DD279317A1 - TEST ASSAY FOR IMMUNE-CYMOMETRIC DETERMINATION OF HUMAN-INTERFERON-ALPHA-1 - Google Patents

Abstract

The invention relates to a test kit for immunoenzymometric determination of human interferon alpha-1 for medical diagnostics, basic biomedical research and process control in biotechnological interferon production. The invention test kit is characterized in that it consists of: the first fixed on the solid phase monoclonal antibodies (mAb) ZIM IVH2 (ZIM-0396) or ZIM VD11 (ZIM-0397) - the second, labeled with an enzyme mAK ZIM -IIG11 (ZIM-0087), an interferon-alpha-1 standard preparation, the chromogen or a chemiluminescent substance and the enzyme substrate. Essential to the invention is the selection of the respective antibody pair, both mAb binding to spatially distinct antigenic determinants. The first mAb against human interferon-alpha-1 is adsorbed to the solid phase and the interferon-containing solution added. The bound interferon-alpha-1 is quantitatively determined via the enzyme activity after reaction with the second MAb, which is enzyme-labeled. The test system according to the invention permits quantitative interferon-alpha-1 detection in the range from 4 IU / ml to 500 IU / ml. The total duration of the assay is 212-4 hours and is specifically possible in body fluids from the fermentation of IFN-producing cells (human cells and genetically engineered animal and microbial cells).

Description

Field of application of the invention

The invention relates to a method for carrying out quantitative determinations of human interferon-alpha-1. The invention can be used in medical diagnostics, basic biomedical research and biotechnological interferon initiation.

Characteristic of the known state of the art

Interferons (IFN) are endogenous substances which are formed in the context of the defense reaction of the organism against viral infections and prevent the spread of infection. In addition to the antiviral effect, the FNs also have proliferation-inhibiting and immunomodulatory activity and are therefore of great medical and biological interest. The heterogeneity of this class of substances makes the availability of molecular-specific determination methods necessary.

So far, the quantitative detection of interferons on the determination of their antiviral activity, also on an international scale. In this case, the inhibition of an infection spread in an in vitro cell culture is measured. The unit is defined as the reciprocal of the IFN dilution which either inhibits virus plaque formation by 50% or protects 50% of the cell lawn from destruction by a test virus. By international standards, the read test units are translated into international units (IU). converted. An IU corresponds to 1-Opg human interferon-alpha.

This method of concentration determination of IFN is very time consuming, labor intensive and requires a costly, susceptible cell culture. The error range of the test is 50% and more.

Since the availability of monoclonal antibodies (mAbs) to human interferon-alpha (eg Secher, DS and DC Burke, Nature 285, 446-450 / 1980 + US Pat. No. 4,423,147), several immunochemical methods have been described to be faster, safer and more accurate than to be able to determine IFN concentrations in solutions with the biological test.

Such immunochemical methods based on the use of monoclonal antibodies may be based on several test principles:

a) Immunoradiometric Methods "IRMA" DSSecher, Nature290, 501-502 (1981) Horovitz et al .: Immunenzymatic Techniques, ed S. Avrameas, Elsevier, Amsterdam 1983, p 193, Stachelin, T. et al., Methods Enzymol 1279, 589 (1981)

b) Competitive binding assays as radioimmunoassay Meurs, E. et al., Infect. Immune. 37, 919-926 (1982); Trotta, P. et al., J. Biol. Resp. Mod. 2,348-359 (1983); Bernier, J. et al., J. Nucl. Medicine 25, 765-772 (1984)

c) enzyme immunoassays

Gallati, H.J., Clin. Chem. Clin. Biochem. 20, 907-914 (1982) Berthold, W. et al .: Arzneimittelforsch. 3G, 364-369 (1985). The methods described utilize mAb or polygonal antibodies to hiiman interferon-alpha in various combinations and assays, using solid-phase systems and methods in solution. The achieved test parameters vary with regard to sensitivity, duration and workload.

The previously published methods concern either human interferon-alpha-2 or leucocyte interferon (human interferon-alpha). The company Cell Tech sells a test kit with the "YOK" mAK with which it could be possible to determine human interferon-alpha-1 without being shown in the prospectus.

For human interferon-alpha-1, there is an immunochemical method of determination wherein a polyclonal antibody as a first layer and a monoclonal antibody labeled with peroxidase as a second layer are used. This test has a sensitivity of 30GIU / ml. The disadvantage of this method is the use of the polyclonal antibody whose standardized production can not be guaranteed in terms of quality and quantity. The sensitivity of 300 IU / ml is too low, since, according to previously available concentrations for IFN in the serum of normal persons, obtained with the biological test, the detection limit below 10IE / ml for an applicable iminur.enzymoitietrischen test.

Object of the invention

The aim of the invention is to provide a test kit with which interferon-alpha-i can be determined specifically, quantitatively, quickly and precisely in still very low concentrations. It sol! this is an environmentally friendly process that eliminates the use of radioactivity.

Explanation of the essence of the invention

The test kit according to the invention for immuno-enzymometric determination of human interferon-alpha-1 is characterized in that

that it consists of:

- The first fixed to the solid phase mAb ZIM-IV H 2 (ZIM-0396) or its Fab fragments

the second enzyme-labeled mAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments

- the buffer for recording the marked MAC

- the IFN standard preparation

- the substrate buffer

- the enzyme substrate

- the chromogen or a luminizing substance with Varstärkerreagenz or a fluorochrome or that it consists of:

the first fixed phase mAb ZIM-VD11 (ZIM-0397) or its Fab fragments

the second enzyme-labeled mAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments

- the buffer for recording the marked MAC

- the IFN standard preparation

- the substrate buffer

- the enzyme substrate

- The chromogen or a luminescent substance with amplifier reagent or a fluorochrome or that it consists of:

- a microplate (MTP)

- the first mAK ZIM-IV H2 (ZIM-0396) or its Fab fragments

the second enzyme-labeled mAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments

- the IFN standard preparation

Coating buffer for coating the MTP with the first MAK

- Buffer for recording the marked mAK

- the substrate buffer

- the enzyme substrate

- The chromogen or a luminescent substance with amplifier reagent or a fluorochrome or that it consists of:

- an MTP

- the first mAb ZIM-V D11 (ZIM-0397) or its Fab fragments

- _# the second, enzyme-labeled mAb ZIM-IIG11 (ZIM-0087) or

its enzyme labeled Fab fragments

- the IFN standard preparation

Coating buffer for coating the MTP with the first MAK

- the substrate buffer

- the enzyme substrate

According to the invention, an immunoenzymometric two-side binding test is used for the quantitative determination of human interferon-alpha-1 using a pair of two monoclonal antibodies directed against spatially distinct epitopes of the human interferon alpha-1 immunoreactive agent. 1 are used, is used. The first monoclonal antibody is adsorbed to the surface of the wells of reaction tubes of plastic or other material, primarily 96-well polystyrene microtest plates, for solid phase immunoassay.

The IFN solutions to be measured are added to the antibody-coated reaction vessels and incubated. The second MAb, which is labeled with an enzyme, preferably with peroxidase, is incubated either together with the IFN-containing solution or after removal of this solution. U: 3 detection reaction is carried out in usually photometrically with o-phenylenediamine and H ₂ O ₂ or luminometrically with luminol / H ₂ O ₂ or its derivatives and an amplifier reagent (iodophenol, phenylphenol) or fluorometrically with a fluorochrome.

The essence of the invention is that a pair of monoclonal antibodies is selected for the test, each of which, due to its properties, such as recognition of different epitopes, sorption capacity on plastic surfaces, affinity constants and markability, a very sensitive, precise and rapid determination of the human interferon -alpha-1. At least one of the MAbs used must react exclusively with human interferon alpha-1.

According to the invention, the high test sensitivity is achieved by the selection of the following antibody pairs or their fragments or derivatives of the cell lines

ZIM-IVH 2 (ZIM-0396) and ZIM-IIG 11 (ZIM-087)

or ZIM VD11 (ZIM-0397) and ZIM-IIG11 (ZIM-0087)

reached. The first mentioned MAb is sorbed to the solid phase, the second is labeled with an enzyme. The used mAb ZIM-IVH2 or ZIM-VD11 are HulFN-alpha-1 subtype specific. They belong to the immunoglobulin class IgGI and recognize the same epitope on the antigen. The mAK ZIM-IIG11 was described in WP G01 N / 295281.7. For labeling any of the known marker enzymes can be used, but preferably peroxidase, because then the detection reaction using appropriate substrates, eg. As o-phenylenediamine, p-hydroxyphenylpropionic acid or luminol can be photometrically, fluorimetrically or luminometrically.

In accordance with the invention, stabilized MAK precoated MTPs are preferably used, the sorbed AK being stabilized by the addition of BSA, sucrose and other substances (bacteriocides). The functionality of this coated MTP can be ensured in the absence of moisture and storage at 4 ° for 1-2 years. The user can also make the coating of the MTP with the first mAK itself.

In both cases, the unspecific adsorption sites on the mAK-coated surface of the reaction vessels are then blocked by buffer solutions containing NH ₂ groups and / or solutions containing prctein, such as gelafusal. "According to the invention, the IFN-containing compounds to be measured are either sequentially or simultaneously After thorough washing with tap water, the photometric, luminomotric or fluorimetric detection reaction is carried out with the aid of a substrate.

The advantageous process characteristics of the immunoenzymometric test kit according to the invention, such as the very high sensitivity, the short test times and the specificity for HulFN-alpha-1 without cross-reactivity with HulFN-alpha-2, are achieved by the monoclonal antibody pairs used according to the invention and the use of the double-sided binding technique, the Enzyme labeling of the monoclonal antibody and the sensitive detection reaction achieved. In order to ensure consistent quality, the use of two monoclonal antibodies is of crucial importance and has enormous advantages over the use of polyclonal antibodies. The test kit with pre-coated MTP is user friendly, easy and quick to perform. In addition, it is environmentally friendly, since no radioactive waste problems occur. It can be carried out in any laboratory without specially trained personnel and without special equipment. The use of these test kits is also material-saving because z. B. all washing steps with tap water can be made.

The test kit is suitable for both natural and recombinant human interferon-alpha-1. In addition, it is possible to selectively human lr, terferon-alpha-1 in different samples, eg. As serum, hybrid interferon preparations and culture solutions from the fermentation of IFN-producing cells (human cells and genetically engineered animal and microbial cells) to determine.

The test system according to the invention permits a quantitative interferon-alpha-1 detection in the range from 4 IU / ml to 500 IU / ml. The total duration of the test procedure is 2V-4 hours.

Exemplary content of the test kit

example 1

• an MTP whose wells were coated with 10ΰμΙ each of a solution of 20pg ZIM-IV H2 / ml coating buffer; the blocking of the non-specific binding sites was carried out with 5% sucrose, 0.5% bovine serum albumin; 0.02% merthiolate

• the second mAb ZIM-IIG11 labeled with horseradish peroxidase

• Human interferon-alpha-1 standard preparation

Dilution Buffer I: 0.5% bovine serum albumin-0.02M K ₂ HPO ₄ / 0.02M KH ₂ PO ₄ -0.15M NaCIpH 7.3 / 0.1% Tween 20 (PBS / Tween)

Dilution Buffer II: 10% Gelafusal ^R -PBS / Tween

Substrate buffer (0.15M Na ₂ HPO ₄ , 0.07M citric acid pH 5.0)

• O-phenylenediamine

• H ₂ O ₂

Example 2

• an MTP whose wells were coated with ΙΟΟμΙ each of a solution of 1 μg ZIM-VD11 / ml coating buffer; the blocking of the non-specific binding sites was carried out with 5% sucrose, 0.5% bovine serum albumin; 0.02% merthiolate

• the second mAb ZIM-IIG11 labeled with horseradish peroxidase

• Human interferon-alpha-1 standard preparation

Dilution buffer I: 0.5% bovine serum albumin - PBS / Tween

Dilution Buffer II: 10% Gelafusal PBS / Tween

Substrate buffer (0.15M Na ₂ HPO ₄ , 0.07M citric acid pH 5.0)

• 0-phenylenediamine

• H ₂ O ₂

Exemplary embodiment 1 for test packets 1 + 2

In the well of the coated plate 100μΙ of a standard solution or the sample to be examined are filled. The dilution is carried out with dilution buffer I. Subsequently, it is incubated in a moist chamber for 1 hour with shaking. The solutions are aspirated with a water jet pump and the microplate is washed 3 times with tap water.

Then, 100 μl of the peroxidase-labeled second monoclonal antibody (dilution 1: 1000 with dilution buffer II) are introduced and incubated for 1 hour at room temperature and with shaking in a moist chamber After washing at least 5 times with tap water, 200 μl of a substrate solution are introduced into the individual wells. eg the following composition

O-Phonylenediamine 0.4 mg / ml H ₂ O ₂ 0.015% Phosphate citrate buffer pH 5.0

pipetted in and after 15 minutes with 50 μl of 2.5N H ₂ SO ₄ the color reaction was stopped. The color intensity in the individual wells is measured photometrically at a wavelength of 492 nm. From the measured absorbance, the human interferon alpha-1 content of a sample can be determined from the standard curve.

Exemplary embodiment 2 for test packets 1 + 2

In the wells (wells) of the coated plate 50μΙ of a standard solution or the sample to be examined and 50μΙ of the peroxidase-labeled second monoclonal antibody (dilution 1: 500) are filled.

Incubate for 1 hour at room temperature in a humidified chamber with shaking. After washing at least 5 times with tap water, each 200μΙ of a substrate solution, e.g. following composition

O-phenylenediamine 0.4 mg / ml H ₂ O ₂ 0.015% phosphate citrate buffer pH 5.0

pipetted in and stopped after 15 minutes with 50μΙ2,5Ν H ₂ SO _4, the color reaction. The color intensity in the individual cuvettes is finally measured photometrically at a wavelength of 492 nm. From the measured absorbance, the standard curve of the human interferon-alpha-1 content of a sample can be determined.

Contents of the test kit Example 3

φ a MTP

• mAK ZIM-IV H2 for coating the MTP

• the second mAb ZIM-IIG11 labeled with horseradish peroxidase

• Human interferon-alpha-1 standard preparation

Dilution buffer I: 0.5% bovine serum albumin PBS / Tween

Dilution Buffer II: 10% Gelafusal "PBS / Tween

Coating buffer: 0.05M Na ₂ CO ₃ ; 0,035M NaHCO ₃ pH 9.6

Substrate buffer: 0.15M NaHPO ₄ ; 0.07 M citric acid pH 5.0

• O-Pheny! Endiamin. H ₂ O ₂

Example 4 an MTP

mAK ZIM-VD11 for coating the MTP

second mAb ZIM-IIG11 labeled with horseradish peroxidase Human interferon-alpha-1 standard preparation Dilution buffer I: 0.5% bovine serum albumin PBS / Tween dilution buffer II: 10% Gelafusal® PBS / Tween coating buffer: 0.05M Na ₂ CO ₃ 0.035M NaHCO ₃ pH 9.6 Substrate Buffer: 0.15M NaHPO ₄ ; 0.07M Citric Acid pH 5.0 O-phenylenediamine H ₂ O ₂

Version3 example 1 for test packs 3 + 4

In each of the wells of the MTP, ΙΟΟμΙ of the AK solution is pipetted. The concentration is 20 μg / ml coating buffer ZIM-IV H2 (Example 3) and 1 μg / ml coating buffer ZIM-V D11 (Example 4).

Drying takes place at 37 ° C for 16-20 hours. The unspecific binding sites are then blocked with 200μΙ Dilution Buffer II for 1 hour. After vigorous deflection, ΙΟΟμΙ of a standard solution or of the sample to be examined are introduced into the wells of the coated plate. The dilution is carried out with dilution buffer I for one hour with shaking. It is then incubated in a humid chamber. The solutions are aspirated with a water jet pump and the microplate is washed 3x with tap water.

Then, 100 μl of the peroxidase-labeled second monoclonal antibody (dilution 1: 1000 with dilution buffer II) are introduced and incubated for 1 hour at room temperature and with shaking in a moist chamber After washing at least 5 times with tap water, 200 μl of a substrate solution are added to the individual wells. eg

O-phenylenediamine 0.4mg / ml H ₂ O ₂ 0.015% phosphate citrate buffer pH 5.0

pipetted in and after 15 minutes with 50 μί 2.5 NH ₂ SO ₄ stopped the color reaction. The color intensity in the individual wells is finally measured photometrically at a wavelength of 492 nm. From the measured absorbance, the human interferon alpha-1 content of a sample can be determined from the standard curve.

Exemplary embodiment 2 for test packets 3 + 4

Each 100 μΙ of an AK solution is pipetted into the wells of the MTP. The concentration is 20 μg / ml coating buffer ZIM-IV H 2 (Example 3) and 1 μg / ml coating buffer ZIM-V D11 (Example 4).

The initial drying is carried out for 16-20 hours at 37 ⁰ C. The nonspecific binding sites were then blocked with 200μΙ Diluent Il for 1 hour.

In the wells of the coated plate 50μΙ of a standard solution or the sample to be examined and 50μΙ of the peroxidase-labeled second monoclonal antibody (dilution 1: 500) are filled. Incubate for 1 hour at room temperature in a humidified chamber with shaking.

After washing with tap water at least 5 times, 200 μΙ each of a substrate solution, e.g. B.

o-phenylenediamine 0.4mg / ml H ₂ O ₂ 0.015% phosphate citrate buffer pH 5.0

pipetted in and after 15 minutes with 50μΙ 2.5NH ₂ SO ₄ stopped the color reaction. The color intensity in the individual wells is finally measured photometrically at a wavelength of 492 nm. From the measured absorbance, the human interferon alpha-1 content of a sample can be determined from the standard curve.

Claims (6)

1. Test kit for the rhino-enzymometric determination of human interferon-alpha-1, characterized in that it consists of: - The first, fixed to the solid phase mAb ZIM-IV H2 (ZIM-0396) or its Fab fragments the second, enzyme-starved MAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments - the buffer for recording the marked MAC - the IFN standard preparation - the substrate buffer - the enzyme substrate - The chromogen or a luminescent substance with amplifier reagent or a fluorochrome or that it consists of: the first fixed phase mAb ZIM-VD11 (ZIM-0397) or its Fab fragments - The second enzyme-labeled mAK ZIM-IIG11 (ZIM-0087) or desse ι enzyme-labeled Fab fragments - the buffer for recording the marked MAC - the IFN standard preparation the substrate buffer. the enzyme substrate - The chromogen or a luminescent substance with amplifier reagent or a fluorochrome or that it consists of: - a microtest plate (MTP) - the first mAK ZIM-IV H2 (ZIM-0396) or its Fab fragments the second, mix enzyme labeled mAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments - the IFN standard preparation Coating buffer for coating the MTP with the first MAK - Buffer for recording the marked mAK - the substrate buffer - the enzyme substrate the chromogen oaer a luminescent substance with amplifier reagent or a fluorochrome or that it consists of: - an MTP - the first mAb ZIM-V D11 (ZIM-0397) or its Fab fragments the second enzyme-labeled mAb ZIM-IIG11 (ZIM-0087) or its enzyme-labeled Fab fragments - the IFN standard preparation Coating buffer for coating the MTP with the first MAK - Buffer for recording the marked mAK - the substrate buffer - the enzyme substrate - The chromogen or a luminescent substance with amplifier reagent or a fluorochrome. 2. Test kit according to claim 1, characterized in that the mAb are used as Fab fragment or as derivatives. 3. Test kit according to claim 1, characterized gekonnzeichnet that the solid phase is a 96-well microtest plate. 4. Test kit according to claim 1, characterized in that the enzyme on the second mAK is Meerrett'ch peroxidase. 5. Test kit according to claim 1, characterized in that the cromogen o-phenylenediamine or ABTS (2,2-azido-di-3-ethylbenzthiazolin-sulfonate). 6. Test kit according to claim 1, characterized in that the luminescent substance luminol and its derivatives and the booster reagent is iodophenol or phenylphenol.