CA1244761A - Process for the determination of a polyvalent antigen and reagent therefor - Google Patents
Process for the determination of a polyvalent antigen and reagent thereforInfo
- Publication number
- CA1244761A CA1244761A CA000471361A CA471361A CA1244761A CA 1244761 A CA1244761 A CA 1244761A CA 000471361 A CA000471361 A CA 000471361A CA 471361 A CA471361 A CA 471361A CA 1244761 A CA1244761 A CA 1244761A
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- receptor
- antigen
- marked
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- 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
- G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
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- Urology & Nephrology (AREA)
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- Food Science & Technology (AREA)
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- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Abstract The present invention provides a process for determining a polyvalent antigen by incubation with three different receptors, the first and third of which are present in liquid phase and with which the antigen is bindable, the second receptor is present in solid phase and is only bindable with a part of the first receptor and the third receptor carries a marking and does not cross-react with the first and second receptor, separ-ation of the solid phase. From the fluid phase and measurement of the marking in one of the phases, wherein a solution containing the antigen is brought together with a first solid carrier material, which contains, impreg-nated or lyophilised, the first and the third receptor in removable form and, after removal of the receptors, the solution obtained is immediately contacted with a second solid carrier material which contains a second receptor in non-removable form, whereafter the liquid is separated from the second solid carrier material and the marking is measured.
The present invention also provides a dry reagent for determining a polyvalent antigen comprising a) a first solid carrier material impregnated or lyophilised.
with two different soluble receptors 1 and 3, bindable with the antigen, of which receptor 3 carrier a marking, and b) a second solid carrier material, which is present physically separated from the first carrier material, the second solid carrier material containing an insoluble second receptor only bindable with a part of the non-marked soluble first receptor, with the proviso that the marked third receptor is a Fab fragment,
The present invention also provides a dry reagent for determining a polyvalent antigen comprising a) a first solid carrier material impregnated or lyophilised.
with two different soluble receptors 1 and 3, bindable with the antigen, of which receptor 3 carrier a marking, and b) a second solid carrier material, which is present physically separated from the first carrier material, the second solid carrier material containing an insoluble second receptor only bindable with a part of the non-marked soluble first receptor, with the proviso that the marked third receptor is a Fab fragment,
Description
7~1 ~;
~ he present in~ention is concerned with an immune chemicaI process for the determination of a polyvalent, i..a.. at least bifunctiona~ ligand ~antigen) in a liquid ~ample ~ he sensitive determination of polyvalent antigens (peptides, proteins) with the use of two antibodies which are directed a~ainst dif~erent antigen determinants t iS
known as a two-~ite immunoradiometric or immunoenz~mo-metric assa~, for example, from J. Clin Chem. Clin.
~iochem " 18, 197-208/1980.. This known met~od of deter-mination is carried out most commonly be first incubating the antigen to be determined with a first antibod~ which is pre~ent in solid phase by binding to an appropriate carrier material, such as Sepharose~ agarose~ s~nthetic ~e~in tubes or the like, During this first incubation, the first antibody,; which, as a rule, must be present in large excess, binds with an antigen determinant of the anti~en to be determined~ ~he samp~e Iiquid is then separated from the ~olid phase in the usual manne~ iin order to avoid di~u~bance~ due to non-speci~ic substance~,~
such as human plasma protein" or due t~ cross-reacting anti~en~ in the ~ubsequent sscond incubation. A definite amount of a second marked antibod~ in liquid ~hase is then incubated.with the so~id phase. ~ne speci~icity of th~
second antibody is thereb~ seIected in ~uch a manner that it is directed against a determinant of the antigen to be determined different from that of the first antibod~ in ~Z~'7~L
order to avoid a competition between the two sntibodies for the same binding place on the antigen to be deter-mined since the sensi`tivit~-of th~ test would thereb~
be impaired, During this second incubation, the marked second antibod~r which is aIso usuallg present in excess, reac~s with aIl binding pIaces on the molecule of the ~mtigen to be determïned, A~ter the second incubation, the act.ivitg ~f the marking substance can be measured eithe~ on the so~id phase or ïn the supernatant~ ~he m~asurement there-b~
usually take~ plaE in the solid phase after washing out the liquid phase. In the most favourable case t the activit.
determined is almost proportional to the amount Q~f the antigen to be determined.~
~his ~wo-~tage sandwich process has the advantage that possibly c~o~s-reacting antigens are aIreadg removed in the ca~e of the fir~t phase separation. ~his is o~
particular imp~r~ance in the case of test systems in whlch the two antibodies must indeed posg~3s differing sp-eci~icitg ` because. of the sensitivï.tD requîrements~ In this ca3e~ the;
high re~uirement o,f the ~pecifici~ onlg exists for one of the two antibodies and the other one must not be abso~ute~
~pecifïc ~owards crQss-react~ng antigens, ~eve~tha~es~,, this proce~ suff~rs ~rom three im~ortant disadvantages:
1`~ In the first incubation step,; one reac.tant is present in the soIid phase and the other in solution~ ~he velocity - 1 2 ~
cpnstant of the reac.tion i8 thuJ smaller than wou~d be the case were both reaction components (antigen and first antibody~ present in soIution. However, it is nece~sar~
to incubate until all antigen molecules are present bound to the solid phase since non-bound antigen would be remGve~ in the case of the phase separation and this would result in a falsification of the result~
~ he present in~ention is concerned with an immune chemicaI process for the determination of a polyvalent, i..a.. at least bifunctiona~ ligand ~antigen) in a liquid ~ample ~ he sensitive determination of polyvalent antigens (peptides, proteins) with the use of two antibodies which are directed a~ainst dif~erent antigen determinants t iS
known as a two-~ite immunoradiometric or immunoenz~mo-metric assa~, for example, from J. Clin Chem. Clin.
~iochem " 18, 197-208/1980.. This known met~od of deter-mination is carried out most commonly be first incubating the antigen to be determined with a first antibod~ which is pre~ent in solid phase by binding to an appropriate carrier material, such as Sepharose~ agarose~ s~nthetic ~e~in tubes or the like, During this first incubation, the first antibody,; which, as a rule, must be present in large excess, binds with an antigen determinant of the anti~en to be determined~ ~he samp~e Iiquid is then separated from the ~olid phase in the usual manne~ iin order to avoid di~u~bance~ due to non-speci~ic substance~,~
such as human plasma protein" or due t~ cross-reacting anti~en~ in the ~ubsequent sscond incubation. A definite amount of a second marked antibod~ in liquid ~hase is then incubated.with the so~id phase. ~ne speci~icity of th~
second antibody is thereb~ seIected in ~uch a manner that it is directed against a determinant of the antigen to be determined different from that of the first antibod~ in ~Z~'7~L
order to avoid a competition between the two sntibodies for the same binding place on the antigen to be deter-mined since the sensi`tivit~-of th~ test would thereb~
be impaired, During this second incubation, the marked second antibod~r which is aIso usuallg present in excess, reac~s with aIl binding pIaces on the molecule of the ~mtigen to be determïned, A~ter the second incubation, the act.ivitg ~f the marking substance can be measured eithe~ on the so~id phase or ïn the supernatant~ ~he m~asurement there-b~
usually take~ plaE in the solid phase after washing out the liquid phase. In the most favourable case t the activit.
determined is almost proportional to the amount Q~f the antigen to be determined.~
~his ~wo-~tage sandwich process has the advantage that possibly c~o~s-reacting antigens are aIreadg removed in the ca~e of the fir~t phase separation. ~his is o~
particular imp~r~ance in the case of test systems in whlch the two antibodies must indeed posg~3s differing sp-eci~icitg ` because. of the sensitivï.tD requîrements~ In this ca3e~ the;
high re~uirement o,f the ~pecifici~ onlg exists for one of the two antibodies and the other one must not be abso~ute~
~pecifïc ~owards crQss-react~ng antigens, ~eve~tha~es~,, this proce~ suff~rs ~rom three im~ortant disadvantages:
1`~ In the first incubation step,; one reac.tant is present in the soIid phase and the other in solution~ ~he velocity - 1 2 ~
cpnstant of the reac.tion i8 thuJ smaller than wou~d be the case were both reaction components (antigen and first antibody~ present in soIution. However, it is nece~sar~
to incubate until all antigen molecules are present bound to the solid phase since non-bound antigen would be remGve~ in the case of the phase separation and this would result in a falsification of the result~
2~ The bi~ding of a specificaIly bindab~e antibod~ for the antigen on to a solid phase re~uires,. because of the low bindin~ gie~ds" relativelg large amounts of the ~irst antibod~, which is usuaI~y an expensive substance~, especially when the antiserum must be obtained, for reasons of specificitg, in smal~ animals t such as rabbits,~
æuinea pigs and the like.. Furthermore, in the cas~ of the binding reaction to the solid phase, affinity deterior-ations arise on the antibod~ which have a~ disadvantageous effect. on the sensitDvity_
æuinea pigs and the like.. Furthermore, in the cas~ of the binding reaction to the solid phase, affinity deterior-ations arise on the antibod~ which have a~ disadvantageous effect. on the sensitDvity_
3~ As a; ~u~a9 the ob~aining Q~ the two antibo~ies of different specificit~ a~ainst the ~ame anti~en i~ labor-ious and f~e~uentI~ i~ onlg possibIe ~ithin limits" for e~am~Ie, bg immunisin~ two differ~nt animal specie~ or b~
att~mpting to separats the antibod~ population~ from one animal into sui.table fra~ments by immune adsorption on the antig.en to be determi~ed.
Various attem~ts are alread~ knoYs to ove~come the abov~ deficiencies. ~hus, according to U.S, Patent Speci~icati~n ~o. 4,098,876" the first incubation of the antigen is carri`ed out with isotope-marked~ di~sol~ed
att~mpting to separats the antibod~ population~ from one animal into sui.table fra~ments by immune adsorption on the antig.en to be determi~ed.
Various attem~ts are alread~ knoYs to ove~come the abov~ deficiencies. ~hus, according to U.S, Patent Speci~icati~n ~o. 4,098,876" the first incubation of the antigen is carri`ed out with isotope-marked~ di~sol~ed
4'~1 antibod~. Onlvv thereafter is the solid phase-bound anti-bod~ added thereto and the second incubation carried out.
In this case, onlg one phase ~eparation is nece~sary, whi.ch resutts in a certain increa~e o~ the sen~itivity and practicabilitgr However,. the specificity problem remains unsolved since bo'h antibodies must be directed highly specifically against the antigen since otherwise.,.
in the pre~sence of comparativel~ large am~un~s of cro~s-reacting antigens, the apparent concentration of the antigen to be determined is increased in the cas~ o~ a possib~e non-specificity o~ the second antibod~ and reduced in the case of non-speci~icity of the first antibod~.
~ederal Republic of Germany Patent ~pecification No~ 29 25 565 describes a process in which the first antibody present in solid phase and the marked, dissolved second antibod~ are incubated simultaneouslg with the a!ntigen~ ~he ~ame prob~ems here exist as in the case of the previ~usly mentioned process~ ~urthermore,. disadvant-a~es 2 and 3 described above for the fundamental process aIso remain unsolved Finally~ pub~;shed Jap~nese Patent Applioation No~
I2~,802 o~ the 6th Oc.tober, 1978`,~ ~escribe~ a proces~ in which a solid phase antibody is used which is specifica~y directed against the seco~d antibody whïch is bindable with the antigen to be mea~u~ed In this ca~e, the bindin~
of an antibody airected against the antigen to the solid phase is avoided, ~owever, the sensitivity is Iowored ~ince thsre ï~ onlg used a sin~le antibody which is . ~ lZ4'~76 .i specifically bindable with the anti~en~ ~he concentration determination of the anti~en to be determined takes pIace via the competitive reaction between the antigen and a definite amount of isotope-marked antigen with the specific antibod~.
A process is known from European Patent Specific-ation No 0,105,714 in the case of which there is used a solid phase antibody, to~ether with two fùrther antibodies, at least one of which must be a monoclonal antibod~
according to the sandwich princip~e for antigen determin-ation~ The antigen is therebg first inc~bated with th~ two antibodie~ present in Iiquid phase and thereafter with the solid phase antibody. ~hiS process suf~ers from the dis-advantage that two incubation steps are necessar~ which hinde~s automation, results in too long ïncubation times and does not permit the use of undiluted samp~es A similar process is known from C~in~ Chem~g 27(6~, 823-827/1981 ~here is there described a radiometric method of determin~
a~tion for cPeatine kinase MB isoenzyme ïn which~ in a first - incubation step, the antigen is reacted simul~aneously with an unmarked and with a marked antibod~ of di~fering specificity and fro~ different animal species~ In the second step, an antibody present in solid phase, with specifïcity against the unmarked an~ibod~ of the first incubation step ~! iS then added thereto. ~he disadvantage of this process is again the fact that there are needed two different antibodies specific for the antigen and three different animal species.
~2'~6~
`
~ he~sfore, it is an object oi the present invention better to avoid ane or more of the above-described disad-vantages of the fundamental ~rocess then the previousl~
known processes. In particu~ar~ there is to be provided such a process whi.ch only requires one incubation and none of the antibodies has to be used in liquid phase, which is suitable for automation and which displays hi~h sensitivity and exactitude~ ~urthermore, the proce~s is ~o make it possible to manage,. if necessar~, wit~ a sin~le lo animal species for obtaining the necessary antibody or to be able to use large animaIs for obtaining the antiserum ~ hus, according to the present invention, there is provided a process for determining a polyvalent antigen b~ inrubation with three different receptors,. the first and third of which are present in li~uid phase and with which the antigen is bindable, the second receptor ïs present in solid phase and ïs on~y bindable with a part of the first receptor and the third receptor carries a marking and does not cross-react with the first and 2~ second receptor, separation of the solid pha~e ~rom the liquid phase and measurement of the markin~ in one of the phases" wherein a solution con~aining the antigen is brou~ht together with a first solid carriex material, which contains,.im~regnated or lyophilised, the f~rst and the third receptor in removable form and, after removaI
of the receptors, the solution obtained is immediately contacted wi~h a second solid carrier material which 4~6~
contains the second receptor in non-removable form, whereafter the li~uid i~ ~eparated from the second solid carrier material and the marking is measured.
~ receptors in the scope of the pre~ent invention, there are used either specifically bindable complete anti-bodies (polyclonal or monoclonal), antibod~ fra~ments thereof or conjugates of antibodies or antibod~ fra~ments with haptens or antigens. As first receptor" there i~
preferabl~ used a complete antibody or an a~tibod~ or antibod~ fr~gment bound or marked with a hapten or antigen, in each ca~e a monoclonal antibodg bein~ especial~y preferred.
As second receptor, there is used an antibod~
which is on~y bindable with a part of the first receptor~
an antibod~ which i5 only bindable with the ~c part of the first receptor being especiall~ preferred. Alterr.ativel~
as second receptor, there can also be used an antibod~
which is only ~pecifically bindable with the hapten part or antigen part of the first receptor~ Ths second receptor can also be generalIy directed against the Fc part of immuno~lobulin.
~he third receptor, which must not cross-reacted with the second receptor, can be a marked antibod~,bindable with the antigen, which is obtained ~rom an animal species different from that from which the first receptor is obtained~
However,~ there is prefer~bly used a third receptor which is a marked Fab fra~ment~ ~ithin ~he scope of the z4~'7~i~
_9_ present invention, bg Fab fragment there are also to be understood (Fab)2. and Fab' fragments. In this case, receptor 3 can also be obtained from the same animal species as the antibody component of the first receptor.
ThiS also appIies when the first receptor consists of an antibody which carries a hapten or antigen and the second receptor is only bindable with this hapten or antiæen, ~specially preferably, the third receptor is a part, especial.~y a Fab fragment, of the first receptor.
lo AIternatively~ as third receptor there can also be used an antibod~ which originates from the same animal species as the second rec.ep:tor~ Both alternatives have the advant,-age that only two animal species are required for the three receptors~ If the second receptor is only bindable~
with the hapten o~r antigen of the first r.ecept.or, onl~
one animal species is needed ~or aIl three receptors.
~speciallg prefbrab~y" the third ana the first receptor con~ain mono.clonal antibodies or fragments thereo~ which a~e directed against different and/or repetitive de~ermln-ants of the antigen.
~he third receptor is preferabl~ used in an amount which is greater than the amounts of the antï~en ~o be determined.. ~n this case,.. i..e. in the case of an e~ces~, an exact dosing or a pre~isely known amount is not necessary.. ~he third receptor is marked in a manner known to the e xper~. ~he marking preferably takes place by coupling with an enzyme or with a fluorescing, chemi--~ ~Z'~'7`~
I.uminescing and radioactive substance. Processes for the marking of such receptors are known to the expert, fcr example from Clin. Chim Acta, 8~ 40/1977, and do not require further explanation here ~ he first and third receptors are applied dissolved to a suitable carrier, for example to an absorbent paper, and, by dr~ing or lgophilising, converted into a form which can easilg be removed therefrom.
~he binding of the second receptor present in so~id phase to an insolub~e carrier material can be carried out according to the conventional methods known to the expert for the fixing of biologically active proteins to solid carrier substances~ Not onI~ a covalent but aIso an adsorptive binding is suitable. However,1 because of the~
hi~her yields which can hereb~ be achieved and of the simplified method of working, there is preferred soIel~
an adsorptive binding, for e~amp-le on a s~nthetic resin or on to a fleece, for examp~e paper~ ~her~ have proved to be suïtab~e,7 for examp~e7 reagent ve~se~s made of polystgrene and similar s~nthetic resins which are adsorptivel~ coated on the inner ~urface with the second receptor~ ~e incubation can then be carried out in the~e tubelet~ or in containera of some oth~r shape and, for the pha~e separation, it suffice~ to remove the liquid phase from the tubelet, for example,. by suckin~ out, shaking out or the like. ~fter wa~hing the tu~elet~
the measurement of the marking therein can be carri0d 12~76~
directly, especially when the marking ha~ taken place with an enzyme. It then suffices simply to measure the activity of the markin~ enz~me, for examp~ peroxidase or ~-~alactosidase, in the manner generally known for this purpose by adding a known detection reagent for this marking enzyme, for example a colour reagent, and to measure either in the presence of the so~id phase or after separation~ The measured enzyme activity is then measure for the amount of polyfunctional antigen to be lo determined, However,: as solid phas~ carrier for the~
second receptor, there can also be considered particulate substances,~ fio~ example ion exchangers, molecu~ar sieve materials~ glass particles, synthetic resin tubes and the l~ke~ ~s carrier for the second receptor~ it is ~speciaIly preferr~d to use~a porous, la~er-like car~iert such as paper If the marking does not take place w~th an enzyme but rather with a radioactive substance, an isotope,; a fluorescing or chemi~uminescing subst~nce, here, too~ the measurement is c.arried out according to the methods which are well known to the expert. ~herefore ~! here a~ain, a aescription of these methods of measurements in not n~c~ssary~
~or carrying out the process 9 the sample solution, which accordin~ to an especially advantageous character-istic of theinventio~ can even be used in undiluted form~
f.or exam~e as blood,. pla~ma or serum,~ is placed together lZ'~ 61 with the first carrier material for a sufficient len~th of time for the removal of the two receptors, then separated off and incubated with the second carrier material.. In ~eneral, the contact time with the first carrier material is from about 10 seconds to 10 minutesO
Advantageously, the process for this purpose is carried out in a device of the t~pe which i`s described in pubIi~hed Federal ~epubIic of Germany Patent Application No~ 34 25 008~
Surprisingly, we have found that according to the present invention there is achi.eved a cl~arly high~r sensitivit~ than in the case of the conventional two-step sandwich method and of the one-step sandwich ~ethod~
If, according to a preferred embodiment. of the present invention, there is used a second receptor directed against the Fc part of the first receptor or against the hapten or antigen which i5 c-oup~ed with the f'irst receptor,, then there can hereb~ be achleved a ~urthe~r incEease of the sensitivit~ since a possible steric hindrance,~ of the binding reaction of the anti~en with the bindin~ place of the f,irst receptor is avoided~
is ïs an,especial,advant.a~e when anti~ens with estpeciall~
low concentration mu~t be determined, such as i~ ~or example, the case in the determination of th~reotropin in serumO
.
~Z~'~'7~1 --~3--he present invention aIso provides a dry reagent f~r determining a polyvalent antigen, comprising a) a first solid carrïer material impre~nated or I~ophilised with two diff~rent soluble receptors 1 and 3, bindable with the antigen,.of which receptor 3 carries a marking, and b) a second solid carrier material, which is present physicall~
separated from the first carrier material, the second ~olid car~ier material containin~ an insoluble second receptor onlg bindable with a part of the non-marked lo solub~e first r.ec.eptor, with the proviso that the marked third receptor is a Fab ~ra~ment.
~ he physical separation can j,. for example, take place in that the insoluble second receptor and the solub~e first receptor and the third receptor aPe applied to separate carrier materials This can be achieved in that receptor I and receptor ~ are l~ophilised or impregnated into or on to a suit~ble carrier. simultan-eousl~ or succes~ively (for example synthetic resin tube~ets or absorbent carriers, such as psper or the ~ike) and the re~eptor 2 is bound adsorp-tivel~- or cova~en~ on to an appropriate carrier (for example ~gnthetic resin tubelets,; paper or particIes)~ ~he sample with the pol~-valent anti~en to be de.termined is first brou~h* into contact wi`th the carrier l, which contains the soluble first and third receptor~,; and thereafter with the carrier-2, on which is fixed the insoluble receptor 2 Alternatively~ the first and third receptor, on the one hand, and the second receptor, on the other hand, can be lZ~
pr~sent on carriers connect.ed with one another which consist, for e~ample, of a- paper- strip whïch contains the receptors at different places separated from one another.
~ ccording to a first pre~erred embodiment, a reagent according to the present invention is characterised in that it contains a first receptor, which is an antibod~, and a t~rd re¢eptor, which is an enæyme-marked ~ab fragment of an antibod~ Preferably, it also contains a buffe~ and a s~stem for the determination of the enz~me lo marking.
According to a further preferred embodiment of the reagent according to the present invention, this is characterised in that the receptors 1 and 3 are monoclonal and are directed ~gainst different determinants of the antigen and originate from the same animal ~pe~ies~
According to another preferred embodiment of the rea~ent according to the present invention, it is characterised in that the second receptor is an antibody agains* the Ec. part of the first receptor and the third receptor originates from.the same animal species as the second receptor, whereas the first receptor originates f rom another animal species.
~ccordin~ to yet another preferred embodiment of the reagent according to the present invention, this is characterised in that all three receptors contain anti-bodies from the same animal species, the first receptor being a hapten-marked monoclonal antibody, w~ereas the second receptor is directed against the hapten of the first receptor lZ4~76~L
~ he receptors l and 3 of the reagent accordin~
to the present invention are present on a solid carrier material impregnated or lyophilised.
~ he present invention is suitable for the deter-mination of all antigens with at least two antigen deter-minants, Examples thereof include thyreotropin (~SH), oetoprotein (AFP) r hCG,. carcinoembr~onal anti~en~
luteinising hormone ~I~), follicle-stimulating hormone (~SH~),. @2-micro~lobulin, acidic prostate phosphatase t lo prolactin, ~erritin and insulin.
In the case of the present invention, recept.or can react homogeneousl~ with the anti~en~ Consequentlg-~
t~is reaction possesses ahi~er velocit~ constant than in the case.of the~use of a solid phase-bound insoluble~
first receptor, Since the antibodg losses in the case of the fixing (makïng insoluble) on to the sol;d phase~ ar~
avoide~, the amount of the first receptnr can be further decreas~d, In cont~adistinction to the process described in C;~in, Ch~mO, 27 (6~, ~981, it is not nec~ssarg to ob~ain the.first and the third receptar from ~wo different animat species which, in addition, have to be so chosen that recep~or.~2 on~y reac~s with receptor 1 but not with recep~or 3~
~inall~, the obtainin~ of the antibod~ for the receptor l and for the receptor 3 from the same animal species also maXes it possible to use lar~e anima~s for obtainin~ the antiserum.
lZ~
FinaIl~ accord~ing to the prese~t invention,.
~he sensitivity i5 also increased. ~hu-s, ~or TSE there is-found a sensitivity of less than 40 pg./ml. (1 ~ U
170 pg~/ml` ), whereas in the case of the two site assa~
~sandwich process) it lies above 100 p~./ml ~he foIIowing ~xamp~Ies are given for the purpose~
of illustrating the present invention, reference being made to the acc:ompan~ing drawings, in whïch:
Fi~ 1 is a calibration curve accordin~ to Example 2 lo ~ig~ 2 is a calibration curve according to Examp~le 3~
Fig. 3 i~ a calibration curve according to Example 4 and Fig. 4 i~ an insert element for a centrifugal analysis automatic appar.atus which i5 used according to Examp~le 2 Examp~e l.
Determination of th~reotropin (~SE)~
Rec:eptor 1 and ~ originate from the same animal species (m~use) and ~ecep*or ~ is-a marked Fab ~agment~
Receptor ~ is a monoclonal antibody and the Fab ~ragment in ~e~ep~r.-3 a~so o-riginates from a monoclonal antibod~
which is directed a~ainst a determinant of the ~ other than that aæainst which the monocIonal antibod~ of receptor 1 i5 direct.ed. Recep~or 2 originat.e~ from sheep and is direct~ a~ains~t the ~c part of the monoclonal mouse. antibod~
Tha. development of monoclonal antibodies take~
pIace acc~rding to the method of ~dhler and Milstein ~Eur~ J. ImmunoL , 6~ 292~1976)~
` ~ 24~'7 a2 ~arrging o~t of the test according to the present inv.ention:
Reagents:
I, Incubation buffe.r (IB):
15 mM sodium phosphat.e buffer (pH 7.4); 154 mM NaCl;
In this case, onlg one phase ~eparation is nece~sary, whi.ch resutts in a certain increa~e o~ the sen~itivity and practicabilitgr However,. the specificity problem remains unsolved since bo'h antibodies must be directed highly specifically against the antigen since otherwise.,.
in the pre~sence of comparativel~ large am~un~s of cro~s-reacting antigens, the apparent concentration of the antigen to be determined is increased in the cas~ o~ a possib~e non-specificity o~ the second antibod~ and reduced in the case of non-speci~icity of the first antibod~.
~ederal Republic of Germany Patent ~pecification No~ 29 25 565 describes a process in which the first antibody present in solid phase and the marked, dissolved second antibod~ are incubated simultaneouslg with the a!ntigen~ ~he ~ame prob~ems here exist as in the case of the previ~usly mentioned process~ ~urthermore,. disadvant-a~es 2 and 3 described above for the fundamental process aIso remain unsolved Finally~ pub~;shed Jap~nese Patent Applioation No~
I2~,802 o~ the 6th Oc.tober, 1978`,~ ~escribe~ a proces~ in which a solid phase antibody is used which is specifica~y directed against the seco~d antibody whïch is bindable with the antigen to be mea~u~ed In this ca~e, the bindin~
of an antibody airected against the antigen to the solid phase is avoided, ~owever, the sensitivity is Iowored ~ince thsre ï~ onlg used a sin~le antibody which is . ~ lZ4'~76 .i specifically bindable with the anti~en~ ~he concentration determination of the anti~en to be determined takes pIace via the competitive reaction between the antigen and a definite amount of isotope-marked antigen with the specific antibod~.
A process is known from European Patent Specific-ation No 0,105,714 in the case of which there is used a solid phase antibody, to~ether with two fùrther antibodies, at least one of which must be a monoclonal antibod~
according to the sandwich princip~e for antigen determin-ation~ The antigen is therebg first inc~bated with th~ two antibodie~ present in Iiquid phase and thereafter with the solid phase antibody. ~hiS process suf~ers from the dis-advantage that two incubation steps are necessar~ which hinde~s automation, results in too long ïncubation times and does not permit the use of undiluted samp~es A similar process is known from C~in~ Chem~g 27(6~, 823-827/1981 ~here is there described a radiometric method of determin~
a~tion for cPeatine kinase MB isoenzyme ïn which~ in a first - incubation step, the antigen is reacted simul~aneously with an unmarked and with a marked antibod~ of di~fering specificity and fro~ different animal species~ In the second step, an antibody present in solid phase, with specifïcity against the unmarked an~ibod~ of the first incubation step ~! iS then added thereto. ~he disadvantage of this process is again the fact that there are needed two different antibodies specific for the antigen and three different animal species.
~2'~6~
`
~ he~sfore, it is an object oi the present invention better to avoid ane or more of the above-described disad-vantages of the fundamental ~rocess then the previousl~
known processes. In particu~ar~ there is to be provided such a process whi.ch only requires one incubation and none of the antibodies has to be used in liquid phase, which is suitable for automation and which displays hi~h sensitivity and exactitude~ ~urthermore, the proce~s is ~o make it possible to manage,. if necessar~, wit~ a sin~le lo animal species for obtaining the necessary antibody or to be able to use large animaIs for obtaining the antiserum ~ hus, according to the present invention, there is provided a process for determining a polyvalent antigen b~ inrubation with three different receptors,. the first and third of which are present in li~uid phase and with which the antigen is bindable, the second receptor ïs present in solid phase and ïs on~y bindable with a part of the first receptor and the third receptor carries a marking and does not cross-react with the first and 2~ second receptor, separation of the solid pha~e ~rom the liquid phase and measurement of the markin~ in one of the phases" wherein a solution con~aining the antigen is brou~ht together with a first solid carriex material, which contains,.im~regnated or lyophilised, the f~rst and the third receptor in removable form and, after removaI
of the receptors, the solution obtained is immediately contacted wi~h a second solid carrier material which 4~6~
contains the second receptor in non-removable form, whereafter the li~uid i~ ~eparated from the second solid carrier material and the marking is measured.
~ receptors in the scope of the pre~ent invention, there are used either specifically bindable complete anti-bodies (polyclonal or monoclonal), antibod~ fra~ments thereof or conjugates of antibodies or antibod~ fra~ments with haptens or antigens. As first receptor" there i~
preferabl~ used a complete antibody or an a~tibod~ or antibod~ fr~gment bound or marked with a hapten or antigen, in each ca~e a monoclonal antibodg bein~ especial~y preferred.
As second receptor, there is used an antibod~
which is on~y bindable with a part of the first receptor~
an antibod~ which i5 only bindable with the ~c part of the first receptor being especiall~ preferred. Alterr.ativel~
as second receptor, there can also be used an antibod~
which is only ~pecifically bindable with the hapten part or antigen part of the first receptor~ Ths second receptor can also be generalIy directed against the Fc part of immuno~lobulin.
~he third receptor, which must not cross-reacted with the second receptor, can be a marked antibod~,bindable with the antigen, which is obtained ~rom an animal species different from that from which the first receptor is obtained~
However,~ there is prefer~bly used a third receptor which is a marked Fab fra~ment~ ~ithin ~he scope of the z4~'7~i~
_9_ present invention, bg Fab fragment there are also to be understood (Fab)2. and Fab' fragments. In this case, receptor 3 can also be obtained from the same animal species as the antibody component of the first receptor.
ThiS also appIies when the first receptor consists of an antibody which carries a hapten or antigen and the second receptor is only bindable with this hapten or antiæen, ~specially preferably, the third receptor is a part, especial.~y a Fab fragment, of the first receptor.
lo AIternatively~ as third receptor there can also be used an antibod~ which originates from the same animal species as the second rec.ep:tor~ Both alternatives have the advant,-age that only two animal species are required for the three receptors~ If the second receptor is only bindable~
with the hapten o~r antigen of the first r.ecept.or, onl~
one animal species is needed ~or aIl three receptors.
~speciallg prefbrab~y" the third ana the first receptor con~ain mono.clonal antibodies or fragments thereo~ which a~e directed against different and/or repetitive de~ermln-ants of the antigen.
~he third receptor is preferabl~ used in an amount which is greater than the amounts of the antï~en ~o be determined.. ~n this case,.. i..e. in the case of an e~ces~, an exact dosing or a pre~isely known amount is not necessary.. ~he third receptor is marked in a manner known to the e xper~. ~he marking preferably takes place by coupling with an enzyme or with a fluorescing, chemi--~ ~Z'~'7`~
I.uminescing and radioactive substance. Processes for the marking of such receptors are known to the expert, fcr example from Clin. Chim Acta, 8~ 40/1977, and do not require further explanation here ~ he first and third receptors are applied dissolved to a suitable carrier, for example to an absorbent paper, and, by dr~ing or lgophilising, converted into a form which can easilg be removed therefrom.
~he binding of the second receptor present in so~id phase to an insolub~e carrier material can be carried out according to the conventional methods known to the expert for the fixing of biologically active proteins to solid carrier substances~ Not onI~ a covalent but aIso an adsorptive binding is suitable. However,1 because of the~
hi~her yields which can hereb~ be achieved and of the simplified method of working, there is preferred soIel~
an adsorptive binding, for e~amp-le on a s~nthetic resin or on to a fleece, for examp~e paper~ ~her~ have proved to be suïtab~e,7 for examp~e7 reagent ve~se~s made of polystgrene and similar s~nthetic resins which are adsorptivel~ coated on the inner ~urface with the second receptor~ ~e incubation can then be carried out in the~e tubelet~ or in containera of some oth~r shape and, for the pha~e separation, it suffice~ to remove the liquid phase from the tubelet, for example,. by suckin~ out, shaking out or the like. ~fter wa~hing the tu~elet~
the measurement of the marking therein can be carri0d 12~76~
directly, especially when the marking ha~ taken place with an enzyme. It then suffices simply to measure the activity of the markin~ enz~me, for examp~ peroxidase or ~-~alactosidase, in the manner generally known for this purpose by adding a known detection reagent for this marking enzyme, for example a colour reagent, and to measure either in the presence of the so~id phase or after separation~ The measured enzyme activity is then measure for the amount of polyfunctional antigen to be lo determined, However,: as solid phas~ carrier for the~
second receptor, there can also be considered particulate substances,~ fio~ example ion exchangers, molecu~ar sieve materials~ glass particles, synthetic resin tubes and the l~ke~ ~s carrier for the second receptor~ it is ~speciaIly preferr~d to use~a porous, la~er-like car~iert such as paper If the marking does not take place w~th an enzyme but rather with a radioactive substance, an isotope,; a fluorescing or chemi~uminescing subst~nce, here, too~ the measurement is c.arried out according to the methods which are well known to the expert. ~herefore ~! here a~ain, a aescription of these methods of measurements in not n~c~ssary~
~or carrying out the process 9 the sample solution, which accordin~ to an especially advantageous character-istic of theinventio~ can even be used in undiluted form~
f.or exam~e as blood,. pla~ma or serum,~ is placed together lZ'~ 61 with the first carrier material for a sufficient len~th of time for the removal of the two receptors, then separated off and incubated with the second carrier material.. In ~eneral, the contact time with the first carrier material is from about 10 seconds to 10 minutesO
Advantageously, the process for this purpose is carried out in a device of the t~pe which i`s described in pubIi~hed Federal ~epubIic of Germany Patent Application No~ 34 25 008~
Surprisingly, we have found that according to the present invention there is achi.eved a cl~arly high~r sensitivit~ than in the case of the conventional two-step sandwich method and of the one-step sandwich ~ethod~
If, according to a preferred embodiment. of the present invention, there is used a second receptor directed against the Fc part of the first receptor or against the hapten or antigen which i5 c-oup~ed with the f'irst receptor,, then there can hereb~ be achleved a ~urthe~r incEease of the sensitivit~ since a possible steric hindrance,~ of the binding reaction of the anti~en with the bindin~ place of the f,irst receptor is avoided~
is ïs an,especial,advant.a~e when anti~ens with estpeciall~
low concentration mu~t be determined, such as i~ ~or example, the case in the determination of th~reotropin in serumO
.
~Z~'~'7~1 --~3--he present invention aIso provides a dry reagent f~r determining a polyvalent antigen, comprising a) a first solid carrïer material impre~nated or I~ophilised with two diff~rent soluble receptors 1 and 3, bindable with the antigen,.of which receptor 3 carries a marking, and b) a second solid carrier material, which is present physicall~
separated from the first carrier material, the second ~olid car~ier material containin~ an insoluble second receptor onlg bindable with a part of the non-marked lo solub~e first r.ec.eptor, with the proviso that the marked third receptor is a Fab ~ra~ment.
~ he physical separation can j,. for example, take place in that the insoluble second receptor and the solub~e first receptor and the third receptor aPe applied to separate carrier materials This can be achieved in that receptor I and receptor ~ are l~ophilised or impregnated into or on to a suit~ble carrier. simultan-eousl~ or succes~ively (for example synthetic resin tube~ets or absorbent carriers, such as psper or the ~ike) and the re~eptor 2 is bound adsorp-tivel~- or cova~en~ on to an appropriate carrier (for example ~gnthetic resin tubelets,; paper or particIes)~ ~he sample with the pol~-valent anti~en to be de.termined is first brou~h* into contact wi`th the carrier l, which contains the soluble first and third receptor~,; and thereafter with the carrier-2, on which is fixed the insoluble receptor 2 Alternatively~ the first and third receptor, on the one hand, and the second receptor, on the other hand, can be lZ~
pr~sent on carriers connect.ed with one another which consist, for e~ample, of a- paper- strip whïch contains the receptors at different places separated from one another.
~ ccording to a first pre~erred embodiment, a reagent according to the present invention is characterised in that it contains a first receptor, which is an antibod~, and a t~rd re¢eptor, which is an enæyme-marked ~ab fragment of an antibod~ Preferably, it also contains a buffe~ and a s~stem for the determination of the enz~me lo marking.
According to a further preferred embodiment of the reagent according to the present invention, this is characterised in that the receptors 1 and 3 are monoclonal and are directed ~gainst different determinants of the antigen and originate from the same animal ~pe~ies~
According to another preferred embodiment of the rea~ent according to the present invention, it is characterised in that the second receptor is an antibody agains* the Ec. part of the first receptor and the third receptor originates from.the same animal species as the second receptor, whereas the first receptor originates f rom another animal species.
~ccordin~ to yet another preferred embodiment of the reagent according to the present invention, this is characterised in that all three receptors contain anti-bodies from the same animal species, the first receptor being a hapten-marked monoclonal antibody, w~ereas the second receptor is directed against the hapten of the first receptor lZ4~76~L
~ he receptors l and 3 of the reagent accordin~
to the present invention are present on a solid carrier material impregnated or lyophilised.
~ he present invention is suitable for the deter-mination of all antigens with at least two antigen deter-minants, Examples thereof include thyreotropin (~SH), oetoprotein (AFP) r hCG,. carcinoembr~onal anti~en~
luteinising hormone ~I~), follicle-stimulating hormone (~SH~),. @2-micro~lobulin, acidic prostate phosphatase t lo prolactin, ~erritin and insulin.
In the case of the present invention, recept.or can react homogeneousl~ with the anti~en~ Consequentlg-~
t~is reaction possesses ahi~er velocit~ constant than in the case.of the~use of a solid phase-bound insoluble~
first receptor, Since the antibodg losses in the case of the fixing (makïng insoluble) on to the sol;d phase~ ar~
avoide~, the amount of the first receptnr can be further decreas~d, In cont~adistinction to the process described in C;~in, Ch~mO, 27 (6~, ~981, it is not nec~ssarg to ob~ain the.first and the third receptar from ~wo different animat species which, in addition, have to be so chosen that recep~or.~2 on~y reac~s with receptor 1 but not with recep~or 3~
~inall~, the obtainin~ of the antibod~ for the receptor l and for the receptor 3 from the same animal species also maXes it possible to use lar~e anima~s for obtainin~ the antiserum.
lZ~
FinaIl~ accord~ing to the prese~t invention,.
~he sensitivity i5 also increased. ~hu-s, ~or TSE there is-found a sensitivity of less than 40 pg./ml. (1 ~ U
170 pg~/ml` ), whereas in the case of the two site assa~
~sandwich process) it lies above 100 p~./ml ~he foIIowing ~xamp~Ies are given for the purpose~
of illustrating the present invention, reference being made to the acc:ompan~ing drawings, in whïch:
Fi~ 1 is a calibration curve accordin~ to Example 2 lo ~ig~ 2 is a calibration curve according to Examp~le 3~
Fig. 3 i~ a calibration curve according to Example 4 and Fig. 4 i~ an insert element for a centrifugal analysis automatic appar.atus which i5 used according to Examp~le 2 Examp~e l.
Determination of th~reotropin (~SE)~
Rec:eptor 1 and ~ originate from the same animal species (m~use) and ~ecep*or ~ is-a marked Fab ~agment~
Receptor ~ is a monoclonal antibody and the Fab ~ragment in ~e~ep~r.-3 a~so o-riginates from a monoclonal antibod~
which is directed a~ainst a determinant of the ~ other than that aæainst which the monocIonal antibod~ of receptor 1 i5 direct.ed. Recep~or 2 originat.e~ from sheep and is direct~ a~ains~t the ~c part of the monoclonal mouse. antibod~
Tha. development of monoclonal antibodies take~
pIace acc~rding to the method of ~dhler and Milstein ~Eur~ J. ImmunoL , 6~ 292~1976)~
` ~ 24~'7 a2 ~arrging o~t of the test according to the present inv.ention:
Reagents:
I, Incubation buffe.r (IB):
15 mM sodium phosphat.e buffer (pH 7.4); 154 mM NaCl;
5 mM ED~A; 0~2~ bovine serum albumen (BSA); pH 7.4, 2~ Receptor ~:
Mouse ~n~ SH.antiserum (receptor 1):
'~he ascites li~uid from mice containing monoclonal anti-~SH~antibo~ie.s is mi~ed ad 1~8M wi~h ~E4S04~ ~he pre.ci~ï~at.e is taken up in a buffer of 15 mM sodium phosphate (pH 7,0) and 50 mM NaCl~ ~he. solution so obtained is subject.ed to a passage over DEAE-cellulose, 3~ Recep,~r 3:
Anti-~SE ~ntibod~ (mono.cIonal) which recognises a determinant different from that recognised by Re.cep.t~r 1, Pero~idalse conjugate (Recept.or~3): mou~e anti-~S~
antiserum is purified ~ke Recep~or l, ~he ~ubsequent obtaining of (~ab~2 from the comp~Iete antibod~
mo~ecu~e~takes place by the method described in Examp~e 2~ ~h~ cou~lin~ with horse radish peroxidas~
takes place acc;ording to the method of Nakan~ (M,B.
Wi~son9. E~E~ Nakane,."Recent development3 in t~
periodate method of cônjugating horseradish pero~ida~e to antibod`ies"9 1978,~ pub~ Elsevier9. North ~olland Biome~tcaI Pres~,~ p~, 215-224 in "Immunofluorescence and related ~tainin~ technïque~").
` ` lZ~'~'761 4.. RecQp~or 2:.
A~sorber material.with fixed sheep anti-mouse-~cY-antibod~ ~R.eceptor 2 adsorber):
Sheep anti-mouse ~cY antiserum ¢~ mixed ad 1,8M w~h NE4SO4. ~he precipitate is taken up in a buffer of 15 mM sodium pho.sphate (pH 7 O) and 5O mM Nal ~he solution so obtained is subjected to a passage over ~AE-cellulose. The lgG-containing fraction (Receptor 2) is coupled with the "affinit~ adsorbent, activated ~lutardialdeh~de~" of Boehrin~er Mannheim GmbH (o~de~
No~ 665 525~ accordin~ to the manufacturer's working instructions~
5.. Indicator rea~ent:
1~8 mM 2,2'-azino-di-(3-eth~lbenzthiazo~ine-6~
sulphonate (AB~S), 3 3 mM sodium perborate ïn lOO mM
phosphate-cit~ate buffer (pH 4 4)~
Carrying out:
5O ~ ReceRtor 1 and yo mU Receptor 3, dissolved in incubation buf~er, are together applled dropwise to a f~eece consisting of commercial polyester paper with a size of 6 x:6 mm~ and l,O mm. thickness and dried at ambient temperature. ~he reagent-containing paper fleec~
is cal~ed rea~ent carrïer l, On ~o reagent carrier 1 are pipet~ed 40 ~ 1~ o~
the samp~e to be de~ermined or known amounts o~ antigen-containin~ standard solution. Subse~uently, the fle~c~
i~ immediatel~ centri~`uged off in an Eppendorf centrifuge, whereb~ reagent càrrier I is placed on an Eppenaorf cup . :~2~6~
and the eluted liquid in the cup impinges during the centrifuging on to the receptor 2, which ïs coupled on an adsorber,~ and.reacts with thïs. Of receptor 2, 2~5 mg~
adsorber material are used per test, ~ he reaction mixture is incubated on a shaking apparatus for 15 minutes at 37C.
A~ter precise~y 15 minutes,! the reaction vess~ls are rem~ved from the apparatus and cen~.rifugea~
An aliquot of the supernatant is removed with a pipette and the coloured materiaI.formed is determined by wa~ of its extinction at ~05 nm, ~ he following e~tinct~on values aere determined with two dif~erent ~SH samp~es:
~S~ E405 nm/cm+
( ~U/ml,) . _ 0 0,74 50 _ 7~5~ .
* ~h~ extinction va~ues were determined at a ~ayer t~ckness of 0~2 cm~ and recalcu~ated to~a ~ay-er thickne-ss- of l cm~
b) Sim~le sandwich (`com~arison).
~he carr~ing out took plac~ with the same reagents and in the same manner as described under a~ but with the following changes:
l_ 2,5 m~ ad~orber-recep~or 2 is pre-incubat:ed for l hour with 20 ~ ~, receptor 1 in 002 mi~, with shaki~g.
lZ~7~i~
-Subsequently~ the supernatant is sucked off and the residue washed three times with, in each case, I ml. r~.
2~ ~dsorber rece~tor 2 pre-treated in this manner wit~
receptor I is incubated at 37C. for 4 hours with sample and receptor ~ on a shakin~ apparatus, the addition of solub~e receptor 1 being omitted in the case of thïs variant, ~ubsequently, the supernatant is sucked o:Ef as usua~., the residue is washed and the fi~ed enzy~e activit~ ïs lo detected with indicator reagent~
The following extinction vaIues were determined with two different T~H samples:
( ~U/ml,) E405 nm/cm+
. 0,20 50 2~87 + The extinction values were determined at a layer thickness of 0~2 cm~ and recalculatea to a la~er thickness o~ 1 cm.
The sensitivit~ is clearly smaller than according to a) aIthouæh in the case of b) more receptor 1 is used than in the case o~ a) ~ratio 1:400)~
Example 2 Determination of human choriogonadotropin (HQG~
Reagents:
z4~761 ~uffer A:
100 mmol sodium phosphate7 pH 7t3 (37 Cr) 2 mmol magnesium chloride 0.9~ NaCl 0.5~ RSA (bovine serum albumin) 120 ~g./ml. anti-HCG monoclonal antibodies from mice (ascites)~receptor 1) 100 mU/ml. anti-HCG antibodg (sheep) Fab fragment-~-~àlactosidase conjugate Receptor 3 The preparation of the Fab fra~ment takes place according to T. Kitagawa in "Enzyme immunoassay, E
Ishikawa, ~ Kawai, K Miyai eds~ Iga~-Shoin, Tok~o/New York, 198l~ pp~ 81-89". ~he covalent coupling of ~-galactosidase to ~ntibodies or antibodg fragments takes place according to the method of R R. Porter~ Biochem. J , 9/1959.
Substrate soDution: as~ buf~er A and additional~y 5 mmoI
o-nitrophen~l-galactoside (O~PG), 1~ ~he production of rea~ent carrIe~ l takes place analogousl~ to Example 1 in such a manner that 40 ~ 1 of solution A is applied dropwise to a fleece consistinæ of commercial polyester paper and subsequentl~ dried at ambient temperature. Until it i5 u~ed, this ~leece ïs is s~o~d at 4 C. and at a relative humidity of ~ 20~.
2. Production of reagent carrier 2: n to a cellulose fleece are fixed, by the cyano~en bromide actavation --` lZ4~'i'~1 process (see pub~ished Federal Republic of German~ Patent ~pplication Noc I7 68 512),< M-FcY> antibodies from sheep (IgG fraction), 10 mg. of antibodies bein~ available for fixing per gram of fibre material~. Non-coupled antibody is removed bg washin~ and the fleece is gentlg dried at ambient temper~ture. St~rage of this fleece takes place analogousIy to reagent carrier 1 The determination of HCG with the help of these two reagent carriers I and 2 takes place with the help lo of the device for carryin~ out anal~tical determinations described in Fe~eral ~epublic of Ge.rman~ Patent Application No. 24 25 008.5. ~his describes a rotor insert element for centrifugal automatic analysers consistin~ of a formed bod~, which has a samp~!e reception chamber which is connected with a pluralit~ of rea~ent zones, each of which contains an absorbent carrier material impre~nated with a particular reagent, at Ieast one mixin~ Y.alve chambe~ and a measurement chamber which togther form a samp~e li~uid transport path which passe~ from radially inwardI~-to radially outwardI~- when the insert eIement is fixed on to the rotor and,. in addition, at Ieast one further chamber for the reception of a liquid and a transport path whïch leads from this chamber to the measurement ~uide and is at Ieast partIg~identical to the sample liquid transport pa~h~ The sample liquid transport path thereb~ passes from a sample reception chambe~ ) via a chamber~ (a) fillea with absorbent material.and containin~ buffer, a chamber (c) and a '7 first val.ve chamber (VKL) arranged between the chambers (:a) and ~c) to a second valve chamber (VK2) and from this, via a chamber (d) and ~i.a a collection chamber (AK) to a measurement chamber (E) For the reception o~ a further liquid, there is provided a substra~e chamber (PK) ~ormed as a pump chamber which is connected wïth the second valve chambe~ (VK2) via a dosing device,consisting of a dosin~ chamber (DK) and capillary (Kap~), and an ove.r~low chamber (UK). Fig. 4 shows schematicallg the rotor insert element used In this case" rea~ent carrie~ l is plac~d on fi~d c of the insert element and the reagent carrier 2 pn field d~ ~h-e carrying out of the reaction corresponds essentially to E~amp~e 1 of this German Patent Ap~ication.
40 ~ ~ of samp~e are thereby pipett;ed through an opening on the upper edge directl~ on to field a ~he-sample is n~t dilut-ed~
B~ means of an appropriate programme~ in which high spee~s of rotation alternate with ~topEing~,samp~e-and substrate are then transp~rted in the direction of the separating matrix and cuvette. In the fol~owing, .
centrifuging means a high speed of ro~ation and inter~
mediate steps with lower speeds of rotation serve for the mora~ssnsiti~ control of the lïquid tran~port, ~h~
~ubstra~e/wash solution is divided up b~ the dosag~
capillar~ (DK) into equal~ large portions~
1st centrif~ging Sample and samp~e bu~f.er are centrifugPd in ~K~, the first sample is in the dosage chamber (:DE) 12447~
,~
.t stopR~n~
Sample runs to field c and dissol~es the receptors I.and 3,. The 1st portion of substrate solution passes into the overflow chamber UK
2nd centrifuging ~CG and rec-ep~o~s l.and 3 pass into YK2 and cen~rï-f~ging is carried out in order to achieve homo-~eneous mixing of the rea~ents. ~he 1st portion of substrate solution is held back in UK and the 2nd lo portion of substrRte solution paqses into the dosage chamber D~
2nd stop~in~
an the samp~e side" the liquid is transportea to fie~d d and there follow~ a stopping for 5 minu~e~
durin~ which time.the comple~ binds to the ca~rler~
Non-comp~exed anti-HG~ is also bound, At the end o~ the reac;tion" not ~t complexed Fab conjuga~e is present in the so~ution, ~he 2nd portion of substrate so.lution pass~s into the UK, 3r~ cent~if~ing T~e Iiquid coming ~rom the samp~e canal is cen~ri-fu~ed in the reception chamb~r (AK),~with it the excess o~ ~ab conjugate~ ~he 2nd substrate portion is retained in UK~ ~he 3rd substxate portion is present in dosa~e chamber DKD
3rd stop~n~
The 3rd substrate portion i5 transported to UK~
4th centrif uging Portion 4 to DK
Portion 3 to VK2 4th stopping Portion 4 to U~
Portion 3 to field ~
The 1st wash portion is present on reagent carrier 2 5th centrifugin~s Portion 5 to DK
Portion 4 to VEC2 Portion 3 to AK
5th stopping Portion 5 to UK
Po.rtion 4 to d 2nd wash portion on reagent carrier 2 6th centrifuging Portion 6 to ~
Portion 5 to V~2 Portion 4 to ~K
6th stopl~in~;
Portion 6 to Ul~
Portion 5 to d 3rd wash portion on reagent carrier 2 7 th c entrif uging Portion 7 to D~
Portion 6 to VK2 Portion 5 to AE
7~ 4'7Çj1 7th stopping Portion 7 to UE
Portion 6 to d 4th wash portion on rea~ent carrier 20 8th centrifu~ing Portion 8 to DK
Portion 7 to VK2 Portion 6 to AK
8th stopping 1o Por~ion 8 to UK
Portion 7 to d Detection portion on reagent carrier 2~ In 5 minutes reaction,, the. substrate is split o.ff by the enz~me bound to the rea~ent carrier 29 i~e,~ by an am~unt of enz~me which, due to the comp~ex formation, is proportional t~
the amount of HCG introduced, and the colour to be measu~ed is formed.
9th centrifuging The li~uid coming from the rea~ent carrie~ 2 comphetel~ fil~s the AK with a first aliquot and the r~mainder is pa~sed into the cu~*.t,e~
~h~ measurement of the col~ur formed takes place in the cu~ette at 578, nmv ~he transfer of the samp~e from fi'eld c to d takes place~ within a ver~ short period of time (~ 60 seconds), ~h~ final substrat~ volume leading to the detection a~so amounts to 40 ~ o that no sample dilution takes place.
up to the signal measurement.
s ~24~
.
With the use of caIib~ation serum, there is obtained ths caIibration curve shown in ~ig. 1 which includes the range from O to 100 mU HCG/ml, (standardised according to the 1st IRP standard for HCG) and makes pos~ible a sufficiently sensitive measurement o HCG in serum and plasma~
~xample 3..
~etermination of ~ foetop~otein (~P), Use of a hapten-marked 1st receptor, all three antibodies ori~inating from the same animal species~
~he carrying out and the buf~ers used correspond to Examp~e 2 As receptor ~ there areu~d anti-A~P antibo.dies ~rom she~p (~OO ng~/ml;~) which are marked w~th d~goxin (according to Federal ~epublic of Germany Patent ~ecific-ation No, 25 37 129) and as receptor 3 there are u-sed anti-A~P antibodie~ from sheep marked with ~-galactosidase.
accordin~ to the method described in the case of HCG
(~amp~e 2~ and as solid phase-coupled receFtor 2 the~e are- use.d sma~ paper discs of po~yester paper on to which are coupled adsorptiveI~ antibodies against di~oxin fr.-om sheep. ~he coating process c~rresponds to the process known for pIastics tube~ets. ~he calibration curve obtained is shown in ~ 2 of the ~cGomp~g.ing drawings, Example 4.
Determination of th~reotro~in (T~H.
Reagents:
lZ~gL'7~i~
1~ Incubation buffer (IB?: 15 mM ~odium phosphate buffer (pH 7.4), 154 mM NaCl, 5 m M ED~A, 0~2~ RSA, pH 7~40 2. Receptor 1: sheep anti-TSH antiserum: the crude serum is mixed ad 1.8M with NH4S04. ~he precipitate is taken up in a buffer of 15 mM sodium phosphate (pH 700) and 50 mM
NaCl and the solution so obtained is su~jected to a passage over DEAE-cellulose. The I~G-containing fraction is subse~uentl~ freed from the portions reacting with the alpha chain of TSH b~ passage over an immune adsorber loaded with HCG~ The eluate is applied to an immun~
adsorber loaded with ~SH. After washing the adsorber, there take~ place the elution of the antibodies immune-reactive a~ainst TSH with lM propionic acid~ ~he eluate is subsequent~y dialysed against IB and optionall~ con-centrated by ultrafiltration.
3~ Receptor 3: antibody-peroxidase conjugate: A further sheep anti-TSH antiserum is purified in the same wa~ as rec~ptor 1 and subse~uently (Fab)2 fragments obtained therefromO ~he prepsration of the (~ab)2 fragments take~
place according to the method of E ~amoge et al., J
Immunol~ Methods" ~,. 235-243~1983~ ~he couplin~ with horse radish peroxidase takes place accordin~ to the method of Nakane (M,B~ Wilson, P~K. Nakane, "Recent developments in the periodate method of conju~ating horseradish peroxidase to antibodies", 1978, pub~
Elsevier9 North Holland Biomedical Press, pp. 215-224, in "Immunofluorescence and relating stai~ing techniqu~s")~
` ~2~'76 4. Receptor 2: Adsorber material with Pixed donkey anti-sheep-FcY antibody: Donke~ anti-sheep-Fc~-antiserum is mixed ad 1~8 M with NH4SO4. The precipitate is taken up in a buffer of 15 mM sodium phosphate (pH 7.0) and 50 mM NaCl and the solution so obtained is subaected to a passage over DEAE-cellulose~ ~he IgG-containing fraction (receptor 2) is coupled with the "affinit~
adsorben~ glutardialdeh~de activated" oP Boehringer Mannheim GmbH (order No~ 665 525) accordin~ to the manufacturer's working instructions.
5~ Indicator reagent: 1~8 mM 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonate (AB~S), 3,3 mM sodium perborate in 100 m~l phosphate-citrate buffer (p~ 4.4), Carrying out:
~he carrying out cDrresponds to the process described in Example 1, whereby, in this case, lOO ng.
of re~eptor 1 and lOO mU of receptor 3~ dissol~ed in 40 ~ 1. of incubation buffer are applied dro~wise to a cellulose fleece with the dimensions of 6 x 6 mm.
After drgingh the Ple~ce is again stored at 4C. until it is used.
The further carryin~ out corresponds to the manner of working described in Example 1. ~he amount of samp~e used is 40 ~ 1~
~he calibration c~rve thus obtained is shown in Fig. 3 of the accompan~ing drawings.
Mouse ~n~ SH.antiserum (receptor 1):
'~he ascites li~uid from mice containing monoclonal anti-~SH~antibo~ie.s is mi~ed ad 1~8M wi~h ~E4S04~ ~he pre.ci~ï~at.e is taken up in a buffer of 15 mM sodium phosphate (pH 7,0) and 50 mM NaCl~ ~he. solution so obtained is subject.ed to a passage over DEAE-cellulose, 3~ Recep,~r 3:
Anti-~SE ~ntibod~ (mono.cIonal) which recognises a determinant different from that recognised by Re.cep.t~r 1, Pero~idalse conjugate (Recept.or~3): mou~e anti-~S~
antiserum is purified ~ke Recep~or l, ~he ~ubsequent obtaining of (~ab~2 from the comp~Iete antibod~
mo~ecu~e~takes place by the method described in Examp~e 2~ ~h~ cou~lin~ with horse radish peroxidas~
takes place acc;ording to the method of Nakan~ (M,B.
Wi~son9. E~E~ Nakane,."Recent development3 in t~
periodate method of cônjugating horseradish pero~ida~e to antibod`ies"9 1978,~ pub~ Elsevier9. North ~olland Biome~tcaI Pres~,~ p~, 215-224 in "Immunofluorescence and related ~tainin~ technïque~").
` ` lZ~'~'761 4.. RecQp~or 2:.
A~sorber material.with fixed sheep anti-mouse-~cY-antibod~ ~R.eceptor 2 adsorber):
Sheep anti-mouse ~cY antiserum ¢~ mixed ad 1,8M w~h NE4SO4. ~he precipitate is taken up in a buffer of 15 mM sodium pho.sphate (pH 7 O) and 5O mM Nal ~he solution so obtained is subjected to a passage over ~AE-cellulose. The lgG-containing fraction (Receptor 2) is coupled with the "affinit~ adsorbent, activated ~lutardialdeh~de~" of Boehrin~er Mannheim GmbH (o~de~
No~ 665 525~ accordin~ to the manufacturer's working instructions~
5.. Indicator rea~ent:
1~8 mM 2,2'-azino-di-(3-eth~lbenzthiazo~ine-6~
sulphonate (AB~S), 3 3 mM sodium perborate ïn lOO mM
phosphate-cit~ate buffer (pH 4 4)~
Carrying out:
5O ~ ReceRtor 1 and yo mU Receptor 3, dissolved in incubation buf~er, are together applled dropwise to a f~eece consisting of commercial polyester paper with a size of 6 x:6 mm~ and l,O mm. thickness and dried at ambient temperature. ~he reagent-containing paper fleec~
is cal~ed rea~ent carrïer l, On ~o reagent carrier 1 are pipet~ed 40 ~ 1~ o~
the samp~e to be de~ermined or known amounts o~ antigen-containin~ standard solution. Subse~uently, the fle~c~
i~ immediatel~ centri~`uged off in an Eppendorf centrifuge, whereb~ reagent càrrier I is placed on an Eppenaorf cup . :~2~6~
and the eluted liquid in the cup impinges during the centrifuging on to the receptor 2, which ïs coupled on an adsorber,~ and.reacts with thïs. Of receptor 2, 2~5 mg~
adsorber material are used per test, ~ he reaction mixture is incubated on a shaking apparatus for 15 minutes at 37C.
A~ter precise~y 15 minutes,! the reaction vess~ls are rem~ved from the apparatus and cen~.rifugea~
An aliquot of the supernatant is removed with a pipette and the coloured materiaI.formed is determined by wa~ of its extinction at ~05 nm, ~ he following e~tinct~on values aere determined with two dif~erent ~SH samp~es:
~S~ E405 nm/cm+
( ~U/ml,) . _ 0 0,74 50 _ 7~5~ .
* ~h~ extinction va~ues were determined at a ~ayer t~ckness of 0~2 cm~ and recalcu~ated to~a ~ay-er thickne-ss- of l cm~
b) Sim~le sandwich (`com~arison).
~he carr~ing out took plac~ with the same reagents and in the same manner as described under a~ but with the following changes:
l_ 2,5 m~ ad~orber-recep~or 2 is pre-incubat:ed for l hour with 20 ~ ~, receptor 1 in 002 mi~, with shaki~g.
lZ~7~i~
-Subsequently~ the supernatant is sucked off and the residue washed three times with, in each case, I ml. r~.
2~ ~dsorber rece~tor 2 pre-treated in this manner wit~
receptor I is incubated at 37C. for 4 hours with sample and receptor ~ on a shakin~ apparatus, the addition of solub~e receptor 1 being omitted in the case of thïs variant, ~ubsequently, the supernatant is sucked o:Ef as usua~., the residue is washed and the fi~ed enzy~e activit~ ïs lo detected with indicator reagent~
The following extinction vaIues were determined with two different T~H samples:
( ~U/ml,) E405 nm/cm+
. 0,20 50 2~87 + The extinction values were determined at a layer thickness of 0~2 cm~ and recalculatea to a la~er thickness o~ 1 cm.
The sensitivit~ is clearly smaller than according to a) aIthouæh in the case of b) more receptor 1 is used than in the case o~ a) ~ratio 1:400)~
Example 2 Determination of human choriogonadotropin (HQG~
Reagents:
z4~761 ~uffer A:
100 mmol sodium phosphate7 pH 7t3 (37 Cr) 2 mmol magnesium chloride 0.9~ NaCl 0.5~ RSA (bovine serum albumin) 120 ~g./ml. anti-HCG monoclonal antibodies from mice (ascites)~receptor 1) 100 mU/ml. anti-HCG antibodg (sheep) Fab fragment-~-~àlactosidase conjugate Receptor 3 The preparation of the Fab fra~ment takes place according to T. Kitagawa in "Enzyme immunoassay, E
Ishikawa, ~ Kawai, K Miyai eds~ Iga~-Shoin, Tok~o/New York, 198l~ pp~ 81-89". ~he covalent coupling of ~-galactosidase to ~ntibodies or antibodg fragments takes place according to the method of R R. Porter~ Biochem. J , 9/1959.
Substrate soDution: as~ buf~er A and additional~y 5 mmoI
o-nitrophen~l-galactoside (O~PG), 1~ ~he production of rea~ent carrIe~ l takes place analogousl~ to Example 1 in such a manner that 40 ~ 1 of solution A is applied dropwise to a fleece consistinæ of commercial polyester paper and subsequentl~ dried at ambient temperature. Until it i5 u~ed, this ~leece ïs is s~o~d at 4 C. and at a relative humidity of ~ 20~.
2. Production of reagent carrier 2: n to a cellulose fleece are fixed, by the cyano~en bromide actavation --` lZ4~'i'~1 process (see pub~ished Federal Republic of German~ Patent ~pplication Noc I7 68 512),< M-FcY> antibodies from sheep (IgG fraction), 10 mg. of antibodies bein~ available for fixing per gram of fibre material~. Non-coupled antibody is removed bg washin~ and the fleece is gentlg dried at ambient temper~ture. St~rage of this fleece takes place analogousIy to reagent carrier 1 The determination of HCG with the help of these two reagent carriers I and 2 takes place with the help lo of the device for carryin~ out anal~tical determinations described in Fe~eral ~epublic of Ge.rman~ Patent Application No. 24 25 008.5. ~his describes a rotor insert element for centrifugal automatic analysers consistin~ of a formed bod~, which has a samp~!e reception chamber which is connected with a pluralit~ of rea~ent zones, each of which contains an absorbent carrier material impre~nated with a particular reagent, at Ieast one mixin~ Y.alve chambe~ and a measurement chamber which togther form a samp~e li~uid transport path which passe~ from radially inwardI~-to radially outwardI~- when the insert eIement is fixed on to the rotor and,. in addition, at Ieast one further chamber for the reception of a liquid and a transport path whïch leads from this chamber to the measurement ~uide and is at Ieast partIg~identical to the sample liquid transport pa~h~ The sample liquid transport path thereb~ passes from a sample reception chambe~ ) via a chamber~ (a) fillea with absorbent material.and containin~ buffer, a chamber (c) and a '7 first val.ve chamber (VKL) arranged between the chambers (:a) and ~c) to a second valve chamber (VK2) and from this, via a chamber (d) and ~i.a a collection chamber (AK) to a measurement chamber (E) For the reception o~ a further liquid, there is provided a substra~e chamber (PK) ~ormed as a pump chamber which is connected wïth the second valve chambe~ (VK2) via a dosing device,consisting of a dosin~ chamber (DK) and capillary (Kap~), and an ove.r~low chamber (UK). Fig. 4 shows schematicallg the rotor insert element used In this case" rea~ent carrie~ l is plac~d on fi~d c of the insert element and the reagent carrier 2 pn field d~ ~h-e carrying out of the reaction corresponds essentially to E~amp~e 1 of this German Patent Ap~ication.
40 ~ ~ of samp~e are thereby pipett;ed through an opening on the upper edge directl~ on to field a ~he-sample is n~t dilut-ed~
B~ means of an appropriate programme~ in which high spee~s of rotation alternate with ~topEing~,samp~e-and substrate are then transp~rted in the direction of the separating matrix and cuvette. In the fol~owing, .
centrifuging means a high speed of ro~ation and inter~
mediate steps with lower speeds of rotation serve for the mora~ssnsiti~ control of the lïquid tran~port, ~h~
~ubstra~e/wash solution is divided up b~ the dosag~
capillar~ (DK) into equal~ large portions~
1st centrif~ging Sample and samp~e bu~f.er are centrifugPd in ~K~, the first sample is in the dosage chamber (:DE) 12447~
,~
.t stopR~n~
Sample runs to field c and dissol~es the receptors I.and 3,. The 1st portion of substrate solution passes into the overflow chamber UK
2nd centrifuging ~CG and rec-ep~o~s l.and 3 pass into YK2 and cen~rï-f~ging is carried out in order to achieve homo-~eneous mixing of the rea~ents. ~he 1st portion of substrate solution is held back in UK and the 2nd lo portion of substrRte solution paqses into the dosage chamber D~
2nd stop~in~
an the samp~e side" the liquid is transportea to fie~d d and there follow~ a stopping for 5 minu~e~
durin~ which time.the comple~ binds to the ca~rler~
Non-comp~exed anti-HG~ is also bound, At the end o~ the reac;tion" not ~t complexed Fab conjuga~e is present in the so~ution, ~he 2nd portion of substrate so.lution pass~s into the UK, 3r~ cent~if~ing T~e Iiquid coming ~rom the samp~e canal is cen~ri-fu~ed in the reception chamb~r (AK),~with it the excess o~ ~ab conjugate~ ~he 2nd substrate portion is retained in UK~ ~he 3rd substxate portion is present in dosa~e chamber DKD
3rd stop~n~
The 3rd substrate portion i5 transported to UK~
4th centrif uging Portion 4 to DK
Portion 3 to VK2 4th stopping Portion 4 to U~
Portion 3 to field ~
The 1st wash portion is present on reagent carrier 2 5th centrifugin~s Portion 5 to DK
Portion 4 to VEC2 Portion 3 to AK
5th stopping Portion 5 to UK
Po.rtion 4 to d 2nd wash portion on reagent carrier 2 6th centrifuging Portion 6 to ~
Portion 5 to V~2 Portion 4 to ~K
6th stopl~in~;
Portion 6 to Ul~
Portion 5 to d 3rd wash portion on reagent carrier 2 7 th c entrif uging Portion 7 to D~
Portion 6 to VK2 Portion 5 to AE
7~ 4'7Çj1 7th stopping Portion 7 to UE
Portion 6 to d 4th wash portion on rea~ent carrier 20 8th centrifu~ing Portion 8 to DK
Portion 7 to VK2 Portion 6 to AK
8th stopping 1o Por~ion 8 to UK
Portion 7 to d Detection portion on reagent carrier 2~ In 5 minutes reaction,, the. substrate is split o.ff by the enz~me bound to the rea~ent carrier 29 i~e,~ by an am~unt of enz~me which, due to the comp~ex formation, is proportional t~
the amount of HCG introduced, and the colour to be measu~ed is formed.
9th centrifuging The li~uid coming from the rea~ent carrie~ 2 comphetel~ fil~s the AK with a first aliquot and the r~mainder is pa~sed into the cu~*.t,e~
~h~ measurement of the col~ur formed takes place in the cu~ette at 578, nmv ~he transfer of the samp~e from fi'eld c to d takes place~ within a ver~ short period of time (~ 60 seconds), ~h~ final substrat~ volume leading to the detection a~so amounts to 40 ~ o that no sample dilution takes place.
up to the signal measurement.
s ~24~
.
With the use of caIib~ation serum, there is obtained ths caIibration curve shown in ~ig. 1 which includes the range from O to 100 mU HCG/ml, (standardised according to the 1st IRP standard for HCG) and makes pos~ible a sufficiently sensitive measurement o HCG in serum and plasma~
~xample 3..
~etermination of ~ foetop~otein (~P), Use of a hapten-marked 1st receptor, all three antibodies ori~inating from the same animal species~
~he carrying out and the buf~ers used correspond to Examp~e 2 As receptor ~ there areu~d anti-A~P antibo.dies ~rom she~p (~OO ng~/ml;~) which are marked w~th d~goxin (according to Federal ~epublic of Germany Patent ~ecific-ation No, 25 37 129) and as receptor 3 there are u-sed anti-A~P antibodie~ from sheep marked with ~-galactosidase.
accordin~ to the method described in the case of HCG
(~amp~e 2~ and as solid phase-coupled receFtor 2 the~e are- use.d sma~ paper discs of po~yester paper on to which are coupled adsorptiveI~ antibodies against di~oxin fr.-om sheep. ~he coating process c~rresponds to the process known for pIastics tube~ets. ~he calibration curve obtained is shown in ~ 2 of the ~cGomp~g.ing drawings, Example 4.
Determination of th~reotro~in (T~H.
Reagents:
lZ~gL'7~i~
1~ Incubation buffer (IB?: 15 mM ~odium phosphate buffer (pH 7.4), 154 mM NaCl, 5 m M ED~A, 0~2~ RSA, pH 7~40 2. Receptor 1: sheep anti-TSH antiserum: the crude serum is mixed ad 1.8M with NH4S04. ~he precipitate is taken up in a buffer of 15 mM sodium phosphate (pH 700) and 50 mM
NaCl and the solution so obtained is su~jected to a passage over DEAE-cellulose. The I~G-containing fraction is subse~uentl~ freed from the portions reacting with the alpha chain of TSH b~ passage over an immune adsorber loaded with HCG~ The eluate is applied to an immun~
adsorber loaded with ~SH. After washing the adsorber, there take~ place the elution of the antibodies immune-reactive a~ainst TSH with lM propionic acid~ ~he eluate is subsequent~y dialysed against IB and optionall~ con-centrated by ultrafiltration.
3~ Receptor 3: antibody-peroxidase conjugate: A further sheep anti-TSH antiserum is purified in the same wa~ as rec~ptor 1 and subse~uently (Fab)2 fragments obtained therefromO ~he prepsration of the (~ab)2 fragments take~
place according to the method of E ~amoge et al., J
Immunol~ Methods" ~,. 235-243~1983~ ~he couplin~ with horse radish peroxidase takes place accordin~ to the method of Nakane (M,B~ Wilson, P~K. Nakane, "Recent developments in the periodate method of conju~ating horseradish peroxidase to antibodies", 1978, pub~
Elsevier9 North Holland Biomedical Press, pp. 215-224, in "Immunofluorescence and relating stai~ing techniqu~s")~
` ~2~'76 4. Receptor 2: Adsorber material with Pixed donkey anti-sheep-FcY antibody: Donke~ anti-sheep-Fc~-antiserum is mixed ad 1~8 M with NH4SO4. The precipitate is taken up in a buffer of 15 mM sodium phosphate (pH 7.0) and 50 mM NaCl and the solution so obtained is subaected to a passage over DEAE-cellulose~ ~he IgG-containing fraction (receptor 2) is coupled with the "affinit~
adsorben~ glutardialdeh~de activated" oP Boehringer Mannheim GmbH (order No~ 665 525) accordin~ to the manufacturer's working instructions.
5~ Indicator reagent: 1~8 mM 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonate (AB~S), 3,3 mM sodium perborate in 100 m~l phosphate-citrate buffer (p~ 4.4), Carrying out:
~he carrying out cDrresponds to the process described in Example 1, whereby, in this case, lOO ng.
of re~eptor 1 and lOO mU of receptor 3~ dissol~ed in 40 ~ 1. of incubation buffer are applied dro~wise to a cellulose fleece with the dimensions of 6 x 6 mm.
After drgingh the Ple~ce is again stored at 4C. until it is used.
The further carryin~ out corresponds to the manner of working described in Example 1. ~he amount of samp~e used is 40 ~ 1~
~he calibration c~rve thus obtained is shown in Fig. 3 of the accompan~ing drawings.
Claims (27)
1. Process for determining a polyvalent antigen by incubation with three different receptors, the first and third of which are present in liquid phase and with which the antigen is bindable, the second receptor is present in solid phase and is only bindable with a part of the first receptor and the third receptor carries a marking and does not cross-react with the first and second receptor, separation of the solid phase from the liquid phase and measurement of the marking in one of the phases, wherein a solution containing the antigen is brought together with a first solid carrier material, which contains, impregnated or lyophilised, the first and the third receptor in removable form and, after removal of the receptors, the solution obtained is immediately contacted with a second solid carrier material which contains the second receptor in non-removable form, whereafter the liquid is separated from the second solid carrier material and the marking is measured. .
2. Process according to claim 1, wherein an antibody is used as the first receptor.
3. Process according to claim 1, wherein an antibody marked with a hapten or antigen is used as the first receptor.
4 Process according to claim 1, wherein, as the first receptor, there is used an antibody fragment which is marked with a hapten or antigen.
5. Process according to claim 2, wherein a second receptor is used which is only bindable with the Fc part of the first receptor.
6. Process according to claim 3 or 4, wherein a second receptor is used which is only bindable with the hapten part or the antigen part of the first receptor.
7. Process according to claim 5, wherein a second receptor is used which is directed against the Fc part of immunoglobulin.
8. Process according to claim 1, wherein said third receptor is a Fab fragment and originates from the same animal species as the antibody com-ponent of the first receptor.
9. Process according to claim 8, wherein a part of the first receptor is used as the third receptor.
10. Process according to claim 3 or 4, wherein the third receptor consists of a marked, complete antibody.
11. Process according to claim 1, wherein the third receptor originates from the same animal species as the second receptor and the first receptor originates from another animal species.
12. Process according to claim 1, wherein the third receptor originates from the same animal species as the second receptor and the first receptor is marked with a hapten or antigen.
13. Process according to claim 11, wherein said first receptor is marked with a hapten or antigen.
14. Process according to claim 3 or 4, wherein all three receptors contain antibodies or fragments thereof which originate from the same animal species.
15. Process according to claim 1, 2 or 3, wherein the third receptor is marked with an enzyme or with a fluorescing, chemiluminescing or radio-active substance.
16. Process according to claim 4, 5 or 7, wherein the third receptor is marked with an enzyme or with a fluorescing, chemiluminescing or radio-active substance.
17. Process according to claim 1, 2 or 3, wherein said solution is an undiluted sample solution.
18. Dry reagent for determining a polyvalent antigen comprising:
a) a first solid carrier material impregnated or lyophilised with two different soluble receptors 1 and 3, bindable with the antigen, of which receptor 3 carries a marking, and b) a second solid carrier material, which is present physically separated from the first carrier material, the second solid carier material contain-ing an insoluble receptor 2 only bindable with a part of the non-marked soluble receptor 1, with the proviso that the marked receptor 3 is a Fab fragment.
a) a first solid carrier material impregnated or lyophilised with two different soluble receptors 1 and 3, bindable with the antigen, of which receptor 3 carries a marking, and b) a second solid carrier material, which is present physically separated from the first carrier material, the second solid carier material contain-ing an insoluble receptor 2 only bindable with a part of the non-marked soluble receptor 1, with the proviso that the marked receptor 3 is a Fab fragment.
19. Reagent according to claim 18, wherein the receptor 1 is an antibody and the receptor 3 is an enzyme-marked Fab fragment of an antibody.
20. Reagent according to claim 19, wherein receptors 1 and 3 are monoclonal and are directed against at least one of different and repetitive determinants of the antigen, and originate from the same animal species.
21. Reagent according to claim 19, wherein the receptor 2 is an antibody against the Fc part of the receptor 1 and the receptor 3 originates from the same animal species as the receptor 2, whereas the receptor 1 originates from another animal species.
22. Reagent according to claim 20, wherein all three receptors 1, 2 and 3 contain antibodies from the same animal species, the receptor 1 being a hapten antigen-marked monoclonal antibody and the receptor 2 being directed against the hapten antigen of the receptor 1.
23. Reagent according to claim 19, wherein the receptor 1 is a complete antibody, the receptor 3 is from the same animal species as the receptor 1 and the receptor 2 is an antibody directed against the Fc part of the receptor 1.
24. Reagent according to claim 18, 19 or 20, containing a buffer and a system for determining the enzyme marking.
25. Reagent according to claim 21, 22 or 23, containing a buffer and a system for determining the enzyme marking.
26. Reagent according to claim 18, 19 or 20, wherein the receptor 3 is marked with peroxidase or with .alpha.- or .beta.-galactosidase.
27. Reagent according to claim 21, 22 or 23, wherein the receptor 3 is marked with peroxidase or with .alpha.- or .beta.-galactosidase.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3400027.5 | 1984-01-02 | ||
| DE19843400027 DE3400027A1 (en) | 1984-01-02 | 1984-01-02 | Method for the determination of a polyvalent antigen and reagent therefor |
| DE19843425008 DE3425008A1 (en) | 1984-07-06 | 1984-07-06 | METHOD AND DEVICE FOR CARRYING OUT ANALYTICAL PROVISIONS |
| DEP3425008.5 | 1984-07-06 |
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| Publication Number | Publication Date |
|---|---|
| CA1244761A true CA1244761A (en) | 1988-11-15 |
Family
ID=25817348
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000471361A Expired CA1244761A (en) | 1984-01-02 | 1985-01-02 | Process for the determination of a polyvalent antigen and reagent therefor |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0147848B1 (en) |
| AU (1) | AU567998B2 (en) |
| CA (1) | CA1244761A (en) |
| DE (1) | DE3485265D1 (en) |
| DK (1) | DK165860C (en) |
| ES (1) | ES539297A0 (en) |
| IE (1) | IE58254B1 (en) |
| IL (1) | IL73938A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3705686C2 (en) * | 1987-02-23 | 1995-11-30 | Boehringer Mannheim Gmbh | Methods for the determination of antibodies |
| US4825648A (en) * | 1987-03-02 | 1989-05-02 | General Electric Company | Turbofan engine having a split cowl |
| US4918025A (en) * | 1987-03-03 | 1990-04-17 | Pb Diagnostic Systems, Inc. | Self contained immunoassay element |
| DE3714147A1 (en) * | 1987-04-28 | 1988-11-17 | Boehringer Mannheim Gmbh | IMMUNCHEMICAL METHOD AND REAGENT FOR DETERMINING A POLYVALENT ANTIGENT IN A LIQUID SAMPLE |
| DE3829245A1 (en) * | 1988-08-29 | 1990-03-01 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A SPECIFICALLY BINDABLE SUBSTANCE |
| EP0368674A3 (en) * | 1988-11-11 | 1991-10-09 | SANYO CHEMICAL INDUSTRIES, Ltd. | Immunoassay and test kits therefor |
| DE4120412C1 (en) * | 1991-06-20 | 1993-01-07 | Henning Berlin Gmbh Chemie- Und Pharmawerk, 1000 Berlin, De | |
| DE4328070C1 (en) * | 1993-08-20 | 1994-11-24 | Henning Berlin Gmbh | Method for the determination of an analyte in a volume of a liquid sample, and its use for the determination of anti-TSH receptor autoantibodies in a patient's serum |
| US5695928A (en) | 1993-12-10 | 1997-12-09 | Novartis Corporation | Rapid immunoassay for detection of antibodies or antigens incorporating simultaneous sample extraction and immunogenic reaction |
| US5589344A (en) * | 1994-06-15 | 1996-12-31 | Johnson & Johnson Clinical Diagnostics, Inc. | Test kit and method for competitive specific binding assay |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4292403A (en) * | 1978-08-24 | 1981-09-29 | Akzona Incorporated | Detection and/or determination of IgM, IgA, IgD and IgE immunoglobulins |
| CA1160566A (en) * | 1980-04-25 | 1984-01-17 | Harald Gallati | Immunological determination method |
| DE3044385A1 (en) * | 1980-11-25 | 1982-06-24 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR CARRYING OUT ANALYTICAL PROVISIONS AND ROTOR INSERT ELEMENT SUITABLE FOR THIS |
| DE3225027A1 (en) * | 1982-07-05 | 1984-01-05 | Boehringer Mannheim Gmbh, 6800 Mannheim | IMMUNCHEMICAL MEASUREMENT METHOD |
| DE3377531D1 (en) * | 1982-09-29 | 1988-09-01 | Serono Diagnostics Ltd | Immunoassay of antigens |
-
1984
- 1984-12-25 IL IL73938A patent/IL73938A/en unknown
- 1984-12-27 EP EP84116335A patent/EP0147848B1/en not_active Expired - Lifetime
- 1984-12-27 DE DE8484116335T patent/DE3485265D1/en not_active Expired - Lifetime
-
1985
- 1985-01-02 ES ES539297A patent/ES539297A0/en active Granted
- 1985-01-02 IE IE785A patent/IE58254B1/en not_active IP Right Cessation
- 1985-01-02 DK DK002085A patent/DK165860C/en not_active IP Right Cessation
- 1985-01-02 AU AU37247/85A patent/AU567998B2/en not_active Ceased
- 1985-01-02 CA CA000471361A patent/CA1244761A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| IE58254B1 (en) | 1993-08-25 |
| ES8601479A1 (en) | 1985-11-16 |
| EP0147848B1 (en) | 1991-11-13 |
| DE3485265D1 (en) | 1991-12-19 |
| AU3724785A (en) | 1985-07-18 |
| DK2085A (en) | 1985-07-03 |
| EP0147848A2 (en) | 1985-07-10 |
| AU567998B2 (en) | 1987-12-10 |
| DK2085D0 (en) | 1985-01-02 |
| IL73938A0 (en) | 1985-03-31 |
| ES539297A0 (en) | 1985-11-16 |
| DK165860B (en) | 1993-01-25 |
| IE850007L (en) | 1985-07-02 |
| DK165860C (en) | 1993-06-21 |
| IL73938A (en) | 1989-09-28 |
| EP0147848A3 (en) | 1988-06-22 |
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