CA1172959A - Process for binding and separating in competitive radioimmunoassay, and reagent for this purpose - Google Patents
Process for binding and separating in competitive radioimmunoassay, and reagent for this purposeInfo
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- CA1172959A CA1172959A CA000381601A CA381601A CA1172959A CA 1172959 A CA1172959 A CA 1172959A CA 000381601 A CA000381601 A CA 000381601A CA 381601 A CA381601 A CA 381601A CA 1172959 A CA1172959 A CA 1172959A
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
- G01N33/541—Double or second antibody, i.e. precipitating antibody
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Abstract
Abstract: HOE 80/F 156 Process for binding and separating in competitive radio-immunoassay, and reagent for this purpose A process is described for binding an antigen, which is specifically bound to an antibody, in an aqueous solution, and for separating off this bound antigen for the determination of the antigen concentra-tion in a body fluid, wherein the antibody with the specifically bound antigen is separated off from the free antigen which is dissolved in the aqueous solution, a reaction accelerator being added to the aqueous solu-tion. Polyethylene glycol, dextran, polyvinyl-pyrrolidone, ammonium sulfate or sodium sulfite serve as reaction accelerators.
Description
117~55~
In competitive radioimm~moassay, a radioactively labeled antigen, an unlabeled antigen and an antibody which is specific for the antigen are subjected to a competitive antlgen/antibody reaction in an aqueous 5 buffer solution, In this reaction, a defined quan-tity of radioactively labeled antigen and unlabeled antigen compete for a defined quantity of antibody, a deficiency, in comparison to the ant~gen, of the anti-body being present, The proportion of the radioacti-10 vely labeled antigen ~hich is bound to the antibodydepends on the quantity of the unla~eled antigen, and decreases with increasing quantity of unlabeled antigen, The principle of the radioimmunometric measure-15 ~ent method is based on the fact that the proportion of the radioactively labeled an-tigen which is bound to the antibody, which is observed in a sample to be ; measured, is compared ~lith the result of calibration - samples with known an-tigen content, and the antigen con-20 tent of the sample to be measured is thereby determined, To measure the proportion of the raàioactlvely labeled an-tigen ~hich is bound to the antibody, it is necessary to separate off the antigen/antibody complex f~om antigen ~hich is no~ bound, The method of 25 com~"etitive radioimmunoassay .is described in detail in the literature (see R,P, Ekins, Br,Med.Bull. ~0, 3-11 (1~74), and C.M, Boyd, D.L. Herzberg, in: Practical radicimmunoassay pages 1-13 (A.J. Mossg G.V, Dalrym.ple, C.M. Boyd, editors) The C,V, Mosby Comp.lïly, Saint Louis 1~ S ~
1~76) The accuracy of the competitive radioimmunoassay essentially depends on the separation of the radio-active]y labeled antigen which is bound to the anti-body, fron free radioactively labeled antigen, afterthe end of the antigen/antibody reaction.
Methods and separation reagents for separatin~
free antigens and antigens which are bo~nd to antibodies are known, for example ~0 1. Precipitati~n of the antigen/antibody complex with protein-precipitating reagents, see B. Desbuquois and G.D. Aurbach,J.Clin.Endocr. 33, 732-738 (1971)
In competitive radioimm~moassay, a radioactively labeled antigen, an unlabeled antigen and an antibody which is specific for the antigen are subjected to a competitive antlgen/antibody reaction in an aqueous 5 buffer solution, In this reaction, a defined quan-tity of radioactively labeled antigen and unlabeled antigen compete for a defined quantity of antibody, a deficiency, in comparison to the ant~gen, of the anti-body being present, The proportion of the radioacti-10 vely labeled antigen ~hich is bound to the antibodydepends on the quantity of the unla~eled antigen, and decreases with increasing quantity of unlabeled antigen, The principle of the radioimmunometric measure-15 ~ent method is based on the fact that the proportion of the radioactively labeled an-tigen which is bound to the antibody, which is observed in a sample to be ; measured, is compared ~lith the result of calibration - samples with known an-tigen content, and the antigen con-20 tent of the sample to be measured is thereby determined, To measure the proportion of the raàioactlvely labeled an-tigen ~hich is bound to the antibody, it is necessary to separate off the antigen/antibody complex f~om antigen ~hich is no~ bound, The method of 25 com~"etitive radioimmunoassay .is described in detail in the literature (see R,P, Ekins, Br,Med.Bull. ~0, 3-11 (1~74), and C.M, Boyd, D.L. Herzberg, in: Practical radicimmunoassay pages 1-13 (A.J. Mossg G.V, Dalrym.ple, C.M. Boyd, editors) The C,V, Mosby Comp.lïly, Saint Louis 1~ S ~
1~76) The accuracy of the competitive radioimmunoassay essentially depends on the separation of the radio-active]y labeled antigen which is bound to the anti-body, fron free radioactively labeled antigen, afterthe end of the antigen/antibody reaction.
Methods and separation reagents for separatin~
free antigens and antigens which are bo~nd to antibodies are known, for example ~0 1. Precipitati~n of the antigen/antibody complex with protein-precipitating reagents, see B. Desbuquois and G.D. Aurbach,J.Clin.Endocr. 33, 732-738 (1971)
2. Adsorption of the free antigen on adsorption agents
3. Binding the antibody (before the antigen/antibody reaction) to surfaces of solids which are insoluble in water, see DOS (German Offenlegungsschrift) 2,322,533
4 A~sorption of the antigen/antibody complex on Staphylococcus aureus cells, see S, Jonsson and G.
Kronvall,Eur J I~nunol 4, 29-33 (1974)
Kronvall,Eur J I~nunol 4, 29-33 (1974)
5. Precipita-tion of the antigen/an-tibody cornplex by means of a second antibody (separation antibody), see M.J. Martin and J. Landon, Radioimmunoassay in Clini^al Biochemistry, pages 269-281 (Pasternak, editor) Heyden, London (1975)
6 Adsorption of the antigen/antibody complex on small particles (solids), which are insGluble in water and which are coated with a second antibody (separation - an-tibody), see F.C Den Hollander and ~.H W.M. Schuurs, Radi~imrnunoassay Me-thods, pa~res 419-4~2 (Kirkhan and t 17~?59 Hunter, editors) Churchill Livingstone, London (1971);
and S Jonsson and ~. Kronvall IAEA Report SM 177/48, 287-298 (1974) The separation of antibody-bound and free anti-gen is ~derta~en by means of a phase separation, ~hichmostly precedes a centrifuging step The abovementioned methods can be described as follows: .
- 1, The precipitation of antigen/antibody complexes by protein precipitation reagents, such as ethanol, ammon um sulfate or polyethylene glycol, is simple to carry out, since a separation by centrifuging is poss-~ ible immediately after mixing the complex with -the : precipi.tation reagent, However, a prerequisite of this method is that a sufficient quanti.ty o~ im~uno-globulins is present in the sample to be measured.
Although this is the case in serum or plasma, it is not so in urine, liquor or gastric juice.
In addition, effective separations are only possible for antigens with a molecular weight which is substantially below the molecular ~ieight of the anti-bodies. Accordln~ly, this process is only applicable for special cases Furthermore, it has -the dis-advant2ge that a proportion, ~hich is of-ten not insub-stantial, of the free antigen is co-precipitated.
This co-precipitation varies wi-th the protein content and the protein composition of the sample, and can interfere ~ith the result of measurement 2 The adsorpti.on of the free antigen on adsorpti.on ~ 17 ~5 agents, such as active charcoal, ion exchangers and silicates, is only suitable fcr small antigens (for example steroids or peptides), and is not applicable generally In addition1 the reaction equilibrium can be disturbed in the case of a relatively lon~ reaction time with the adsorption agent.
3. On us.ing an antibody ~hich is bound to surfaces of water-insoluble solids (for example test tube inner . wall, paper strips, polymer balls of various sizes, glass beads or bacteria cells), the antigen/antibody complex formed during the incubation is likewise pre-sent bound to the solid, and is separated off, wi.th the solid, from the free antigen which is contained in the reaction solution The same quantity of antibody in each case, ~ith whi.ch such solids are coated, is a prerequisite for the accuracy of the competitive radioimrnunoassay, and is - attainable only with great effort and care. In addi-tion, undesired interferences occasionally occur through adsorption of proteins, from the sample, on the solid, and the blocking, which i.s associated therewith, of the antibodies,~hich mustbe effec-tive in a defined quantity.
4. Because of the specific adsorp1;ion properties of the Staphylococcus aureus cells for a few classes of immunoglobulins of various animal s~ecies - the an-ti-bod,es used in radioirrmunoassay are lmmunoglobulins -Staphylococcus aureus cells can serve as adsorbents for antigen/alltibody complexes. The rne-thod can only be applie(~ i.f the antibody belongs to an immv.noglobulin 1~7;~S5 cl~ss of an animal species, the corresponding immuno-globulin of which is specifically bound by Staphylo-coccus aureus Accordingly, the process is not applicable for antibodies of any desired animal spccies.
If, in addition to the antibody used as the reagent, the sample to be measured (for example serum or plasma) also contains, in addition, relatively large quantities of immunoglobulin which bind Staphylococcus aureus, a correspondingly greater quantity of Staphylo-coccus aureus cel'.s must be employed This has the disadYantage that, owing to non-specific adsorptions of free antigen, the separation is not complete The precipitation of the antigen/antibody com-plex by a second antibody (separation antibody), which is directed against the antigen-specifi.c first antibody, is time-consuming, since two antigen/antibody reactions have to be carried out The second a~tigen/antibody reaction can be shortened by mean.s of reaction acceler-ators This method has the disadvan-tage that undesired co-precipitations of free antigen occur, these co-precipitations being determined by the varying pro-~ein content of the sample In addition, the deposit is poorly visible after the precipitation, and can be lost, unnoticed, on separating off t~e li~uid abcve the deposit 6. The adsorption of the antigen/antibody comple~
~y the second antibody (separation antibody), ~hich is direc-ted against -the antigen-specific first antibody, and is covalently bonded to a wa-tcr-insoluble solid 1~7;~59 (for example cellulose or S~aphylococcus aureus), has the disad~antage that a few hours are necessary for - complete adsorption of the antigen/antibody complex, during which time, in addition, a homo~eneous suspen-sion of t~.e solid has to be produced by continuous agitat.ion of the reaction vessel.
; The object of the present invention is the provision of a separation process and a separation rea~ent for the separation~ in competitive radioimmuno-assay, of free antigen from an antigen bound to a first antibody, the separation process and the separation re-agent avoiding the disadvantages of the separation pro-cesses and separation reagents mentioned above under 1 to 6.
The invention relates to a process for binding, in an aqueous solution, an antigen which is specifically bound to a first antibody, and for separaiing this bound antigen from antigen which is not bound, for the quali-tative or quantitative determina~ion oî thc antigen concentration in a body liquid with ~he aid of competi-tive radioi.~munoassay, whereby the first antibody with the specifi.cally bound antigen is separated from the free antigen which is dissolved in the aqueous soluti.on, the separation being carried out by means of a second antibody, ~hich is bound, on the one hand, to a Staphylo-coccus aureus cell, and, on the other hand, specifically binds the first antibody, and the process com~rises adding a reaction acce3erator to the aqueous solution, the reaction accelerator accelerati.ng the specific 1~7;~59 binding of the first antibody by the second antibody, so that the binding reaction is completed in a few minutes.
The antigens used in the process according to the invention are peptides, proteins or glycol proteins, which functionally can be hormones, enzymes, trans-port proteins or structural proteins, and which, in the immunization of various vertebrate species, preferably mammals, produce a formation of antigen-specific first antibodies The determination of antigen in body fluids, such as blood plasma, blood serum, urine, liquor, gastric juice, duodenal juice, amniotic fluid or s~Jnovial fluid, is undertaken with the aid of competitive radio-immunoassay, and the body fluids mentioned can originatefrom man or from an animal.
As the second antibodies, those various a~imal classes, principally of vertebrates, preferably of mammals, are used, in which this second antibody was obtained by immun.ization of the animals~rith immuno~obul~s of adifferent animal speciesfrom~hich the first antibodyori-ginates. Staphylococcus aureus cellsbind gamma-globulins of manorofvarious animal species, such as donkey, goat, pig, dog, cat, gvinea pig or rabbit, specifically via their Fc-fragment, and the antigen-specific Fab-fragment of the gamma~globulins remains undisturbed by t;he binding to the Staphylococcus aureus cell (see J.~. Goding, J.
Immunol.Meth 20, 241-253 (1978)) The use of a second antibody of a relat;ively 117;~59 g large anlmal, such as a goat or donkey, is advantageous The second antibody can be bound to the serum of the immuni~ed animal by incubation of the Staphylococcus aureus cell 9 whereby, in particular, the gamma-globulins are specifically bound, and the non-binding serum proteins and othe~ serum constituents can easily be separated off by washing the cells, without a sub-stantlal amount of the second antibody being desorbed again.
Fcr the determination of an antigen in body fluids, such as blood serum or blood plasma, which con-^
tain relatively large amounts of immunoglobulins, particularly gamma-globulins, it is advantageous co-valently to bind the second antibody to the Staphylo-coccus aureus cell, in addition to the specific (adsorptive) bond mentioned For this purpose, ~he second antibody is covalently bonded, after the fipecific adsorption, to the Staphylococcus aureus cell, under mild conditions, by means of a bifunctional re-agent, the reactivity of the second antibody not dimin--lshing substantially ~ing to the covalent bondS
the second anti.body cannot detach itself from the cell, and cannot be displaced by gamma-globuli.ns of the sample.
By means of covalent bonding ~7ith a bifunctional reagentS a~.ino groups of the second antibody, on the one hand, and amino groups of ce].l ~Jall proteins of the StaphylocGccus aureus, on the other hand, are bonded ~ with one another Dialdehydes, d-iimidates, diesters : ' or diisoc~ranales, for example ~lutaric dialdehyde, dimethyladipin~diimidate, dime-thyl suberate or hexa-methylelle diisocyanate, are use~ as bifunctional re-agents, Before being used in the process accordina to the invention, tne S-taphylococcus aureus cells are advantageously ki.lled by heat treatment or treatment with formaldehyde. The cells are advan-tageously warrned to 80C for 30 minutes, and are incubated in a 1,5% strength formaldehyde solution, whereby the bond--ing capability of the cells for gar~na-globuli.ns does not decrease substantially, Reaction accelerators according to the inventicn are substances ~hich accelerate the specific bonding reaetion of a first antibody by a second antibody, ~ithout substantially changing the spatial polypeptide strueture of the im~unoglobulins, particularly the garnma-globulins, and thereby not destroying the s~ecific reactivity of the antiboc~.es, Examples of.such sub-stances arehydrophilicpolyme~s, such as de~tran orpolyvinylpyrrolidone. Substances which, in hi~h concentrati.ons, impart; insolubility to, or precipit~te, preferably immunoglobulins, parti.cularly gar~a-globulins, are advantageously used, Exa~nples of such sub-stances are hydrophilic polymers, such as polyethyleneglycc,~l, or sal~ts, such as am~onium sul.fate anc~ sodium sulfite, In this process, a lo~er concentration of these substances is to be used than ~rculd lead to the prccipitat:ion o:. the f~rst antibody ancl of the antigen, 117~59 The concentratioll of the reaction accelerators in the reaction mixture consisting OI the first antibody and the antigen and the second antibody which is bou-nd to the Staphylococcus aureus cell, is chosen in such a manner that the polyethylene glycol concentration is, in general, three to ten % by weight, preferably four to eight % by weight, and particularly 5 to 6% by weight, or the dextran concentration is, in general, three to twenty % by ~eight, preferably five to ten /0 by weight, and particularly seven % by weight, or the polyvinylpyrrolidone concentration is, in general, three to fifteen % by ~7eight, preferably fi~e to tcn % by weight, and particularly seven ~ by weight, or the ammonium sulfate concentration is, in general, 0 3 to 1 2 molar, preferably 0 5 to 0,9 molar, and particularly 0,7 to 0.8 molar, or the sodium su]fi-te concentration is, in general, 0 5 to 1,5 molar, preferably 0.8 to 1 2 molar, and particularly 1 0 rnolar.
The molecular weigh-t of the polymeric reaction accelerators in the case of polyethylene glycol is, in '~ 25 general, between 1,000 and 20,000, preferably bet~een 2,000 and 10,000, and part.icularly ~,000, in the case of dex-tran, in gcneral, between 2,000 and 50,000, preferably betweerl 4,C00 and 20,000, and particularly between ~,000 and 12,000, and il7;~59 in the case of polyvinylpyrrolidone, in general,between 2,000 and 25,000, preferably between 5,000 and 15,000, and particularly 10,000.
The reaction accelerator can already be added to the reaction mixture before the reaction of the first antibody with the antigen? and before the second anti-body which is bound to the Staphylococcus aureus is added for the purpose of separation, However, a separation reagent which is ready for use is advanta-geously added to the reaction mixture consisting ofantigen and first antibody, The separation reagent which is ready for use, contain~, .in an aqueous buffer soluti.on, the second antibody, whic~ is bound to Staphylococcus aureus, and the reacticn acoelerator, the concentration of which is selectedso that, after the addition of the separation ~eagent which is ready for use, to the reaction mixture consisting of ~ntigen and first antibody, the reaction accelerator is present in the concentrations mentioned above, The quantity of second antibody which is bound to the Staphylococcus aureus (~lith the useofO.l ml ofthe body fluid to be de-termined) corresponds to the quan-tity which is customarily required in a precipitation of the anti.gen/antibody complex by a second antibody, which is not bour.d, ~ he quantity of Staphylococcus (with the use of 0,lml ofthebody fluidtobe determined) is, in general, 0,5 to 10 r,~g, preferably 1 to 5 mg, and particularly 1 ~7~59 2,5 mg, To carry out the rad1.oim~unometric determirlation, radioactively labeled antigen, unlabeled an-tigen and antigen-specific first antibody are first incubated together, In this process, either a solution;of known antigen content for use as a calibration sample, or a body fluid wi-ch unkno~m antigen content, as the sample to be determined, is used as the unlabeled antigen, The incubation is undertaken, in general, at temperatures of between 0 and 4~C, preferably between 17 and 27C and usually at room temperature, The separation reagent is shaken before addi-tion to the reaction mixture, so that a uni~orm cell suspension is obtained, The ternperature of the separation reagent is, in general, between 0 and +45C, and it is advantageously adapted to the temperature of the preceding incubation, After the sep~ration reagent 'nas b~cn mixed with the reac-tion mixture, the binding of the first antibody ~ by the second antibody is co~.plete in a few minutes, ,'~ During this time, the cells, owi~g to their small size, remain uni~ormly distri.buted in the solution, so t,hat they are not lost, through sedimAentation, for binding, By .subsequent centrifuging, the antigen bound to the first antibody, and the Staphylococcus aureus cell charged with the second antibody5 are separa-ted off, as a deposit, from the free antigen ir. the superna-tan-t solution, To determine the ~uantity o~ labeled antigen bound to the first antibody, the supernatant solution . is decanted or filtered off under suction, the deposi-t then advantageously being washed wlth a buffer solu-tion In the case of a large volume of supernatantliquid the washing can be omitted The determina+ion of the radioacti.vity is undertaken according to known processes, by means of radiation detectors A. The separation process according to the inven-tion is suitable for the determination of antigens with low or high molecular weight, particularly for peptides and proteins, such as follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, chorionic gonadotropin, galactin, placental lactogen, growth hormone, adrenocorticotropic hormone, glucagon, ~-chain of chorlonic gonadotropin, parathyroid hormone, alpha~fetoprotein, carcinoembryogenic antigen, prostate-specific acid phosphates, and pregnancy-specific ~1-20 glycoprotein, which, in the absence of antigen-specific antibodies, are no-t bound to Staphylococcus aureus cells B The separation process is applicable generally for the separation of free antigen and anti.gen ~rhich is bound to any desired first antibody, the first anti-body originating from any desired first animal species, and the second antibody being obtained, in each cas~, by immuni~ation of a second animal speci.es with immuno-globulin ol the first animal species, and the second animal speci.es is chosen so that the gamma-globulins 1 ~7~ 59 - thereof are largely bound by the S-tap~lylococcus aureus cell.
C. By means of the specific bi.nding of the firs-t antibo&y, which is charged with antigen, to the second 5 antibody, which is bound to the Staphylococcus aureus cell, the separation process yields a largely quantita-tive separation of the free antigen fro~ the antigen bound to the first antibody, and separation is not sub-stantially disturbed even by greatly varying protein 10 content of the sample to be determined D. The reaction accelerator for the binding cf the first an-tibody, char~ed with antigen, by the second antibody, bound to the Staphylococcus aureus, requires only a very short second incubation time, during ~-hich ].5 a continuous agitation of the r~action mixture for homogeneous distribution of the cells is superfluous, t so that, after the mixture has been mixed with the separation reagent, the mixture can be centrifuged without a further operational step and without great 20 delay, E, The cell deposit after centrifuging is clearly visible, and adheres well to the bo-t-tom of the centri-fuge tube, so that separating off the supernatant liquid, by means of filtration under suction or decanta 25 tion, present,sno problem, and can easily be monitorc-d visu~lly.
The invention is illustrated in more detail by tne example.swhich follow In thc examples, all data in percent refer to percen-t by ~eight.
i~7~{~59 Example 1 Determination of the h~.an luteini?in~ horl~.cne (h~h) in blood plasma or blood serum a) Preparation of the separation reagent 16 ml of a goat antiserum, which ccntained antibodies against rabbit gamma-globulin, were added to 50 ml of a 10% strength suspension of Staphy]ococcus aureus cells in a 0 05 molar aqueous sodium phosphate buffer solution, containi.ng 0 ~/0 of sodium chloride, of pH 7 4. The suspension was stirred for one hour.
The cells charged with antibodies were then separated off from the supernatant liquid, by means of centri-fuging and decantation The cells were resuspended in 45 ml of the abovementioned buffer, and, for co-valent bonding of the antibody to 'che cells, 50 ml of aO, 2% strength glutaric dialdehyde solutlon in the above-mentioned buffer was added to the suspension, whilst the lat-ter was continuously stirred, and, after the suspen-sion had been stirred for one hour, 50 ml of a 5%
strength sodiun sulfi.te solution in t'ne abovementioned buCfer ~Jas added -to it, in order -to inactivate the excess of unreacted glutaric dialdehyde. After the suspension had been stirred for a further hour, the cells were separa-ted off, by centrifuging and decanta--tion, from the supernatant liquid. The cells ~rere was~ed by resuspending them in 200 ml of 0 05 mo].ar aqueous sodiu~, phosphate buffer of pH 7 4, and were separated off from the washing b~f:Cer solution by cen-trifuging and decantation. The separa-ti.on ~L~'7~59 reagent, ready for use, was prepared by tnen resuspending the cells thus treated, with the second antibody, ir~ 500 ml of a l~o strength solution of polyethylene glycol, with a molecular weight of 6,ooo, in 0.05 molar aqueous 5 sodium phosphate buffer of pI~ 7.4.
b) Carrying out the determination The analyses were carried ou-t in centrifuge tubes of 3 to 5 ml, made of polystyrene or polypropyl-ene 0 1 rnl of the human serum or plasma sarnple to be investigated was introduced into a tube.
0 1 ml of a calibration sample with various hlh concentrations, for example, 0, 4, 7, 1ll, 27, 54 and 100 molar units of hlh/ml was introduced, in each case, into a tube 0 2 ml of a solution containing 125I-hlh (about 0 5 ng of hlh/ml of a specific radi.oactivity of 100 nCi of iodine-]25/ng hlh) wa.s introduced into all tubes.
0 2 ml of an antibody solution against h:Lh, which, in the absence of unlabeled hlh, could bind 30 to 5~/0 of the 125I-hlh,was introducedinto allthc tubes.
The incubation took place during the course of 20 hours at room -temperature 0 5 ml of the separa~ti.on reagent, ready for use, corresponding to Example la), was shaken before use i.n order -to obtai.n the homogeneous cel' suspensionS and was introduced into all the tubes.
All the tubeswere shal~entomixthe contents,and, ~fter a-t leasttenminutes, were centri.,.llged for ten 1:~'7~?59 minutes at about 2,000 g.
The deposit was separated off, by means of decantation or filtration under suction, from the supernatant liquid, and was washed with a 0,05 molar aqueous sodium phosphate buffer solution of pH 7,4, After the supernatant liquid had been separated off, the gamma radiation of the labeled hlh contained in the deposit was measured in all the -tubes.
The pulses of the gamma radiation of the calibration samples per time unit was plotted linearly, on a graph, against the hlh concen-tration of the calibra^tion samples, The hlh concen-tration o the unkno~n salnple was then determined from this graph.
RY~
Determillatior, of hum3n alpha-feto~rotein (AFP~ in amniotic fluid ..
a) Preparation of the separation rea~ent 50 ml of a donkey antiserum, which con~tained antibodies against rabbit gamma-globulin, was added to 50 ml ol a 10~/o strength suspension of Staphylococcus aureus cells in a 0,~/0 strength aqueous sodiurn ch]oride, The suspension was stirred for Gne hour, The cells were then separated off, by cen-trifuging and decanta-t.ion, from the supernatant liquid, The cells were washed by r~suspendir,g them in 200 ml O:r o. ~0 strength aqueous sodiu~ chloride solution, and were separa-ted off ~rom tihe wash solution by means of centrifuging and decantation, The separatiorl reagen-t, ready Ior use, was 1~ 59 prepared by then resuspending tne cells thus treated, with the second antibody, in 500 ml of an aqueous buffer solution of pH 7 4, which contained 1.6 molar sodiurn sulfite and 0. 05 molar sodiurn phosphate 5 b ) Carrying out the determination The analyses were carried out in centrifuge tubes of 3 to 5 ml, made of polystyrene or polypropyl-ene, 0,1 ml of the amniotic fluid sample which was 10 to be investigated and which had previously been diluted . by 1 :. 100, was introduced into a tube 0,1 ml of a calibration sarnple with various ~FP
concentrations, for example, 0, 25, 50, 100, 200 and 400 units of AFP/ml, was introduced, in each case, into a tube, 0, 2 ml of a solution containing 125I-AFP (about 1 urit of AFP/ml of a specific radioactivity of 40 nCi iodine-125/unit AFP) was introduced into all ihe tubes .
0, 2 ml of an antibody solution against AFP, which, in the absence of ~mlabel ed . AFP, could bind 40 to 60~/o of the 125I~AFP, was introduced into all the tubes.
. The incubation took place during the course of twenty hours at room ternperature 0 5 ml of the separation reagent, ready for use, - 25 corresponding to Example 2a ), was shaken for a short time before use, in order -to obtain a homog-neous cell suspension, and was introduced into all the tubes.
All the tubes were shaken for a short time to rnix the contents, and were centri:Euged five -to ten minu-tes s9 later for t.en minutes at about 2,000 g, The deposit was separated off from the super-natant liquid by decantation or filtration under suc-tion.
After the supernatant liquid had been separated off, the gamna radiation of the labelled AFP contained in the deposit was measured in all the tubes.
The p~Lses of the gamma radiation of the calibration samples per time unit were plot-ted linearly, on a graph, against the AFP concentration of the calibration samples. The AFP concentration of the - unknown sample could then bedetermined fromthis graph.
Examp~Le 3 Determination of human al~ha-fetoprotein (AFP) in amniotic fluid a) Preparation of the separation reagent The preparation of the separation reagent was carried out, including the washing step, according to Example la).
The separation reagent, ready for use, was pre-pæred by then resuspending the cells thus treated, with the second antibody, in 500 ml of a 10% strength solu-tion of polyvinylpyrrolidone, w.ith an average molecular wei~ht of L0,000, in 0,05 molar aqueous sodi7~m phosphate buffer of p~ 7.4.
b) ~arr~ing out -the determination The analyses were carried out in centrifuge tubes of 3 to 5 ml$7r,ade of polystyrene or polypropylene.
Ool ml of the a7-~niotic fluid sa7,nple ~h.i.ch was ., .
~ 17~59 -- 21 ~
to be investigated and which had previously been diluteà by 1 : 100, was introduced into a tube.
0.1 ml of a calibration sample with various AFP
concentrations, for example 0, 25, 50, 100, 200 an~ 400 units of AFP/ml, was introduced, in each case, into a tube, o 2 ml of a solution containing 125I-AFP (about 1 unit of AFP/ml of a specific radioactivity of 40 nCi -of iodine-]25/unit AFP) was introduced into all thetubes.
0.2 ml of an antibody solution, again~t AFP, which, in the absence of unlabeled AFP, could bind 40 to 6~/o of the 125I-AFP, was introduced into all thetubes.
The i~cubation took place during the course of twenty hours at room temperature.
1.0 ml of the separation reagent, ready for use, corresponding to Example 3a), was shaken for a short time before use, in order to obtain a homogeneous cell suspension, and was introduced into all the tubes.
All the tubeswere shaken for ashorttime to rni~
the contents, and were centrifuged ten to thirty min-utes later for ten minutes at about 2,000 g.
The deposit was separated off by decantation or fil-tration under suction of the supernatant liquid, After the supernatant liquid had been separated off, the gamma radiation of the labeled AFP contained in the deposit ~as measlired in all the tubes.
The pulses of the gamma radia-tion of the calibraticn samples per -time ~nit were plotted linearly, on a gr~ph agains-t -the A~P concentration of the 1;~7~59 calibration samples, The AFP concentration of the unknown sa~.ple could thenbedetermined fromthis graph.
Example 4 Determination of human_thyroid-stimulating hormone (htsh) in blood serum or bloo~3 ~lasma a~ Preparation of the separation reagen-t The preparation of the separation reagent was carried out, including tne washing step, according to Example la), The separation reagent, ready for use, was prepared by then resuspending the cells thus treated, with the second antibody, in 500 r~ of an aqueous buffer solution OI pH 7,4, which contained 1,3 molar al~onium sulfate and 0.05 molar sodium phosphate, b) Carrying out the determination 0,1 ml of the human serum or plasma sample to be investigated was introduced into a tube, 0,1 l~l of a calibration sa~ple with various htsh concentrations, for example 0, 6, 12, 25, 50 and 100 ~1 units of htsh/ml, was introduced, in e~ch case, into a tube, 0,~ ml of a solution containing 125I-h-tsh (abov.t 0,~ ng of htsh/ml ol a specific radioac~ivlty of ~0 nC.i of iodine-125/ng htsh) was introduced into all the tubes.
0,1 ml of an antibody soluti.on, a~in~ htsh, which, in the absence of unlabeled ht-sh, could bind 30 to 50~ of the 125I-h-tsh, was in-troducecl into all t'ne tubes.
The incubation took place during the course of ; twenty ho~lrs at room -temperature, ~,5 r,l' of the separation reagent, ready for use, ~:~'7~959 corresponding -to Example 4a), was shaken for a short time before use, in order to obtain a homogeneous cell suspension, and was introduced into all the tubes.
All the tubeswere shakenforashorttime to mix the contènts, and were centrifuged fi.fteen to thirty minutes later for ten minutes at about 2,00~ g, The deposit was separated off by decantation or filtration under suction of the supernatant liquid, and was washed with a 0,05 molar aqueous sodium phosphate buffer solution of pH 7.4 After the supernatant liquid had been separated off, the gamma radiation of the labeled htsh contained in the deposit was measured in all the tubes.
The pulses of the gamm~ radiation of the calibration samples per time unit were plotted linearly, on a graph against the htsh concentration of the calibrati.on samples The htsh concentration of the unknown sample 30 could then be determined from this graph.
Example 5 Determination of human follicle-stimulatin~ hormone (hfsh) in blcod serum or blood ~asma a) Preparation of the separation reagent The preparation of the scparation reagent was carried out, including the washing step, according to Ex~mple la) The separation reagent, ready for use, was pre-pared. by then resuspending the cells thus treated, with the second antibodv, in 500 ml of a 15% strength ~17'~C~SY
` - 24 -soluti.on of pol.yvinylpyrrolidone,~lavinganaveragerl-.ole-cular ~eight of 10,000, in 0.05 molar aqueous sodiurn phosphate buffer of pH 7.4.
b) Carrying out the determination - The analyses were carried out in c~ntrifuge tubes of 3 to 5 ml, maae of polystyrene or polypropylene.
O 1 ml of the hurnan serum or plasma sample to be i.nvestigated W2S introduced into a tube.
0.1 ml of a calibration sample with various hfsh concentrations, for example 0, 5, 10, 20, 40 and 80 molar units of hfsh/ml, was introduced, in each case, - into a tube.
0 2 ml of a solution contai.ning 125I-hfsh (about 0.5 ng of hfsh/ml of a specific radi.oactivity of 100 nCi of iodine-125/ng hfsh) was introduced into all the tubes.
0 2 ml of an antibody solu-tion, against hfsh, ~ich, in the absence of unlabeled hîsh, could bind 30 to 5~0 of the 125I-hfsh, was ~ntroduced into all the tubes.
The incubation took place during the course of twenty hours at room temperature 0.5 Ml of the separation rea~ent, ready for use, corresponding to Exarnple 5a), was shaken for a short time before use, in order to obtain a horrlogeneous cell suspens:ion, and was introduced into all the tubes.
hll-the tubeswere shaken fora short t.irne -to mix the con-lents, and were centrifuged fiften to thirty mi.nutes later fo~. ten minvtes a-t about 2,000 g.
The depos~t was separated off by decan~tation or `. filtration u-nd.el suction of the su~erna-tant liquid, and 3~'7~59 . . , - 2~ -~as washed with a 0,05 molar aqueous scdium phosphate buffer solution of pH 7.4, After the supernatant liquid had been separated off, the gamma radiation of the labeled hfsh contained in the deposit was measurcd in all the tubes.
The pulses of the garnma radiation of the calibration samples per time unit were plotted linearly, on a graph against the hfsh concentration of the calibration samples. The hfsh concentration of the unkno~m sample could then be determined from this graph.
and S Jonsson and ~. Kronvall IAEA Report SM 177/48, 287-298 (1974) The separation of antibody-bound and free anti-gen is ~derta~en by means of a phase separation, ~hichmostly precedes a centrifuging step The abovementioned methods can be described as follows: .
- 1, The precipitation of antigen/antibody complexes by protein precipitation reagents, such as ethanol, ammon um sulfate or polyethylene glycol, is simple to carry out, since a separation by centrifuging is poss-~ ible immediately after mixing the complex with -the : precipi.tation reagent, However, a prerequisite of this method is that a sufficient quanti.ty o~ im~uno-globulins is present in the sample to be measured.
Although this is the case in serum or plasma, it is not so in urine, liquor or gastric juice.
In addition, effective separations are only possible for antigens with a molecular weight which is substantially below the molecular ~ieight of the anti-bodies. Accordln~ly, this process is only applicable for special cases Furthermore, it has -the dis-advant2ge that a proportion, ~hich is of-ten not insub-stantial, of the free antigen is co-precipitated.
This co-precipitation varies wi-th the protein content and the protein composition of the sample, and can interfere ~ith the result of measurement 2 The adsorpti.on of the free antigen on adsorpti.on ~ 17 ~5 agents, such as active charcoal, ion exchangers and silicates, is only suitable fcr small antigens (for example steroids or peptides), and is not applicable generally In addition1 the reaction equilibrium can be disturbed in the case of a relatively lon~ reaction time with the adsorption agent.
3. On us.ing an antibody ~hich is bound to surfaces of water-insoluble solids (for example test tube inner . wall, paper strips, polymer balls of various sizes, glass beads or bacteria cells), the antigen/antibody complex formed during the incubation is likewise pre-sent bound to the solid, and is separated off, wi.th the solid, from the free antigen which is contained in the reaction solution The same quantity of antibody in each case, ~ith whi.ch such solids are coated, is a prerequisite for the accuracy of the competitive radioimrnunoassay, and is - attainable only with great effort and care. In addi-tion, undesired interferences occasionally occur through adsorption of proteins, from the sample, on the solid, and the blocking, which i.s associated therewith, of the antibodies,~hich mustbe effec-tive in a defined quantity.
4. Because of the specific adsorp1;ion properties of the Staphylococcus aureus cells for a few classes of immunoglobulins of various animal s~ecies - the an-ti-bod,es used in radioirrmunoassay are lmmunoglobulins -Staphylococcus aureus cells can serve as adsorbents for antigen/alltibody complexes. The rne-thod can only be applie(~ i.f the antibody belongs to an immv.noglobulin 1~7;~S5 cl~ss of an animal species, the corresponding immuno-globulin of which is specifically bound by Staphylo-coccus aureus Accordingly, the process is not applicable for antibodies of any desired animal spccies.
If, in addition to the antibody used as the reagent, the sample to be measured (for example serum or plasma) also contains, in addition, relatively large quantities of immunoglobulin which bind Staphylococcus aureus, a correspondingly greater quantity of Staphylo-coccus aureus cel'.s must be employed This has the disadYantage that, owing to non-specific adsorptions of free antigen, the separation is not complete The precipitation of the antigen/antibody com-plex by a second antibody (separation antibody), which is directed against the antigen-specifi.c first antibody, is time-consuming, since two antigen/antibody reactions have to be carried out The second a~tigen/antibody reaction can be shortened by mean.s of reaction acceler-ators This method has the disadvan-tage that undesired co-precipitations of free antigen occur, these co-precipitations being determined by the varying pro-~ein content of the sample In addition, the deposit is poorly visible after the precipitation, and can be lost, unnoticed, on separating off t~e li~uid abcve the deposit 6. The adsorption of the antigen/antibody comple~
~y the second antibody (separation antibody), ~hich is direc-ted against -the antigen-specific first antibody, and is covalently bonded to a wa-tcr-insoluble solid 1~7;~59 (for example cellulose or S~aphylococcus aureus), has the disad~antage that a few hours are necessary for - complete adsorption of the antigen/antibody complex, during which time, in addition, a homo~eneous suspen-sion of t~.e solid has to be produced by continuous agitat.ion of the reaction vessel.
; The object of the present invention is the provision of a separation process and a separation rea~ent for the separation~ in competitive radioimmuno-assay, of free antigen from an antigen bound to a first antibody, the separation process and the separation re-agent avoiding the disadvantages of the separation pro-cesses and separation reagents mentioned above under 1 to 6.
The invention relates to a process for binding, in an aqueous solution, an antigen which is specifically bound to a first antibody, and for separaiing this bound antigen from antigen which is not bound, for the quali-tative or quantitative determina~ion oî thc antigen concentration in a body liquid with ~he aid of competi-tive radioi.~munoassay, whereby the first antibody with the specifi.cally bound antigen is separated from the free antigen which is dissolved in the aqueous soluti.on, the separation being carried out by means of a second antibody, ~hich is bound, on the one hand, to a Staphylo-coccus aureus cell, and, on the other hand, specifically binds the first antibody, and the process com~rises adding a reaction acce3erator to the aqueous solution, the reaction accelerator accelerati.ng the specific 1~7;~59 binding of the first antibody by the second antibody, so that the binding reaction is completed in a few minutes.
The antigens used in the process according to the invention are peptides, proteins or glycol proteins, which functionally can be hormones, enzymes, trans-port proteins or structural proteins, and which, in the immunization of various vertebrate species, preferably mammals, produce a formation of antigen-specific first antibodies The determination of antigen in body fluids, such as blood plasma, blood serum, urine, liquor, gastric juice, duodenal juice, amniotic fluid or s~Jnovial fluid, is undertaken with the aid of competitive radio-immunoassay, and the body fluids mentioned can originatefrom man or from an animal.
As the second antibodies, those various a~imal classes, principally of vertebrates, preferably of mammals, are used, in which this second antibody was obtained by immun.ization of the animals~rith immuno~obul~s of adifferent animal speciesfrom~hich the first antibodyori-ginates. Staphylococcus aureus cellsbind gamma-globulins of manorofvarious animal species, such as donkey, goat, pig, dog, cat, gvinea pig or rabbit, specifically via their Fc-fragment, and the antigen-specific Fab-fragment of the gamma~globulins remains undisturbed by t;he binding to the Staphylococcus aureus cell (see J.~. Goding, J.
Immunol.Meth 20, 241-253 (1978)) The use of a second antibody of a relat;ively 117;~59 g large anlmal, such as a goat or donkey, is advantageous The second antibody can be bound to the serum of the immuni~ed animal by incubation of the Staphylococcus aureus cell 9 whereby, in particular, the gamma-globulins are specifically bound, and the non-binding serum proteins and othe~ serum constituents can easily be separated off by washing the cells, without a sub-stantlal amount of the second antibody being desorbed again.
Fcr the determination of an antigen in body fluids, such as blood serum or blood plasma, which con-^
tain relatively large amounts of immunoglobulins, particularly gamma-globulins, it is advantageous co-valently to bind the second antibody to the Staphylo-coccus aureus cell, in addition to the specific (adsorptive) bond mentioned For this purpose, ~he second antibody is covalently bonded, after the fipecific adsorption, to the Staphylococcus aureus cell, under mild conditions, by means of a bifunctional re-agent, the reactivity of the second antibody not dimin--lshing substantially ~ing to the covalent bondS
the second anti.body cannot detach itself from the cell, and cannot be displaced by gamma-globuli.ns of the sample.
By means of covalent bonding ~7ith a bifunctional reagentS a~.ino groups of the second antibody, on the one hand, and amino groups of ce].l ~Jall proteins of the StaphylocGccus aureus, on the other hand, are bonded ~ with one another Dialdehydes, d-iimidates, diesters : ' or diisoc~ranales, for example ~lutaric dialdehyde, dimethyladipin~diimidate, dime-thyl suberate or hexa-methylelle diisocyanate, are use~ as bifunctional re-agents, Before being used in the process accordina to the invention, tne S-taphylococcus aureus cells are advantageously ki.lled by heat treatment or treatment with formaldehyde. The cells are advan-tageously warrned to 80C for 30 minutes, and are incubated in a 1,5% strength formaldehyde solution, whereby the bond--ing capability of the cells for gar~na-globuli.ns does not decrease substantially, Reaction accelerators according to the inventicn are substances ~hich accelerate the specific bonding reaetion of a first antibody by a second antibody, ~ithout substantially changing the spatial polypeptide strueture of the im~unoglobulins, particularly the garnma-globulins, and thereby not destroying the s~ecific reactivity of the antiboc~.es, Examples of.such sub-stances arehydrophilicpolyme~s, such as de~tran orpolyvinylpyrrolidone. Substances which, in hi~h concentrati.ons, impart; insolubility to, or precipit~te, preferably immunoglobulins, parti.cularly gar~a-globulins, are advantageously used, Exa~nples of such sub-stances are hydrophilic polymers, such as polyethyleneglycc,~l, or sal~ts, such as am~onium sul.fate anc~ sodium sulfite, In this process, a lo~er concentration of these substances is to be used than ~rculd lead to the prccipitat:ion o:. the f~rst antibody ancl of the antigen, 117~59 The concentratioll of the reaction accelerators in the reaction mixture consisting OI the first antibody and the antigen and the second antibody which is bou-nd to the Staphylococcus aureus cell, is chosen in such a manner that the polyethylene glycol concentration is, in general, three to ten % by weight, preferably four to eight % by weight, and particularly 5 to 6% by weight, or the dextran concentration is, in general, three to twenty % by ~eight, preferably five to ten /0 by weight, and particularly seven % by weight, or the polyvinylpyrrolidone concentration is, in general, three to fifteen % by ~7eight, preferably fi~e to tcn % by weight, and particularly seven ~ by weight, or the ammonium sulfate concentration is, in general, 0 3 to 1 2 molar, preferably 0 5 to 0,9 molar, and particularly 0,7 to 0.8 molar, or the sodium su]fi-te concentration is, in general, 0 5 to 1,5 molar, preferably 0.8 to 1 2 molar, and particularly 1 0 rnolar.
The molecular weigh-t of the polymeric reaction accelerators in the case of polyethylene glycol is, in '~ 25 general, between 1,000 and 20,000, preferably bet~een 2,000 and 10,000, and part.icularly ~,000, in the case of dex-tran, in gcneral, between 2,000 and 50,000, preferably betweerl 4,C00 and 20,000, and particularly between ~,000 and 12,000, and il7;~59 in the case of polyvinylpyrrolidone, in general,between 2,000 and 25,000, preferably between 5,000 and 15,000, and particularly 10,000.
The reaction accelerator can already be added to the reaction mixture before the reaction of the first antibody with the antigen? and before the second anti-body which is bound to the Staphylococcus aureus is added for the purpose of separation, However, a separation reagent which is ready for use is advanta-geously added to the reaction mixture consisting ofantigen and first antibody, The separation reagent which is ready for use, contain~, .in an aqueous buffer soluti.on, the second antibody, whic~ is bound to Staphylococcus aureus, and the reacticn acoelerator, the concentration of which is selectedso that, after the addition of the separation ~eagent which is ready for use, to the reaction mixture consisting of ~ntigen and first antibody, the reaction accelerator is present in the concentrations mentioned above, The quantity of second antibody which is bound to the Staphylococcus aureus (~lith the useofO.l ml ofthe body fluid to be de-termined) corresponds to the quan-tity which is customarily required in a precipitation of the anti.gen/antibody complex by a second antibody, which is not bour.d, ~ he quantity of Staphylococcus (with the use of 0,lml ofthebody fluidtobe determined) is, in general, 0,5 to 10 r,~g, preferably 1 to 5 mg, and particularly 1 ~7~59 2,5 mg, To carry out the rad1.oim~unometric determirlation, radioactively labeled antigen, unlabeled an-tigen and antigen-specific first antibody are first incubated together, In this process, either a solution;of known antigen content for use as a calibration sample, or a body fluid wi-ch unkno~m antigen content, as the sample to be determined, is used as the unlabeled antigen, The incubation is undertaken, in general, at temperatures of between 0 and 4~C, preferably between 17 and 27C and usually at room temperature, The separation reagent is shaken before addi-tion to the reaction mixture, so that a uni~orm cell suspension is obtained, The ternperature of the separation reagent is, in general, between 0 and +45C, and it is advantageously adapted to the temperature of the preceding incubation, After the sep~ration reagent 'nas b~cn mixed with the reac-tion mixture, the binding of the first antibody ~ by the second antibody is co~.plete in a few minutes, ,'~ During this time, the cells, owi~g to their small size, remain uni~ormly distri.buted in the solution, so t,hat they are not lost, through sedimAentation, for binding, By .subsequent centrifuging, the antigen bound to the first antibody, and the Staphylococcus aureus cell charged with the second antibody5 are separa-ted off, as a deposit, from the free antigen ir. the superna-tan-t solution, To determine the ~uantity o~ labeled antigen bound to the first antibody, the supernatant solution . is decanted or filtered off under suction, the deposi-t then advantageously being washed wlth a buffer solu-tion In the case of a large volume of supernatantliquid the washing can be omitted The determina+ion of the radioacti.vity is undertaken according to known processes, by means of radiation detectors A. The separation process according to the inven-tion is suitable for the determination of antigens with low or high molecular weight, particularly for peptides and proteins, such as follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, chorionic gonadotropin, galactin, placental lactogen, growth hormone, adrenocorticotropic hormone, glucagon, ~-chain of chorlonic gonadotropin, parathyroid hormone, alpha~fetoprotein, carcinoembryogenic antigen, prostate-specific acid phosphates, and pregnancy-specific ~1-20 glycoprotein, which, in the absence of antigen-specific antibodies, are no-t bound to Staphylococcus aureus cells B The separation process is applicable generally for the separation of free antigen and anti.gen ~rhich is bound to any desired first antibody, the first anti-body originating from any desired first animal species, and the second antibody being obtained, in each cas~, by immuni~ation of a second animal speci.es with immuno-globulin ol the first animal species, and the second animal speci.es is chosen so that the gamma-globulins 1 ~7~ 59 - thereof are largely bound by the S-tap~lylococcus aureus cell.
C. By means of the specific bi.nding of the firs-t antibo&y, which is charged with antigen, to the second 5 antibody, which is bound to the Staphylococcus aureus cell, the separation process yields a largely quantita-tive separation of the free antigen fro~ the antigen bound to the first antibody, and separation is not sub-stantially disturbed even by greatly varying protein 10 content of the sample to be determined D. The reaction accelerator for the binding cf the first an-tibody, char~ed with antigen, by the second antibody, bound to the Staphylococcus aureus, requires only a very short second incubation time, during ~-hich ].5 a continuous agitation of the r~action mixture for homogeneous distribution of the cells is superfluous, t so that, after the mixture has been mixed with the separation reagent, the mixture can be centrifuged without a further operational step and without great 20 delay, E, The cell deposit after centrifuging is clearly visible, and adheres well to the bo-t-tom of the centri-fuge tube, so that separating off the supernatant liquid, by means of filtration under suction or decanta 25 tion, present,sno problem, and can easily be monitorc-d visu~lly.
The invention is illustrated in more detail by tne example.swhich follow In thc examples, all data in percent refer to percen-t by ~eight.
i~7~{~59 Example 1 Determination of the h~.an luteini?in~ horl~.cne (h~h) in blood plasma or blood serum a) Preparation of the separation reagent 16 ml of a goat antiserum, which ccntained antibodies against rabbit gamma-globulin, were added to 50 ml of a 10% strength suspension of Staphy]ococcus aureus cells in a 0 05 molar aqueous sodium phosphate buffer solution, containi.ng 0 ~/0 of sodium chloride, of pH 7 4. The suspension was stirred for one hour.
The cells charged with antibodies were then separated off from the supernatant liquid, by means of centri-fuging and decantation The cells were resuspended in 45 ml of the abovementioned buffer, and, for co-valent bonding of the antibody to 'che cells, 50 ml of aO, 2% strength glutaric dialdehyde solutlon in the above-mentioned buffer was added to the suspension, whilst the lat-ter was continuously stirred, and, after the suspen-sion had been stirred for one hour, 50 ml of a 5%
strength sodiun sulfi.te solution in t'ne abovementioned buCfer ~Jas added -to it, in order -to inactivate the excess of unreacted glutaric dialdehyde. After the suspension had been stirred for a further hour, the cells were separa-ted off, by centrifuging and decanta--tion, from the supernatant liquid. The cells ~rere was~ed by resuspending them in 200 ml of 0 05 mo].ar aqueous sodiu~, phosphate buffer of pH 7 4, and were separated off from the washing b~f:Cer solution by cen-trifuging and decantation. The separa-ti.on ~L~'7~59 reagent, ready for use, was prepared by tnen resuspending the cells thus treated, with the second antibody, ir~ 500 ml of a l~o strength solution of polyethylene glycol, with a molecular weight of 6,ooo, in 0.05 molar aqueous 5 sodium phosphate buffer of pI~ 7.4.
b) Carrying out the determination The analyses were carried ou-t in centrifuge tubes of 3 to 5 ml, made of polystyrene or polypropyl-ene 0 1 rnl of the human serum or plasma sarnple to be investigated was introduced into a tube.
0 1 ml of a calibration sample with various hlh concentrations, for example, 0, 4, 7, 1ll, 27, 54 and 100 molar units of hlh/ml was introduced, in each case, into a tube 0 2 ml of a solution containing 125I-hlh (about 0 5 ng of hlh/ml of a specific radi.oactivity of 100 nCi of iodine-]25/ng hlh) wa.s introduced into all tubes.
0 2 ml of an antibody solution against h:Lh, which, in the absence of unlabeled hlh, could bind 30 to 5~/0 of the 125I-hlh,was introducedinto allthc tubes.
The incubation took place during the course of 20 hours at room -temperature 0 5 ml of the separa~ti.on reagent, ready for use, corresponding to Example la), was shaken before use i.n order -to obtai.n the homogeneous cel' suspensionS and was introduced into all the tubes.
All the tubeswere shal~entomixthe contents,and, ~fter a-t leasttenminutes, were centri.,.llged for ten 1:~'7~?59 minutes at about 2,000 g.
The deposit was separated off, by means of decantation or filtration under suction, from the supernatant liquid, and was washed with a 0,05 molar aqueous sodium phosphate buffer solution of pH 7,4, After the supernatant liquid had been separated off, the gamma radiation of the labeled hlh contained in the deposit was measured in all the -tubes.
The pulses of the gamma radiation of the calibration samples per time unit was plotted linearly, on a graph, against the hlh concen-tration of the calibra^tion samples, The hlh concen-tration o the unkno~n salnple was then determined from this graph.
RY~
Determillatior, of hum3n alpha-feto~rotein (AFP~ in amniotic fluid ..
a) Preparation of the separation rea~ent 50 ml of a donkey antiserum, which con~tained antibodies against rabbit gamma-globulin, was added to 50 ml ol a 10~/o strength suspension of Staphylococcus aureus cells in a 0,~/0 strength aqueous sodiurn ch]oride, The suspension was stirred for Gne hour, The cells were then separated off, by cen-trifuging and decanta-t.ion, from the supernatant liquid, The cells were washed by r~suspendir,g them in 200 ml O:r o. ~0 strength aqueous sodiu~ chloride solution, and were separa-ted off ~rom tihe wash solution by means of centrifuging and decantation, The separatiorl reagen-t, ready Ior use, was 1~ 59 prepared by then resuspending tne cells thus treated, with the second antibody, in 500 ml of an aqueous buffer solution of pH 7 4, which contained 1.6 molar sodiurn sulfite and 0. 05 molar sodiurn phosphate 5 b ) Carrying out the determination The analyses were carried out in centrifuge tubes of 3 to 5 ml, made of polystyrene or polypropyl-ene, 0,1 ml of the amniotic fluid sample which was 10 to be investigated and which had previously been diluted . by 1 :. 100, was introduced into a tube 0,1 ml of a calibration sarnple with various ~FP
concentrations, for example, 0, 25, 50, 100, 200 and 400 units of AFP/ml, was introduced, in each case, into a tube, 0, 2 ml of a solution containing 125I-AFP (about 1 urit of AFP/ml of a specific radioactivity of 40 nCi iodine-125/unit AFP) was introduced into all ihe tubes .
0, 2 ml of an antibody solution against AFP, which, in the absence of ~mlabel ed . AFP, could bind 40 to 60~/o of the 125I~AFP, was introduced into all the tubes.
. The incubation took place during the course of twenty hours at room ternperature 0 5 ml of the separation reagent, ready for use, - 25 corresponding to Example 2a ), was shaken for a short time before use, in order -to obtain a homog-neous cell suspension, and was introduced into all the tubes.
All the tubes were shaken for a short time to rnix the contents, and were centri:Euged five -to ten minu-tes s9 later for t.en minutes at about 2,000 g, The deposit was separated off from the super-natant liquid by decantation or filtration under suc-tion.
After the supernatant liquid had been separated off, the gamna radiation of the labelled AFP contained in the deposit was measured in all the tubes.
The p~Lses of the gamma radiation of the calibration samples per time unit were plot-ted linearly, on a graph, against the AFP concentration of the calibration samples. The AFP concentration of the - unknown sample could then bedetermined fromthis graph.
Examp~Le 3 Determination of human al~ha-fetoprotein (AFP) in amniotic fluid a) Preparation of the separation reagent The preparation of the separation reagent was carried out, including the washing step, according to Example la).
The separation reagent, ready for use, was pre-pæred by then resuspending the cells thus treated, with the second antibody, in 500 ml of a 10% strength solu-tion of polyvinylpyrrolidone, w.ith an average molecular wei~ht of L0,000, in 0,05 molar aqueous sodi7~m phosphate buffer of p~ 7.4.
b) ~arr~ing out -the determination The analyses were carried out in centrifuge tubes of 3 to 5 ml$7r,ade of polystyrene or polypropylene.
Ool ml of the a7-~niotic fluid sa7,nple ~h.i.ch was ., .
~ 17~59 -- 21 ~
to be investigated and which had previously been diluteà by 1 : 100, was introduced into a tube.
0.1 ml of a calibration sample with various AFP
concentrations, for example 0, 25, 50, 100, 200 an~ 400 units of AFP/ml, was introduced, in each case, into a tube, o 2 ml of a solution containing 125I-AFP (about 1 unit of AFP/ml of a specific radioactivity of 40 nCi -of iodine-]25/unit AFP) was introduced into all thetubes.
0.2 ml of an antibody solution, again~t AFP, which, in the absence of unlabeled AFP, could bind 40 to 6~/o of the 125I-AFP, was introduced into all thetubes.
The i~cubation took place during the course of twenty hours at room temperature.
1.0 ml of the separation reagent, ready for use, corresponding to Example 3a), was shaken for a short time before use, in order to obtain a homogeneous cell suspension, and was introduced into all the tubes.
All the tubeswere shaken for ashorttime to rni~
the contents, and were centrifuged ten to thirty min-utes later for ten minutes at about 2,000 g.
The deposit was separated off by decantation or fil-tration under suction of the supernatant liquid, After the supernatant liquid had been separated off, the gamma radiation of the labeled AFP contained in the deposit ~as measlired in all the tubes.
The pulses of the gamma radia-tion of the calibraticn samples per -time ~nit were plotted linearly, on a gr~ph agains-t -the A~P concentration of the 1;~7~59 calibration samples, The AFP concentration of the unknown sa~.ple could thenbedetermined fromthis graph.
Example 4 Determination of human_thyroid-stimulating hormone (htsh) in blood serum or bloo~3 ~lasma a~ Preparation of the separation reagen-t The preparation of the separation reagent was carried out, including tne washing step, according to Example la), The separation reagent, ready for use, was prepared by then resuspending the cells thus treated, with the second antibody, in 500 r~ of an aqueous buffer solution OI pH 7,4, which contained 1,3 molar al~onium sulfate and 0.05 molar sodium phosphate, b) Carrying out the determination 0,1 ml of the human serum or plasma sample to be investigated was introduced into a tube, 0,1 l~l of a calibration sa~ple with various htsh concentrations, for example 0, 6, 12, 25, 50 and 100 ~1 units of htsh/ml, was introduced, in e~ch case, into a tube, 0,~ ml of a solution containing 125I-h-tsh (abov.t 0,~ ng of htsh/ml ol a specific radioac~ivlty of ~0 nC.i of iodine-125/ng htsh) was introduced into all the tubes.
0,1 ml of an antibody soluti.on, a~in~ htsh, which, in the absence of unlabeled ht-sh, could bind 30 to 50~ of the 125I-h-tsh, was in-troducecl into all t'ne tubes.
The incubation took place during the course of ; twenty ho~lrs at room -temperature, ~,5 r,l' of the separation reagent, ready for use, ~:~'7~959 corresponding -to Example 4a), was shaken for a short time before use, in order to obtain a homogeneous cell suspension, and was introduced into all the tubes.
All the tubeswere shakenforashorttime to mix the contènts, and were centrifuged fi.fteen to thirty minutes later for ten minutes at about 2,00~ g, The deposit was separated off by decantation or filtration under suction of the supernatant liquid, and was washed with a 0,05 molar aqueous sodium phosphate buffer solution of pH 7.4 After the supernatant liquid had been separated off, the gamma radiation of the labeled htsh contained in the deposit was measured in all the tubes.
The pulses of the gamm~ radiation of the calibration samples per time unit were plotted linearly, on a graph against the htsh concentration of the calibrati.on samples The htsh concentration of the unknown sample 30 could then be determined from this graph.
Example 5 Determination of human follicle-stimulatin~ hormone (hfsh) in blcod serum or blood ~asma a) Preparation of the separation reagent The preparation of the scparation reagent was carried out, including the washing step, according to Ex~mple la) The separation reagent, ready for use, was pre-pared. by then resuspending the cells thus treated, with the second antibodv, in 500 ml of a 15% strength ~17'~C~SY
` - 24 -soluti.on of pol.yvinylpyrrolidone,~lavinganaveragerl-.ole-cular ~eight of 10,000, in 0.05 molar aqueous sodiurn phosphate buffer of pH 7.4.
b) Carrying out the determination - The analyses were carried out in c~ntrifuge tubes of 3 to 5 ml, maae of polystyrene or polypropylene.
O 1 ml of the hurnan serum or plasma sample to be i.nvestigated W2S introduced into a tube.
0.1 ml of a calibration sample with various hfsh concentrations, for example 0, 5, 10, 20, 40 and 80 molar units of hfsh/ml, was introduced, in each case, - into a tube.
0 2 ml of a solution contai.ning 125I-hfsh (about 0.5 ng of hfsh/ml of a specific radi.oactivity of 100 nCi of iodine-125/ng hfsh) was introduced into all the tubes.
0 2 ml of an antibody solu-tion, against hfsh, ~ich, in the absence of unlabeled hîsh, could bind 30 to 5~0 of the 125I-hfsh, was ~ntroduced into all the tubes.
The incubation took place during the course of twenty hours at room temperature 0.5 Ml of the separation rea~ent, ready for use, corresponding to Exarnple 5a), was shaken for a short time before use, in order to obtain a horrlogeneous cell suspens:ion, and was introduced into all the tubes.
hll-the tubeswere shaken fora short t.irne -to mix the con-lents, and were centrifuged fiften to thirty mi.nutes later fo~. ten minvtes a-t about 2,000 g.
The depos~t was separated off by decan~tation or `. filtration u-nd.el suction of the su~erna-tant liquid, and 3~'7~59 . . , - 2~ -~as washed with a 0,05 molar aqueous scdium phosphate buffer solution of pH 7.4, After the supernatant liquid had been separated off, the gamma radiation of the labeled hfsh contained in the deposit was measurcd in all the tubes.
The pulses of the garnma radiation of the calibration samples per time unit were plotted linearly, on a graph against the hfsh concentration of the calibration samples. The hfsh concentration of the unkno~m sample could then be determined from this graph.
Claims (4)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Process for binding, in an aqueous solution, an antigen which is specifically bound to a first antibody, and for separating this bound antigen from antigen which is not bound, for the determination of the antigen con-centration in a body liquid with the aid of competitive radioimmunoassay, whereby the first antibody with the specifically bound antigen is separated from the free antigen which is dissolved in the aqueous solution, the separation being carried out by means of a second anti-body, which is bound, on the one hand, to a Staphylo-coccus aureus cell, and, on the other hand, specifically binds the first antibody, and the process comprises adding a reaction accelerator to the aqueous solution, the reaction accelerator accelerating the specific binding of the first antibody by the second antibody.
2. Process as claimed in claim 1, in which poly-ethylene glycol, dextran, polyvinylpyrrolidone, ammonium sulfate or sodium sulfite is used as the reaction accelerator.
3. Reagent for the separation of free antigen from an antigen which is bound to a first antibody in the determination of an antigen with the aid of competitive radioimmunoassay, the reagent consisting of an aqueous buffer solution, a reaction accelerator and Staphylo-coccus aureus cells, to which a second antibody is specifically bound, the reacton accelerator accelerating the specific binding of the first antibody by the second antibody.
4. Reagent as claimed in claim 3 , in which poly-ethylene glycol, dextran, polyvinylpyrrolidone, ammo-nium sulfate or sodium sulfite is used as the reaction accelerator.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19803026665 DE3026665A1 (en) | 1980-07-14 | 1980-07-14 | METHOD FOR BINDING AND SEPARATING THE COMPETITIVE RADIOIMMUNOASSAY AND REAGENT THEREFOR |
| DEP3026665.0 | 1980-07-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1172959A true CA1172959A (en) | 1984-08-21 |
Family
ID=6107171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000381601A Expired CA1172959A (en) | 1980-07-14 | 1981-07-13 | Process for binding and separating in competitive radioimmunoassay, and reagent for this purpose |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0044041B1 (en) |
| JP (1) | JPS5749861A (en) |
| AT (1) | ATE12316T1 (en) |
| AU (1) | AU7281281A (en) |
| CA (1) | CA1172959A (en) |
| DD (1) | DD202073A5 (en) |
| DE (2) | DE3026665A1 (en) |
| DK (1) | DK310781A (en) |
| ES (1) | ES8205465A1 (en) |
| FI (1) | FI812187L (en) |
| IL (1) | IL63291A0 (en) |
| NO (1) | NO812397L (en) |
| PL (1) | PL232148A1 (en) |
| PT (1) | PT73359B (en) |
| ZA (1) | ZA814741B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU647428B2 (en) * | 1989-12-01 | 1994-03-24 | Dade Behring Marburg Gmbh | Immunoassay for antibodies to infectious disease agents |
| DE4202924A1 (en) * | 1992-02-01 | 1993-08-05 | Behringwerke Ag | METHOD FOR DETERMINING RHEUMA FACTORS AND MEANS FOR IMPLEMENTING THE METHOD |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3981982A (en) * | 1974-09-11 | 1976-09-21 | Abbott Laboratories | Radioimmunoassay for determining the digoxin content of a sample |
| US3995019A (en) * | 1975-03-04 | 1976-11-30 | Baxter Travenol Laboratories, Inc. | Diagnostic reagent system |
| US4018883A (en) * | 1975-03-10 | 1977-04-19 | Nichols Institute For Endocrinology | Thyroxine (T4) radioimmunoassay |
| US4139604A (en) * | 1977-09-16 | 1979-02-13 | Becton, Dickinson And Company | Separation technique for assays |
| FI792576A (en) * | 1979-08-20 | 1981-02-21 | Orion Yhtymae Oy | METHOD OF ENZYME IMMUNOLOGICAL ASSESSMENT |
-
1980
- 1980-07-14 DE DE19803026665 patent/DE3026665A1/en not_active Withdrawn
-
1981
- 1981-07-08 ES ES503763A patent/ES8205465A1/en not_active Expired
- 1981-07-09 DE DE8181105350T patent/DE3169371D1/en not_active Expired
- 1981-07-09 AT AT81105350T patent/ATE12316T1/en not_active IP Right Cessation
- 1981-07-09 EP EP81105350A patent/EP0044041B1/en not_active Expired
- 1981-07-10 FI FI812187A patent/FI812187L/en not_active Application Discontinuation
- 1981-07-10 DD DD81231666A patent/DD202073A5/en unknown
- 1981-07-10 PL PL23214881A patent/PL232148A1/xx unknown
- 1981-07-13 AU AU72812/81A patent/AU7281281A/en not_active Abandoned
- 1981-07-13 IL IL63291A patent/IL63291A0/en unknown
- 1981-07-13 CA CA000381601A patent/CA1172959A/en not_active Expired
- 1981-07-13 DK DK310781A patent/DK310781A/en not_active Application Discontinuation
- 1981-07-13 ZA ZA814741A patent/ZA814741B/en unknown
- 1981-07-13 PT PT73359A patent/PT73359B/en unknown
- 1981-07-13 NO NO812397A patent/NO812397L/en unknown
- 1981-07-14 JP JP56108929A patent/JPS5749861A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| PL232148A1 (en) | 1982-03-15 |
| EP0044041B1 (en) | 1985-03-20 |
| DK310781A (en) | 1982-01-15 |
| ES503763A0 (en) | 1982-06-01 |
| DD202073A5 (en) | 1983-08-24 |
| IL63291A0 (en) | 1981-10-30 |
| PT73359A (en) | 1981-08-01 |
| ZA814741B (en) | 1982-07-28 |
| NO812397L (en) | 1982-01-15 |
| DE3169371D1 (en) | 1985-04-25 |
| DE3026665A1 (en) | 1982-02-11 |
| EP0044041A1 (en) | 1982-01-20 |
| FI812187L (en) | 1982-01-15 |
| AU7281281A (en) | 1982-01-21 |
| JPS5749861A (en) | 1982-03-24 |
| ES8205465A1 (en) | 1982-06-01 |
| ATE12316T1 (en) | 1985-04-15 |
| PT73359B (en) | 1983-01-13 |
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