FR2631349A1 - ENZYME IMMUNOLOGICAL ASSAY SYSTEM - Google Patents

Abstract

This invention relates to an enzyme immunoassay system for use in the detection and/or quantitation of one or more antibodies in a sample. In use, an antibody containing sample is drawn over a solid phase support (1), which is coated with an antigen, wherein the antibody containing sample reacts. Unbound antibody containing sample is washed from the system and an immunoassay conjugate, consisting of an active receptor for immunoglobulin and an active marker enzyme is drawn over the previously reacted antigen coated solid phase support (1). Unbound conjugate is washed from the system and an assay for enzymatic activity of the marker enzyme is performed by incubating the previously reacted antigen coated solid phase support (1) with a suitable substrate.

Description

 The present invention relates to a new immunoLogical assay method, the reagents used in this assay and their use, and the kits for carrying out this assay.

 More particularly, the present invention relates to a new reagent for binding and detecting antibodies, and the kits and methods incorporating said reagent.

Enzyme Immunoassay (EIA) systems are
Widely used in the serodiagnosis of a large number of diseases, in particular those involving viral agents. These assay systems not only have the many initial benefits of radioimmunoassay (RIA) systems, but are also easier, safer and more economical than RIA systems. Although the EIA system offers potential for high quality serological testing, it has not been exploited to an extensive degree so far. Such a test should ideally be rapid, economical, simple to conduct and interpret, should use robust reagents and be safe to use outside the laboratory. No configuration of the EIA system currently available meets all these criteria.,
A number of enzymes have been widely used in the EIA system including horseradish peroxidase, alkaline phosphatase and p-galactosidase. However, many recent reports (Yolken et al., J. Imm. Methods 73, 109-123, 1984; Wagla et al., Indian J. Med.Res. 81, 275-280, 1985) show p-lactamase as a marker for enzyme immunoassays (EIA). Its high availability, its low price, its high renewal speed at ambient temperature, its substrate on and its total absence of mammalian tissues, compare it favorably with the high cost, the limited availability and the potentially carcinogenic substrates of other enzyme systems. Likewise, the activity of p-lactamase can be determined visually by the rate of discoloration of the polyvinyl alcohol (PVA) / dark blue iodine complex as with the iodination of penicilloic acid, the final product of the hydrolysis of penicitline with p-lactamase.

The synthesis of an enzyme conjugate with a molecule which reacts specifically with immunoglobulins offers wide application. Previous reports have described the covalent binding of ss-lactamase to polyclonal antibodies, monoclonal antibodies and avidin and the use of these enzyme protein conjugates in EIA systems (Yolken et al., J. Imm. Methods 73, 109-123, 1984, Wagle et al., Indian
J. Med. Res 81, 275-280, 1985). However, an enzyme conjugate with staphylococcus A protein or streptococcal protein
G, which specifically binds to the Fc region of immunoglobulins from a large number of species, could provide a universal tool for the detection of these antibodies. In addition, the lactamase / protein A or G conjugates retain affinity significant for the four human IgG subclasses. Although these two proteins specifically bind to the Fc region of immunoglobulins, they do not interfere with the ability of said immunoglobulins to bind to an antigen. In addition, ss-lactamase can be linked to protein A or protein G to give conjugates which retain a significant proportion of their enzymatic and immunological activity.

 The concept of using protein A together with ss-lactamase in an enzyme immunoassay system was presented by Ambedkar et al. (Hindustan Antibiotics Bulletin 29, 4-5, 1987) and Citri Israeli patent application no. 80313 filed October 15, 1986).

The present invention relates to a novel EIA system which uses an immunoglobulin binding protein conjugated to an enzyme suitable for the detection and / or quantification of antibodies against one or more specific antigens in a sample. The EIA system implements :: (a) a solid phase support, the surface of which must be exposed
sée to the sample containing the antibody, is coated with
The specific antigen, (b) the reaction of the sample containing the antibody with the anti
gene linked to the support, so that the antibodies present in
sample that are specific for the bound antigen bind
audit related antigen,
and washing to remove excess unbound antibody, and (c) Incubating the bound antibody using a conjugate of
immunoassay consisting of an active receptor for
The immunoglobulin that binds to the bound antibody, and a
active enzyme marker,
and washing with an aqueous solution to remove excess
unbound conjugate, and (d) Assaying the enzyme activity of the enzyme label by
incubation with a suitable substrate, for example, a
substrate which leads to a visible color change of a
suitable reagent, in which the enylhatic substance is a
enzyme marker for bound immunoglobulin and indicates the
presence of the antibody.

 The present invention therefore provides an immunoassay reagent for binding and detecting antibodies, said reagent comprising an active receptor for immunoglobulin and an active enzyme marker.

 In addition, the specific binding assay method in accordance with the present invention can be applied to the detection and / or quantification of a hapten or an antigen in a sample, consisting of a liquid medium, by an assay. sandwich immunoassay according to which the "capture antibody" is immobilized on the solid surface to capture the hapten or the antigen present in the sample. A "second" antibody to the hapten or the antigen is then applied to form a "sandwich", and the binding of this second antibody can be revealed by incubation using the immunoglobulin receptor. labeled enzymatically (for example protein G / protein A) and by determination of the enzymatic activity (as in steps (c) and (d) mentioned above. The only requirement of this method is that the binding receptor of the enzyme labeled immunoglobulin does not bind to the capture antibody.

This requirement can be satisfied by using capture antibodies which do not bind to the immunoglobulin binding receptor (for example in the case of protein G, IgM is used and in the case of protein A, using IgY from chicken serum or egg yolk.

 An alternative method for detecting and / or quantifying the hapten or the antigen in a sample consisting of a liquid medium, consists in adding to the sample an antibody specific for the hapten or the antigen to be detected, and incubating the reaction mixture in contact with a solid support on the surface of which is fixed the same hapten or antigen to be assayed.

The amount of antibody available for binding to hapten or
The antigen attached to the solid surface is proportionally reduced. After separation of said sample from the solid support by rinsing with an aqueous solution, steps (c) and (d) described above are carried out and the enzymatic activity is inversely proportional to the concentration of hapten or of antigen in the sample. By comparison with a series of reference samples having known standard concentrations of hapten or antigen, it is possible to calculate the concentration of hapten or antigen in the sample.

 Another object of the present invention is to provide an autonomous EIA system in the form of a kit configuration. The kit configuration consists of a syringe or a specially designed capillary system, which, taken together with the immunoglobulin binding enzyme-protein conjugate, provides a rapid self-test which is inexpensive, easy to use, does not require no instrument, implements a minimum number of steps, is readable visually, is to be discarded after use, minimizes contact with samples and reagents, and generally meets all the criteria for a rapid test.

 Immunoglobulin binding proteins which can be conjugated to the EIA enzyme include all immunoreagents which can be conjugated to said enzyme. Suitable immunoreagents include the following: immunoglobulins, avidin, biotin and other immunoglobulin binding proteins, for example the L protein of Peptococcus magnus which has a strong affinity for the light chains of immunoglobulins (Lars Bjorck et al. , J. Imm. Methods 140 (4), 1194, 1988). Preferred immunoglobulin binding proteins which can be used in accordance with the present invention include protein A and protein G, more preferably protein G.

 Enzymes suitable for EIA are those which provide visual endpoint detection by reaction with colored reagent substrates to give a clearly visible and distinct color change. In a preferred embodiment of the invention, p-lactamase is the conjugated enzyme which catalyzes the hydrolysis of penicillin into penicilloic acid, the presence of which is indicated by a distinct color change in the solutions of PVA / iodine or d starch / iodine.

The EIA system described herein can be used for the detection of antibodies to any antigen, in particular, viral or bacterial antigens. This system may also be suitable for the detection of fungal or parasitic diseases, autoimmunity diseases, snake venom, serum components, steroids, hormones or any other agent found in the biological fluids for which you conventional EIA system is insufficient. Biological fluids include whole blood, serum, plasma, saliva and spinal brain fluids, so antigens that can be applied to the solid phase support can include those derived from the virus.
Hepatitis B, rubella virus, Epstein-Barr virus, cyto-megalovirus and human immunodeficiency virus CHIV).

 Solid phase carriers which can be used include any suitable Liai antigen carrier, especially capillary systems and syringes, including disposable systems, which allow the assay to be carried out in accordance with invention and which are also capable of giving a detection of the usual end point.

 The advantages of the EIA system in accordance with the present invention include the following: 1. The discoloration of PVA / iodine by the protein G-p-lactamase conjugate compares well with other detection systems from a sensitivity perspective.

2. The preparation of the conjugate is simple and inexpensive.

3. Ss-lactamase has a high turnover rate, a low price and is absent in mammalian tissues.

4. Reports indicate that the conjugates are stable for long periods of time, i.e. at least one year (IgG / p-Lactamase, Wagle et al. 1985 and Protein A / alkaline phosphatase, Paln et al. 1981), indicating a possible long storage of the protein G- and protein A-ss-lactamase conjugates and possibly other conjugates.

5. Possibility of dosing at an alternative site, said dosing being autonomous, rapid and easy to conduct, comprising few steps, using robust, non-toxic reagents which can be discarded safely and giving rapid visual results which are easy to to interpret.

6. The ability to conduct multiple tests in a single system, the same controls being included in the system regardless of the coating antigen.

 The invention will be described in more detail with reference to preferred embodiments, in order to make the invention fully understandable. In particular, the following description describes the invention developed specifically for detecting antibodies to HIV. However, the invention is not limited to the preferred embodiments described here and must cover all variants, modifications and equivalents, which are included within the scope of the invention as defined by the appended claims.

 The following protocols and examples, which include preferred embodiments, serve to illustrate the practice of the present invention and are given only to present the most useful process description as well as the principles and conceptual aspects of the present invention.

Antigen coating on solid phase supports
The solid phase supports are coated overnight with 10 μg / ml of random copolymer of L-Lysine, of L-phenylalanine (PLP, Cat. NO P 3150, Sigma Chemical Co.), in distilled water, rinsed with phosphate buffered saline (PBS) and activated for 24 h with 1 X of glutaraldehyde (Cat. n G 6257,
Sigma Chemical Co.) in PBS. The supports are rinsed with
PBS and then coated overnight with 50-100 µg / ml whole viral lysate in 0.1 M NaHCO3, pH 9.6 or with 100 µg / ml synthetic peptides in borate buffered saline (BBS ) at pH 8.4. The control supports are coated with the buffer alone.

An alternative coating process may include mixing the protein or peptide C1 mg / ml in PBS, pH 6.4) with PLP (1 mg / ml in distilled water) and then adding 100 ug of l-ethyl-3-C3-dimethylaminopropyl) .- carbodiinide (EDAC,
Cat. n E 6383, Sigma Chemica-1 Co., or any similar cross-linking agent) freshly dissolved, Incubation for 6 h at 250C then dilution 1 to 10 in PBS and coating the surfaces overnight.

After application of the antigen coating, the solid phase supports are coated with Coffee white at X (non-lactate) or BLOTTO at 1 x (skim milk powder) (Johnson et al. Gene
Anal. Techn. 1, 3-8, 1984) in PBS to block Nonspecific protein binding during EIA. However, fetal calf serum, gelatin or a mixture of the above proteins can provide suitable alternatives.

Conjugation of protein G-ss-lactamase (Yolken et al., J. Imm.

Methods 73, 109-23, 1984).

 Protein G is supplied by the firm Pharmacia and plactamase (EC 3.5.2.6) is supplied by Sigma Chemical Co. (Cat.

n P 0389). Lactamase is dialyzed against three changes of distilled water at 40C. A 4: 1 or 2: 1 molar ratio of lactamase to protein G is conjugated via glutaraldehyde: 2.13 mg of lactamase in 0.5 ml of 0.1 M PBS are added to 0.65 mg of protein G (2: 1) or 0.33 mg of protein G (4: 1), and 0.025 ml of 1% glutaraldehyde is added dropwise to the solution. After incubation for 2 h at room temperature, the solution is dialyzed against three changes of PBS, pH 7.4.

Purification of the conjugates by gel exclusion chromatography does not lead to an increase in the sensitivity of the reaction compared to the unpurified conjugates (Yolken and probably stick due to the high rate of incorporation into the conjugate.

Endpoint detection reagent
Penicillin G (benzylpenicillin, PEN-K, Sigma Chemical
Co.) or penicillin V (phenoxymethyl-penicilLinic acid, Cat.

 n P 1382, Sigma Chemical Co.) is used at 5 mg / ml in PBS.

The PVA / iodine / penicillin substrate reagent comprises 63 mM KI / 12 mM I2 and 0.5 X PVA (polyvinyl alcohol, BDH, Cat. No. 30573), in distilled water. Just before use, penicillin is added at 5 mg / ml at a 1: 2 dilution of PVA reagent / iodine in PBS.

Kit configuration
Polyethylene transfer pipettes to be discarded after CDPTP use,
Style M Cat. n 223-9563, BIO-RAD)
They are initially used as a prototype syringe. The working capacity of the DPTP is 0.65 ml, the volume of the cylinder being approximately 0.02 ml. The immunoassay is conducted in the DPTP cylinder, while the bulbs are used as a reservoir for the washing solutions.
The cylinders are coated with antigen, as described above and subsequently blocked with white Coffee at 1 i. The test serum, diluted with 1 × Coffee white in PBS / O, OS X of tween 20, is incubated in DPTP and the unbound anti-serum is washed off with several volumes of PBS / tween. After incubation using the protein G / ss-lactamase conjugate, the unbound conjugate is removed by washing with PBS / tween. The presence of the antibody is indicated by discoloration of the PVA / iodine / penicillin substrate from a blue color to a colorless solution.

Each test includes a positive and negative serum control and a
"Antigen-free" DPTP for each sample.

Disposable syringe
The drawing of a disposable syringe in accordance with the present invention is illustrated schematically in the single figure, in which the polypropylene syringe (5) has in the front part an inlet orifice (3) and a polycarbonate insert (2 ) through which are molded three capillaries (1) (with the possibility of more than 3). The transparent or translucent quality of the syringe and of the insert allows easy reading of any color change inside the capillaries. By actuation of the piston (4), the serum, the conjugate, the washing solutions and the substrate / indicator can be successively aspirated by the inlet orifice (3) through the antigen-coated capillaries (1) of the patch (2) and collect behind the patch in the syringe barrel (6). It is assumed that a 10 ml syringe is necessary to have an adequate volume for all reagents. The advantages of this system is that, apart from being autonomous and easy to discard, the positive control, the negative control and the serum to be tested can be incorporated in the same syringe: The positive control can include a coated capillary immunoglobulin while the negative control will be free of antigen. It is also possible to use the syringe in a multi-test system with a number of capillaries coated with different antigens, for example Hepatitis B surface antigens and
HIV, or positive and negative controls are used for what is to be tested.

 Unlike the DPTP which requires the taking and evacuation of the serum to be tested, the washing solutions and the conjugate, the syringe system is based on taking the above reagents in one direction only, that is to say say, in The syringe barrel, which can then be discarded safely at the end of the test. A ratchet system can be envisaged to deliver fixed volumes, but this increases the cost of production and is not essential. However, the syringe can be easily changed to allow the plunger to move in one direction, which eliminates the back flow problem.

 Minimum incubation times and minimum wash volumes should be estimated in order to meet the requirements for a rapid test. This includes optimizing the dilutions of serum and conjugate and Achieving saturation coating conditions without compromising the sensitivity and specificity of the assay. The preparation of the conjugate must be checked severely to ensure uniformity between batches.

 The following examples are given to illustrate the methods and apparatus described above. The protocol described below is used for all the examples and includes the negative controls (without antigen) and the positive controls (antibody activity known by standard ELISA techniques) 1. Incubation with serum at various dilutions in 1 × Coffee white in PBS / tween for 5 min at 250C.

2. Wash with PBS / tween.

3. Incubation with the protein G-ss-lactamase conjugate at various dilutions in 1 × Coffee white in PBS / tween for 5 min at 250C.

4. Washing with iBS / tween.

5. Incubation with the PVA / iodine / penicillin substrate.

6. Time measurement for the color change from dark blue to a light color, indicating a positive reaction.

Example 1
DPTP - Serum from a donor with a high tetanus toxoid (T / T) titer is diluted 1: 1 with 1% white coffee in PBS / tween and the lactamase conjugate is used at dilution of 1: 1000 in white Coffee to 1 X in PBS / tween. The plastic surfaces are coated with a 1/100 dilution of tetanus toxoid stock solution (100Lf / ml, Commonwealth serum laboratories (CSL)) in PBS, pH 7.2, overnight at 250C . The negative controls are still colored dark blue after 72 h while the test sample takes on a light color after 4 min.

Example 2
DPTP - Positive and negative sera for HIV antibodies are tested by dilution to 1: 1 with white Coffee at 1 X in
PBS / tween; The CSL virus isolate is coated at 50 µg / ml; the lactamase conjugate is used at a dilution of 1: 1000. Positive controls change to a light color after 5 min, while the negative controls change to a light color after 60 min.

Example 3
DPTP - Positive and negative sera for HIV antibodies are tested by dilution to 1: 1 with white Coffee at 1 X in
PBS / tween; peptide 2 (specific strain HIV1) and peptide 5 (specific strain HIV2) are coated as described above.

The negative controls are still colored in dark blue after 16 h, the DPTP coated with HIV2 are still colored in dark blue after 16 h, indicating the absence of cross-reactivity between these epitopes; The DPTP coated with HIVI turns a light color after 3 min.

Example 4
Syringes - Positive and negative sera for Antibody
HIV are tested by 1: 1 dilution with 1% white coffee in PBS / tween; peptide 2 (specific for HIV1) is coated on the capillaries of the patches. Positive controls clear after 3-6 min depending on the antibody titer of the individual controls. The negative controls clear after 30 min.

Claims (22)

 1. An enzyme-linked immunoassay system for use in the detection and / or quantification of one or more antibodies in a sample, characterized in that it comprises (a) a solid phase support, the surface of which must be exposed  The sample containing the antibodies is coated with a  specific antigen, (b) the reaction of the sample containing the antibodies with  the antigen linked to the support, so that the antibodies present  in the sample that are specific for the bound antigen get  bind to said bound antigen, and  washing to remove any excess of unbound antibody, and (c) incubation of the bound antibody using a conjugate of  immunoassay consisting of an active receptor for  the immunoglobulin that binds the bound antibody and a marker  active enzyme, and  washing with an aqueous solution to remove excess  unbound conjugate, and (d) assaying the enzyme activity of the enzyme label by  incubation using an appropriate substrate.  2. An enzyme immunoassay system according to claim 1, characterized in that the solid phase support consists of capillaries or syringes.  3. An enzyme immunoassay system according to claim 1, characterized in that said receptor active for immunoglobulin is Protein A.  4. An enzyme immunoassay system according to claim 1, characterized in that the immunoassay conjugate consisting of a receptor active for the immunoglobulin is Protein G.  5. An enzyme immunoassay system according to claim 1, characterized in that the active enzyme marker is p-lactamase.  6. An enzyme immunoassay system according to claim 1, characterized in that the antibody is determined by the enzyme activity which is indicated by a distinct color change in the substrate / indicator solution.  7. An enzyme immunoassay system according to claim 1 or 6, characterized in that the substrate / indicator solution contains the reagents iodine and polyvinyl alcohol, or iodine and starch.  8. An enzyme immunoassay system according to claim 1, characterized in that it consists of a solid phase support on the surface of which is fixed a capture antibody or a hapten or an antigen of the specific antibody which is to be dosed.  9. An enzyme immunoassay system according to claim 1, to be used for detection and / or quantification of a hapten or an antigen in a sample, characterized in that on the surface of said support in solid phase is fixed an antibody specific for the antigen or the hapten to be assayed, and in that a sample containing the antigen or the hapten to be assayed is incubated in contact with the support in solid phase, and after washing to remove all the traces of hapten or unbound antigen from the solid support, a second antibody which is specific for said hapten or said antigen but which does not bind to the capture antibody, is incubated in contact with the support in solid phase and the excess of the second unbound antibody is removed by rinsing, so that the amount of the second antibody available for binding to the conjugate is directly proportional to the concentration of hapten or antigen in the sample.  10. An enzyme immunoassay system according to claim 1 for use in the detection and / or quantification of a hapten or an antigen in a sample, characterized in that on the surface of said solid phase system is fixed the same hapten or antigen to be assayed, and in step (a) an antibody specific for said hapten or antigen is added to the test sample, and as a result of the binding of said hapten or antigen in said sample, the amount of antibody available for binding to hapten or antigen attached to the solid surface is proportionally reduced.  11. An enzyme immunoassay system according to claim 10, characterized in that it also contains standardized solutions of an antibody specific to the hapten or to the antigen which is to be assayed.  12. An enzyme immunoassay system according to claim 1, characterized in that the antibodies to be detected are specific for viral, bacterial, mycotic, parasitic or autoimmunity antigens.  13. An enzyme-linked immunoassay system according to claim 1 or 11, characterized in that the antigens or peptides applied as a coating on the solid phase support include those derived from the hepatitis B virus, the rubella virus , Epstein-Barr virus, Cyto-megalovirus, human immunodeficiency virus (HIV).  14. A test kit comprising an enzyme immunoassay system according to claim 7 or 8.  15. A kit to be used for your detection and / or the quantification of the antibodies according to claim 1, characterized in that it consists of solid phase supports coated with a specific antigen or with a capture antibody, a positive control. and a negative control, and an immunoassay conjugate.  16. A kit according to claim 15, characterized in that it is contained inside a closed system.  17. A kit according to claim 16, characterized in that the closed system is a transparent or translucent syringe.  18. A kit according to claim 16 or 17, characterized in that the closed dosing system is disposable.  19. A kit according to claim 15, characterized in that it is used in the simultaneous detection of several specific antigens and / or of specific antibodies.  20. A kit according to claim 17, characterized in that the syringe comprises an insert in the front part of the syringe through which are molded one or more supports in solid phase.  21. A kit according to claim 20, characterized in that the solutions used in the assay including the sample are aspirated over the solid phase supports coated with antigen or coated with antibodies molded in the insert and are collect and are contained behind the patch in the syringe barrel.  22. A kit according to claim 20 or 21, characterized in that the solid phase supports are capillaries.