CA1304683C

CA1304683C - Immunometric determination method

Info

Publication number: CA1304683C
Application number: CA000570360A
Authority: CA
Inventors: Gerd Schnorr; Helmut Strecker; Peter Molz; Guido Simons; Heinz Jurgen Skrzipczyk; Hans-Peter Harthus; Gotz Walter
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1987-06-24
Filing date: 1988-06-24
Publication date: 1992-07-07
Anticipated expiration: 2009-07-07
Also published as: AU620811B2; EP0296544A2; AU1836688A; PT87810A; NO882806L; EP0296544A3; DK349288D0; NO882806D0; PT87810B; FI883057A; JPS6428560A; FI883057A0; DK349288A; DE3720983A1

Abstract

Abstract of the Disclosure 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 determined, an excess of a substance (suppressor sub-stance) which, by reason of its great similarity to the reagent antibodies used and by reason of its great excess, selectively traps the substance which interferes with the antibodies is added to the unlabeled and/or the labeled antibody, and thus rules out an influence on the assay.

Description

HOECHST AKTIENGESELLSC~AFT HO~ 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 determined, an excess of a substance (suppressor substance~
~hich, by reason of its great similarity to the reagent antibodies used ancl by reason of its great excess, selec-tively traps the substance which interferes with the anti-bodies is added to the unlabeled and/or the labeled anti-body, and thus rules out an ;nfluence on the assay.

It is known that immunometric methods are be;ng employedto an ever increasing extent for the qualitative and quan-titative detection of antigens. These methods are based on the formation of a complex of the antigen with one or more antibodies, with one of the partners in the binding being labeled in such a way that it can be qualitatively and/or quantitatively measured using physical or chemical detec-tion methods~ Th;s makes it possible to establish whether, 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 the introduction of monoclonal antibodies (Milstein and Kghler, 1975), the use of which in ;mmunometric assays is described in detail in German Offenlegungsschrift 3,130,834.

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

Particular interest has been attracted to ;mmunometric methods in which one of the antibodies which is used i ~3~83 labeled. These entail the antigen being bound in the form of a ternary complex resembling a sandwich and, after the incubation, the unbound labeled antibody be;ng removed by decantation or washing out. These embodiments are called, depending on the nature of the labeling, 3 two-s;ded ;mmunoradiometric assay (IRMA), an immunoenzymonletric assay (IEMA) or an immunochemiluminometric assay (ICMA).
As a rule, these entail the unlabeled antibody being bound to a solid phase.
Different var;ants of embodiments of the abovementioned sandwich assays are possible, these differing by the reac-tion steps resulting in the formation of the ternary com-plex. Either the antigen can be mixed s;multaneously with the labeled and the unlabeled antibody in a one-step method, or a sequential procedure is possible and the antigen is reacted initially with the unlabeled antibody and, after a sufficient incubation time, then also with the labeled antibody. Finally, it is also possible for 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 direct;on 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 direc-tly proportional to the antigen concentration, and the signal/dose plot has a steep gradient, it is possible even for the weak signals caused by low antigen concentrations still to be clearly recognized. For these reasons, the sandwich assays which operate with labeled antibodies are considerably more sensitive than assays which are based on the use of labeled antigens.

:~3~ 3 FinaLly, another point in favor of the sandw;ch assays which operate with labeled antibod;es is the considerably larger dynamic measurement range, i.e. there is a larger range in which the strength of the signal em;tted by the ternary complex changes in a sens;tive manner when there is also a change ;n concentrat;on of the ant;gen. Another important point for the routine use of commercial ;mmuno-metric determination methods is that they allow the incu-bation times to be m;nimized. This is ensured when ;t ;s possible to employ h;gher reagent concentrations than is the case when labeled ant;bodies are employed in sandwich assays.

aoth types of assay (those with labeled antibodies and those with labeled antigens) may be subject to interfer-ence by serum constituents wh;ch ;ntervene in the primary antigen/ant;body react;on and/or separat;ng react;on. The meaning of separating reaction in this connection ;s the separat;on of bound and free tracer. Th;s may result, depending on the nature of the ;nterference and the prin-ciple of the assay, in either falsely high or falsely low values. Separations based on non-specific precipitation reactions such as, for example, IgG prec;pitation by alcohols such as polyethylene glycol (PEG) have, of course, the greatest degree of robustness in respect of interfer-ing immunological effects, but, on the other hand, as a consequence of the ~ide variation in the;r NSB (non-specific binding) (misclass;f;cation of free tracer) they frequently give results which are analyticallytdiagnosti-cally unreliable. The double-ant;body separating tech-niques, which are distingu;shed by low and precise NSB, are subject to interference by endogenous heteroph;lic antibodies which are directed against the primary (antigen-binding) antibodies in such a way that the separating antibody can no longer attack the ant;gen/antibody complex.
The consequence of this in competitive assays is that the antigen content ;n the pat;ent's sample is overestimated (A. Sain et al. 71, 540 (1979)).

~3~

Interference in the primary antigen/antibody reaction is often observed when auto-antibodies against the anaLyte occur. In doubLe-antibody RlAs this effect gives a faLseLy high result~ ~hereas in the PEG assays falsely low vaLues are obta;ned. Sample preparation by preliminary PEG precip-;tat;on avo;ds th;s type of interference.

Very recentLy interference has also been observed in the sandw;ch assays, this being manifested by falsely h;gh measurements on pat;ents' sera due to an ;ncrease ;n the NSB. The cause is supposed to be ant;bod;es aga;nst the reagents in the assays (R.J. Thompson et al., Clin. Chem, _ , 476 (1986)) (Figs. 1 and 2). A representation of this is shown in Figure 1, using the way of representing anti-bodies customary in the literature. An antibody (1) boundto a solid phase is bound via a non-analyte-specific bind-ing (NSB), for example by an ;nterfer;ng substance (3), to the epitope (5) of a labeled antibody (2). The diagram in Figure 2 represents how such non-specific b;nd;ng (NSB) adds to the spec;fic binding (SB) between ant;body (1) bound to a solid phase, antigen or analyte (4) and labeled antibody (2) to s;mulate excess;vely high, often patholog;-cal analyte concentrations. Since the interfering substan-ces are spec;es-spec;fic ant;bodies, they cannot be bound by just any IgG (as is the case with rheumatoid factors), but species-specific carrier gamma-globulin must be 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-gnostR hCG, Behring-Werke, Marburg, since 1.9.1984). However, despite this measure, it is repeatedly founcl that sera have false-ly high values because of the high titer of antl-mouse lgG. If these samples are tested again ind;vidually with addit;on of a larger amount of mouse or rat serum, gener-ally a lowering of the ;nitial value occurs. The large amounts necessary for this purpose rule out employment as a prophylactic measure in the assay because an effect on ~3 1)~

the assay is to be expected.

It is possible according to the invention to eliminate the disadvan~ages described above by using as carrier (sup-pressor) substance (6) a protein which has great ;mmuno-logical similarity to the IgGs used in the assay and, in the ideal case, di~fers from the latter only in the anti-gen binding structure (7) and thus does not b;nd the analyte (Fig. 3). A large excess of these substances can be admixed to the labeled reagent antibody without inter-vening 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 sub-stance. Moreover, it might not be possible to rule out an effect on the kinetics and position of equilibrium of the analyte reaction.
~ence the invention reLates to an immunometric determina-tion method for an antigenic substance which has at least two antibody-binding sites, in which a liquid sample con-taining the antigenic substance (a) is incubated, sequen-t;ally or in one step, with an unlabeled antibody (b)which is immobilized on a solid phase and is specific for (a)~ and another, labeled antibody (c) which is specific for (a), there being formation of a ternary complex of (a), (b) and (c) which is bound to a solid phase and emits a detectable signal corresponding to the amount of (a), which comprises addition, in the first and/or second incu-bation step in the sequential method and to the reaction mixture in the one-step method, of protein which is immu-nologically 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 bind-ing w;th the assay reagents, in order in this way to rule out an adverse effect on the assay.

- SA _ The invention will now be described in relation ko the drawings, in which:

Figure 1 is a schematic diagram showing binding of antibody via non-analytic specific binding (NSB) to a labeled antibody;
Figure 2 is a schematic diagram showing how NSB adds to the specific binding (SB) between antibody bound to a solid phase, antigen or analyte and labeled antibody;
Figure 3 is a schematic diagram showing the use of a carrier (suppressor) substance to block ~SB in accordance with the present invention;
Figure 4 is a graph showing the binding affinity of an interfering substance (goat anti-mouse IgG) for the reagent antibodies;
lS Figure 5 is a graph showing the binding affinity of an interfering substance (rabbit anti-mouse IgG) for the reagent antibodies;
Figure 6 is a graph showing percentage binding of reagent antibodies to an interfering substance (goat anti-mouse IgG) as a function of varying amounts of suppression medium ~rat IgG);
Figure 7 is a graph showing percentage binding of the reagent antibodies to an interfering substance (rabbit anti-mouse IgG) as a function of varying amounts of suppression medium (rat serum);
Figure 8 is a graph showing percentage binding of the reagent antibodies to an interfering substance (patient's Serum A) as a function of varying amounts of suppression medium (mouse IgG); and Figure 9 is a graph showing percentage binding of the reagent antibodies to an interfering substance (patient's Sexum B) as a function of varying amounts of suppression medium ~monoclonal anti-idiotype antibodies to monoclonal antibody against carcinoembryonic antigen).

, .

~l3~4~i83 The proteins which are employed are pre-ferably one or more different antibodies, in particular one or more from the same species (animal species) as that of the reagent anti-bodies used~ Particularly pre~erred are monoclonal anti-bodies, and very particularly preferred are ant;idiotypemonoclonal antibodies. The meaning of protein hereinbefore and here;nafter is a macromolecular organic compound which is constructed of amino acid units and has antigenic prop-erties.
1û
Anti-idiotype antibodies are defined as those directed against the idiotypic region of the antibody which has elicited their formation.

The 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 ~ay that although they still retain their specifi-city agains~ the interfering substance they no longer react ZO - or at least react d;stinctly less - with the antigen which is to be analyzed. The large excess of the suppressor substances which is expediently added to the immunometric assay means that of the two competing reactions - reagent antibody + ;nterfering substance and suppressor substance ;nterfering substance - the latter is given far greater preference, so that the interfering substances are virtu-ally 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 ~l3~i83 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.
s Antibodies can be cleaved for variant a) by methods known from the literature, for example using enzymes. It is pos-sible 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 thereof means almost complete loss of the epitope-recogniz-ing 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 interfer-ing substance are located on the Fc part of the assay antibody, then an ideal suppression medium would be the pure Fc part of this antibody since, although it reacts ~ith 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 ant;gen-binding structures of the reagent antibody used, as in b), can be effected in general by every sub-stance ~hich is directed against the idiotypic region ofthe reagent antibody and whose binding affinity is suf-ficiently 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. lsolation of such mutants ~;th an altered Ig structure is made possible by the instability of the Ig genes in cultivated hybridoma cells (Morrison et al., CRC Crit. Rev. Immunol~ 3: 1 - 2Z, 1981).
3 Types of structural mutants of particular importance for hybridoma technology are known: (1) class and subclass switch variants (Cebra et al., Ann~ Rev. Immunol~, 2: 493 - 548, 1984; Sublitzky et al., Immunol. Rev., 67: 59 - 72, 1982; Shimizu et al., Cell~ 36: 801 - 803, 1984; Tilley et ai. Proc. Natl. Acad. Sci., USA, 80: 6967 - 6971, 1983);

(2) deletion or point mutations in the constant reg-ion with changes in effector function (Yelton et al.~ J.
Exp. Med., 156: 1131 - 1148, 1982; Kenter et al., Science, 206: 1307 - 130~, 1979; Teillaud et al., J. Immunol, 1984 in press); and ~3) mutants with altered antigen-binding propert;es (Dildrop et al., EM~0 J., 1: 635 - 640, 1982;
Cook et al., Proc. Natl. Acad. Sci. USA, 74: 5687 - 5691, 1~77). Mutations which finalLy result in complete loss of the ability to bind a specific antigen have a very high spontaneous occurrence in some clones and derive from the replacement of individual amino acids ;n 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 su;table mutants it is merely necessary to carry out a fe~ e%periments to elucidate and establish ;n each case ~hether 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 ant;-bodies by immunization ~ith a monoclonal antibody direc-ted against a non-analyte antigen. These substances have epitope homology in respect of the constant regions of the reaoent antibody and comply ~ith the requirement of non-reaction ~ith the other serum constituents.

The suppressor substances prepared as in a-d can be employed in immunometric determination methods ~ith the aim of selectively trapping interfering substances (for example antibodies or the like). They can be employed ;n immunoassays for the determination of a very wide variety of antigenic substances, for example for human thyroid-stimulating hormone (hTSH) or for the oncofetal ~3~ 3 proteins such as alpha-fetoprotein (AFP), human chori-onic gonadotropin (hCG) or carcinoembryonic antiyen (CEA). Use is particuLarly advantageous in the case of analytes (antigens) which occur in low concentrations or S are virtually absent ;n healthy people and ~here even low concentrat;ons ind;cate a pathological state. In these cases, even smaLl increases ;n concentration brought about by ;nterference would then ;nd;cate a patholog;cal state.
The immunometric determ;nat;on method according to the invention is rel;able, techn;cally stra;ghtforward, rapid and robust. It can be used in a large number of commercial sandwich kits. It is also possible for sup-pressor substances with the properties described aboveto be introduced as supplements to existent commercial assay kits.

For this purpose, it ;s exped;ent for them to be added to the convent;onal assays ;n concentrations which are at least 10, preferably 50, particularly preferably 80, and not more than 1000, preferably S00, particularly preferably 120, times those of the reagent antibodies.

The commercial assay kits are preferably those in wh;ch the polyclonal, but preferably monoclonal, antibody which is bound to the solid phase is unlabeled. This antibody is preferably adsorbed onto or covalently bond-ed to the soLid phase, with the solid phase preferably be;ng composed of a plastic tube, a microtiter plate, plastic articles such as plastic beads or plastic pro-pellers~ or else of microscopically small plastic beads which, for example, are suspended in a liquid. The labeled antibodies are likewise polyclonal, but prefer-ably monoclonal, antibodies. The labeling is effected bymethods known from the literature, us;ng a radioactive isotope, an enzyme or a fluorescent or chemilum;nescent group.

~ 3 ~

~he invention is explained in more detail by the examples which follou.

Example 1 Preparation of somatic mutants of monoclonal antibodies Hybridoma ~ell clones ~hich produce antibodies of kno~n specificiey 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 ~ere then tested first for their Ig content and then for their antigen-binding properties.
~hen 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 ;nsufficiencyO

xample 2 Preparat;on of monoclonaL anti-idiotype antibodies A monoclonal antibody ~as used as immunization antigen for the preparation of monoclonal anti-idiotype anti-bodies. The monoclonal antibody used in this case was one directed against carcinoembryonic antigen (CEA) and of syngeneic origin, and ~as administered as the whole antibody to female 8ALP/c mice 6 - 8 weeks old. This entailed about 10 ~9 per mouse of the whole antibody, emuLsified in complete Freund s adjuvan~, being injected subcutaneously and, in a second case, intraperito-neally. A second and a third immunization followed 4 and8 weeks~ respectively, later. Immediately before the actual fus;on, the experimental animals were addition-ally 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 1~7 cells of a myeloma cell line (SP 2/0) and were then inoculated on 24-well plates (Costar~ in a concentration of 106 cellstwell in a .. r ~ * Denotes trade-mark Ç;83 select;on medium (DMEM (Dulbecco's minimal essential nedium) + 20% FCS (fetal calf serum); 0.1 mM hypo-xanth;ne; 0.4 mM am;nopterin; 16 mM thym;dine). 2 - 3 ~eeks later ;ndiv;dual cell colonies were isolated from the wells and each was transferred into another weLl of ne~ culture plates t24-well, Costar). After a further 2 - 3 days~ these culture supernatants were examined in an en~yme immunoassay, using the immun;zation anti-body conjugated with peroxidase (POD), for the presence of anti-idiotype antibodies, and these in turn were exam;ned in a further enzyme immunoassay for their antigen inhibitability. Hybrids producing antigen-inhib;table anti-idiotype antibodies ~ere selected and cLoned using a single-cell manipulator~
Example 3 In analogy to Example 2, anti-idiotype antibodies were prepared using for the immunization in place of the uhole antibody the Fab' fragment of the ant;-CEA ant;-body, coupled to 8SA.

Example 4In analogy to Example 2, ant;-id;otype antibodies were prepared using for the immun;zat;on ;n place of the ~hole antibody the Fab' fragment of the anti-CEA anti-body, coupled to KLH.

The following comparison experiments were carried out to detect the suppressor properties of the substances accord;ng to the ;nvention:

Example 5 The b;nd;ng aff;nity of interfer;ng substances (Fig. 4:
goat anti-mouse IgG; F;g~ 5: rabb;t ant;-mouse IgG) for the reagent ant;bod;es used - expressed in % b;nd;ng (1û0% b;nding - 100% of reagent antibodies bound to the interfering substance) - was determined with a constant amount of suppress;on med;um as a funct;on of the amount of interfer;ng substance added~ The RIA-gnost~R) hTSH

~3~ 3 assay kit (~ehringwerke, Marburg) was used. The folLow-ing compar;sor, suppression media were used:

Curve No. (in Figs. 4 and 5) Mult;ple *
rat serum 1 43004300 rat IgG 2 100 100 subs~ance according to 3 100 100 the invention (from 1û Example 2) * based on the specific tracer IgG used Two other curves are drawn in Figures 4 and 5 for com-parison, 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 RIA-gnost assay kit which contains no suppress;on medium whatever (curve No. 5, multiple = 0; standard).
The binding (in %) to the reagent antibody increases with increasing concentration of interfering substance (the following were used: goat anti-mouse IgG (Fig. 4) and rabbit anti-mouse IgG (Fig. 5)). Analyte is simula-ted. 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 5).

xample 6 The percentage binding of the reagent antibodies to an ;nterfer;ng substance whose concentration (titer) was constant was determined as a function of varying amounts of suppression medium. The following ;nterfering sub-stances were used:

~30~

Titer __ Goat anti-mouse IgG 1.5 ~g/ml (Fig. 6) Rabbi~ anti-mouse IgG 1.5 ~g/ml (Fig. 7) Patient's serum A (Fig. 8) Patient's serum a (Fig. 9) The fol~owing comparison suppression substances were used:
Curve No. in Figs. 6 to 9 10 rat IgG 6 rat serum 7 mouse IgG 8 substance according 9 to the invention 15 (from Example 2)

Claims

1. An immunometric determination method for an antigenic substance which has at least two antibody-binding sites, in which a liquid sample containing the antigenic sub-stance (a) is incubated, sequentially or in one step, with an unlabeled antibody (b) which is immobilized on a solid phase and is specific for (a), and another, labeled antibody (c) which is specific for (a), there being formation of a ternary complex of (a), (b) and (c) which is bound to a solid phase and emits a det-ectable signal corresponding to the amount of (a), which comprises addition, in the first and/or second incubation step in the sequential method and to the re-action mixture in the one-step method, of protein which is immunologically related to the antibodies used.

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

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

4. The method as claimed in claim 3, wherein the antibodies are in the form of one monoclonal or several monoclonal antibodies.

5. The method as claimed in claim 4, wherein the monoclonal antibody or antibodies is or are monoclonal anti-idio-type antibodies.

6. The method as claimed in claim 3, wherein the mono-clonal antibody or antibodies correspond(s) to the re-agent 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 mono-clonal 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 due to mutation in cell cultures, and these antibodies are sought out by suitable screening.

10. 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. The method as claimed in claim 1, wherein the un-labeled antibody (b) immobilized on the solid phase is a monoclonal or polyclonal antibody.

13. The method as claimed in claim 12, wherein the un-labeled antibody (b) 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, microtter plates, plastic beads or propellers or of micro-scopically small plastic beads which are suspended in a liquid.

15. The method as claimed in claim 1, wherein the label-ed antibody (c) is a monoclonal or polyclonal anti-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-stimulating hormone (hTSH) or the oncofetal proteins alpha-fetoprotein, hchorionic gonadotropin or carcinoembryonic antigen.