so th~t the volume o~
~he matrix ~ormed ~a~ be assessed b~ measuring its surface area o~ by measur~g its diameter or radius if it is of cir~ul~r con~igura~ion~

lS NevRr~hel~ss it is to be ~derstood ~hat ~ile the inve~tion may well employ ~els in ~he ~orm of la~inae i~to ~kich ~he sample ~s ~llowed ~o di~fu~e radially, it ~a~ e~plo~ gels o~ o~her shapes. ~o~ example a s~mple may be caused to diff~se along a narro~ strip or col~n of gel~

The nvention is base~ o~ the dis~overy that use~l resul~s ~an fol ow when sai~ seco~d agent ~s atta<:~ed to ca~rier means. Before that discove~y W2S
made it had been ~houg~ ~hat no such results could a~i~e fro~ ~ha~

In particular 1~ had previousl~ been t~ought t~at a necessary re~uiremen~ ~or t~e su¢~essful fo~mation o~
~ ~atrix in a gel ~a~ that bo~h o~ the ~atrix-formihy 2gents shoula be free to di~f~se th~oug~ t~e interstices of the gel~ t had ~here~ore been a~sumed ~at attaching the seco~d matrix-fo~mL~ age~t to ca~rier means wo~l~ lrlter~ere Wi ~ ~ fonnatio~ or would wholly preve~t ma~rix-~ormation. It ha~ now been found ~hat ~his is no~ so and ~hat m~t~ices ~an readily.

.~_". , , . , ..................................... , ,.. ~., ~ , . .
:~. . . .: . .: . ' 25 I:J~ '90 1~ 21 ~56 13~`3 ~ARI~ER BRETTELL ~ IACRAE ~()()7/()15 2~

fonll even if said second a~ent is ~ttached to ~rrier mea~s and is therefore rende~ed less ~apable or even substanti~lly i~capable or ~olly lncapable of difiusing thro~h the interstices of ~he gel.

It will b~ appreciated t~a~ ~hen a partic~late carrier material is used, t~at ~texial consti~utes part of any ~ih~lly-produoed light-sca~tering aggregation and that the concentration o~
10 matrix-fo~ming agents in the aggregation is less than that ~hich occuxs in an aggregation o~ si~ilar size - incorpora~ion no carrier material. Consequently, the second of ~he known methods descri~ed abo~e can usually be carried ou~ with samples tha~ are less concentrated than those need for carrying out ~he first of t~ose known methods. ~n other words, ~he use of particulate c~rrier can lead to an înc~ease in sensitivit~ On the o~er hand, as ihdi~ted abo~e, it was expe~ted ~hat ~he use of a particula~e carrier in a ZO gel rat~er than ~ free liquid would not lead to m~trix-for~ation or at least would no~ lead to satisfa~tory matrix-~ormation in view of ~he fact that the particles of the carrier would ~e pre~ented from moving in the ~el or ~ould be hamp~red in ~heir movement. -~n ~act, ho~evex, the use.o~ a particulate ~arrier in ~ gel can yield ~seful results. It is postulated that the reason for t~is are as follows. ~i~st, it will be appreciated th~t in carrying out ~e firs~ o~
t~e kno~n methods~ an~ ~isible ag~regation reg~ires there t~ be a relati~el~ large concen~ration of link~ges ~etween ~ti~en . and antibody m~lecules, ~:
whereas in c~rr~in~ out t~e secon~ of the known methods, in ~Aich the aggregation in~orporates a partioulate car~ier, the ~oncentra~ion of such li~kages .,~, ,~:~.

26 1)~ PU21 ~5G 13~ BARKER BRETTELL ~ IAC,RAE ~ Or)8/01~
~53~'~

is less. ~oreover, as t~e particle-size increases, the concentration of link~es i~ a visiblë ~ggre~ion is pxogressi~e~y reduced. It is suggested t~a~ when rela~i~ely sm~ll p~rticles ~re employed ~o~ility o~
S those particles in only partially hampered by the gel and t~t the conseq~e~t red~ction in speed ~f matri~-~ormation is a~ least partial~y counterbalahced by the red~ction in the concen~ration o~ linkages rey~i~ed. ~hen rela~i~el~ large particles are used, 10 they ma~ ~e l~el~ ~r w~olly preventea b~ their size from di~fusing through the gel. Ne~e~theless it appeaxs t~lat oo~ponent strands o~ ~he ~el are su~iciently flexible to enable neigh~ou~ing particles to approa~h one anoth~ sufficie~tly closely ~o enab~e lS the matrix-~orming agent at~a~hed to those parti~les t~
~eco~e linked to the complementary ayent dif~sing throug~ t~e gel~ Th~s it see~s likely that the decreasing mobility that arises w~th i~creasing part~cle-size is adequately cou~ter~alanced by t~e reduction in the concentratio~ of linkages re~uired in matri~-~ormation ~nd ~he unexpected ~lexi~ility o~ the co~pohent strands of ~he gel.

Similarlyt when the carrie~ me~n~ is constituted ~5 b~ the gel itself, ~e ~ight-sca~tering a~regatio~s t~at are ~ormed incorporate pOrtiohs of ~he ~el. In order to increase the sensitivi~y of the method, us~
m~y be ~ade of a gel with ~elatively thick co~ponen~
strands; a la~ex gel with relati~ely thick co~ponent strands may be suitable.

Another way o~ considering the ad~an~ages of the present invention îs to co~p~re two ~e~hods which dif~e~ fro~ ea~h othe~ only in this, namely that in the ~ir5t method ~he second agent is f~ee in ~he ~el, in a manne~ oharacteristic of the first o~ the kno~n .x.

- 2~5;~,7 me~lods, while in the second me~hod the second agent is at-tached to carrier m~a~s, in ~he manner ~haracteristic o~ the present in~en~ion~ The con~entr~tion of the second aqent is the .~m~ ; n ~oth mQt~odo -~d thc con~en~ation of the firs~ agen~ in the sample is the same in both methods When the samples are applied, ma~rix-~ormation occurs in the two gels at substantially the s~me rate bu~ in the first m~thod visible aggregations are formed more slowly ~han in the second me~hod~ Indeed, if the concentr~tions o f agents are low, ~isible aggregations may ~ot be formed ~t all in t~e irst me~hod~ Consequently, use of ~he second method, in accordance with ~he invention, enables ~reater sensitivity to be ~chie~ed ~t low concentrations o~ agents and ~t higher concentrativns enables readings to be obt~ined more rapidly.

A me~hod in acGordan~e with the present invention may be used not o~l~ for detecting the presence of said ~irst matrix-~orming agent bu~ also for ~ssessi~g the quantity of th.e ~irst agent in the sample~ ~his being done ~y p~o~iding i~itially 2 known con~entration of said se~ond ~gent in ~he gel and ~y determini~g ~he extent o~ the matrix when i~ has ceased to increase in ~5 size When ~he test body is in th~ form of a lamina wi~h ~ hole or well in i~ into which a sample of predetermined ~olu~e is introd~ed, the quantity o~ the firs~ age~ in ~he sample is propor~ional to the diameter of the rin~ or ~isc formed ~round ~he hole.
If r on ~he ot~er hand the tes~ body is in the shape of ~ narrow strip or ~olumn, ~he qu~nti~y of ~he first agent in the colbmn is propo~tional to ~he le~th of the visible matrix formed in the st~ip or column.

35Conversely a method in accordan~e with the present invention may be ~sed i~ ca7ibrating t~e concentration :i.~ : : . . , :

2 t3~36'7 of said second agent in a gel To this end, use is made of a sample ~ontaining a known concentration of said firs~ agent One of the complementary m~trix-forming agents is preferably an antibody, ~he other a~en~ ~eing an antigen. ~he antibody prefera~ly constitutes said ~econd agent and i5 initially present in the gel.

As is well known, anti~ens ~come releasably ~ound ~a antibodies b~ a mechanism such that ~he association cons~ant is Gonsiderably greater th~n the disociation oonstan~. Conse~uently, the rate at ~hich ~ matrix ~rm~ is largely dependant on the value ~f the disociation constant. In the foxmation of a matrix inco~porating all or substantîally all of the available mole~ules of said ~irs~ agent, the ~irst agent diffuses through the gel and ~eco~es repeatedly bound to and released from said seeond ag~nt until a mat~ix is formed.

It ~s fo~nd that for the present invention to operate satisfactorily the association constant must he relati~ely high, and it is thou~ht that this is a conseque~ce o~ the fact ~h~t the binding forces ~etween the age~ts m~s~ be great enough to resist ~he elastic ~or~es exerted ~y the ~omponent s~rands of ~he gel which may ~end to urge the agents apart~ ~hether or not any par~icular pair of complementary agents c~n be 3 0 used in ~arryi~g out a method în a~cordance with the present invention can best ~e de~ermined by e.Ype~iment. As indicat~d above, antibodie~ ~nd proteina~eous anti~ens ~an generally be employed satis~acto~ily~ In general it is considered that ~or 3~ sa~isfactory operation the ass~ciation ~onstant should be ~elatively high, or e~ample ~he equili~rium JI;~ 'U21 .151~ 1313~ BARRER BRETTELL ~ IACRAE 1~00~/01,;
2~5~7 constant ~eq should normally be at least 108 moles A method in accordahce with the present invention S Inay employ antiboaies 7.nd pro~einaceous antigens as eomplementary a~en~s. AlternAtively it may ~mplo~ any of a wide r~nge o~ other complementa~y agents. For exa~ple one of the agehtS ~ay be avidi~ and the other agent biotin. Either one ma~ oonstitu~e said ~irst 10 agent bu~ in a preferred method bio~in would cohsti tute the first ~ge~t and that avidin would constitute ~he second agent and be attached to a par~ioulate carrier.
O~her complementary a~en~s ~hat may be employed are lectin and car~o~y & a~es, par~icula~ly sugaxs In a 15 preferred method lectin constitutes the second agent, bei.ng a~tached to a particulate carrier in the gel, while the carbohydra~e constitutes the ~ixst agent Tllrnin~r now to the nature of t~e matrices th~t are fo~ed when employi~g the first o~ the kn~wn methods and wheh e~ploying ~he p~ese~ inven~ion, it ~as been obse~ed ~hat in general, when t~e first of t~e kno~n methods is employed, a ~isible disc is first forme~;
its diame~er progressi~ely increases to a maximum but 2$ at ~he same ti~ ah inne~ paxt of the disc progressively dis~ppears ~ain so that t~e final visible m~t~ix is in the shape of a ring. This is ~hought to he a conse~uenc~ of the fact ~hat bot~
~on~tituent agents can dif~use through ~he gel. I~
o~nt~ast, ~hen the prese~ inve~tion is employed, t~e matrix ~enerall~ ~orms a visible disc of ~hi~h t~e inner part ~oes not disappear. Thi~ is ~hough~ to res~lt from the fa~t ~ha~ the se~o~d ~geht is generally immobile or subs~antially Lm~o~ile i~ the gel.

, 2s,~u~ 13 ~J21 ~56 1368 BARhER :~RETrELL ~ lACRAE [j~10~ J1;
2~S3~7 In its pa~sage through the ge7 towards the rim of t~le ring or disc a molecule o~ said fixst agent is likely to become bound to and released from a large number o~ molecules of said second agent and to follow a non-rectilinear p~th, its gradual, o~erall movement occurring as a result ohly of the ~oncentration gradien~ in ~he gel. ~n carrying out a method in accordance with the ~irst known method referred to above, a gxea~ num~er of linkages must be formed before visi~le ag~regations are formed so the me~hod is relatively slow. In using a method in ac~ordance with the pres~n~ invention, howe~er, the concentratio~ of mutually bound agen~s necessary to yield a ~isible aggregation is reduced, owing ~o the presence o~ the lS carrier means, with the co~sequen~e that results can gener~lly be obtained more rapidly.

~ hen ~he ca~rier means is of p~rticulate form it compxiseS a pluxality o~ m4~ute particles to each o~
which is at~ached a plurality of the molecules of said seco~d agent. The particles ~ay be particles o~ a latex such as a pol~s~yrene latex. ~he m~nuf~cture o~
colloidal polystyrene particles coated wi~h a~tibody molecules is ~lready ~ known tec~niq~e ~s such ~oated ~5 parti~les are used in ~he second o~ the kno~ methods refer~ed to abo~e~ Coated pa~ticles o~ ~hat existin~
kind may also ~e use~ in gels embodying ~he present invention. Alternatively the pa~icles may ~omprise a metal su~h ~s gold. ~ot~ polystyrene particles and gold parti~les are s~ch that when th~ form par~ of an aggxega.tion ~f the kind des~ibed ~hey are relatively transparen~. ~he matri~es of ~7hich they for~ a part are visible because light is refrac~d a~d internally reflected hy the Gonsti~uent particles. ~se of inhexently more opaque s~bs~ances may i~c~ease the ~isibility of the matri~es. ~or example, use may be ~ ,~ . . - . . : -... ..
.. ,., , . ~ - ~ .

_ 25, 01 ' 9~ 1.3 ~021 ~5~ 13~ 8 BARhER BRETTELL ~ IACRAE ~() 11~015 m~de of, a ~er ~o,~p~i"s~g; pi~c~s ,e~, of ~h'' ' compr~ses a co~e'oE iro~,'~d-~' coa~n :of s~ . ' -~ e~er, ~e' ~ o ,, ~e :p~icles, '~e.. ~a~e . '.of e~ch part~ le ~o~.,c~ es.. ~ largé '~umber of' ':
: mo1eclIles , of sa:~ secontI ' 'ma~,~ g ' agent, ' the ' ' ' , .
overall cor~ceht~i~io I .o~ hat ' ~LgeA~ gel '; ~ia,'y ',. ' the~e~ore,. still . ~e:- ~ia~lvel~y ,hig~ so'- t~a~ e ~,:

10 r~l~'~ y~e~r~L ~ h geLt ~e.ce saI~ ~, i "';
Wlll also be co~resp ~ ylhl~h. . Co~se~uen'~ly ',~he;'. "-~a~e o f,,o~matio~ of ~he. ~ple~ed~a~ lX is l~kel~ to : '" '' be ,relatively ,.slo~ ,th.'on~ si~le'''aggrega~io~s' :m~
~tar~ ,~o app ~ " ~ei'a~ ,lyC.qui~kly~ ,..' ~owevRr,'.b~ , ' lS red~c ~ ~,;.~ e' ~ ,e~ .Of:'~eSr 0~ s~ d"secoRd. agen . ", on t~e ~ , of:':each~ -particle~ le ~e~aL~ng .t~ë
same. .con~ent'ratio~ ;o~ p ~ ~les, t~e o~ëra~
ConCR~tratiO~ 'of:,~ha,'.~ sec~ ' = , in ~he ~ 1S~ '', .;
~educed o'.~a~ hë~ once,~ ~ ion~ of-s~id':~lrst a~ent. ''-.' necess ~ -~or:,t e re ~ c'~,ce ' a~ons'o~i~ e ' " ' "
, . . ,. , so...rei~u~ed. ,...,Conse~ue~ he'~" .:' ' S~l~Vi~` O :..~ e 'e o'd~,'is,,.,~c.ré~ ed,'~ d ~ e e' co ~ etio~ of: the m-tr~x~'s:jal'so ~ea~c,ed'~,.: T;h~-'s~ze `o~'~'''" ' ~e par lés :is:su ~ ~ ,eve~. ~ ~ole ~ es S
" ot~e~ ~ .~.~.",,~ e,a~ a ~ ei~'.,o";l~;.~ ,,~, ~ arë;.~oo ~ all tO'I'''''." :
- s~at~er }~h~ of.; '~he';'~a ~ ~, ~,~sëd. ,.Ne~e~t~eless'~. ' .
they ~2~;be'.o~-':a slze: s~.' ~ only'a r.ëla~ivel~. ew''~ ,-; '' par~icles, for'exa~ple~ *ë.:parti~ies,'~ n~. .' ,. ' -scatter ~ ~ eno~gh t't''t ~ . ~ 00 lu=~ b,~gh: ~ cle~ " -The ac~ .t s, a~~.p ~sp ~ ~,ve o one p~ ~. o~ p1a~e fo''~se i ~ ' 1 .

'v ~11 Jl.~ .J~ LI~ l~h~llLLL ~ IA~ A~ f)~
2~53~i7 out a method in accord~nce wit~ the present invention,~his bein~ illustrated ~erely by way of example.

The ~est plate comprises a ~igid tra~ 1 wi~h a fla~ base in t~e shape o~ a rectansle with rouhded oor~ers and ~n upstanding pe~ipheral flange 2. ~he ~ray ca~ries ~ . lamiha ~ of u~i~orm t~ickness constitu~ing a test bod~ in aocord~nce wit~ the present inven~ion. 8n ar~ay of th~o~gh ~oles 4 is ~ormed i~
the lamina, ~s illustrated.

The lamina 3 comp~ises a gel inoorporating polystyrene particles coated wi~h ~ntibodies~ The particles ~re of uni~orm size, each typic~lly ha~ing a diameter of 100 nm. S~c~ polystyrene par~icles are a~ail~le on the market and ~re usually supplied in an a~ueous suspension containing S0~ solids and a~ a relati~ely hi~h p~, typic~lly ~.5~ When ~hR paxticles are to be coated the suspension is diluted and subjected to ultrasonic radiation to ensure that the paxticles are fully separated from each ot~e~. A
sol~tion con~ining the desired anti~odies is introduced, and the antibodies attach themselves to t~e pa~ticles, this occuring naturally as ~oth t~e ~5 an~ibodie~ and the par~icles are ~ydrophobic. T~is action ~y be assisted i~ desired by t~e addition of a relatively small a~ou~t of albumin.

~he lamina is for~ed from an ag~o~e gel ~ith a relatively long ~hain-length. T~is is ~eated to cause i~ to melt, melting ~s~all~ occuri~g ~t about 100C
The melted agarose is then cooled in con~rolled conditions, t~e aqarose h~ving the property t~at ~t does not solidif~ again until it reaches ~bout 40C.
~he coatea particle-c are in~rodu~ed into t~e l~quid agaro~e when it is 2~ a te~perat~re a~ ita mel~iuy ~ .

2.;,1~ 11 t~:15 ~021 ~56 1.36~ BAR~ER BRETTELL ~ IACRAE ~ 01.3/~15 21~5367 point b~t below 56~C, at which te~perature the antibodies would s~a~ to become damaged. ~hen the particles have been thoro~ghl~ ~ixed wit~ the liquia~
the mixture is poured i~to the t~ay 1 and ~llo~ed to S set to ~o~m a lamina of uni~or~ thickness. After it h2s set the lamina is te3llporarily removed from t~e tray ~ld t~e ~oles 4 ~re punc~ed throug~ it before it is retuxned to ~e ~ray. An identifioa~ion label ~ is attached ~o t~e gel.
In use a predetermined vol~me of a liquid sample cont~i~ing an antigen complementa~y to the antibodies i~ introd~ced ~n~o one o~ the holes and the antigen dif~uses ~hrough the ~el in the manner described abo~e so that a visib~e matrix is progressively for~ed. This ~o~m~lly takes the for~ of a disc concentric with the hole, one typical disc of ~his kind being indicated at ~. ~he.dia~eter of the disc i~ ~easured to enable the ~oncentration of antiyen ih the sa~ple to be deduced.
~ here are several ways in which the plate ~a~ be used~ ~n one ~ethod, a test sa~ple of u~k~o~n strength is introdu~ed into o.ne of the holes 4 while several ~tandard samples, each of a known strength, are int~oduced i~to other holes. When the di~ around the samples have reac~ed sui~ably large si~es, ~eir dia~e~ers ~re measured. Those ~esul~i~g fro~ t~e sta~d~rd samples are plot~ed as points on a graph of diameter against streng~h. A line is dra~n through those pOihtS a.nd the streng~h of the test sample is assessed fro~ the diame~er of the matrix visible around t~e hole 3nto ~hic~ it was i~roduced. Using that ~ethod it }llAy be possil:~le ~o o~t~in useful results in a 3S single day. ~n a so~ew~at simil~r method, whic~ may lead to gre~te- ~cu~ac~ but ~ay well take longer, the ~.
. . .

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~ 5~ 136~ ~ARIs~K ~RI:l'lLL ~ IA~,RAI~ ol4/~

2~53~ 7 method is contin~ed until ~e discs ~ave reached their maximum di~me~ers. This may take ~wo days or even more. ~n another ~ethod, the plate is calibrated by the m~nufa~turer and a ta~le is provided sho~ing the 5 maxim~m diameters o~ the ma~rices ~hat will be ob~ained wit~ samples of given streng~hs.

Test plates of the kind descr~bed ~an be used by relatively unskilled people and wi~hout the neea for ~o~plex or expensive measuring d~vices. In par~icular they enab71e doctors to carr~ out ~ests quickl~ ahd simply ~ithout the need to make use of equipmen~ ahd expertise normally a~ailable only at a hospital.

The first of the known methods ~eferred to above enabled meAsurements to be made wi~h sa~ples containing concentrations of antigens down to about 5 mgJl but the present 7nven~io~ readily enables meas~reme~ts to be made ~it~ samples con~aining cohcen~ra~ions of an igens 20 do~n to O.5 mg/l, and in some cases the limi~ may be reduced to 0.15 or 0.~ mg/l. ~onse~uentl~, many conditions t~at have hit~er~o ~een detectable and measurable only after ~eference to a hospital can no~
~e detected an~ measured by a local doGtor~

.,.~ ~: - , : .

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Claims (11)

1. A method of detecting the presence of the first of a pair of complementary matrix-forming agents in a sample, in which method the sample is applied to a test body comprising a gel in which there is present the second of said pair of complementary matrix-forming agents, said second agent being attached to carrier means, allowing said first agent (if present) to diffuse through the gel until it is incorporated in a light-scattering matrix, the light-scattering properties if the matrix differing from those of the test body, and using a change in the light-scattering resulting from the formation of the matrix to detect the presence of the matrix and consequently the presence of said first agent in the sample.

2. A method according to claim 1 in which the carrier means comprises the gel.

3. A method according to either of claims 1 and 2 in which the carrier means comprises particulate material.

4. A method according to any one of claims 1 to 3 in which one of the matrix-forming agents is an antibody and the other is an antigen.

5. A test body for use in carrying out a method in accordance with any one of the preceding claims and comprising a gel in which there is present said second matrix-forming agent attached to carrier means.

6. A test body according to claim 5 in which the carrier means comprises the gel.

7. A test body according to either of claims 5 and 6 in which the carrier means comprises particulate material.

8. A test body according to claim 7 in which the particulate carrier comprises polystyrene particles.

9. A test body according to either of claims 7 and 8 in which each particle is of about 100 nm in diameter.

10. A test body according to any one of claims 5 to 9 in which the test body is in the shape of a lamina of uniform thickness with at least one hole or well in it into which a sample can be introduced.

11. A test body substantially as hereinbefore described with reference to the accompanying drawing.