CN115505575B

CN115505575B - Two hybridoma cell strains secreting canine IL-31 monoclonal antibody and application thereof

Info

Publication number: CN115505575B
Application number: CN202211463230.2A
Authority: CN
Inventors: 校海霞; 高福; 郭天玲; 郑予馨; 高峰; 庞雪飞
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2022-11-16
Filing date: 2022-11-16
Publication date: 2023-03-24
Anticipated expiration: 2042-11-16
Also published as: CN115505575A

Abstract

The invention discloses two hybridoma cell strains combined with a canine IL-31 monoclonal antibody and application thereof. The preservation number of the hybridoma cell strain TIBGAO-2D10-2 is as follows: CGMCC No.45175, and the preservation number of the hybridoma cell strain TIBGAO-1C6 is as follows: CGMCC No.45174, both can secrete monoclonal antibody binding to canine IL-31. The invention establishes a double-antibody sandwich ELISA detection method by taking an antibody which is subjected to ascites purification after a hybridoma cell strain TIBGAO-2D10-2 is injected into a mouse as a capture antibody and an antibody which is labeled by biotin and is subjected to ascites purification after the hybridoma cell strain TIBGAO-1C6 is injected into the mouse as a detection antibody. The method has the advantages of simple and quick operation and clear result, provides a new method for in vitro diagnosis of the atopic dermatitis of the dog, and has application value.

Description

Two hybridoma cell strains secreting canine IL-31 monoclonal antibody and application thereof

Technical Field

The invention belongs to the field of veterinary medicine and medical biology, and particularly relates to two hybridoma cell strains secreting canine IL-31 monoclonal antibodies and application thereof.

Background

Atopic Dermatitis (AD) is an inflammation that causes allergic itching of the skin. In dogs, AD can cause severe itching, accompanied by secondary alopecia and erythema. About 450 million dogs worldwide suffer from this chronic lifelong disease. The incidence of disease also increases year by year.

Interleukin-31 (IL-31) is a cytokine that was cloned in 2004. It is produced mainly by activated T helper cells (Th 2). As a neuromodulatory factor, it plays an important role in skin inflammation and itching. IL-31 binds to a heterodimer consisting of IL-31 receptor alpha (IL-31 RA) and Oncoinhibin M receptor beta (OSMR beta), and IL-31 signaling activates three important signaling pathways: janus kinase (JAK)/activator of transcription (STAT), phosphoinositide 3-kinase (P13K)/AKT and mitogen kinase (ERK)/MAPK, resulting in cellular biological changes. These changes include immune cell chemotaxis, proinflammatory cytokines and chemokine secretion, cutaneous pruritus response, and disruption of the skin barrier due in part to cell proliferation, differentiation. The expression of IL-31 is obviously increased in AD patients, and can induce chemokines CCL1, CCL17 and CCL22. These chemokines recruit more T cells to the infected skin, thereby secreting more IL-31. Researchers have found that the IL-31RA/OSMR β co-receptor is expressed in macrophages, mast cells, eosinophils, basophils, keratinocytes and the dorsal root ganglia of peripheral neurons [ Gibbs BF, patsinakidis N, raap U.E. Role of the pruritic cytokine IL-31in autoimmune skin diseases. Front Immunol. 2019.

IL-31 is involved in dermatitis, pruritic lesions, allergies and respiratory allergies. IL-31 is produced in increased amounts in pruritic skin areas and can induce scratching behavior in various species, such as mice, monkeys, and dogs. It has been shown that IL-31 induces strong pruritic behaviour in dogs when administered intradermally, subcutaneously or intravenously. Transgenic mice overexpressing IL-31 develop skin inflammation, itching, severe dermatitis, and hair loss. Subcutaneous injection of IL-31 causes infiltration of inflammatory cells, neutrophils, eosinophils, lymphocytes and macrophages, resulting in thickening of the epidermis and hyperplasia of the dermis. Further studies have shown that IL-31 is associated with skin inflammation and itching caused by atopic dermatitis in humans [ Lai T, wu D, li W, et al. Interleukin-31expression and relationship to a disease selectivity in human asthma. Sci Rep. 2016 (1): 22835.; west NR, hegazy AN, owens BMJ, et al. Oncostatin M drive specific information and expression response to a tissue promoter-receptor promoter book. Nat Med. 2017 (5): 579-589.].

At present, the companies have developed therapeutic drugs aiming at the signaling caused by canine IL-31, such as JAK small molecule inhibitor Epoke and canine IL-31 monoclonal antibody drug Cytopoint, which are introduced by Shuiteng. However, these drugs for IL-31 signaling are expensive, and require precise treatment, so that they can be used without any problem, thereby reducing the cost of pet owners. In addition, it has been found that in vitro recombinant IL-31 can be used as a vaccine to prevent atopic dermatitis by inoculating the vaccine into animals in small amounts and many times [ Olomski F, fetterschoss V, jonsdottir S, et al. Interleukin 31in induced hypertension-inducing closed system by active vaccination in allergy 2020 Apr 75 (4): 862-871 ]. Accurate symptomatic treatment of the atopic dermatitis of the dogs caused by the IL-31, yield determination of the recombinant canine IL-31 protein in an expression system and the like need to be matched with corresponding IL-31 detection reagents.

Disclosure of Invention

The first purpose of the invention is to provide two hybridoma cell strains for secreting monoclonal antibodies of canine IL-31, namely a hybridoma cell strain TIBGAO-2D102 and a hybridoma cell strain TIBGAO-1C6. Has been preserved in China general microbiological culture Collection center (address No. 3 of Xilu No. 1 of Beijing Kogyo area, china) at 26.5.2022, wherein the preservation number of the hybridoma cell strain TIBGAO-2D102 is CGMCC No.45175, and the preservation number of the hybridoma cell strain TIBGAO-1C6 is CGMCC No.45174.

The second purpose of the invention is to provide two monoclonal antibodies, 2D102 and 1C6, which are combined with canine IL-31, and the two antibodies are respectively secreted and produced by the two hybridoma cell strains. ELISA results show that both monoclonal antibodies can specifically bind to canine IL-31 protein.

The invention provides application of the monoclonal antibody in detection or diagnosis of related diseases caused by canine IL-31.

Specifically, the canine IL-31 related disease is atopic dermatitis with pruritus, skin lesion, erythema and allergy as symptoms. Preferably, the detection method is a double antibody sandwich ELISA detection method.

The invention also provides application of the monoclonal antibody in preparation of preparations for detecting canine IL-31 protein.

The fifth purpose of the invention is to provide a double antibody sandwich ELISA detection kit, which is used for a double antibody sandwich ELISA detection method and comprises a monoclonal antibody 2D102 which is secreted by a hybridoma cell strain TIBGAO-2D102 and is combined with canine IL-31, and a biotin-labeled monoclonal antibody 1C6 which is secreted by a hybridoma cell strain TIBGAO-1C6 and is combined with canine IL-31.

Specifically, monoclonal antibody 2D102 secreted by hybridoma cell strain TIBGAO-2D102 is used as a capture antibody, and monoclonal antibody 1C6 which is labeled by biotin and is secreted by hybridoma cell strain TIBGAO-1C6 is used as a detection antibody.

Preferably, in the above-mentioned double antibody sandwich ELISA detection method, the capture antibody is coated on the ELISA plate, the optimal coating amount of the capture antibody is 2 μ g/well, and the working concentration of the detection antibody is 2 μ g/ml.

Compared with the prior art, the invention has the following beneficial effects:

1. monoclonal antibodies 2D102 and 1C6 bind to canine IL-31, have higher affinity and slower off-rate than the marketed caninized antibody Cytopoint which binds to canine IL-31, and are useful in the diagnosis of canine IL-31 or diseases associated with IL-31, as well as the detection of the production of canine IL-31 protein in different expression systems, and in laboratory assays for canine IL-31.

2. The two hybridoma cell strains can stably secrete the monoclonal antibodies, the pairing effect of the two secreted antibodies is good, and the method is suitable for the double-antibody sandwich ELISA detection method.

3. The two antibodies secreted by the two hybridoma cell strains are not interfered by canine serum by double antibody sandwich ELISA, and have higher sensitivity when being used for detecting canine IL-31 related diseases.

4. The mouse hybridoma cell strain and the monoclonal antibody secreted by the mouse hybridoma cell strain have important application prospects in the aspects of development of canine IL-31 detection reagents and development of disease diagnosis reagents related to canine IL 31.

5. The double-antibody sandwich ELISA provided by the invention has the advantages of simple operation, clear result and the like.

Drawings

FIG. 1 shows that antibodies 2D102 (A) and 1C6 (B) prepared by ascites from two hybridoma cells (TIBGAO-2D102, TIBGAO-1C 6) of the present invention were subjected to Superdex ^TM 200 Purifying by 10/30 GL molecular sieve chromatography;

FIG. 2 is a graph showing the ELISA binding of two antibodies prepared according to the present invention and a caninized antibody Cytopoint to recombinant canine and feline IL-31, respectively. Wherein, A is the combination of 2D102, 1C6, cytopoint and irrelevant control antibody and recombinant canine IL-31 protein, B is the combination of 2D102, 1C6 and irrelevant control antibody and recombinant feline IL-31 protein;

FIG. 3 shows the affinity of two antibodies 2D102 and 1C6 obtained by purifying ascites in mice and the commercial Cytopoint antibody to recombinant canine IL-31, as determined by surface plasmon resonance. Wherein A is the binding constant (ka) of the three antibodies to recombinant canine IL-31; b is the dissociation constant (kd) of the three antibodies to recombinant canine IL-31; c is the affinity constant (KD) of the three antibodies to recombinant canine IL-31. All results are statistical values of the data of the triplicate experiments. Wherein represents p <0.05, represents p <0.01, represents p <0.001.

FIG. 4 is a binding diagram of sandwich ELISA of two antibodies obtained by purifying ascites fluid of mice from two hybridoma cells according to the present invention. Wherein, A shows that 2D102 is an immobilized antibody, and biotin-labeled 1C6 is a detection antibody; b shows that 1C6 is an immobilized antibody and biotin-labeled 2D102 is a detection antibody;

FIG. 5 shows the concentration of recombinant canine IL-31 detected by the double antibody sandwich ELISA method used in the present invention. Wherein, A is a standard curve made after the recombinant canine IL-31 with different concentrations obtained by PBS dilution is detected by using the double-antibody sandwich ELISA method of the invention; b is a standard curve made by diluting different concentrations of recombinant canine IL-31in a double-antibody sandwich ELISA method for detecting canine serum (which is negative to detect the recombinant canine IL-31 by using a commercial kit).

Biological material preservation information:

the mouse hybridoma cell strain TIBGAO-1C6 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation unit is abbreviated as CGMCC, and the address is as follows: western road No. 1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101. The preservation date is 26 months at 2022 years, and the preservation number is CGMCC No.45174; the classification of the strain was named: mouse hybridoma cells.

The mouse hybridoma cell strain TIBGAO-2D102 is preserved in China general microbiological culture Collection center (CGMCC), the preservation unit is CGMCC for short, and the address is as follows: western road No. 1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101. The preservation date is 2022 years, 5 months and 26 days, and the preservation number is CGMCC No.45175; the classification of the strain was named: mouse hybridoma cells.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following detailed description is given to further illustrate the technical solutions of the present invention with reference to the accompanying drawings, and all reagents used in the examples are commercially available or can be prepared according to texts or known methods.

EXAMPLE 1 preparation and purification of murine monoclonal antibodies

The purified recombinant canine IL-31 protein was used as an antigen to immunize female BALB/c mice in five, subcutaneous, multiple immunizations for 6-8 weeks. The immunization dose is 30 mug/time, and the interval time of the first four immunizations is 14 days. For the first immunization, freund's complete adjuvant is used; freund's incomplete adjuvant was used for the 2 nd to 4 th immunization. On the 7 th day after the 3 rd immunization, tail vein blood is taken to detect the titer of the serum antibody, and the mouse with the highest titer is selected for fusion. The spiking immunization was performed 3 days before the fusion, i.e., the fifth immunization, in which 100. Mu.g of the immunization stock was injected into the abdominal cavity of the mouse. The spleen cells and myeloma cells SP2/0 of the mice were taken as follows: fusing at the ratio of 1, selectively culturing in HAT screening culture medium, and diluting to obtain hybridoma cell strain. Recombinant canine IL-31 is coated in an enzyme label plate at 25 ng/hole in a carbonate buffer solution with pH9.6, 15 monoclonal antibody cell strains with high antigen binding capacity are screened by an ELISA method, and the 15 monoclonal antibody strains are subjected to competitive ELISA pairing to obtain two monoclonal antibodies, namely 2D102 and 1C6, which have the competitive binding with the canine IL-31.

The preparation of monoclonal antibody is carried out by using retired BALB/c mouse. First, the adjuvant special for ascites was injected intraperitoneally at 0.4 ml/tube. Hybridoma cells were injected intraperitoneally 10-14 days after adjuvant inoculation, with an inoculation dose of 30 ten thousand cells/cell. 10-15 days after cell injection, ascites from mice were collected, enriched and purified for IgG by Protein A, and then subjected to Superdex ^TM 200 Further purification with 10/300 GL molecular sieves showed IgG peaking at 16-17 ml (A, B in FIG. 1) to obtain homogeneous 2D102 and 1C6 IgG proteins. SDS-PAGE showed that these two antibodies were approximately a 50 kDa heavy chain and a 25 kDa light chain under reducing conditions (dithiothreitol, + DTT was added to the loading buffer) (A, B in FIG. 1). Under non-reducing conditions (no DTT added to the loading buffer, -DTT), the heavy and light chains are linked by disulfide bonds to form a heterodimer of tetrapeptide chain molecules, i.e., igG, with a molecular weight of 150 kDa (A, B in FIG. 1).

EXAMPLE 2 determination of the binding Activity of monoclonal antibodies to recombinant canine IL-31 and recombinant feline IL-31

The purified recombinant canine IL-31 and recombinant feline IL-31-Fc fusion proteins were immobilized on 96-well ELISA plates using a carbonate coating solution pH9.6, 200 ng/well, overnight at 4 ℃ by an indirect ELISA method. Then, the microplate was placed at room temperature, shaken for 30 min to return to room temperature, the liquid in the microplate was discarded, and washed three times with PBS for 5 min each time. Then, blocking solution (PBS +2% BSA) was added and incubated for 1 h on a shaker. The 96-well plate was washed three times with PBST (PBS +0.05% Tween-20) for 5 min each. Subsequently, adding different dilution concentrationsMonoclonal antibody, 100 u l/hole, room temperature, shaking table oscillation for 1 h. The microplate was washed three times with PBST for 5 min each. Then, HRP-labeled goat anti-mouse IgG or rabbit anti-dog IgG diluted 1. Washing the enzyme-linked plate with PBST for three times, adding TMB, developing for about 5 min, and adding 2M H ₂ SO ₄ The reaction was terminated. Readings were taken with a microplate reader at a wavelength of 450 nm. The results show that 2D102 binds specifically to recombinant canine IL-31, EC ₅₀ 0.01. Mu.g/ml, 1C6 specifically binds to the recombinant canine IL-31 protein, EC ₅₀ 0.02 μ g/ml, cytopoint and recombinant canine IL-31 protein EC ₅₀ It was 0.14. Mu.g/ml (A in FIG. 2). While both 2D102 and 1C6 did not bind to the recombinant feline IL-31-Fc fusion protein (B in FIG. 2). The results show that the antibodies secreted by the two hybridoma cell strains can be specifically combined with the recombinant canine IL-31 protein, and the binding force is higher than that of the commercialized antibody Cytopoint.

Example 3 binding kinetics assay of monoclonal antibodies to recombinant canine IL-31

And (3) measuring the kinetic parameters of the combination of the antibody and the antigen by adopting a surface plasmon resonance method. First, SA was immobilized on the surface of a CM5 chip by means of amino coupling, and then biotin-labeled recombinant canine IL-31 was immobilized on the chip by binding to SA. The immobilized recombinant canine IL-31 protein was then conjugated with varying concentrations of 2D102, 1C6 and Cytopoint antibodies as mobile phase. As shown in FIG. 3 and Table 1, the binding constants (ka) of 2D102, 1C6 and Cytopoint were close to each other and 1.27. + -. 0.02X 10 ⁵ M ^-1 s ^-1 、1.64±（0.41）×10 ⁵ M ^-1 s ^-1 And 1.78 + - (0.89). Times.10 ⁵ M ^-1 s ^-1 . The kinetics of dissociation of 2D102 and 1C6 binding to recombinant canine IL-31 were such that the dissociation constants (kd) were 8.58 + - (6.18). Times.10, respectively ^-7 s ^-1 And 2.93 + - (1.42). Times.10 ^-6 s ^-1 . The dissociation constant of Cytopoint antibody is 1.22 + - (0.45) × 10 ^-4 s ^-1 . The rate of re-dissociation of the commercial antibody Cytopoint after binding to antigen is significantly different compared to 2D102, 1C6 (p)<0.05 Indicating that the dissociation rate of the binding of Cytopoint and antigen is higher than that of the present inventionTwo antibodies in the invention are fast. The affinity constants (KD) of 2D102, 1C6 and Cytopoint with recombinant canine IL-31 are respectively 6.81 + - (4.96) x 10 ^-12 M、2.80±（1.55）×10 ^-11 M and 8.28 + - (3.01) × 10 ^-11 And M. The affinity KD value of the 2D102 and the recombinant canine IL-31 has significant difference (p) compared with that of the commercialized antibody Cytopoint<0.05 Indicating that the antibody has higher affinity for antigen binding than Cytopoint.

TABLE 1 detailed values after statistics of the ka, KD and KD data of three tests

The results show that 2D102 and 1C6 and recombinant canine IL-31 are fast binding, slow dissociation mode, and compared with the commercial antibody Cytopoint, in the dissociation rate of higher advantage. And 2D102 has a higher affinity compared to Cytopoint.

Example 4 establishment of double antibody Sandwich ELISA method

A monoclonal antibody is immobilized on an ELISA plate with 96 holes by carbonate coating liquid with pH9.6, and the optimal immobilization amount of the capture antibody is 2 mug/hole and stays overnight at 4 ℃ by verification. The ELISA plate was then placed at room temperature, shaken for 30 min to return to room temperature, the liquid in the ELISA plate was discarded, and washed three times with PBS for 5 min each time. Then, blocking solution (PBS +2% BSA) was added and incubated for 1 h on a shaker. The 96-well plates were washed three times with PBST for 5 min each. Subsequently, 2. Mu.g/ml of recombinant canine IL-31 as a detection antigen was added to the microplate at 100. Mu.l/well at room temperature, and shaken on a shaker for 1 h. The ELISA plates were washed three times with PBST, 5 min each time. Then, another monoclonal antibody labeled with biotin of different concentration gradients was added thereto at 100. Mu.l/well and shaken on a shaker at room temperature for 1 hour. The microplate was washed three times with PBST for 5 min each. Then add 1 10000 diluted HRP labeled streptavidin (streptavidin) at room temperature and shake the shaker for 1 h. After the microplate was washed three times with PBST, TMB was added to 50. Mu.l of each well and developed for about 5 min, and then 2M H was added to 50. Mu.l of each well ₂ SO ₄ The reaction was terminated. Reading with a microplate reader at a wavelength of 450 nmAnd (4) counting. The results show that when 2D102 is the capture antibody and biotin-labeled 1C6 is the detection antibody, the value of OD450 increases with increasing 1C6 concentration (a in fig. 4). Similarly, when 1C6 was used as the capture antibody and biotin-labeled 2D102 was used as the detection antibody, the OD450 value increased with increasing concentration of 2D102 (B in fig. 4). These results indicate that the two monoclonal antibodies 2D102 and 1C6 do not bind the same epitope as canine IL-31, and that there is no competition for binding to canine IL-31 between the two antibodies. According to the detection result of the double-antibody sandwich ELISA, the optimal working concentration of the two paired antibodies is also determined, namely the optimal coating amount of the capture antibody is 2 mug/hole, and the optimal working concentration of the biotin-labeled detection antibody is 2 mug/ml.

Example 5 determination of detection line for double antibody sandwich ELISA

In this example, the lowest detection value for canine IL-31 detected by this method was determined using the double antibody sandwich ELISA method determined in example 3. First, capture antibody 2D102 was immobilized with a coating solution at 2. Mu.g/well overnight at 4 ℃. Then, the solution was blocked with a blocking solution for 1 hour. Subsequently, different concentrations of recombinant canine IL-31 protein, 25.6 ng/ml,12.8 ng/ml,6.4 ng/ml,3.2 ng/ml,1.6 ng/ml, 0.8 ng/ml,0.4 ng/ml,0.2 ng/ml, respectively, diluted in PBS were added. 100 μ l/well, room temperature, shaking table for 1 h. The ELISA plates were washed three times with PBST, 5 min each time. Then 2. Mu.g/ml biotin-labeled 1C6 detection antibody was added thereto at 100. Mu.l/well, and shaken on a shaker at room temperature for 1 hour. The microplate was washed three times with PBST for 5 min each. Then, 1:10000 dilutions of HRP-labeled streptavidin, room temperature, shaking for 1 h on a shaker. Washing the enzyme labeling plate with PBST for three times, adding 100. Mu.l TMB into each well, developing for about 5 min, and adding 100. Mu.l H into each well ₂ SO ₄ (2M) the reaction was terminated. Readings were taken with a microplate reader at a wavelength of 450 nm. The result shows that when the concentration interval of the canine IL-31 protein is 0.2 to 25.6 ng/ml, the detection result of the double-antibody sandwich ELISA method established by the invention is in a linear relation with the concentration of the canine IL-31 protein, so that a standard curve is drawn, and the standard curve equation is y = -0.0037x ² +0.1891x+0.0731，R ² =0.9973. The lowest detected canine IL-31 concentration was 400 pg/ml (OD 450 of 0.13).

To determine whether the double antibody sandwich ELISA method established in the invention has background or interference caused by serum itself when determining clinical canine serological samples, canine serum (negative for recombinant canine IL-31 detected by a commercial kit) is used in the invention, but PBS buffer is not used as a diluent, and recombinant canine IL-31 protein with final concentrations of 25 ng/ml, 12.5 ng/ml,6.25 ng/ml,3.125 ng/ml,1.56 ng/ml,0.78 ng/ml and 0.39 ng/ml is added. Then, the response value of the samples is measured by using the double-antibody sandwich ELISA method established by the invention. The result is shown in B in figure 5, the detection result of the double-antibody sandwich ELISA method established by the invention is still in a linear relation with the concentration of the recombinant canine IL-31 protein diluted by the canine serum, and the standard curve equation is y = -0.0044x ² +0.2063x+0.1097，R ² =0.9918. The lowest detected concentration of recombinant canine IL-31 was 390.6 pg/ml (OD 450 value of 0.1). The result shows that the result obtained by using the canine serum as the diluent (B in figure 5) is consistent with the result obtained by using the PBS as the diluent (A in figure 5), and the double-antibody sandwich ELISA method established by the invention is proved to be free from the interference of the canine serum and have better sensitivity.

Claims

1. A hybridoma cell strain TIBGAO-2D102 with a preservation number of: CGMCC No.45175.

2. A monoclonal antibody that binds canine IL-31, said monoclonal antibody being secreted from the hybridoma cell strain TIBGAO-2D102 of claim 1.

3. A hybridoma cell strain TIBGAO-1C6, which has a preservation number of: CGMCC No.45174.

4. A monoclonal antibody that binds canine IL-31, said monoclonal antibody being secreted from the hybridoma cell strain TIBGAO-1C6 of claim 3.

5. The use of the monoclonal antibody of claim 2 or 4 for the preparation of a reagent for detecting or diagnosing a disease associated with canine IL-31.

6. The use according to claim 5, wherein the canine IL-31 associated disease is atopic dermatitis with symptoms of pruritus, lesions, erythema, and allergy.

7. The use of claim 6, wherein the related disease is detected by a double antibody sandwich ELISA assay.

8. Use of the monoclonal antibody of claim 2 or 4 in the preparation of a formulation for detecting recombinant canine IL-31 protein.

9. The use of claim 8, wherein the sample is assayed for canine IL-31 by an enzyme-linked immunosorbent assay.

10. The use according to claim 8 for the quantitative determination of canine IL-31 protein expressed in mammalian cells, e.coli, yeast at various stages of miniprep, pilot.

11. A double antibody sandwich kit comprising the monoclonal antibody of claim 2 or 4 and further comprising additional reagents for a double antibody sandwich assay.

12. The double antibody sandwich kit according to claim 11 wherein the monoclonal antibody of claim 4 is labeled with biotin as a detection antibody and the monoclonal antibody of claim 2 as a capture antibody.

13. The double antibody sandwich kit of claim 12 wherein the capture antibody is coated on an elisa plate.

14. The double antibody sandwich kit of claim 12 wherein the capture antibody is coated in an amount of 2 μ g/well and the detection antibody is present at a working concentration of 2 μ g/ml.