AU620811B2 - An immunometric determination method - Google Patents

Description

COMMONWEALTH OF AUSTRALIA 6 2O Ivo 1 PATENTS ACT 1952-69 COMPLETE SPEC IFICATION,,

(ORIGINAL)

Class I t. Class Application Number: Lodged: "Complete Specif lcatmin Lodged: Accepted: Published: ~4 00 00 4 Priority 4 44 04 4 o 40 4 444400 belated Art 44 0 4 4 4 00 0 44 44 4 4~ Name of Applicant: Addr~qs of ApplicLuat :ctual Inventor: HOECHST AKTIENGESELLSCHAFT 45 Br'iningstrasse, D-6230 Frankfurt/Main 80, Federal Repuh'ilc of Germany GERD SCHNORR, HELMUT STRECKER, PETER MOLZ, GUIDO SIMONS, HEINZ JURGEN SKRZIPGZYK, HANS-PETER HARTHUS, GOTZ WALTER Address for Service EDWD. WATERS SONS, 50 QUEEN STREET, ME&,BOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled-, AN IMMUNOMETRIC DETERMINATION METHOD The following statement is a full description of this invention, including the best miethod of performing it known to *-US HOECHST AKTIENGESELLSCHAFT HOE 87/F 178 Dr. SW/sch Description An immunometric determination method The invention relates to an immunometric determination method in which, in order to avoid non-specific increases or decreases in the values for the antigen which is to be K 10 determined, an excess of a substance (suppressor substance) which, by reason of its great similarity to the reagent antibodies used and by reason of its great excess, selecit tively traps the substance which interferes with the antii r 4 bodies is added to the unlabeled and/or the labeled antii 15 body, and thus rules out an influence on the assay.

I t It is known that immunometric methods are being employed to an ever increasing extent for the qualitative and quantitative detection of antigens. These methods are based on 2 the formation of a complex of the antigen with one or more Santibodies, with one of the partners in the binding being I labeled in such a way that it can be qualitatively and/or quantitatively measured using physical or chemical detection methods. This makes it possible to establish whether, I o 25 and in what amount, a complex has formed from the antigen ;and one or more antibodies. Crucial improvements in the immunometric determination methods were achieved with thintroduction of monoclonal antibodies (Milstein and Kohler, 1975), the use of which in im'unometric assays is described in detail in German Offenlegungsschrift 3,130,834.

The immunometric determination methods can be divided into two man types depending on whether the antigen or the antibody is labeled. The labeled partner in the binding is always used in excess.

Farticular interest has been attracted to immunometric methods in which one of thk antibodies which is used is 2 Labeled. These entail the antigen being bound in the form of a ternary complex resembl'ng a sandwich and, after the incubation, the unbound Labeled antibody being removed by decantation or washiig out. rhese embodiments are called, depending on the nature of the Labeling, a two-sided immunoradiometric assay (IRMA), an immunoenzymomeLric assay (IEM') or an immunochemiluminometric assay (ICMA).

As a rule, these entail the unlabeLed antibody being bound to a solid phase.

Different variants of embodiments of the abovementioned sandwich assays are possible, these differing by the reaca tion steps resulting in the formation of the ternary compo 1 plex. Either the antigen can be mixed simultaneou-sly with 15 the labeled and the unlabeled antibody in a one-step method, or a sequential procedure is possible d the antigen is reacted initially with the unlabelea antibody and, after a sufficient incubation time, then also with the labeled antibody. Finally, it is also possible for S 20 these reaction steps to be carried out in the reverse sequence.

The sandwich assays based on the use of labeled antibodies have crucial analytical advantages compared with the assays carried out using labeled antigens: It is possible to employ the antibodies in excess and, owing to the increase in their concentration, to displace the equilibrium in the direction of formation of the ternary complex. It is possible in this way for even small amounts of antigens to be bound and coupled to the label.

Since, moreover, the signal emitted by the label is directly proportional to the antigen concentration, and the signal/dose plot has a steep gradient, it is possible even for weak signals caused by low antigen concentrations still to be clearly recognized. For these reasons, the sandwich assays which operate with labe!ed antibodies are considerably more sensitive than assays which are based on the use of Labeled antigens.

15 B sought out by suitable screening.

The method as claimed in claim 6, wherein the antigen-bindina sites of the reaaent antibodies are

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i 3 Finally, another point in favor of the sandwich assays which operate with Labeled antibodies is the considerably larger dynamic measurement range, i.e. there is a larger range in which the strength of the signal emitted by the ternary complex changes in a sensitive manner when there is also a change in concentration of the antigen. Another important point for the routine use of commercial immunometric determination methods is that they a'low the incubation times to be minimized,, This is ensured when it is possible to employ higher reagent concentrations than is the case when labeled antibodies are employed in sandwich assays.

Both types of assay t 'hose with labeled antibodies and those with labeled -itigens) may be subject to interference by serum constituents which intervene in the primary antigen/antibody reaction and/or separating reaction. The meaning of separating reaction in this connection is the separation of bound and free tracer. This may result, depending on the nature of the interference and the principle of the assay, in either falsely high or falsely low values. Separations based on non-specific precipitation reactions such as, for example, IgG precipitation by alcohols su:h as polyethylene glycol (PEG) have, of course, the greatest degree of robustness in respect of interfering immunological effects, but, on the other hand, as a consequence of the wide variation in their NSB (nonspecific binding) (misclassifination of free tracer) they frequently give results which are analytically/diagnostically unreliable. The double-antibody separating techniques, which are distinguished by low and precise NSB, are subject to interference by endogenous heterophilic antibodies which are directed against the primary (antigenbinding) antibodies in such a way that the separating antibody can no longer attack the antigen/antibody complex.

The consequence of this in competitive assays is that the antigen content in the patient's sample is overestimated Sain et al. 71, 540 (1979)).

-i 4 Interference in the primary antigen/antibody reaction i.

often observed when auto-antibodies against the anaLyte occur. In doubLe-antibody RIAs this effect gives a falsely high result, whereas in the PEG assays falsely Low values are obtained. Sample preparation by preliminary PEG prec: itation avoids this type of interference.

Very recently interference has also been observed in ehe sandwich assays, this being manifested by falsely high measurements on patients' sera due to an increase in the NSB. The cause is supposed to be antibodies against the reagents in the assays Thompson et al., Clin. Chem, 32, 476 (1986)) (Figs. 1 and A represernt tion of this is shown in Figure 1, using the way of representing antibodies customary in the Literature. An antibody bound to a solid phase is bound via a non-analyte-specific binding (NS6), for example by an interfering substance to the epitope of a Labeled antibody The diagram in Figure 2 represents how such non-specific binding (NSB) S 20 adds to the specific binding (SB) between antibody (1) So bound to a solid phase, antigen or analyte and labeled S antibody to simulate excessively high, often pathological analyte concentrations. Since the interfering substances are species-specific antibodies, they cannot be bound by just any IgG (as is the case with rheumatoid factors), but species-specific carrier gamma-globulin must be t l employed.

In the present state of the art, non-specific mouse or rat serum is added to assays in which monoclonaL anti-bodies from mice are used (EP-A 0,174,026 or RIA-gnost R hCG, Behring-Werke, Marburg, since 1.9.1984). However, despite this measure, it is repeatedly found that sera have falsely high values because of the high titer of anti-mouse IgG. If these samples are tested again individually with addition of a Larger amount of mouse or rat serum, gererally a lowering of the initial value occurs. The large amounts necessary for this purpose rule out employment as a prophylactic measure in the assay because an effect on

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5 the assay is to be expected.

It is possible according to the invention to eliminate the disadvantages described above by using as carrier (suppressor) substance a protein which has great immuno- Logical similarity to the IgGs used in the assay and, in the ideal case, differs from the latter only in the antigen binding structure and thus does not bind the analyte (Fig. A Large excess of these substances can be admixed to the Labeleo reagent antibody without intervening in the analyte reaction.

Another prerequisite for this is immunological inertness to all the other serum constituents. Non-compliance with this requirement might result in network formation in the serum sample and reduce the suppressive action of the substance. Moreover, it might not be possible to rule out an effect on the kinetics and position of equilibrium of the analyte r'eaction.

Hence the invention relates to an immunometric determination method for an antigenic substance which has at least two antibody-binding sites, in which a liquid sample containing the antigenic substance is incubated, sequen- 25 tially or in one step, with an unlabeled antibody (b) Swhich is immobilized on a solid phase and is specific for S and another, labeled antibody which is specific for there being formation of a ternary complex of and which is bound to a solid phase and emits a detectable signal corresponding to the amount of which comprises addition, in the first and/or second incubation step in the sequential method and to the reaction mixture in the one-step method, of protein which is immunologically related to the antibodies used and which is thus able to bind components which are present in the patient's serum and which would enter into undesired binding with the assay reagents, in order in this way to rule out an adverse effect on the assay.

i 6 a o oa a <C 'aV a at a I I a.

The proteins which are employed are preferably one or more different antibodies, in particular one or more from the same species (animal species) as that of the reagent antibodies used. Particularly preferred are m 'onoclonal antibodies, and very particularly preferred are antiidiotype monoclonal antibodies. The meaning of protein hereinbefore and hereinafter is a macromolecular organic compound which is constructed of amino acid units and has antigenic properties.

Anti-idiotype antibodies are defined as those directed against the idiotypic region of the antibody which has elicited their formation.

15 'he preparation of the suppressor substances according to the invention preferably starts from the reagent antibodies which are used, and entails modification of the latter in such a way that although they still retain their specificity against the interfering substance they no Linger react 20 or at least react distinctly less with the a'ltigen which is to be analyzed. The large excess of the suppressor substances which is expediently added to the imrunometric assay means that of the two competing reactions reagent antibody intertf ring substance and suppressor substance interfering substance the latter is given far greater preference, so that tie interfering substances are virtually quantitatively removed from the reaction equilibrium.

Examples of possible ways of generating these suppressor substances starting from the reagent antibodies are the following: a) Selective elimination of the antigen-binding structures from the reagent antibody used b) Blockade, by a complementary antigenic structure, of the antigen-binding structures of the reagent antibody used c) Alteration of the antigen-binding structure of the reagent antibody used, by external intervention, for 7 example by mutation in the cell culture d) Preparation of anti-idiotype antibodies (see US Patent 4,536,479) which belong to the same IgG subclass as the reagent antibodies.

Antibodies can be cleaved for variant a) by methods known from. the literature, for example using enzymes. It is possible in this connection to eliminate individual fragments from the light and the heavy chains. As is known, the antigen-binding structures are Located on the N-terminal end of the polypeptide chains so that the elimination S ,thereof means almost complete loss of the epitope-recognizing constituents of the antigen. It is likewise possible to separate the two Fab parts from the Fc part. If, for example, the epitopes which are recognized by the interfering substance are Located on the Fc part of the assay V antibody, then an ideal suppression medium would be the pure Fc part of this antibody since, although it reacts with the interfering substance, it does not react with the antigen (analyte) because of the absence of the Fab parts.

The blockade, by a complementary antigenic structure, of the antigen-binding structures of the reagent antibody used, as in can be effected in generaL by every substance which is directed against the idiotypic region of the reagent antibody and whose binding affinity is sufficiently high, for example by an anti-idiotype antibody.

The alteration by mutation in the cell culture as in c) can be carried out, for example, as follows: Somatic mutants of monoclonaL antibodies are isolated from cell cultures and then the effects of these mutations on antibody function are examined. Isolation of such mutants with an altered Ig structure is made possible by the instability of the Ig genes in cultivated hybridoma cells (Morrison et at., CRC Crit. Rev. Immunol, 3: 1 22, 1981).

3 Types of structural mutants of particular importance for hybridoma technology are known: class and subclass S- 8 switch variants (Cebra et at., Ann. Rev. ImmunoL., 2: 493 548, 1984; Sublitzky et at., Immunol. Rev., 67: 59 72, 1982; Shimizu et at., CeLL, 36: 801 803, 1984; Tilley et at. Proc. Natl. Acad. Sci., USA, 80: 6967 6971, 1983); deletion or point mutations in the constant region with changes in effector function (Yelton et al., J.

Exp. Med., 156: 1131 1148, '1982; Kenter et al., Science, 206: 1307 1309, 1979; Teillaud et at., J. Immunol, 1984 in press); and mutants with altered antigen-binding properties (Dildrop et aL., EMBO 1: 635 640, 1982; Cook et at., Proc. Natl. Acad. Sci. USA, 74: 5687 5691, 1977). Mutations which finally result in complete Loss of the ability to bind a specific antigen have a very high i spontaneous occurrence in some clones and derive from the replacement of individual amino acids in the heavy chain of the V region (Rudikoff et aL., Proc. NatL. Acad. Sci.

USA, 79: 197 1983, 1982).

Variant 3 is suitable and preferred for employment as suppression medium. To choose the most suitable mutants it is merely necessary to carry out a few experiments to elucidate and establish in each case whether 1. the antigen-binding properties are still present and 2. the interfering substance still recognizes, and thus binds, "its" epitopes on the mutant.

For d) it is possible to generate anti-idiotype antibodies by immunizat. n with a monoclonal antibody directed against a non-analyte antigen. These substances have epitope homology in respect of the constant regions of the reagent antibody and comply with the requirement of non-reaction with the other serum constituents.

The suppressor substances prepared as in a-d can be employed in immunometric determination methods with the aim of selectively trapping interfering substances (for example antibodies or the like). They can be employed in immunoassays for the determination of a very wide variety of antigenic substances, for example for human thyroid-stimulating hormone (hTSH) or for the oncofetal I~ U- 9 proteins such as aLpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) or carcinoembryonic antigen (CEA). Use is particularly advantageous in the case of anaLytes (antigens) which occur in Low concentrations or are virtually absent in healthy people and where even low concentrations indicate a pathological state. In these cases, even small increases in concentration brought about by interference would then indicate a pathological state.

The immunometric determination method according to the invention is reliable, technically straightforward, rapid and robust. It can be used in a large number of commercial sandwich kits. It is also possible for suppressor substances with the properties described above to be introduced as supplements to existent commercial assay kits.

For this purpose, it is expedient for them to be added to the conventional assays in concentrations which are at least 10, preferably 50, particularly preferably and not more than 1000, preferably 500, particularly t preferably 120, times those of the reagent antibodies.

The commercial assay kits are preferably those in which the polyclonal, but preferably monoclonal, antibody which is bound to the solid phase is unlabeled. This antibody is preferably adsorbed onto or covalently bonded to the solid phase, with the solid phase preferably being composed of a plastic tube, a microtiter plate, plastic articles such as plastic beads or plastic propellers, or else of microscopically small plastic beads which, for example, are suspended in a Liquid. The Labeled antibodies are likewise polyclonal, but preferably monoclonal, antibodies. The Labeling is effected by methods known from the Literature, using a radioactive isotope, an enzyme or a fluorescent or chemiluminescent group.

10 The invention is explained in more detail by the examples which follow.

Example 1 Preparation of s .,atic mutants of monoclonal antibodies Hybridoma cell clones which produce antibodies of known specificity were recloned in microtiter plates using a single-cell manipulator or the Limiting dilution method. The culture supernatants from the wells with positive cell growth were then tested first for their Ig content and then for their antigen-binding properties.

When the cloning rate was sufficiently high the cell clones obtained were distinguished by a high production of Ig molecules with, at the same time, antigen-binding insufficiency.

Example 2 Preparation of monoclonal anti-idiotype antibodies A monoclonal antibody was used as immunization antigen for the preparation f monoclonal anti-idiotype antibodies. The monoclonal antibody used in this case was one directed against carcinoembryonic antigen (CEA' and of syngeneic origin, and was administered as the whole antibody to female BALB/c mice 6 8 weeks old. Th 4 entailed about 10 pg per mouse of the whole antibody, emulsified in complete Freund's adjuvant, being injected subcutaneously and, in a second case, intraperitoneally. A second and a third immunization followed 4 and 8 weeks, respectively, later. Immediately before the actual fusion, the experimental animals were additionally intravenously boosted on 4 consecutive days. On the day of fusion, the spleens were removed under sterile conditions and converted into suspensions of single cells. Hybrid cells were produced by fusion of 108 spleen cells with 2 x 107 cells of a myeloma cell line (SP 2/0) and were then inoculated on 24-well plates (Costar) in a concentration of 106 .IlI,/well in a llc~-u~.xuraPin=u;rr;*-Pmr;sra;i~9La^' 11 selection medium (DMEM (Dulbecco's minimal essential medium) 20% FCS (fetal calf serum); 0.1 mM hypoxanthine; 0.4 mM aminopterin; 16 mM thymidine). 2 3 Weeks later individual cell colonies were isolated from the wells and each was transferred into another well of new culture plates (24-well, Costar). After a further 2 3 days, these culture supernatants were examined in an enzyme immunoassay, using the immunization antibody conjugated with peroxidase (POD), for the presence of anti-idiotype antibodies, and thes in turn were examined in a further enzyme immunoassay for their antigen inhibitability. Hybrids producing antigeninhibitable anti-idiotype antibodies were selected and cloned using a single-cell manipulator.

S Example 3 In analogy to Example 2, anti-idiotype antibodies were prepared using for the immunization in place of the whole antibody the Fab' fragment of the anti-CEA antibody, coupled to BSA.

Example 4 In analogy to Example 2, anti-idiotype antibodies were I prepared using for the immunization in place of the whole antibody the Fab' fragment of the anti-CEA antibody, coupled to KLH.

The following comparison experiments were carried out to detect the suppressor properties of the substances according to the invention: Example The binding affinity of interfering substances (Fig. 4: goat anti-mouse IgG; Fig. 5: rabbit anti-mouse IgG) for the reagent antibodies used expressed in binding

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(100% binding 100% of reagent antibodies bound to the interfering substance) was determined with a constant amount of auppression medium as a function of the amount of interfering substance added. The RIA-gnost(R) hTSH ;i 12 assay kit (Behringwerke, Marburg) was used. The foLLowing comparison suppression media were used: Curve No. (in Figs. 4 and Multiple rat serum 1 4300 4300 rat IgG 2 100 Ann substance according to 3 100 1-r the invention (from Example 2) based on the specific tracer IgG used Two other curves are drawn in Figures 4 and 5 for comparison, one (curve No. 4) describing the RIA-gnost hTSH assay kit currently on the market (contains 4400 times the amount of a non-specific suppressor IgG relative to the tracer IgG used) and the other describing a RIAgnost assay kit which contains no suppression medium whatever (curve No. 5, multiple 0; stancard).

The binding (in to the reagent antibody increases with increasing concentration of interfering substance I (the following were used: goat anti-mouse IgG (Fig. 4) and rabbit anti-mouse IgG (Fig. Analyte is simuleted. The increase in non-specific binding is least when the substances according to the invention from Example 2 are used (curve No. 3 in Figs. 4 and Example 6 The percentage binding of the reagent antibodies to an interfering substance whose concentration (titer) was constant was determined as a function of varying amounts of suppression medium. The following interfering substances were used: 1 13 Goat anti-mouse IgG Rabbit anti-mouse IgG Patient's serum A Patient's serum B Titer 1.5 pg/mL (Fig. 6) 1.5 pg/mL (Fig. 7) (Fig. 8) (Fig 9) I The following comparison suppression substances were Sused: Curve No. in Figs. 6 to 9 rat IgG 6 rat serum 7 mouse IgG 8 substance according 9 to the invention v 15 (from Example 2)

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Claims (16)

1. An immunometric determination method for an antigenic substance having at least two antibody binding sites, comprising the steps of: a) Immunobilizing an unlabeled antibody specific to said antigenic substance to a solid-phase support; b) Incubating a liquid sample containing an interfering substance and said antigenic substance with said immunobilized antibody; c) Further incubating, in the same step as above or sequentically, said liquid sample 1) with a suppressor protein which has an immunological inertness to all other serum constituents, a great immunological similarity to the antibodies used in the assay and differs from the latter only 'n the antigen binding icture and thus does not bind the antigenic substance and 2) with a labeled reagent antibody specific for said antigenic S, substance, said suppressor protein being added in at least a ten-fold excess in relation to 1" the labeled reagent antibody there being forimed a ternary complex which emits a detectable signal corresponding to the amount of antigenic substance in said liquid sample. it

2. The method as claimed in claim 1, wherein the suppressor protein takes the form of one or more different antibodies.

3. The mothod as claimed in claim 2, wherein the suppressor antibody or antibodies is or are from the same species as the reagent antibodies.

4. The method as claimed in claim 3, wherein the suppressor protein antibodies are in the form of one monoclonal or several monoclonal antibodies. The method as claimed in claim 4, wherein the si:ppressor protein monoclonal antibody or antibodies is or are monoclonal anti-idiotype antibodies.

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6. The method as clairr,- in claim 3, wherein the suppressor protein monoclonal antibody or antibodies correspond(s) to the reagent antibodies apart from the antigen-binding site.

7. The method as claimed in claim 6, wherein the antigen-binding sites of the reagent antibodies are blocked immunologically.

8. The method as claimed in claim 7, wherein a suppressor protein monoclonal anti-idiotype antibody is used for blockade.

9. The method as claimed in claim 6, wherein the antigen-binding sites of the reagent antibodies are lost tldu to mutation in cell cultures, and these antibodies are sought out by suitable screening.

The method as claimed in cLaim 6, wherein the anti- gen-binding sites of the reagent antibodies are chemically altered or are selectively eliminated.

11. The method as claimed in claim 2, wherein the added antibody or antibodies are used in at least a ten- fold excess compared with the reagent antibodies.

12. TL? method as claimed in claim 1, wherein the un- labeled antibody immobilized on the solid phase is a monoclonal or polyclonal antibody.

13. The method as claimed in claim 12, wherein the un- labeled antibody is adsorbed onto or covalently bonded to the solid phase.

14. The method as claimed in claim 1, wherein the solid phase is composed of a plastic tube, microtiter plates, plastic beads or propellers or of micro- scopically small plastic beads which are suspended in a Liquid.

The method as claimed in claim 1, wherein the label- ed antibody is a monoclonal or polyclonal anti- t body, and it carries a radioactive isotope, an enzyme or a fluorescent or chemiluminescent group as indicator.

16. The method as claimed in claim 1, wherein the antigenic substance determined is human thyroid- i stimulating hormoine (hTSH) or the oncofetal proteins alpha-fetoprotein, hchorionic gonadotropin or carcinoembryonic antigen. DATED this 23rd day of June 1988. HOECHST AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3000.