DD292720A5 - METHOD FOR PRODUCING TEST KITS FOR DETECTING IMMUNOCOMPONENTS - Google Patents

Abstract

Test kits for the detection of immune components; Enzyme immunoassay; Test kit-assembly base; beta-lactam hydrolase as an enzyme component in the conjugate; beta-lactam as a substrate; beta-lactam hydrolase inhibitor as a stop reagent; Iodine inclusion compound as a chromogenic substance; Color change; Reaction according to the all-or-nothing principle; Omission of the spectrophotometric evaluation; medical diagnostics; Detection of viral infections (HIV1, HBV, rotavirus); Graviditätsnachweis

Description

For this 3 pages drawings

Field of application of the invention

The invention relates to a process for the preparation of test kits of the basic type of the enzyme immunoassay which can be used for the detection of immune components (antigens, antibodies, antigen-antibody complexes).

Such test kits can be used, inter alia, both for the detection of antiviral antibodies, as they occur in an HBV, an HIV or a rotavirus infection, as well as, for example, for the detection of trophoblastic beta-glycoprotein, which occurs when pregnancy occurs in the serum , use.

The field of application of the invention lies in those areas of the pharmaceutical and chemical industry which make test kits, especially enzyme immunoassays, for immunodiagnostics in human and veterinary medicine.

Characteristic of the known state of the art

The detection principle known since 1971 as enzyme immunoassay (EIA) is primarily linked to the use of conjugates of an immunologically active reaction partner and an enzyme, so that after immunological binding to the immune partner to be detected by enzymatic reaction with a corresponding to the substrate to be selected, to which a chromogenic substance can be added as an indicator system, color reactions are triggered (Engvall, E. and Perlmann, P., Immunochem., 1971, 8, 871-874; Van Veemen, BK and Schuurs, AHWM FEBS Letters (US Pat. 1971], 15,332-336, Engvall, E. and Perlmann, P., J. Immun. Meth. (1971), 10, 161-170, see also patents,). This chromogenic reaction, which has hitherto always turned from colorless to color, then becomes In the following, with the aid of spectrophotometric detection and evaluation systems (extinction measurement), the immunoassay was evaluated ym component preferably a peroxidase or an alkaline phosphatase in covalent coupling to an immunologically active reactants used (see "Nonisotopic immunoassay", ed. by Ngo.T.T., 1988, Plenum Press, New York, London; Patents). As reactants (immune components) of the above labeling, in each ELISA modification (simple competitive assays or sandwich assays, differentiated into competitive or non-competitive variant), primarily mono- or polygonal antibodies, more rarely also antigens, are used.

Hydrogen peroxide with ortho-phenylenediamine (OPD) or with 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) for peroxidase and 4-nitrophenyl phosphate for alkaline phosphatase are preferably established as substrates in the known test kits , Upon binding of the conjugate to each immuno-component to be detected, the substrate used is cleaved by the coupled enzyme, whereby the above-mentioned color change (from virtually colorless to color) is triggered (see again "Nonisotopic Immunoassay", ed. By Ngo TT, aa 0. ).

For the methods outlined above, there is the general disadvantage that, for the evaluation of the immunological reaction, it is always necessary to make use of differential measurements of extinction values (from positively reacting sample to extinction average from at least three negative control standards). This is constantly linked to objectivating spectrophotometric measuring devices. All currently known enzyme immunoassays are thus technically complex and in terms of the evaluation of their cut-off value, i. of the actual immune reaction underlying negative control value of the extinction, not always beyond doubt.

Object of the invention

The invention aims to provide novel test kits for sensitive detection systems in the various basic variants of the enzyme immunoassay technique.

Explanation of the essence of the invention

The invention has for its object to describe a method for the preparation of novel test kits that can be used in the main variants of the enzyme immunoassay technique.

According to the invention the object is solved in its basic trait in such a way that for the detection of immune components test kits of the basic type of an enzyme immunoassay (more precisely: solid phase enzyme immunoassay [ELISA]) with the components

Reaction vessel (microtiter plate) with adsorbed antigen or monoclonal antibody,

- conjugate of antibody and enzyme,

Substrate with addition of a chromogenic substance and

- Optional stop-coagulation plus various washing and dilution buffers and optionally various positive and negative control standards

are constructed, each having a conjugate with a beta-lactam hydrolase as the enzyme component and thus a beta-lactam as a substrate and an iodine-inclusion compound as the chromogenic substance (Cramer, F., "inclusion compounds", Springer-Verlag, Berlin, Göttingen , Heidelberg, 1954.) In this new basic version of enzyme-immunoassay-based test kits, preference is given to using such a conjugate which comprises a beta-lactamase as enzyme component in combination with a monoclonal antibody or with an anti-antibody as immunologically active reaction partner The antibody used for this labeling may be mouse, human or other monoclonal antibodies to any antigen or anti-gammaglobulin antibody.

In addition, a beta-lactam hydrolase inhibitor, preferably a beta-lactamase inhibitor, is advantageously added to the test kit.

With recourse to the above-mentioned components, the proposed new basic version includes both simple competitive solid-phase enzyme immunoassay type test kits and solid phase sandwich enzyme immunoassay type test kits, preferably non-competitive sandwich type, produced.

In its further embodiment, the invention is additionally characterized in that in the kit, a stable conjugate is used, which is a beta-lactamase as an enzyme component in combination with an anti-gamma globulin antibody or with a monoclonal antibody against selected Proteinf actor as a reactant and containing a polysaccharide as a carrier material.

As support materials can hereby

Sucrose polymers of different degrees of polymerization,

- Other sugars and their derivatives of different degrees of polymerization or

- Include dextranes and their derivatives of different degrees of polymerization einbezogben.

Of the total of the anti-gammaglobulin Antlkörper be in leather embodiment advantageously various anti-IgG Antlkörper, so primarily anti-Fab antibody, anti-Fc fragment antibody or anti-gamma chain antibody, used; in contrast, all of the monoclonal antibodies of the above labeling are advantageously those against viral antigens to be detected or fragments thereof, e.g. against the surface antigens of the virus hepatitis B or rotavirus), or against biogenic factors, such as the trophoblastic betal glycoprotein or against the tumor necrosis factor. The finished conjugate is advantageously provided as a lyophilisate or as an aqueous solution for the test kits according to the invention.

The substrate used here is a penicillin, an ampicillin or a derivative of these beta-lactams; as chromogenic substance either an iodine-starch or an iodine-polyalcohol complex is used.

As adsorbed antigens to the solid phase, native and recombinant or synthetic proteins (or immunologically reactive fragments thereof), such as. Components of HBV, HIV, Rota and other viruses, the trophoblastic beta 1 glycoprotein, the tumor necrosis factor and other biogenic factors (or again immunologically reactive fragments thereof). Reaction vessels are coated with the antigen component or a monoclonal antibody of the above labeling as solid phases, primarily microtiter plates, in a manner known per se. It has proved advantageous, as already mentioned above, to add in each case one stop reagent to the test kit according to the invention in addition to the conjugate, substrate and reaction vessel with adsorbed antigen or monoclonal antibody as the fourth component. For this purpose, preferably a synthetic beta-lactamase inhibitor is used at the level of the further embodiment of the invention. With this inhibitor, the enzymatically catalyzed reaction of the cleavage of the beta-lactam ring by the beta-lactamase either by enzyme inhibition or by substrate competition reaction is suppressed or at least greatly limited. Through this procedure, a test result that is stable over several hours and thus reliably evaluable is achieved in all previously tested variants of the kit.

The proposed test kits are based on the following reaction principle: After immunological reactions of substances, haptens or antibodies of the sample to be examined or the respectively used standards with the immune components on the solid phase as well as in the conjugate, the beta-lactam substrate used is replaced by the beta-lactamase of the conjugate hydrolytically cleaved. The cleavage product, in turn, interacts with the blue-colored iodine inclusion compound (iodine-starch or iodine-polyalcohol complex) and, as a result of the reaction, decolorizes it (color change from deep blue to colorless according to the all-or-nothing principle). Based on the usual practice, the new test kits thus produced for the diagnosis of z. B. Antibodies in serum samples as follows: The samples to be tested are introduced in a conventional manner, optionally after appropriate dilution, in the wells of the microtiter plates prepared according to the method and incubated in a conventional manner. In this first incubation step, the antibody is bound from the positive control serum or from the testosterone serum to the correlating antigen. In a second incubation step, the affinity chromatographically purified and beta-lactamase-labeled anti-antibodies of the conjugate react with the antibodies bound to the antigen from the respective serum sample. Unbound serum proteins are removed after the first incubation step and excess antibody-enzyme conjugate is removed after the second incubation step by rinsing with washing buffer. The wash buffer used is preferably phosphate-buffered saline with the addition of 0.05% polyoxyethylene sorbitan monolaurate (n = 20). After addition of the procedure, deep blue colored substrate solution (from penicillin, from ampicillin or derivatives thereof, mixed with an iodine-starch or an iodine-polyalcohol complex) takes place in the third incubation phase, the enzymatic reaction between this solution and the now beta-lactamase bound to the immune complex. In the presence of antibodies in the sample, the color change described above occurs. Positive sera can be recognized by the color change from deep blue to colorless. If no antibodies were bound to the antigens adhered to the microtiter plate, the blue coloration of the substrate solution remains unchanged for a long time (at least 6 hours).

Since the detection system beta-lactamase-beta lactam-iodine inclusion compound is constructed on the all-or-nothing principle, i. H. after the initial initiation, the decolorization reaction proceeds inexorably until complete destruction of the light-absorbing iodine inclusion compound, can be visually recognized by the color change from deep blue to colorless, whether in the serum investigated antibodies or other biogenic factors of each type to be detected are present. After carrying out the process in the further embodiment of the invention, test kits of the type of a solid-phase enzyme immunoassay, in particular of the type of a non-competitive sandwich ELISA, but in principle also test kits of the type of simple competitive ELISA can be produced. In the context of the present invention, it is also possible to construct suitable test kits for quantitative determination. In this case, a normative standard should be added to the kit so that calibration curves showing the dependence of the reaction time on the standard concentration used can be generated.

In the preferred embodiments of the invention, in each case conjugates are used in which polysaccharides of the type of copolymers of sucrose and epichlorohydrin (common name Ficoll) or polysaccharides of the dextran type are included as support materials. These conjugates each contain Bacillus licheniformis beta-lactamase as an enzyme component in combination with

an anti-IgG antibody, preferably in combination with one of the collection of anti-Fab antibodies, anti-Fc fragment antibodies or anti-gamma chain antibodies,

a monoclonal antibody to the HBV surface antigen,

a monoclonal antibody against the trophoblastic beta 1 -glycoprotein (TBG),

a monoclonal antibody against the rotavirus surface antigen or

- a monoclonal antibody against the tumor necrosis factor (TNF) and others as reactants.

From these components, stable conjugates are prepared in each case by

- The selected carrier material in dissolved form (solvent: distilled water.) With sodium periodate and dialyzed overnight against a sodium acetate solution (1 mM, pH 4.4),

mixed the respectively selected antibody with Bacillus licheniformis beta-lactamase in a weight ratio of 1.6: 1 and likewise dialyzed overnight (against phosphate-buffered saline),

the components antibody-beta-lactamase-Gomlsch on the one hand and oxidized carrier material on the other hand in a weight ratio of 5: 1,

- Immediately adjust the pH of the resulting mixture with Notrlumcarbonatlösung to pH 9.6,

- Then incubated at room temperature for 3 to 4 hours and reduced remaining free oxidized groups of the support material with sodium borohydride, '.

- The resulting product overnight dialysiort (against phosphate-buffered saline) and

- The resulting liquid conjugate optionally added to stabilize with bovine serum albumin and lyophilized or treated for preservation with thiomersal and glycerol.

According to this sub-procedure, stable conjugates which are well reproducible in their immunological and enzymatic properties are obtained for at least 6 months.

In this context, the sucrose derivative known as Ficoll 400 proved to be a polysaccharide which was favorable in its application.

The preferred embodiments of the invention are further characterized in that in the test kits to be produced benzylpenicillin as a substrate, an iodine-polyvinyl alcohol Komplox, preferably a complex of elemental iodine, potassium iodide, polyvinyl alcohol and distilled water, as chromogeno substance and sulbactam as a stop reagent been used.

For each microtiter plate (96 wells), 4 mg benzylpenicillin dissolved in 7.5 ml phosphate-buffered saline solution, which has been mixed immediately before 2.5 ml iodine-polyvinyl alcohol complex of the above labeling, are used.

In addition, the test kits produced according to the method are

Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer,

- Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and optionally with 0.1% bovine serum albumin or 5% gelafusal as dilution buffer, and

- gepoite positive sera completed as positive control standard and polar negative sera as negative control standard.

Following the above-described substeps, in the preferred embodiments of the invention, non-competitive sandwich EUSA subtype test kits are prepared. In particular, test kits for HIV1 antibody diagnostics (test kit "RECOMBIII"), for HBV surface antigen diagnostics, for TBG antigen diagnostics, for rotavirus surface antigen diagnostics and for TNF antibodies are used according to this methodology. These tests can also be used for the monitoring of the respective disease or indication, using the RECOMBI II test kit for HIV 1 antibody diagnostics

recombinant (gp41 + gag) HIV protein as antigen absorbed on the microtiter plates,

- Bacillus licheniformis beta-lactamase in combination with rabbit anti-human IgG antibody or rabbit anti-gum chain antibody and Ficoll 400 as stable conjugate,

Benzylpenicillin as substrate, an iodine-polyvinyl alcohol complex as chromogenic substance and sulbactam as stop reagent,

Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer,

- Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 5% Gelafusal as dilution buffer and

- Positive serum poled by 20 patients as a positive control standard and negative serum pooled by 50 healthy volunteers as a negative control standard

constructed and provided for use as a sieve test.

In HBV surface antigen diagnostics, i. for the detection of infectious jaundice, which can be used as a sieve test procedural test kit is resorting to

monoclonal antibody to the HBV surface antigen as monoclonal antibody absorbed on the microtiter plate,

- Bacillus licheniformis beta-lactamase in combination with monoclonal antibody to the HBV surface antigen and Ficoll 400 as a stable conjugate,

Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance,

Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer,

- Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 0.1% bovine serum albumin as dilution buffer and

- Positive serum poled by 8 patients with H3patitis B as a positive control standard and negative serum poled by 20 healthy volunteers as a negative control standard

built up.

The test kit which can be used for the pregnancy indication is used in this embodiment

monoclonal antibody to the trophoblastic beta 1-glycoprotein (TBG) as monoclonal antibody adsorbed to the microtiter plate,

- Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against TBG and Ficoll 400 as stable conjugate,

Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance,

- Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer

- Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 0.1% bovine serum albumin as dilution buffer and

- Serum polarized by pregnant women as a positive control standard and serum biased by non-pregnant women as a negative control standard

compiled.

The kit, which can be used to detect rotavlus infectonon as a sieve test, is based on

monoclonal antibodies against the rotavlus surface antagonist as monoclonal antibody adsorbed to the microtiter plate,

Bacillus lichoniformis beta-Lactamaso in combination with monoclonal antibody against the Rotavirua surface antigen and Ficoll 400 as a stable conjugate,.

Benzylpenicillin as substrate and an iodine-polyvinyl alcohol complox as chromogenic substance,

Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer,

- Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and 0.1% bovine serum albumin as dilution buffer and

- Supergnatante oiner 10% suspension of faeces in phosphate buffered saline, centrifuged as a positive control standard, from 10 patients with rotavirus infections and an equivalent supernatant of the faeces of 20 healthy volunteers as a negative control standard

built up.

Finally, the test kit that can be used in TNF diagnostics will be used

monoclonal antibodies against tumor necrosis factor (TNF) as monoclonal antibodies absorbed on the microtiter plate,

- Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against TNF and Ficoll 400 as stable conjugate,

Benzylpenicillin as substrate and an iodine polyvinyl alcohol complex as chromogenic substance,

Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer,

- Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 5% gelafusal as dilution buffer and

- optionally recombinant TNF compiled as Normativstandard.

It will undoubtedly be possible for a person skilled in the art on the basis of the above-given description of the invention to construct further test kits of the disclosed new basic type for a variety of other indications. The advantages of the present technical solution are mainly seen in that

it is within the methodology set forth for the preparation of enzyme immunoassays, preferably of Fesiphasen enzyme immunoassay (ELISA), with fundamentally new combination of enzyme component, substrate and chromogenic substance in a simple manner, stable conjugates of bota-lactam hydrolases with to gain the respective antibodies,

the color change indicating the detection of an immune reaction which has taken place takes place according to the all-or-nothing principle, so that a spectrophotomeric evaluation can be dispensed with,

- When using a stop reagent from the proposed class of substance, the reaction result, d. H. the color change from deep blue to colorless, remains unchanged over several hours and

- Each previously developed concrete test kit with the proposed detection principle in the result of the above-mentioned benefits proved to be extremely reliable means of verification.

embodiments

example 1

Production of the test kit "Recombi II" for HIV 1 antibody diagnostics

1.1. Preparation of a beta-lactamase anti-IgG antibody conjugate using Ficoll 400

1 mg of Bacillus licheniformis beta-lactamase is mixed in a weight ratio of 1: 1.5 with rabbit anti-human IgG antibodies or rabbit anti-human gamma chain antibodies and overnight at 4 degrees Celsius against phosphate buffered saline Dialyzed (PBS).

To 5 mg of this beta-lactamase antibody mixture is added 1 mg Ficoll 400 (MW: 400,000) oxidized with sodium periodate. Immediately thereafter, the pH of the reaction mixture is adjusted to pH 9.6 with a 0.1 M sodium carbonate solution (pH 11). For 500μΙ reaction mixture 100μΙ 0.1 M sodium carbonate solution is needed.

Oxidized Ficoll 400 is prepared by mixing 1 ml of 1% Ficoll 400 solution with 400 μl of a 0.5M sodium periodate solution. The Ficoll 400 sodium periodate solution thus obtained is then dialyzed overnight against a 1 mM sodium acetate solution having a pH of pH 4.4.

After adding the oxidized Ficoll 400 to the beta-lactamase antibody mixture, it is shaken at room temperature for 3 to 4 hours, after which 50 μl of 0.8% sodium borohydride solution is added. After incubation for 1 hour at room temperature with shaking, the conjugate is dialyzed against PBS overnight at 4 degrees Celsius.

The resulting beta-lactamase-anti-antibody conjugate is optionally purified by gel chromatography and then stabilized by the addition of bovine serum albumin to the final concentration of 0.05%, glycerol to the final concentration of 10% and sodium azide to the final concentration of 0.02%.

1.2. Construction of the "RECOMBI II" kit for HIV 1 antibody diagnostics and test implementation

Microtiter plates coated with recombinant rgp41 and rgag HIV1 antigens are spiked with serum samples previously diluted 1:50 with PBSTG (PBS with O, 05% polyoxyethylene sorbitan monolaurate [n = 20] and 5% gelafusal). Per cavity 100 μ! pipetted into the diluted serum sample. In the cavity A1 is nu. 100μΙ pure dilution medium pipetted, while the cavities A2 to A4 with 100μΙ negative control serum, the cavities A5 and A6 with 100μΙ positive control serum 1 and the cavities A7 and A8 with 100 μΙ positive, compared to the positive control serum 1 but much weaker control serum. 2 be occupied.

After completion of the pipetting process, the plate is incubated for 60 minutes at 37 degrees Celsius. Subsequently, the cavities of rows A to H are successively filtered off with suction and filled with 200 .mu.l washing buffer (PBS with 0.05% polyoxothylon-sorbitan monolaurate [n = »20]), shaken for 3 to 6 minutes and sucked off again. This process is still wlodorholt twice. Following the washing process, the wells are filled with KX-lyordünntor conjugate solution and also incubated for 60 minutes at 37 degrees Celsius. After completion of the second incubation, the liquid is aspirated analogously to the Orston incubation step and the cavities are washed three times with dom o.g. Washing buffer washed.

After this washing process, in addition to the well A1, 100 μl of the directly prepared substrate solution of penicillin in a concentration of 40μΙ per well and iodine-polyvinyl alcohol complex, consisting of 100 mg iodine, 300 mg potassium iodide and 900 mg polyvinyl alcohol in 100 ml Aqua dost. (Iodinol), diluted 1: 4 with PBS, pipetted. After filling the substrate solution, the plate is incubated at room temperature. After 20 to 30 minutes, the substrate solution is completely decolorized in wells A5 and A6 with dom positive control serum 1. The substrate solution in don Kavitöton A2 to A4 with the negative control serum, however, has retained its deep blue color unchanged.

As soon as the o.g. Incubation, wells A7 and A8, containing the very weak positive control serum 2, are completely decolorized, 50μΙ stop reaction is pipetted into each well in the form of aqueous 0.1 mM sulbactam solution. The evaluation of the test is based on the Farbumsi / ilages from deep blue to colorless, it can thus be vorgenomen. The color difference between positive and Noga ivseren remains after addition de. Stop reagonz boi room temperature at least 6 hours, boi get 4 degrees Celsius at least 24 hours.

(The piiotometric evaluation in the spectral range 570nm to 630mn is possible, the extinction of the negative controls must be above 1.5, while the positive control should have an extinction of less than 0.05.) The reliability of the "RECOMBI ΙΓ test kit was in a field trial of 141 HIV-positive and 670 HIV negative sera. A sensitivity of 100% and a specificity of 99.85% could be achieved.

Example 2

Production of a test kit for HBV surface antigen diagnostics

2.1. Preparation of the specific beta-lactamase-labeled conjugate using Flcoll 400

For the o.g. Test kit in the present example, a conjugate is required, which contains on the carrier material Ficoll 400 Bacillus licheniformis beta-Lactamaso in combination with a monoclonal antibody against HBV surface on-Antigon. For this purpose, any monoclonal antibody of this kind can be used. In the preparation of the specified conjugate, the procedure is as in point 1.1. of Example 1, where now! mg beta-lactamase of the above labeling in a weight ratio of 1: 1.5 is mixed with a monoclonal antibody of the type mentioned.

2.2. Construction of the complete kit and feedthrough

Microtiter plates coated with monoclonal antibody against hepatitis B surface antigen (HBsAg) are each precontracted with optionally diluted serum samples. For each well 100 μΙ of the respective serum probu are pipetted. The assignment of the cavities A1 to A6 is carried out according to the same scheme as in item 1.2 of Example 1. After incubation of the filled with the serum sample microtiter plates for one hour at 37 degrees Celsius, first followed by the washing process. Now that the bota-lactamase-labeled conjugate, prepared according to the procedure of section 2.1, has been added, the incubation step 2 is carried out as in point 1.2 of example 1. The detection of the antigens bound to the monoclonal antibody from the serum samples is carried out in the same way carried out as in item 1.2 of Example 1, d. H. in the case of HBV infection, antigones present in serum were also indicated by a color change from deep blue to colorless.

This issue has been verified in field trials with HBV-infected patients and healthy blood donors, each testing more than 500 subjects.

Example 3

Production of a test kit for the detection of the trophoblastic beta 1-glycoprotein in pregnancy diagnostics

3.1. Preparation of the specific beta-lactamase-labeled conjugate using Ficoll 400

For the o.g. In the present example, the test kit requires a conjugate which contains on the carrier material Ficoll 400 Bacillus licheniformis beta-lactamase in combination with a monoclonal antibody against the trophoblastic beta 1-glycoprotein. For this purpose, any monoclonal antibody of this specificity can be used.

For the preparation of the specified conjugate, the procedure is as in item 1.1 of Example 1, wherein now 1 mg of beta-lactamase above labeling in a weight ratio of 1: 1.5 with a monoclonal antibody of the type mentioned is mixed.

3.2. Construction of the complete kit and test execution

Microplates coated with monoclonal antibody to trophoblastic beta 1 -glycoprotein (TBG) were each treated with optionally diluted serum sample. For each well 100 μΙ of the respective serum sample are pipetted. The assignment of the cavities A1 to A6 is carried out according to the same scheme as in item 1.2 of Example 1. After incubation of the occupied with the serum sample microtiter plates for one hour at 37 degrees Celsius, after the subsequent washing process and after addition of 100 μΙ of the corresponding In accordance with the prescription in point 3.1 and diluted beta-lactamose conjugate per well, incubation step 2 is carried out analogously to point 1.2 of example 1. The safe method of binding of the TBB bound to the monoclonal antibody from the serum samples and thus confirmation of pregnancy is from the second day given and carried out in the same manner as described in item 1.2 of Example 1 (discoloration of the chromogenic substance)

 The predictability of the test was confirmed in field trials with approximately 500 pregnants and approximately 800 non-pregnant women.

Example 4

Production of olnos test kits for rotavirus surface antigen testing

4.1. Preparation of Specific Bete-Lactamae Labeled Conjugates Using Flcoll 400

For don o.g. Test kit in the present example, a conjugate is required, which contains on the carrier material Ficoll 400 Bacillus Hchonlformls beta-lactamase in combination with a monoclonal antibody against Rotavlrus Oborflächen antigen. For this purpose, any monoclonal antibody of this type can be used. For the preparation of the specified conjugate, the procedure is as in point 1.1 of Example 1, wherein now 1 mg of beta-lactamase obigor labeling in a weight ratio of 1: 1.5 is mixed with a monoclonal antibody of the gonannten type.

4.2. Construction of the complete kit and test execution

10% Suspensions of Faeces In PBS, centrifugation is performed at 10000 rpm, and 100 μl of the supernatant are pipetted into the wells of a monoclonal antibody to rotavirus surface antigen coated microtiter plate. The occupancy of Kavlti th A1 to A6 and also the following Inkubatlons- and the subsequent washing steps urfolgon in the same Wolso as in item 1.2 of Example 1. After now per cavity 100μΙ of the prepared and diluted according to the rule of Section 4.1 conjugate were added , the incubation step 2 described in point 1.2 of Example 1 takes place.

If rotavirus infection is present, the rotavirus surface antigen present in the faeces is bound to the monoclonal antibody adhered to the microtiter plate and described in the same manner as in item 1.2 of Example 1, ie by decolorization of the chromogenic substance. demonstrated.

This test kit was also used in field trials in approximately 600 children with rotavirus infections and

healthy children were included, checked for reliability.

Claims (39)

1. A method for the preparation of test kits for the detection of immune components, each kit as an enzyme immunoassay with the components Reaction vessel (microtiter plate) with adsorbed antigen or monoclonal antibody, - conjugate of antibody and enzyme, Substrate with addition of a chromogenic substance and - Optional stop reagent plus various washing and dilution buffers as well as various control or normative standards is to be classified, characterized in that one a) a conjugate with a beta-lactam hydrolase as enzyme component and a beta-lactam as substrate and b) uses an iodine inclusion compound as a chromogenic substance. 2. The method according to claim 1, characterized in that one uses a conjugate with a beta-lactamase as the enzyme component in combination with a monoclonal antibody or with an anti-antibody as a reactant and with an organic polymer as a carrier material. 3. The method according to claim 1 and 2, characterized in that one uses a conjugate with a beta-lactamase in combination with an anti-gamma globulin antibody and with a polysaccharide. 4. The method according to claim 1 to 3, characterized in that a conjugate with a beta-lactamase in combination with an anti-IgG antibody and with a polysaccharide from the collection Sucrose polymers of different degrees of polymerization, - Other sugars and their derivatives of different degrees of polymerization or - Dextrans and their derivatives of different degrees of polymerization begins. 5. The method according to claim 2 to 4, characterized in that a conjugate with Bacillus licheniformis beta-lactamase, in combination with an anti-IgG antibody from the collection anti-Fab antibodies, anti-Fc fragment antibodies or - anti-gamma chain antibodies as well as with a sucrose polymer from the collection - Ficoll 400 resp. - Ficoll 70
starts. 6. The method according to claim Γ to 4, characterized in that a conjugate with Bacillus licheniformis beta-lactamase, in combination with an anti-IgG antibody from the collection anti-Fab antibodies, anti-Fc fragment antibodies or - anti-gamma chain antibodies as well as with a dextran derivative from the collection - Dextran 70, - Dextran 234 or - Dextran 500
starts. 7. The method according to claim 2 to 6, characterized in that one prepares a stable conjugate of the above labeling in each case by - The selected support material is oxidized with sodium periodate and dialyzed overnight against a sodium acetate solution (1 mM, pH 4.4), the selected antibodies are mixed with beta-lactamase in a ratio of 1.5: 1 and dialyzed overnight against phosphate buffered saline, - the components antibody-beta-lactamase mixture to oxidized carrier material! in a weight ratio of 5: 1, immediately adjust the pH of the resulting mixture to pH 9.6 with sodium carbonate solution, incubated for 3 to 4 hours at room temperature and then reduced free oxidized groups of the support material with sodium borohydride, the resulting conjugate is dialyzed overnight against phosphate buffered saline and - The liquid product obtained hereafter optionally stabilized with bovine serum albumin and lyophilized or treated with thiomersal and glycerol for preservation. 8. The method according to claim 1 to 7, characterized in that one additionally uses a beta-lactam hydrolase inhibitor. 9. The method according to claim 1 to 8, characterized in that one - penicillin, ampicillin or a derivative of these beta-lactams as substrate, - an iodine-starch complex as a chromogenic substance as well - Uses a synthetic beta-lactamase inhibitor as a stop reagent. 10. The method according to claim 1 to 8, characterized in that one - Penicillin, ampicillin or a derivative of this beta-Laktamö as a substrate, - An iodine-polyalcohol complex as a chromogenic substance and a synthetic beta-lactamase inhibitor
starts. 11. The method according to claim 10, characterized in that one Benzylpenicillin as substrate, - An iodine-polyvinyl alcohol complex as a chromogenic substance as well - Sulbactam as a stop reagent
starts. 12. The method according to claim 9 to 11, characterized in that per microtiter plate 4mg Benzylpenicillin dissolved in 7.5 ml of phosphate-buffered saline, the immediately before 2.5 ml of iodine-polyvinyl alcohol complex have been added. 13. The method according to claim 1 to 12, characterized in that one phosphate buffered saline solution containing 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer - as dilution buffer phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and optionally with 0.1% bovine serum albumin or 5% gelafusal and - Pooled or correspondingly concentrated positive samples as positive control standard and negative samples pooled as negative control standard or natural or recombinant antigens highly purified as normative standard starts. 14. The method according to claim 1 to 13, characterized in that one builds up using the above-mentioned components test kits of the type of solid-phase sandwich enzyme immunoassay. 15. The method according to claim 14, characterized in that one builds up using the above-mentioned components test kits of the type of non-competitive solid-phase sandwich enzyme immunoassay. 16. The method according to claim 1,4,5,7,11 to 13 and 15, characterized in that one resorting to recombinant (gp41 + gag) HIV1 protein as antigen adsorbed to the microtiter plates, Baccillus licheniformis be ^ a-lactamase in combination with rabbit anti-human IgG antibody or rabbit anti-human gammat chain antibody and with Ficoll 400 as a stable conjugate, Benzylpenicillin as substrate, an iodine-polyvinyl alcohol complex as chromogenic substance and sulbactam as stop reagent, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 5% Gelafusal as dilution buffer and - positive serum pooled by at least 8 HIV-1 infected patients as a positive control standard and negative serum pooled by 50 healthy volunteers as a negative control standard builds a non-competitive solid-phase sandwich enzyme immunoassay kit that can be used as a screening test in HIV 1 antibody diagnostics. 17. The method according to claim 1,4,5,7,10 to 13 and 15, characterized in that one resorting to monoclonal antibodies to the HBV surface antigen as monoclonal antibodies adsorbed to the microtiter plate, Bacillus Hcheniformis beta-lactamase in combination with anti-HBV surface antigen antibody and with Ficoll 400 as a stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 0.1% bovine serum albumin as dilution buffer and - Positive serum pooled by 8 patients as a positive control standard and negative serum pooled by 20 healthy volunteers as a negative control standard builds up a non-competitive solid-phase sandwich enzyme immunoassay kit that can be used as a screening test in HBV surface antigen diagnostics. 18. The method according to claim 1,4,5,7,10 to 13 and 15, characterized in that one resorting to monoclonal antibody to the tropophilic glial glycoprotein (TBG) as monoclonal antibody adsorbed to the microtiter plate, Bacillus licheniformis beta-latamase in combination with monoclonal antibody against TBG and with Ficoll 400 as stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 0.1% bovine serum albumin as dilution buffer and - serum pooled by pregnant women as a positive control standard and serum pooled by non-pregnant women as a negative control standard builds a non-competitive solid phase sandwich enzyme immunoassay type kit useful in TBG antigen diagnostics. 19. The method according to claim 1,4,5,7,10 to 13 and 15, characterized in that one resorting to monoclonal antibodies to the rotavirus surface antigen as monoclonal antibodies adsorbed to the microtiter plate, - Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against the rotavirus surface antigen and with Ficoll 400 as a stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolauratln = 20) and with 0.1% bovine serum albumin as dilution buffer and - the supematant of a 10% solution of faeces in phosphate buffered saline after centrifugation as positive control standard, pooled by 10 patients with rotavirus infections, and an equivalent supernatant of the faeces of 20 healthy volunteers as a negative control standard builds up a non-competitive solid phase sandwich enzyme immunoassay kit to be used as a sieve test in rotavirus surface antigen diagnostics. 20. The method according to claim 1,4,5,7,10 to 13 and 15, characterized in that one resorting to monoclonal anti-tumor necrosis factor (TNF) antibodies as monoclonal antibodies adsorbed to the microtiter plate, Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against TNF and with Ficoll 400 as stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as a washing puff, - Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (r, = 20) and 5% gelafusal as dilution buffer and recombinant TNF protein as standard builds a non-competitive solid phase sandwich enzyme immunoassay type kit useful in tumor diagnostics and therapy monitoring. 21. Test kit for the detection of immunocomponents of the basic type of an enzyme immunoassay with the components Reaction vessel (microtiter plate) with adsorbed antigen or monoclonal antibody, - conjugate of antibody and enzyme, Substrate with addition of a chromogenic substance, optionally stop reagent plus various washing and dilution buffers as well as various control or normative standards, characterized in that it a) a conjugate with a beta-lactam hydrolase as enzyme component and a beta-lactam as substrate and b) has an iodine inclusion compound as a chromogenic substance. 22. Test kit according to claim 21, characterized in that it comprises in the conjugate a beta-lactamase as enzyme component in combination with a monoclonal antibody or an anti-antibody as a reactant and with an organic polymer as a carrier material. 23. Test kit according to claim 22, characterized in that it comprises in the conjugate Bacillus lichenichenisbeta lactamase in combination with an anti-gamma globulin antibody and with a polysaccharide. 24. Test kit according to claim 22 and 23, characterized in that it contains in the conjugate Bacillus licheniformis beta-lactamase in combination with an anti-IgG antibody and with a polysaccharide from the following collection Sucrose polymers of different degrees of polymerization, - other sugars and their derivatives of different degrees of polymerization or - Dextrans and their derivatives have different degrees of polymerization. 25. Test kit according to claim 24, characterized in that it contains in the conjugate Bacillus licheniformis beta-lactamase in combination with an anti-IgG antibody and either with one of the sucrose polymers Ficoll 400 or Ficoll 70 or with one of the dextran derivatives Dextran 70, Dextran 234 or Dextran 500. 26. Test kit according to claim 22 to 24, characterized in that it comprises in the conjugate Bacillus licheniformis beta-lactamase in combination with an anti-Fab, an anti-Fc fragment or an anti-gamma chain antibody and with a polysaccharide following collection Sucrose polymers of different degrees of polymerization, - Other sugars and their derivatives of different degrees of polymerization or - Dextranes and their Derrivate different degree of polymerization has. 27. Test kit according to claim 25 and 26, characterized in that it in the conjugate Bacillus licheniformis beta-lactamase in combination with an anti-Fab, an anti-Fc fragment or an anti-gamma chain antibody and either with one of the sucrose polymers Ficoll 400 or Ficoll 70 - or with one of the dextran derivatives dextran 70, dextran 234 or dextran 500 has. 28. Testkit according to claim 21 to 27, characterized in that it additionally comprises a beta-lactam hydrolase inhibitor. 29. Test kit according to claim 21 to 28, characterized in that br penicillin, ampicillin or a derivative of these beta-lactams as a substrate, an iodine-starch complex as a chromogenic substance and a synthetic beta-lactamase inhibitor as a stop reagent , 30. Test kit according to claim 21 to 28, characterized in that it comprises penicillin, ampicillh or a derivative of these beta-lactams as a substrate, an iodine-polyalcohol complex as the chromogenic substance and a synethetic beta-lactamase inhibitor as a stop reagent , 31. Testkist according to claim 30, characterized in that it comprises benzylpenicillin as a substrate, an iodine-polyvinyl alcohol complex as a chromogenic substance and sulbactam as a stop reagent. 32. Testkit according to claim 29 to 31, characterized in that it phosphate buffered saline solution containing 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer - as dilution buffer phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and optionally with 0.1% bovine serum albumin or 5% gelafusal and - pooled as positive control standard or correspondingly concentrated positive samples and negative samples pooled as a negative control standard or natural or recombinant antigens highly purified as a normative standard having. 33. Test kit according to claim 21 to 32, characterized in that it has the structure of a solid phase sandwich enzyme immunoassay. 34. Test kit according to claim 33, characterized in that it has the structure of a non-competitive solid-phase sandwich enzyme immunoassay. 35. Test kit according to claim 21,22,25 or 26,28,31,32 and 34, characterized in that it is used as a non-competitive sandwich enzyme immunoassay recombinant (gp41 + gag) HIV1 protein as antigen adsorbed to the microtiter plates, Bacillus licheniformis beta-lactamase in combination with rabbit anti-human IgG antibody or rabbit anti-human gamma chain antibody and with Ficoll 400 as a stable conjugate, Benzylpenicillin as substrate, an iodine-polyvinyl alcohol complex as chromogenic substance and sulbactam as stop reagent, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 5% Gelafusal as dilution buffer and - Positive serum pooled by 20 patients as a positive control standard and negative serum pooled by 50 healthy volunteers as a negative control standard and can be used as a sieve test in HIV 1 antibody diagnostics. 36. Test kit according to claim 21, 22, 25, 31, 32 and 34, characterized in that it is used as a non-competitive sandwich enzyme immunoassay monoclonal antibodies to the HBV surface antigen as monoclonal antibodies adsorbed to the microtiter plates, Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against HBV surface antigen and with Ficoll 400 as stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, Phosphate buffered saline solution containing 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and 0.1% bovine serum albumin as diluent buffer and - Positive serum pooled by 8 patients as a positive control standard and negative serum pooled by 20 healthy volunteers as a negative control standard and can be used as a sieve test in HBsAg antigen diagnostics. Test kit according to claims 21, 22, 25, 31, 32 and 34, characterized in that it is used as a non-competitive sandwich enzyme immunoassay monoclonal antibodies to the trophoblastic beta 1-glycoprotein (TBG) as monoclonal antibodies adsorbed to the microtiter plate, - Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against TBG and with Ficoll400 as stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate-buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and 0.1% bovine serum albumin as dilution buffer and serum pooled by pregnant women as a positive control standard and serum pooled by non-pregnant women as a negative control standard and can be used in TBG antigen diagnostics. Test kit according to claims 21, 22, 25, 31, 32 and 34, characterized in that it is used as a non-competitive sandwich enzyme immunoassay monoclonal antibodies to the rotavirus surface antigen as monoclonal antibody adsorbed to the microtiter plate, - Baccillus licheniformis beta-lactamase in combination with monoclonal antibody to the rotavirus surface antigen and with Ficoll400 as a stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 0.1% bovine serum albumin as dilution buffer and - the supernatant of a 10% suspension of faeces in phosphate buffered saline, pooled by 10 patients with rotavirus infections, after centrifugation as a positive control standard and an equivalent supematant of faesces from 20 healthy volunteers as a negative control standard and can be used in rotavirus surface antigen diagnostics. Test kit according to claims 21, 22, 25, 31, 32 and 34, characterized in that it is used as a non-competitive sandwich enzyme immunoassay monoclonal anti-tumor necrosis factor (TNF) antibodies as monoclonal antibodies adsorbed to the microtiter plate, Bacillus licheniformis beta-lactamase in combination with monoclonal antibody against TNF and with Ficoll400 as stable conjugate, Benzylpenicillin as a substrate and an iodine-polyvinyl alcohol complex as a chromogenic substance, Phosphate buffered saline with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) as wash buffer, - Phosphate-buffered saline solution with 0.05% polyoxyethylene sorbitan monolaurate (n = 20) and with 5% Gelafusal as dilution buffer and recombinant TNF protein as normative standard and can be used in tumor diagnostics and therapy monitoring.