AU679921B2

AU679921B2 - Immunochemical method for detecting an analyte

Info

Publication number: AU679921B2
Application number: AU44628/93A
Authority: AU
Inventors: Stefan Brust; Udo Krupka; Michael Noah; Hans-Erwin Pauly
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1992-08-17
Filing date: 1993-08-16
Publication date: 1997-07-17
Anticipated expiration: 2013-08-16
Also published as: EP0583689A1; DE4227102C2; ES2161221T3; DE4227102A1; JPH06160386A; CA2104176A1; JP3398189B2; EP0583689B1; ATE203328T1; DE59310190D1; AU4462893A

Abstract

The invention relates to an immunochemical method for the detection of an analyte in a sample of a biological material which is in liquid form, in which the reaction of the analyte with a specific binding partner is detected by another specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as label.

Description

R/UtIUaln 2(2I) Regulatilon 3.2(2)

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: o *o 0 oooe Invention Title: IMMUNOCHEMICAL METHOD FOR DETECTING AN ANALYTE

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a oe ft oeo The following statement is a full description of this invention, including the best method of performing it known to :-US Ib I- L MEMOl BEHRINGWERKE AKTIENGESELLSCHAFT 92/B 018-Ma 945 Auslandstext Immunochemical method for detecting an analyte The invention relates to an immunochemical method for detecting an analyte in a sample of a biological material, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label.

Immunological methods have attained great importance, in particular for clinical diagnosis, because of their extraordinary sensitivity and specificity. In these methods, as a rule, one of the immunological binding partners is labeled, i.e. is bound to a signal donor.

Common labels are, for example, radioactive isotopes, 15 fluorescent, phosphorescent or luminescent substances, substances having stable unpaired electrons, erythrocytes, latex particles, metal sols and enzymes.

Within the group of enzymes used for the labeling, the peroxidase obtained from horseradish (donor: H202 oxido- 20 reductase, EC 1.11.1.7) is particularly suitable because it is readily detectable and highly stable. Reagents, such as, for example, bifunctional N-hydroxysuccinimide esters, and processes, such as, for example, periodate activation of the protein-bound carbohydrates, for 25 coupling peroxidase to the immunologically active reaction partners are known to the person skilled in the art. These coupling products are designated conjugates, which are distinguished, in particular, by the fact that both the enzymatic properties of the peroxidase and the receptor properties antigen or antibody) are preserved to the greatest possible extent.

It has emerged that correc'. results are obtained for the majority of samples which are investigated by means of such an immunoassay using peroxidase conjugates. However, ~Y 2 in a certain number of samples both falsely positive and falsely negative findings do occur. These sera are designated below as interfering factor sera.

These matrix-based interferences with the enzyme immunoassay can be caused by a wide variety of factors. Effects caused by rheumatoid factors or heterophilic antibodies, in this case human anti-mouse antibodies in particular, which are contained in the interfering factor sera, are known to the person skilled in the art. These effects do not, as a rule, depend on the signal donor used.

Some examples of interferences which are caused directly by the enzyme label are described in the literature and in patents. Kohno et al. Clin. Lab. Anal., 5, pp 197- 205 (1991)) describe interferences by anti-p-D-galactosidase antibodies in an enzyme immunoassay which contains p-D-galactosidase as the label enzyme. This interference is eliminated by the addition of recombinant enzymically inactive, P-D-galactosidase.

EP 0 209 155 discloses a further possibility: false 20 results which are caused by interactions of serum components with carbohydrate residues of label enzymes, in particular peroxidase, can be eliminated by oxidative removal of these sugars. In this method, the reduction .in the enzyme reactivity by about 50 and the aggregation of the oxidized enzyme or conjugate, are disadvantageous for the function of the enzyme immunoassay. In addition, interferences which have their origin in the protein moiety of the peroxidase are not avoided by this method.

The invention therefore relates to an immunochemical method for detecting an analyte in a sample of a biological material, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding 1 -C1~-r I 9-97 11:44 AM ;WATERMARK 8138198010;# 6/13 2a partner to which horseradish peroxidase is coupled (conjugate) directly Or indirectly as the label, wherein the reaction mixture additionally tcontains paroxidase inactivated by hydroxymethylhydroperoxidase hereinafter referred to rIn this description as "inactive peroxidase".

3 partner tn owhi ch horserar-dish---perox-idas- is coupled- (conJjugate) directly or indi-reet-Ly-as-the label, wheretir the reaction miture additionally contains "inactive- -perexidae".

In an advantageous method the "inactive peroxidase" is added to the test mixture before the labeling peroxidase.

In a further advantageous method the "inactive peroxidase" is added at the same time as the labeling peroxidase.

In a particularly advantageous method the "inactive peroxidase" is removed again before the labeling peroxidase is added.

Advantageously, the "inactive peroxidase" is added in such a quantity that it is present in the reaction 15 mixture at a concentration of 0.002 to 10 mq/ml.

Preferably, the "inactive peroxidase" is present in a concentration range of 0.02 to 5 mg/ml, particularly preferably in a concentration range of 0.2 to 2 mg/ml.

The preferred detection method is a solid phase 20 immunoassay; a microtitration plate is very particularly preferred as the solid phase in this context.

The invention furthermore relates to the use of "inactive peroxidase" in an immunochemical detection method.

For the purposes of this invention detection denotes both qualitative detection and quantitative determination.

The invention also relates to a reagent containing "inactive peroxidase" inter alia.

I~II Irrr 4 The invention is therefore based on an immunochemical process for developing reagents which avoid the interactions between serum interfering factors and the marker enzyme peroxidase without having to tolerate the abov mentioned disadvantages.

It has been found, surprisingly, that, by means of a suitable treatment, enzymically inactive peroxidase can be prepared, after whose addition to the conjugate, interfering factor examples, which exhibit interferences with peroxidase conjugates, produce correct results.

It is advantageous that the agent according to the invention is readily available, i.e. horseradish peroxidase, which is economical and available in large quantities, can be used as the starting material and "inactive 15 peroxidase" can be prepared from it, for example by treatment with hydroxymethyl hydroperoxide (Marklund, Arch. Biochem. Biophys. 154, p. 614 622 (1973)). It is advantageous that in this way an inactive protein is obtained which is very similar to the native protein with regard to its antigenic character (tertiary structure, quaternary structure and glycosylation).

Without thereby limiting the invention to a particular mode of action, a possible explanation for the efficacy of the "inactive peroxidase" is that it captures potential interfering factors which are consequently not able to bind to the peroxidase.

The addition of receptors, which are directed against sugar residues or epitopes of the peroxidase, has a comparable effect, namely that non-specific reactions with interfering factor samples are prevented. In this context, a monovalent antibody or a lectin, which reacts with the peroxidase, can, for example, serve as the receptor, without the reaction with the receptors leading to any precipitation or agglutination.

-e I ,-I 5 Preferred antibodies or lectins are those which do not impair the enzymic activity of the peroxidase.

The reagents according to the invention are distinguished, in particular, by the fact that they neither influence the enzymic activity of the peroxidase nor impair the immunological behavior of the conjugate by aggregation.

The reagents according to the invention may be employed in human and veterinary diagnostics as a buffer additive in a multiplicity of immunoassays using peroxidase conjugates as an essential test component. Examples which may be cited are one-step or multi-step tests for the detection of antibodies of various immunoglobulin classes against structural features of viruses viruses of 15 hepatitis A, B and C, as well as various HIV types), of bacterial and parasitic pathogens, and of allergic diseases. Further examples are the detection in one-step or multi-step detection methods of disease-causing agents such as viruses hepatitis B virus), bacteria, parasites or allergens, as well as of disease markers tumor markers).

The following examples illustrate the subject matter of the invention without limiting it.

Example 1: I 25 a) Preparation of enzymically inactive peroxidase The preparation of the peroxidase, whose enzymic activity was irreversibly inactivated, took place by treating commercial peroxidase with hydroxymethyl hydroperoxide according to the method of Marklund (Arch. Biochem. Biophys. 154, p. 614-622 (1973)).

According to protein determination, the yield was The inactive peroxidase was stored at a ~I I- 6 concentration of 20-30 g/l in 50 mM Tris, pH 8.0, at -29°C.

b) Sequentially competitive detection of anti-HIV 1 100 pl of the sample to be investigated were added to microtitration plates from the commercial test kit "Enzygnost Anti-HIV 1" (Behringwerke AG, Marburg, FRG, product number OUVB), which were coated with purified, inactivated HIV 1 lysate, and the plates were then incubated for 60 min. After that, the addition of 25 pl of anti-HIV (human)/peroxidase conjugate (Behringwerke AG, Marburg, FRG, product number OWSB), together with inactive peroxidase (concentration in the conjugate: 1 mg/ml) prepared according to Example 1 takes place 15 without any washing step. After a further 30 min., the solution is sucked off, the plates are washed and the bound peroxidase is detected by means of the commercially available chromogen system (Behringwerke AG, Marburg, FRG, product number OUVF/OUVG). The reaction was then stopped with dilute sulfuric acid after the substrate had been 9 allowed to react for 30 min., and the reacted chromogen was measured at 450 nm. All the samples were considered to be reactive which exhibited o 25 extinctions below the cut-off value (extinction of the negative control The extinctions which were obtained in testing anti- HIV positive and negative sera, as well as interfering factor sera, are summarized in Table 1. It can be seen that the power of the test to correctly recognize "normal" anti-HIV positive and negative samples is not affected by the additive according to the invention. Anti-HIV 1 positive interfering factor sera, which react in a falsely negative manner without the addition of inactive peroxidase,

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7 are correctly found to be positive in the ELISA according to the invention.

Without the additive according to the invention, interfering factor-containing anti-HIV negative sera give extinction values in the test which are in some cases above the measurement range. In the ELISA according to the invention, the extinction values of these sera are in the range of the normal negative sera.

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8- Table 1: does 0..

Sample Conjugate without Conjugate with additive according additive according to the invention to the invention cut-off 0.574 0.579 value Control serum, 1.189 1.121 negative 1.109 1.193 Control serum, 0.007 0.011 positive 0.009 0.015 Anti-HIV neg. sera 12 1.090 1.258 45 1.247 1.147 216 1.198 1.111 Anti-HIV 1 pos. sera WT 17 0.015 0.005 WT 19 0.058 0.051 WT 22 0.021 0.009 Interfering factor sera Anti-HIV 1 positive 40 1.864 0.032 42 2.500 0.060 43 2.500 0.056 44 1.023 0.026 51 2.500 0.053 Interfering factor sera Anti-HIV negative 00181 2.500 1.029 00202 2.500 1.254 Reactive samples are printed in bold type.

1 0 9 Example 2: Semi-monoclonal sandwich assay for the detection of HBsAg 100 Al of the serum to be investigated were incubated for min. with 25 il of anti-HBs (mouse)/peroxidase conjugate (Behringwerke AG, Marburg, FRG, product number OWNF) in a microtitration plate coated with anti-HBs (sheep) ("Enzygnost HBsAg monoclonal", Behringwerke AG, Marburg, FRG, product number OWNE). One part of the investigations was carried out without additive according to the invention, another part with the addition of inactive peroxidase (1 mg/ml), and a further part with the addition to the conjugate of the lectin Tetragonolobus purpureas (Sigma, Deisenhofen, FRG) at a concentration of 1 mg/ml.

Subsequently, the plate was washed and the bound enzyme 15 activity was determined as explained in Example 1. All samples were considered to be reactive whose extinctions were at least 0.050 E above the average value of the negative controls.

The results of the experiments are summarized in Table 2.

20 While the additives according to the invention do not influence the correctness of the results in the case of "normal" HBsAg positive and negative samples, HBsAg negative interfering factor-containing sera, which react in a falsely positive manner without conjugate additive according to the invention, are found to be correctly negative both by the addition of inactive peroxidase and by the lectin Tetragonolobus purpureas.

The signal behavior of two HBsAg negative samples is depicted in Fig. 1 in relation to the quantity of inactive peroxidase added to the test mixture.

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10 Table 2: Sample Conjugate Conjugate with addiwithout tive according to additive the invention according to the Inactiinvention vated per- Lsectin oxidase cut-off value 0.072 0.062 0.082 Control serum 0.0260.1003 negative 0.0170.1003 Control serum 1.2661.5109 positive 1.2791.4094 HBsAg neg. sera 20 0.025 0.016 0.022 21 0.028 0.014 0.036 24 0.031 0.011 0.012 HBsAg pos. sera 88 2.500 2.500 2.5i00 107 2.500 2.500 2.500 209 2.500 2.500 2,1100 Interfering factor sera HB sAg negative 487 0.167 0.018 0.028 N 640 0.139 0.022 0.039 642 0.187 0.009 0.023 674 0.256 0.016 0.013 687 0.272 0.006 1 .029 e Reactive samples are printed in bold type.

Claims

8- 9-97 11:44 AM ;WATEWM1ARK (3316OO1 /13 THF LAIM DEINING THE f MVENIT1ONA ASFLO 1. An imrnunochemical method for detecting an analyie in a sample of a biological material, which is present in liquid form, in which method Mei reacion of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradIsh peroxidase is coupled (conjugate) directly or indirectly as the label, wherein the reaction mixture additionally contains peroxidase inactivated by hydroxymnethyihydroperoxide. 2. The method as claimed in claim 1, wherein it is a solid-phase enzyma~ immunoassay, 3. The method as claimed in claim 1, wherein the peroxidase inactivated by hydroxymnethylhydi'operoxide is added to the test mixture before the labelling paroxidase. 4. The method as claimed In claim 1, wherein the peroxidase inactivated by hydroxymethyihydropeiroxide is added at the same time as the labelling peroxidas. The method as climed in claim 1, wherein the peroxidase inactivated by hydroxy methylihyd rope roxide is removed again before the addition of the labelling peroxidase. S. The method as claimed in at least one of claims 1 to 4, wherein the peroxidase inactivated by hyd roxy methiyl hyd rope roxide is present at a concentration of 0.002 to 10 mg/mI of lest mixture, 7. The method as claimed in claim 1, wherein the peroxidase inactivated by hydroxymethylhydroperoxide is present in a concentration range of 0.02 to mg/mi. 6- 9-7 11:44 AM ;WATERMARK 6138198010;# 81 12 a. The method as claimed In claim 1, wherein the peroxidase Inactivated by hydroxymethylhydroperoxide is present in a concentration range of 0.2 to 2 mg/mi.
9. Use of peroxidase inactivated by hydroxym ethyl hydrope roxide in imrnunochemical detection methods wherein an analyte is present in liquid form and- the reaction of said analyte with a specific binding partner is detected by a further specific binding partner to which horseradish paroxidase is coupled. A reagent including peroxidase inactivated by hydroxy- met-hylhydroperoxide when used in im munochemical detection methods wherein an analyte is present in liquid form and the reaction of said analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled.
11. The method as claimed in claim 2, wherein the -solid phase is a microtitration plate.
12. An immunoohemical method for detecting an analyte in a sample of a biological material, which is present in liquid form, in which methoe the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label, wherein the reaction mixture additionally contains at least one antibody directed against an epitope of the peroxidase, or an appropflate lectin. DATED this 9th day of May, 1997. BEHRINGWIEHIE AK11EN3E;*ELL=HI-AE1 WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA 141S:JZ (DOC. 0) AU)4462893,WPG I I Abstract of the disclosure Immunochemical method for detecting an analyte The invention relates to an immunochemical method for detecting an analyte in a sample of a biological mater- ial, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label. t 0*4t o 0 U.C W I 0 *ee Q t d