Immunological determination method

The present invention relates to an immunochemical determination method in accordance with the competitive principle, wherein the concentration of the analyte is determined in a one-step method and thereby the analyte competes with a first and a second specific binding partner for binding to a third specific binding partner, wherein the first specific binding partner is bound to a water-insoluble carrier and provided the second specific binding partner with a signal-generating label.

Immunological detection methods have gained paramount importance since the first description of a radioimmunoassay (1959) and the first enzyme immunoassays driving in many areas of clinical diagnostics.

In the case of an enzyme immuno driving for determining an analyte binding and immunological reactants are used, such as. For example haptens, antigens, antibodies, or fragments of antibodies. The binding and reactants used can be present for a bound to a solid phase or with a signal-generating label, z. B. be conjugated to a marker enzyme on z. B. a covalent bond. As solid phase concave moldings are used, such. As tubes or wells in the form of microtiter plates, but also convex moldings such. B. balls. Planar solid phase such. As test strips, also apply. As labeling enzymes often alkaline phosphatase, β-galactosidase and horseradish peroxidase are used, wherein as substrates chromogenic, fluorogenic or luminescent compounds can be used. The composition of sample, incubation and washing buffers, and the different substrate / chromogen reagents are known in the art.

Unlike homogeneous enzyme immunoassays driving unbound reactants from bound reactants are removed by phase separation and subsequent washing steps in heterogeneous enzyme immunoassays driving. A distinction is made between the 1-step and the 2-step process. While in the 1-step-procedure as a whole only one separation step ( "bound / free" -Separation) is performed, this is done at the 2-step procedure after each incubation and reaction step.

Generally enzyme immunoassays driving are classified according to the immunological reaction principles in non-competitive and competitive techniques. The non-competitive techniques ( "sandwich" -Teste) are distinguished in that solid phase bound and signal-producing reactants are present as compared to the analyte to be determined in large molar excess. For the formation of the "sandwich" complex at least two binding sites of the analyte are necessary, which are respectively detected by the solid-supported or signal producing reactants. The measured signal of the type formed "sandwich" complex is directly proportional to the analyte concentration.

In the competitive techniques on the other hand one of the reactants is limiting due to its low concentration for Immunkomplexbiidung so that competition between reactants and analyte takes place by at least one binding site of the common binding partner. Within the competitive techniques, these in turn are divided into two groups. In the first group, the number of insolubilized binding partner is less than the number of the signal generating reagents and the to be determined analyte molecules, while in the second group, the concentration of the signal producing reaction partner is less than the number of insolubilized binding partner and free analyte molecules. In both cases, the signaling activity of the complex formed is inversely proportional to the measured analyte concentration.

When comparing the two immunological reaction principles can be stated that the known competitive technologies noncompetitive techniques are inferior in terms of sensitivity, range, specificity, robustness and incubation (EKINS R. (1985) CURRENT CONCEPTS AND FUTURE DEVELOPMENTS: IN ALTERNATIVE IMMUNOASSAYS ).

The differential affinity of the detected serum antibodies therefore essentially determined whether the 1-step or 2-step process is used. When detecting antibodies niederaffiner the 2-step process over the 1-step method found to be advantageous, especially if one carries out the Seruminkubationszeit as a first step overnight. However, it is important to ensure that there is no balance between the bound and unbound signal generating reactants adjusts the second incubation step, which would lead to a displacement niederaffiner analyte antibody. Evidence niederaffiner antibody is thus with the 2-step process in principle better than feasible with the user more easily performed and shorter 1-step process.

The purity of the solid phase bound antigen to compete the binding in a competitive enzyme immunoassays driving both specific antibodies as well as the signal-generating reactants, plays an important role. Critical to the sensitivity of the test is the epitope of insolubilized antigen (KENNY G. et al. (1983) J. CLIN. MICROBIOL. 17, 655-665). If a protein mixture used as such. As in a purified HAV virus antigen is the case, the sensitivity of detection can be significantly affected if the virus-specific protein shares represent only a small share of the total protein used. To avoid this, a corresponding highly purified antigen must therefore in the known methods are used, the preparation of the required purity is associated with considerable effort and correspondingly difficult, expensive and time-consuming customization (PURCELL R. et al. (1976) JOURNAL OF IMMUNOLOGY 116, 349-356). Specifically, in the detection of low-affinity anti-HAV antibodies, which are formed immediately after vaccination, show the various commercially available diagnostics tests a poor sensitivity. Only the use of purified HAV antigen and the vaccine antigen, the sensitivity could be improved in the detection of low-affinity antibodies (DELEM A. (1992) BIOLOGICALS 20, 289-291).

The object of the present invention is based was to develop a method that allows the detection of low-affinity antibodies using the 1-step process, while a minimally purified antigen can be used.

This object is achieved essentially by the embodiments provided in the claims.

The immunochemical method of the invention for determining an analyte by means of a heterogeneous competitive determination process, the following steps including:<ul><li>a) incubating the analyte is provided with a first, a second and a third specific binding partner, the first specific binding partner is bound to a water-insoluble solid phase (solid phase-bound reactant) and the second specific binding partner with a signal-generating label (signal-generating reactants), and the analyte and the first and the second specific binding partner for binding to the third specific binding partner (common binding partner) compete</li><li>b) separating the bound to the solid phase via the third specific binding partner signal-generating label from the unbound fraction,</li><li>c) measurement of which can be generated by the bound portion of the marker signal, and</li><li>d) determining the analyte concentration by comparing the detected in step c) values ​​with a list drawn up under the same conditions or theoretically calculated standard curve.</li></ul>

Analyte can be determined by the method according to the invention are known in the art per se. Advantageously, the analyte is an antibody, especially an antibody of human or animal origin, in microbiological diagnostics, in particular in the field of infectious disease diagnostics as it is done in blood banks, is relevant, such as anti-HAV antibody, anti-HIV antibody, anti- HCV antibodies or antibody in hepatitis B diagnosis.

The first and the second specific binding partner must be able to react specifically with the third specific binding partner.

Advantageously, the first and the second specific binding partner antibodies, monoclonal or polyclonal or antibody fragments. To carry out the inventive method but also lectins or synthetic / recombinant antibodies can be used.

In principle, the inventive method for the detection of antigens other than antibodies is usable, in which case the first and the second specific binding partner is an antigen and the third specific binding partner can be an antibody.

Solid phase as such are known to the skilled worker. Advantageously, water-insoluble solid phase are used, such as. For example, latex particles, magnetically attractable particles or microtiter plates.

Signal generating labels, as such are known to the skilled worker. Such a marking may be either directly conjugated to the respective specific binding partner or a the person skilled in the known reversible coupling such., Biotin-streptavidin, fos-jun or antibody-antigen bonds.

When signal generating label preferably components which are capable of chemiluminescence or fluorescence are used or enzymes can convert the luminogenic, fluorogenic or chromogenic substrates. The enzymes is particularly preferably horseradish peroxidase. In the case of chemiluminescent labels, the European patent applications in the particularly preferred are EP-A-0257541 and EP-A-0330050 described compounds.

Typically, in the described immunochemical methods after the separation of the solid and the liquid phase of the signal is measured at either the solid phase or in the separated supernatant. From the measured signal to the conventional manner is determined by a so-called standard curve the analyte concentration in itself. To establish the standard curve, the signals using the respective determination method of known analyte concentrations measured and implemented either graphically or mathematically in a curve shape. Such standard curve can be calculated theoretically with some accuracy because of the known physical and chemical properties of the specific binding partner.

The inventive method is applicable in immunochemical methods in which a competition between the analyte to be determined and the signal-generating reactants and / or insolubilized reaction partners takes place for binding to a common binding partner.

The inventive method is further characterized in that the common binding partner must be minimally purified. It has been found to be essential that the common binding partner should have at least two different binding sites, wherein one binding site of the insolubilized reaction partners is detected, while the signal-generating reaction partner bind to the second binding site. In the presence of analyte to be detected then a specific competition of the analyte with the signal-generating and / or insolubilized reaction partners for binding to the common binding partner. As much for the novel Ver-drive has arisen here is that when an analyte absence the formation of a signal-generating "sandwich" complex is not prevented by the competition between insolubilized and signal-generating reactants.

It was surprisingly found in the process for the detection of antibodies against the hepatitis A virus of the invention that when an analyte absence (= negative control) no competition between the signal-generating reactants (= monoclonal anti-HAV-specific antibody conjugate) and the insolubilized reactants (= polyclonal anti-HAV-specific antibody) occurred for binding to the common binding partner (= HAV antigen) and thereby the formation of a signal producing "sandwich" complex is made possible. This was surprising, because the known monoclonal antibody to HAV can almost completely block the binding of polyclonal antibodies to the virus in various competitive immunoassay Over Drive (LEMON S. et al (1993) VIROLOGY 4, 285-295;. HUGHES J. et al . (1984) J. VIROL 52, 465-473;. STAPLETON J. et al (1987) J. VIROL 61, 491-498)... By contrast, in the presence of analyte (= anti-HAV specific antibodies = positive control) was found as surprising that a specific competition can take place at the common binding partner between the analyte and the insolubilized reactants and / or signal-generating reactants.

Also surprising was the finding that the common binding partner had only minimally or not at all be purified in order to prove this with the 1-step process low affinity antibodies that can occur in the early phase of infection or after vaccination. In the novel process conditions are created possibly due to the presence of solid phase-bound reactants that allow an especially effective and specific competition between the analyte and the signal producing and / or solid phase bound reaction partners for the binding sites of the common binding partner. Minimal purified according to the present invention in this context means that the amount of specific protein is less than 80, preferably less than 50%, most preferably less than 20%.

Preferably, the invention as a reactant antibody or defined fragments of antibodies may be used in the frame. The preparation of polyclonal or monoclonal antibodies (Kohler G. and Milstein C. (1975) Nature 256, 495-497) is carried out by a known method per se to the skilled person. In addition to polyclonal antibodies and monoclonal antibodies or their fragments (F (ab ')₂ or Fab ') are used. The method according to the invention while a reactant is bound to the water-insoluble solid phase, while the second reactant is used as a signal-generating component. Preferably, in the inventive method a polyclonal antibody is bound to the solid phase and a monoclonal antibody that is conjugated to a marker enzyme, used as a signal-generating reactants. The preparation of monoclonal conjugate used in the invention to those skilled also known (review article:. ISHIKAWA E. et al (1983) J. IMMUNOASSAY 4, 209-327).

The suitability of the antibodies used can, for. Example, be determined by the person skilled in the known experiments.

As a binding partner may be used in the context of the invention, any macromolecule which has at least two separate binding sites for the insolubilized and said signal-generating reactants. Suitable macromolecules are proteins - optionally modified by carbohydrates and / or lipids -, carbohydrates, lipids, synthetic polymers and nucleic acids. Preferably, the molecular weight of the binding partner is from 50,000 to 2 million

The inventive method can be applied to all immunological detection methods in which a specific competition between the analyte and solid-phase reactants and / or signal-generating reactants can take place at least two binding sites of a common binding partner in heterologous immunoassays.

The following examples illustrate the invention:

Abbreviations:

<dl id="dl0001" compact="compact"><dt>HAV:</dt><dd>Hepatitis A virus</dd><dt>POD:</dt><dd>peroxidase</dd><dt>SH:</dt><dd>sulfhydryl</dd><dt>ATCC:</dt><dd>American Tissue Cell Culture</dd></dl>

Examples

example 1

a): conjugation of antibodies

Monoclonal antibodies to HAV are reacted with a heterobifunctional reagent (TARRIMORE et al. (1983) J. IMM. METH. 62, 123-131), followed by SH-activated peroxidase (KING et al. (1978) BIOCHEMISTRY 17, 1499- 1506) incubated and then purified by gel chromatography.

b): Preparation of HAV antigen

To prepare the HAV antigen are commercially available cells such. B. humandiploide embryonic lung fibroblasts with a characterized hepatitis A virus strain, such. As ATCC HM-175, infected. After several days the detached cell supernatant is centrifuged, the cell pellet obtained in a conventional storage buffer and inactivated, the antigen according to any method known in the art. The inactivated antigen can be used without additional purification on. The inventive method is not restricted to the above method for the preparation of HAV antigen, but also others, known to the expert preparation process for antigen isolation can be used. In addition, commercially-available HAV antigen preparations can be used in the process according to the invention.

c) coating the wells of microtiter plates

To a coating solution (sodium carbonate, 0.01 mol / l pH 9.6), a certain amount of human polyclonal anti-HAV antibody (16 ug / ml) was added and homogenized for about 30 minutes under gentle stirring. The coating of the individual wells of a microtiter plate was then carried out with a coating volume of 150 ul. After an overnight incubation at room temperature, the coating solution is aspirated and the individual wells of the microtitration plate are washed twice with a washing solution (0.25 mol / l citric acid / 0.05 mol / l Tris pH 7.4). Following the final wash, the individual wells of the microtiter plate are sucked dry and packed with desiccant (z. B. Silica Gel) sealed in aluminum foil. The coated Miktotitrationsplatten be stored at 4 ° C until use.

d) Enzyme immunoassays driving for the determination of anti-HAV antibodies

The inventive method is a competitive enzyme immunoassay for the 1-step process. In the wells of a microtitration plate coated with human polyclonal anti-HAV specific antibodies, successively test sample (25 ul), conjugate (= POD-conjugated anti-HAV-specific monoclonal antibody, 50 ul) and HAV antigen (50 ul) was added. The beschichtetete microtiter plate, conjugate and HAV antigen (OQEC Behringwerke AG, Marburg Best. Nr.) Were the Enzygnost® anti HAV test kit removed. The test sample containing the to be determined anti-HAV antibodies, they will compete with the conjugate molecules and / or insolubilized polyclonal anti-HAV specific antibodies for binding to the HAV antigen. After an incubation period of 2 hours at 37 ° C to excess conjugate and unbound reactants by aspiration and washing four times, z. B. with the Behring ELISA Processor II or Behring ELISA Processor III (Behringwerke AG, Marburg), removed, and the amount of bound conjugate by the addition of 100 ul of substrate / chromogen solution (Behringwerke AG, Best. Nr. OUVP) determined (30 min, room temperature, protected from light). The enzymatic conversion of the chromogen tetramethylbenzidine dihydrochloride was suspended 0.5 N sulfuric acid by adding 100 .mu.l and absorbance at 450 nm was determined by photometry. The measured absorbance is the anti-HAV antibody concentration contained in the sample is inversely proportional

e) Comparison of the inventive method with an alternative, vollmonoklonalen method.

The inventive method is an enzyme immunoassay for the 1-step process that includes a bound on the solid phase polyclonal anti-HAV antibody specific for human origin and as conjugate antibodies a monoclonal anti-HAV antibody specific for mouse. The comparison process differs from the process of the invention is that the polyclonal solid phase antibody is replaced with a monoclonal antibody.

In Table 1, the inventive method with the alternative, vollmonoklonalen method is shown in relation to the signal intensity of the negative control and determined detection limit. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center"><b>Comparison of the process according to the invention with a method vollmonoklonalen</b></entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Absorbance of the negative control</entry><entry namest="col3" nameend="col3" align="center">detection limit</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Inventive Process</entry><entry namest="col2" nameend="col2" align="right">1427 mE</entry><entry namest="col3" nameend="col3" align="right">13 IU / l</entry></row><row><entry namest="col1" nameend="col1" align="left">Vollmonoklonales method</entry><entry namest="col2" nameend="col2" align="right">543 mE</entry><entry namest="col3" nameend="col3" align="right">28 IU / l</entry></row></tbody></tgroup></table></tables>

The higher signal intensity of the negative control and the improved detection limit of the built up of a polyclonal and a monoclonal anti-HAV antibody method of the invention clearly demonstrate the superiority of the present process over an alternative, vollmonoklonalen process.

DESCRIPTION OF THE FIGURES

The detection limit of the method of the invention was determined using defined dilutions of anti-HAV standards (WHO standard). In Fig. 1, the measured absorbance values ​​of different antibody concentrations are shown in a semi-logarithmic representation. Due to the competitive test structure take the absorbance values ​​with increasing antibody concentration.

The calculated analytical sensitivity is 13.4 IU / L, with a cut-off value of the absorbance of the negative control was halved.

Using Impfserokonversionen was shown that low-affinity antibody to HAV can be detected with the inventive method in a very early stage. In FIG. 2, the time course of a typical anti-HAV seroconversion (0193) is shown. Serum samples were taken at various times from inoculation subjects and those studied by the method according to the invention. The ratio values ​​shown entprechen this quotient values ​​from the measured absorbances and the cut-off value. Ratio values ​​&lt;1 indicate that the detectable appearance of anti-HAV antibodies has occurred seroconversion. As shown in FIG. 2, seroconversion could already be detected from the second week after vaccination with the inventive method.

In Figure 3, the measured absorbance values ​​of the examined seroconversion 0193 using the calibration curve of Fig. 1 were quantified. Already from the 2nd week an antibody concentration was determined that significantly exceeds Impfschutzgrenze of 20 IU / l.