A monoclonal antibody composition against haptoglobin and its application

Abstract

The present invention relates to the field of biological detection technology, and in particular to an anti-haptoglobin monoclonal antibody composition and application thereof. Two paired antibodies are provided, wherein the antibodies include a heavy chain variable region VH and a light chain variable region VL, wherein the VH and VL include a determinant complementary region, and the determinant complementary regions of the VH and VL are both composed of CDR1, CDR2 and CDR3. The two antibodies HY2B4 and HY6D7 obtained by the present invention can specifically bind to haptoglobin, and thus a double antibody sandwich ELISA kit for detecting HP content is prepared by using the antibodies, which has the advantages of a wide detection linear range, a low detection limit, a high recovery rate, a strong anti-interference ability and the like, and thus can accurately, highly sensitively and highly specifically detect trace amounts of haptoglobin in a sample.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an anti-haptoglobin monoclonal antibody composition and application thereof. Background Art

[0002] Haptoglobin (HP), also known as haptoglobin, has a gene located on human chromosome 16q22. The Hp molecule is a tetramer composed of two β chains and two α chains connected by disulfide bonds. The β chains are identical, and the polymorphism of Hp is mainly caused by genetic variation of the α chain. There are two alleles of the α chain, Hp1 and Hp2, forming three different genotypes, namely Hp1-1, Hp2-1 and Hp2-2. There are two types of α chains, a1 and a2, of which a1 has two subtypes, a1S and a1F, which differ only by one amino acid. The 54th amino acid a1F is lysine, and a1S is glutamic acid. The α1 chain contains 83 amino acids and has a relative molecular mass of about 9,100. α2 contains 1.7kb of internal repeats, contains 142 amino acids, and has a relative molecular mass of about 16,000. The protein product of the homozygous Hp1 allele, Hp1-1, is a monovalent with a relative molecular mass of 86,000. The Hp2 allele products, when heterozygous (Hp2-1) or homozygous (Hp2-2), form polymers of multiple Hp molecules: Hp2-1 is a variety of linear homodimers and polymers, and Hp2-2 forms a circular polymer.

[0003] Hp is an acidic glycoprotein in the serum α2 globulin component, which is widely present in the blood and other body fluids of humans and many mammals. The concentration in human plasma circulation is 0.3-3g / L. Hp was originally isolated in the liver as an acute phase response protein of inflammatory response. The main site of synthesis is the liver. Later, it was found that it also exists in immune cells including neutrophils and monocytes. In addition, studies have confirmed that renal proximal tubular epithelial cells can synthesize and secrete Hp under the stimulation of inflammatory response factors, and white adipose tissue and some other tissues (stomach, intestine, adrenal gland, muscle, brain, epididymis, esophagus, placenta, ovary, ureter, etc.) also express Hp mRNA.

[0004] The main function of Hp is to bind to free hemoglobin (Hb) in plasma and be cleared, thereby preventing further redox reactions of free Hb and corresponding tissue damage. Many clinical causes of red blood cell destruction, such as hemolysis and trauma, can cause a significant increase in free Hb in the blood. Free Hb is a potent oxidative protein, and the heme component in it can cause cell damage and tissue damage after oxidation. The Hb binding site on each β chain of the Hp molecule can bind to free Hb in plasma to form a high-affinity, irreversible Hp-Hb complex, which is then recognized by the clearance receptor CD163 and eliminated in the cell to prevent free Hb from damaging tissues. In addition, free Hb can pass through the glomerular filtration membrane, causing oxidative damage to the kidneys, while Hp in plasma can quickly bind to free Hb and be cleared, thereby preventing free Hb from filtering through the glomerulus and preventing possible tubular damage caused by Hb. In addition, Hp has long been considered a marker of inflammatory response and has an antioxidant effect. It plays an important role in resisting infection, repairing damaged tissues, and maintaining the stability of the internal environment. Its serum content increases significantly in pathological conditions such as infection, trauma, inflammatory response, tumors, and myocardial infarction. During inflammatory response and vascular damage, Hp in serum increases, and continuous changes in blood flow can also induce Hp expression in the arterial wall and participate in vascular reconstruction and repair by promoting fibroblast migration. Hp also plays an important role in post-transplant inflammatory response and initiates the process of graft rejection. In addition, Hp has antioxidant activity, inhibits prostaglandin synthesis, inhibits bacteria, and participates in cell apoptosis and immune response. In many cases, Hp is upregulated to respond to potential harmful conditions, which can usually indicate disease or other pathological changes early. Therefore, the detection of Hp is of great significance. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide an anti-haptoglobin monoclonal antibody composition and application, which can detect trace amounts of haptoglobin with high sensitivity and high specificity.

[0006] To this end, the present invention provides the following technical solutions:

[0007] An antibody, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH and VL comprise a determinant complementary region, and the determinant complementary regions of the VH and VL are both composed of CDR1, CDR2 and CDR3; the antibody comprises at least one of the following:

[0008] (1) Antibody HY2B4:

[0009] The CDR1 of the VH has an amino acid sequence as shown in SEQ ID NO.1, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0010] The CDR2 of the VH has an amino acid sequence as shown in SEQ ID NO.2, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0011] The CDR3 of the VH has an amino acid sequence as shown in SEQ ID NO.3, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0012] The CDR1 of the VL has an amino acid sequence as shown in SEQ ID NO.4, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0013] The CDR2 of the VL has an amino acid sequence of: YAS, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0014] The CDR3 of the VL has an amino acid sequence as shown in SEQ ID NO.5, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0015] (2) Antibody HY6D7:

[0016] The CDR1 of the VH has an amino acid sequence as shown in SEQ ID NO.6, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0017] The CDR2 of the VH has an amino acid sequence as shown in SEQ ID NO.7, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0018] The CDR3 of the VH has an amino acid sequence as shown in SEQ ID NO.8, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0019] The CDR1 of the VL has an amino acid sequence as shown in SEQ ID NO.9, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0020] The CDR2 of the VL has an amino acid sequence of GTN, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0021] The CDR3 of the VL has an amino acid sequence as shown in SEQ ID NO. 10, or has at least 90%, 95% or 99% similarity to the amino acid sequence.

[0022] Optional, including at least one of the following:

[0023] (1) The antibody is selected from any one of monoclonal antibodies mAb, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary determining region fragment, and single-chain antibody;

[0024] (2) The heavy chain constant region of the antibody HY2B4 is of the IgG1 subtype, and the heavy chain constant region of the antibody HY6D7 is of the IgG2a subtype;

[0025] (3) The light chain constant region of the antibody HY2B4 is of the Kappa subtype, and the light chain constant region of the antibody HY6D7 is of the Lambda subtype;

[0026] (4) The antibody is a mouse antibody.

[0027] Optional, including at least one of the following:

[0028] The full-length amino acid sequence of the heavy chain of the antibody HY2B4 is as shown in SEQ ID NO.11 or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0029] The full-length amino acid sequence of the light chain of the antibody HY2B4 is as shown in SEQ ID NO.13 or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0030] The full-length amino acid sequence of the heavy chain of the antibody HY6D7 is as shown in SEQ ID NO.15 or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0031] The full-length amino acid sequence of the light chain of the antibody HY6D7 is as shown in SEQ ID NO.17 or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0032] Optionally, the antibody HY2B4 (mouse anti-human haptoglobin monoclonal cell line) shown is prepared from a hybridoma cell line deposited in the General Microbiology Center of the China National Microorganism Culture Collection Administration with a deposit number of CGMCC NO.45850, and the antibody HY6D7 (mouse anti-human haptoglobin monoclonal cell line) shown is prepared from a hybridoma cell line deposited in the General Microbiology Center of the China National Microorganism Culture Collection Administration with a deposit number of CGMCC NO.45851.

[0033] The mouse hybridoma cell line secretes and produces the antibody, and its deposit numbers are CGMCC NO.45850 and CGMCC NO.45851.

[0034] Any of the following biological materials:

[0035] (1) Humanized antibodies are obtained by humanizing the above antibodies;

[0036] (2) a nucleic acid molecule encoding the antibody;

[0037] (3) an expression cassette, recombinant vector, recombinant cell or recombinant bacteria containing the nucleic acid molecule described in (2);

[0038] (4) Antibody conjugates, obtained by coupling the antibody with a marker or protein; the marker is selected from one or more of ELISA dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling;

[0039] (5) A kit comprising the antibody.

[0040] The antibody has any of the following uses:

[0041] (1) Use in detecting haptoglobin or preparing products for detecting haptoglobin;

[0042] (2) Used for immunohistochemistry of tissues containing haptoglobin;

[0043] (3) Used to detect haptoglobin secreted in cell culture supernatant.

[0044] An ELISA kit for detecting trace haptoglobin comprises the antibody, wherein one of the antibody HY2B4 and the antibody HY6D7 is used as a coating antibody, and the other is used as an enzyme-labeled antibody.

[0045] Optionally, the antibody HY6D7 is used as a coating antibody, and the HY2B4 is used as an enzyme-labeled antibody;

[0046] And / or, the enzyme-labeled antibody is labeled with HRP or alkaline phosphatase;

[0047] And / or, the working concentration of the enzyme-labeled antibody is that the enzyme-labeled antibody stock solution is diluted with the diluent at a dilution ratio of 1:1000-8000, and the concentration of the enzyme-labeled antibody stock solution is 1.77 mg / ml;

[0048] And / or, further comprising an ELISA plate, a standard solution, a substrate color developing solution, a stop solution and / or an antibody diluent;

[0049] The ELISA plate is coated with antibodies.

[0050] Optionally, the preparation of the ELISA plate comprises the following steps: coating, blocking, drying and / or sealing;

[0051] In the coating step, the coating antibody is added to the wells of the ELISA plate for coating;

[0052] In the blocking step, the coated ELISA plate is washed, and a blocking solution is added to the wells for blocking;

[0053] In the drying step, the blocked ELISA plate is washed and dried;

[0054] In the sealing step, the dried ELISA plate is sealed;

[0055] Optionally, the coating concentration of the coating antibody is 1-9ug / ml, preferably 3ug / ml;

[0056] And / or, the blocking solution is a PBS solution containing 1-5 w / v% BSA or 5 w / v% skim milk powder, preferably a PBS solution containing 1 w / v% BSA;

[0057] And / or, the sealing condition is 35°C-38°C for 1-2 hours, preferably 37°C for 2 hours.

[0058] A method for detecting trace haptoglobin, comprising using the ELISA kit for detecting trace haptoglobin to perform qualitative or quantitative detection:

[0059] Quantitative detection: add the sample to be tested or the standard solution to the antibody-coated ELISA plate, set a blank well at the same time, incubate, wash the plate, then add the working concentration of the enzyme-labeled antibody, incubate, wash the plate, add the substrate color development solution, color, then add the stop solution to stop the color development reaction, and detect the optical density value; draw a standard curve based on the haptoglobin content of the standard solution and the corresponding optical density value of the test, substitute the luminescence value of the sample to be tested into the standard curve, and calculate the haptoglobin content of the sample to be tested;

[0060] Qualitative detection: add the sample to be tested, positive control solution, and negative control solution to the ELISA plate coated with the antibody, incubate, wash the plate, then add the working concentration of the enzyme-labeled antibody, incubate, wash the plate, add the substrate color development solution, color, then add the stop solution to stop the color development reaction, and detect the optical density value;

[0061] Optionally, the incubation condition of the sample to be tested or the standard solution is 35°C-38°C for 30 minutes-1.5 hours, preferably 37°C for 1 hour;

[0062] And / or, the incubation condition of the enzyme-labeled antibody is 35°C-38°C for 30 minutes-1.5 hours, preferably 37°C for 30 minutes;

[0063] And / or, the sample to be tested is urine, serum, saliva or cerebrospinal fluid;

[0064] Optionally, add the sample to be tested or the standard solution to the antibody-coated ELISA plate, 100ul per well, incubate, wash the plate, then add 100ul per well of the enzyme-labeled antibody at the working concentration, then incubate, wash the plate, add 50ul of substrate color development solution A and B to each well, develop the color at 37°C in the dark for 15 minutes, and then add 50ul of stop solution to each well to terminate the color development reaction.

[0065] The technical solution of the present invention has the following advantages:

[0066] 1. Two antibodies provided by the present invention, the antibodies include a heavy chain variable region VH and a light chain variable region VL, the VH and VL include a determinant complementary region, and the determinant complementary regions of the VH and VL are composed of CDR1, CDR2 and CDR3; (1), antibody HY2B4: the CDR1 of the VH has an amino acid sequence as shown in SEQ ID NO.1, or has at least 90% similarity to the amino acid sequence; the CDR2 of the VH has an amino acid sequence as shown in SEQ ID NO.2, or has at least 90% similarity to the amino acid sequence; the CDR3 of the VH has an amino acid sequence as shown in SEQ ID NO.3, or has at least 90% similarity to the amino acid sequence; the CDR1 of the VL has an amino acid sequence as shown in SEQ ID NO.4, or has at least 90% similarity to the amino acid sequence; the CDR2 of the VL has an amino acid sequence of: YAS, or has at least 90% similarity to the amino acid sequence; the CDR3 of the VL has an amino acid sequence as shown in SEQ ID NO. NO.5, or has at least 90% similarity with the amino acid sequence; (2), antibody HY6D7: the CDR1 of the VH has the amino acid sequence shown in SEQ ID NO.6, or has at least 90% similarity with the amino acid sequence; the CDR2 of the VH has the amino acid sequence shown in SEQ ID NO.7, or has at least 90% similarity with the amino acid sequence; the CDR3 of the VH has the amino acid sequence shown in SEQ ID NO.8, or has at least 90% similarity with the amino acid sequence; the CDR1 of the VL has the amino acid sequence shown in SEQ ID NO.9, or has at least 90% similarity with the amino acid sequence; the CDR2 of the VL has the amino acid sequence: GTN, or has at least 90% similarity with the amino acid sequence; the CDR3 of the VL has the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in NO.10, or has a similarity of more than 90% with the amino acid sequence; the above two antibodies HY2B4 and HY6D7 obtained by the present invention can specifically bind to haptoglobin, and the pairing combination of these two antibodies can detect haptoglobin with a concentration as low as 1ng / ml, and the detection sensitivity is very high, and can detect body fluid samples containing haptoglobin such as urine, saliva, serum, and cerebrospinal fluid. At the same time, the ELISA detection method is simple to operate, does not require special equipment, and is easy for professionals to operate.

[0067] 2. The present invention provides an ELISA kit for detecting trace amounts of haptoglobin, comprising the antibodies, one of the antibodies HY2B4 and HY6D7 as a coating antibody, and at least one as an enzyme-labeled antibody. Due to the advantages of the two antibodies HY2B4 and HY6D7, the above antibodies are used to prepare a double antibody sandwich ELISA kit for detecting HP content, which has the advantages of a wide linear detection range, a low detection limit, a high recovery rate, and a strong anti-interference ability, and can accurately, highly sensitively, and specifically detect trace amounts of haptoglobin in samples.

[0068] 3. The present invention provides an ELISA kit for detecting trace haptoglobin, wherein the antibody HY6D7 is used as a coating antibody and the antibody HY2B4 is used as an enzyme-labeled antibody. The present invention verifies that HY6D7 as a coating antibody and HY2B4 as an enzyme-labeled antibody are the optimal combination for determining Hp content.

[0069] 4. A method for detecting trace amounts of haptoglobin, comprising using the ELISA kit for detecting trace amounts of haptoglobin to perform qualitative or quantitative detection. The above detection method is based on the double antibody sandwich ELISA to detect HP. The principle is to physically adsorb one of the antibodies on the solid surface and maintain its immune activity, bind to the antigen in the sample to be tested, and then add a second enzyme-labeled monoclonal antibody to bind to the bound antigen. A solid-phase antibody-antigen-HRP-antibody complex is formed, which then catalyzes the color development of the substrate. By selecting two antibodies HY2B4 and HY6D7 as the coating antibody and enzyme-labeled antibody, respectively, trace amounts of haptoglobin in the sample can be accurately, highly sensitively, and highly specifically detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0071] Figure 1 is the electrophoresis diagram of the affinity-purified haptoglobin antigen in Example 1 of the present invention;

[0072] Figure 2 is the standard curve diagram in Example 15 of the present invention;

[0073] Figure 3 It is the standard curve diagram in the comparative example. DETAILED DESCRIPTION

[0074] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0075] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0076] Example 1 Preparation of Antibody HY2B4 and Antibody HY6D7

[0077] 1. Antigen preparation:

[0078] Taking advantage of the specific binding characteristics of haptoglobin and hemoglobin, 1 ml of NHS-activated agarose gel was coupled to 20 mg of hemoglobin to prepare affinity purification columns. Since HP2-1 phenotype haptoglobin contains a1, a2 and β chains at the same time, 10 serum samples with HP2-1 phenotype were selected by molecular biological technology, and 20 ml of serum samples containing natural HP2-1 phenotype haptoglobin were obtained by mixing. After column separation and purification, 5 ml of natural HP2-1 phenotype haptoglobin with a concentration of 1.5 mg / ml was obtained. Its purity and molecular weight were identified by SDS-polyacrylamide gel electrophoresis. See Figure 1 The molecular weight of the intact antigen protein is greater than 100 kD, showing multiple bands, corresponding to the polymers corresponding to the Hp2-1 phenotype; the dissociated protein forms 3 bands, corresponding to the α1, α2 and β chains unique to the Hp2-1 phenotype; the above molecular characteristics are consistent with known literature reports. At the same time, no mixed bands were found in the electrophoresis, and the protein purity was ≥95%. It was used as an immunogen for mouse immunization.

[0079] 2. Antibody Preparation:

[0080] (1) Animal immunization

[0081] Two 8-week-old male Balb / c mice weighing about 28g were selected. The specific immunization scheme was as follows: for the initial basic immunization, 1 mg / mL of Hp protein was thoroughly mixed with an equal volume of Freund's complete adjuvant, and 0.2 mL was injected intraperitoneally into each mouse. Two booster immunizations were performed on the 14th and 28th days after the initial immunization, using an immunogen of an equal volume of fully mixed Hp protein (1 mg / mL) and incomplete Freund's adjuvant, and 0.2 mL was injected intraperitoneally. On the 42nd day, 0.1 mL of 1 mg / mL Hp protein was injected directly into the spleen.

[0082] On the 46th day after the first immunization, mice with higher antibody titers detected by indirect ELISA were selected, their eyeballs were removed to collect blood, and they were killed by cervical dislocation. The spleen was isolated under sterile conditions for cell fusion.

[0083] (2) Cell fusion

[0084] Grind the spleen of mice to collect spleen cells, wash with GKN buffer and resuspend the cells in 10mL DMEM. Splenocytes and mouse Sp2 / 0 myeloma cells were mixed at a ratio of 1.6:1 and centrifuged. The supernatant was removed and the centrifuge tube was tapped to make the precipitate into a paste. 1ml of 50% PEG3500 pre-warmed at 37°C was added dropwise, shaking while adding. After adding within 1 minute, shake for another 1 minute. 2ml of serum-free DMEM pre-warmed at 37°C was added dropwise, and the addition was completed within 2 minutes. Continue to add 7ml of serum-free DMEM, adding within 2-3 minutes, shaking while adding. Centrifuge the cell suspension and discard the supernatant, add pre-warmed HAT medium containing 20% ​​bovine serum, mix the cells, and then add to a 96-well culture plate and culture in a cell culture incubator with 5% CO2 and 37°C.

[0085] (3) Positive clone screening and subcloning

[0086] The Hp protein was diluted to 3 μg / mL with carbonate buffer and coated on a 96-well ELISA plate. The plate was placed in a refrigerator at 4°C overnight. After washing and blocking, the cell culture supernatant was added for incubation and detected with HRP-labeled rabbit anti-mouse IgG antibody. After three subclones, two highly specific and high-affinity antibodies, HY2B4 and HY6D7, were finally obtained. The two monoclonal antibodies obtained by Western blotting verification and screening can specifically recognize human haptoglobin. The two antibodies are as follows:

[0087] Antibody HY2B4: includes a heavy chain variable region VH and a light chain variable region VL, wherein the VH and VL include a determinant complementary region (CDR1-CDR3) and a framework region. The amino acid sequences of CDR1-CDR3 of VH are shown in SEQ ID NOs. 1 to 3, the amino acid sequences of CDR1 and CDR3 of VL are shown in SEQ ID NOs. 4 to 5, and the amino acid sequence of CDR2 of VL is: YAS. The full-length amino acid sequence of the heavy chain of antibody HY2B4 is shown in SEQ ID NO. 11, wherein positions 118 to 441 are constant regions. The nucleotide sequence of the heavy chain of antibody HY2B4 is shown in SEQ ID NO. 12. The full-length amino acid sequence of the light chain of antibody HY2B4 is shown in SEQ ID NO. 13, wherein positions 108 to 214 are constant regions. The nucleotide sequence of the light chain of antibody HY2B4 is shown in SEQ ID NO. 14.

[0088] Antibody HY6D7: includes a heavy chain variable region VH and a light chain variable region VL, wherein the VH and VL include a determinant complementary region (CDR1-CDR3) and a framework region. The amino acid sequences of CDR1-CDR3 of VH are shown in SEQ ID NOs. 6 to 8, the amino acid sequences of CDR1 and CDR3 of VL are shown in SEQ ID NOs. 9 to 10, and the amino acid sequence of CDR2 of VL is: GTN. The full-length amino acid sequence of the heavy chain of antibody HY6D7 is shown in SEQ ID NO. 15, wherein positions 118 to 447 are constant regions, and the nucleotide sequence of the heavy chain of antibody HY6D7 is shown in SEQ ID NO. 16. The full-length amino acid sequence of the light chain of antibody HY6D7 is shown in SEQ ID NO. 17, wherein positions 108 to 215 are constant regions, and the nucleotide sequence of the light chain of antibody HY6D7 is shown in SEQ ID NO. 18.

[0089] (4) Preparation of mouse ascites and antibody purification

[0090] Ten days before inoculation of hybridoma cells, mice were intraperitoneally injected with 0.5 mL of liquid paraffin. Hybridoma cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 1×10 6 / mL, 0.5mL was injected into each mouse. After 7 to 9 days, when the abdomen of the mouse bulged, the mouse was killed to collect the ascites, centrifuged and frozen. The hybridoma cells producing HY2B4 antibody (mouse anti-human haptoglobin monoclonal cell line) and the hybridoma cells producing HY6D4 antibody (mouse anti-human haptoglobin monoclonal cell line) were sent to the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address of No. 2, Yard No. 1, Beichen West Road, Changyangqu, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit numbers are CGMCC NO.45850 and CGMCC NO.45851, respectively, and the deposit date is April 29, 2024.

[0091] Antibody purification was performed by affinity chromatography using a protein G-agarose gel pre-packed column. The pre-packed column was balanced with binding buffer for 5 to 10 column volumes. After centrifugation of mouse ascites, it was diluted 10 times with binding buffer and filtered with a 0.45um filter membrane. After balancing 5 to 10 column volumes with binding buffer, eluted with eluent, and the elution peaks were collected in each tube. 60 to 100 μl of neutralization buffer was pre-placed in the collection tube to adjust its pH to neutral. The purified antibody protein was dialyzed three times with 0.02M PBS 1000ml, and the antibody concentrations were calculated to be HY2B4 5.8mg / ml and HY6D4 3.55mg / ml, respectively.

[0092] Example 2 HRP labeled screening antibody ELISA detection titer

[0093] Take 5mg HRP and dissolve it in 0.5mL distilled water, add 0.5mL of freshly prepared 0.06mol / L NaIO4 solution, stir at room temperature in the dark for 20min, and then place at 4℃ for half an hour; add 0.5mL of 0.16M ethylene glycol aqueous solution, mix well and place at room temperature for 30 minutes; add 1mL of the antibody solution prepared in Example 1, mix well and put it into a dialysis bag, dialyze overnight at 4℃ in 0.05M, pH=9.6 carbonate buffer; add 0.2mL of 5mg / mL NaBH4 solution to the dialyzed solution and let it stand at 4℃ for 2 hours; dialyze overnight at 4℃ with 500ml of 0.02M, pH7.4 PBS; take it out and add appropriate amount of neutral glycerol to make it up to 5mL for use. The labeled antibodies are HRP-HY2B4 (1.2mg / ml) and HRP-HY6D7 (1.4mg / ml). The titer of enzyme-labeled antibodies is detected based on the double antibody sandwich ELISA method as follows:

[0094] Purified haptoglobin (3ug / ml) was coated on the ELISA plate, 100ul per well, overnight at 4°C, the plate was washed, 200ul of PBS solution containing 1% BSA (weight / volume) was added to each well and blocked at 37°C for 2 hours, the plate was washed, and dried for use. 100ul of enzyme-labeled antibody diluents of different dilution multiples were added, incubated at 37°C for 30 minutes, the plate was washed 5 times with PBST, 50ul of substrate color development solution A and B (same as Example 3) were added to each well, mixed, color developed at 37°C in the dark for 15 minutes, and then 50ul of stop solution (same as Example 3) was added to stop the color development reaction, and the OD450nm detection value was read by an ELISA reader.

[0095] The titer of the enzyme-labeled antibody was tested with purified antigen and is shown in Table 1.

[0096] Table 1. ELISA titer of HRP-HY2B4 and HRP-HY6D7 antibodies

[0097]

[0098] Example 3 ELISA kit for detecting trace amounts of haptoglobin

[0099] This embodiment provides an ELISA kit for detecting trace amounts of haptoglobin, comprising: an ELISA plate and an enzyme-labeled antibody;

[0100] The ELISA plate is coated with antibody HY6D7;

[0101] The enzyme-labeled antibody was HRP-HY2B4 in Example 2, and the working concentration was HRP-HY2B4 stock solution (concentration was 5.8 mg / ml) diluted at a dilution factor of 1:2000, and the diluent was PBS.

[0102] Furthermore, it also includes a standard solution, a substrate color developing solution, a stop solution and an antibody diluent. The standard solution is a prepared series of gradient concentration haptoglobin solutions, which are diluted with 0.02M pH7.4 PBS diluent containing 1% BSA (bovine serum albumin) to concentrations of 0, 1, 5, 25, 50, 100, and 200 ng / ml, respectively.

[0103] The substrate color developing solution includes color developing agent A and color developing agent B. Color developing agent A is 4.2g citric acid, 13.6g sodium acetate, 0.5g urea peroxide dissolved in 100mL deionized water, fixed to 1L, and the pH value is adjusted to 5.0. Color developing agent B is 1g TMB color developing agent dissolved in 50mL dimethyl sulfoxide, and then 100mL formaldehyde and 16.0g polyvinylpyrrolidone are added after dissolution, and the volume is fixed to 1L with deionized water after dissolution. The stop solution is 2M H2SO4.

[0104] The antibody diluent was 0.05 M carbonate buffer at pH 9.6.

[0105] The ELISA plate preparation method is as follows:

[0106] Use 0.05M PH9.6 carbonate buffer to dilute the coating antibody to 3ug / ml for coating, 100ul per well, coat overnight at 4℃, wash the plate 5 times with PBST, add 1% BSA and PBS blocking solution respectively, 200ul per well, block at 37℃ for 2 hours, wash the plate 5 times, and dry at 33℃.

[0107] Example 4

[0108] The difference between this embodiment and embodiment 3 is that the coating concentration of the coating antibody is 1 ug / ml and the dilution multiple of the HRP-HY2B4 stock solution is 1:1000.

[0109] Example 5

[0110] The difference between this embodiment and embodiment 3 is that the coating concentration of the coating antibody is 9ug / ml and the dilution multiple of the HRP-HY2B4 stock solution is 1:8000.

[0111] Example 6

[0112] The difference between this embodiment and Example 3 is that the blocking solution is a 3% BSA PBS solution. The blocking condition is 37°C for 3 hours.

[0113] Example 7

[0114] The difference between this embodiment and embodiment 3 is that the blocking solution is a PBS solution of 5% skimmed milk powder. The blocking condition is 37° C. for 3 hours.

[0115] Example 8

[0116] This embodiment provides a method for detecting trace amounts of haptoglobin, using the kit of embodiment 3, comprising the following steps:

[0117] In the ELISA plate coated with the antibody, the sample to be tested or the standard solution is added to the positive well (P), and the standard solution containing 0 ng / ml haptoglobin is added to the negative well (N), 100ul per well, incubated at 37°C for 1 hour, washed 5 times with PBST (phosphate buffer), 100ul of enzyme-labeled antibody diluent (working concentration: dilution is 1:2000), incubated at 37°C for 30 minutes, washed 5 times with PBST, 50ul of substrate color development solution A and B are added to each well, mixed, color developed at 37°C in the dark for 15 minutes, and then 50ul of stop solution is added to stop the color development reaction, and the optical density value of OD450nm is read by a microplate reader; a standard curve is drawn based on the haptoglobin content of the standard solution and the corresponding optical density value of OD450nm, the optical density value of the sample to be tested is substituted into the standard curve, and the haptoglobin content of the sample to be tested is calculated.

[0118] Example 9

[0119] The difference between this embodiment and embodiment 8 is that, using the kit of embodiment 4, the incubation conditions of the sample or standard solution to be tested are 37° C. for 30 minutes, and the incubation conditions of the enzyme-labeled antibody diluent are 37° C. for 1.5 hours.

[0120] Example 10

[0121] The difference between this embodiment and embodiment 8 is that, using the kit of embodiment 5, the incubation conditions of the sample to be tested or the standard solution are 37° C. for 1.5 hours, and the incubation conditions of the enzyme-labeled antibody diluent are 37° C. for 1 hour.

[0122] Example 11 Optimal Pairing Combination of Coated Antibody and Enzyme-labeled Antibody

[0123] This example investigates the optimal pairing combination of coating antibody-enzyme-labeled antibody, including the following methods:

[0124] The method in Example 3 was followed, except that HY6D7 and HY2B4 were selected as coating antibodies, and HRP-HY2B4 and HRP-HY6D7 were selected as enzyme-labeled antibodies.

[0125] The reagent obtained above was used to detect the standard solution containing different concentrations of haptoglobin according to Example 8. The minimum detection limit, standard curve, and specificity (specificity refers to the specificity of the combination of the antigenic determinant on the antigen molecule and the hypervariable region of the antibody molecule, which is determined by the complementarity of the spatial structure between the two molecules. In this test, the same concentration of antigen, different antibody pairing combination, the higher the detection OD value, the better the specificity). The results are shown in the table below. It can be determined that HY6D7 as the coating antibody and HRP-HY2B4 as the enzyme-labeled antibody are the optimal combination for the determination of Hp content.

[0126] Table 2 Summary of antibody pairing results

[0127] Coating antibody Enzyme-labeled antibody Minimum detection limit Specificity Linear HY6D7 HRP-HY6D7 Difference Difference Difference HY2B4 HRP-HY6D7 Poor Poor Poor HY6D7 HRP-HY2B4 good excellent better HY2B4 HRP-HY2B4 better better Poor

[0128] Example 12 Determination of the optimal coating antibody concentration and the optimal enzyme-labeled antibody dilution

[0129] The method in Example 3 was followed, except that the coating antibody HY6D7 was diluted to 1ug / ml, 3ug / ml, 6ug / ml, and 9ug / ml for coating. The working concentrations of the enzyme-labeled antibody HRP-HY2B4 were selected at dilutions of 1:1000, 1:2000, 1:4000, and 1:8000, respectively.

[0130] The optimal concentration was screened by chessboard titration. On ELISA plates with different coating antibody concentrations, a standard solution containing 50 ng / ml of haptoglobin was added to the positive wells (P), and a standard solution containing 0 ng / ml of haptoglobin was added to the negative wells (N). 100 ul was added to each well, incubated at 37°C for 1 hour, washed 5 times with PBST, and enzyme-labeled antibody HRP-HY2B4 was added at dilutions of 1:1000, 1:2000, 1:4000, and 1:8000, respectively, incubated at 37°C for 30 minutes, washed 5 times with PBST, and 50 ul of substrate color development solution A and B were added to each well, mixed, and color was developed at 37°C in the dark for 15 minutes, then 50 ul of stop solution was added to stop the color development reaction, and the OD450 nm detection value was read by an enzyme reader, and P / N was calculated as OD450 nm detection value of the positive well (P) / OD450 nm detection value of the negative well (N). The P / N values ​​of paired tests with different dilutions were calculated. The larger the P / N value, the corresponding concentration is the optimal working concentration of the coating antibody and the enzyme-labeled antibody for detecting haptoglobin. Through calculation, it was finally determined that the optimal coating concentration of the capture antibody was 3ug / ml, and the optimal dilution multiple of the enzyme-labeled antibody was 1:2000. The results are shown in Table 3.

[0131] Table 3 Analysis of the pairing of coated antibodies at different concentrations and enzyme-labeled antibodies at different dilutions

[0132]

[0133] Example 13 Optimization of the best sealing conditions

[0134] The method in Example 3 was followed, except that the blocking solutions were PBS containing 1% BSA, 3% BSA, and 5% skimmed milk powder, respectively, and the blocking conditions were 37° C. for 1 hour and 2 hours, respectively.

[0135] On the different ELISA plates obtained, 50 ng / ml of calibrator was added to the positive well (P), and 0 ng / ml of calibrator was added to the negative well (N), 100 ul to each well, incubated at 37°C for 1 hour, washed 5 times with PBST, added enzyme-labeled antibody diluted with PBS 1:2000, incubated at 37°C for 30 minutes, washed 5 times with PBST, added 50 ul of substrate color development solution A and B to each well, mixed, and color developed at 37°C in the dark for 15 minutes, then added 50 ul of stop solution to stop the color development reaction, read the OD450nm detection value with an enzyme reader, and calculated P / N=OD450nm detection value of the positive well (P) / OD450nm detection value of the negative well (N). The P / N values ​​corresponding to the detection under different blocking conditions were calculated. The larger the P / N value, the corresponding blocking condition is the best blocking condition for detecting haptoglobin. The calculation results are shown in the following table. From the data in the table, it can be seen that when the same concentration of haptoglobin is detected with different types of blocking solutions, the P / N value of 1% BSA as the blocking solution is significantly better than the other two groups. In addition, blocking at 37°C for 2 hours is better than 1 hour, so 1% BSA at 37°C for 2 hours is selected.

[0136] Table 4 Detection analysis of different blocking solutions and different blocking times

[0137]

[0138] Example 14 Optimization of the best incubation conditions

[0139] HP was detected using the kit in Example 3, 50 ng / ml of the calibrator solution was added to the positive wells (P), and 0 ng / ml of the haptoglobin calibrator solution was added to the negative wells (N), 100 ul to each well, incubated at 37°C for 30 minutes, 1 hour, and 1.5 hours, respectively, washed 5 times with PBST, and enzyme-labeled antibodies diluted with PBS 1:2000 were added, incubated at 37°C for 30 minutes, 1 hour, and 1.5 hours, respectively, washed 5 times with PBST, 50 ul of substrate color development solutions A and B were added to each well, mixed, and color developed at 37°C in the dark for 15 minutes, then 50 ul of stop solution was added to stop the color development reaction, and the OD450 nm detection value was read using an enzyme reader. The P / N values ​​corresponding to the detection under different incubation conditions were calculated. The incubation conditions with larger P / N values ​​were the optimal incubation conditions for detecting haptoglobin. The calculation results were as shown in the following table. From the data in the table, it can be seen that under different antigen incubation times and different enzyme-labeled antibody incubation times, the P / N value was significantly better than that of other groups when the antigen incubation time was 1 hour and the enzyme-labeled antibody incubation time was 30 minutes. Therefore, the optimal antigen incubation time was 1 hour and the enzyme-labeled antibody incubation time was 30 minutes.

[0140] Table 5 Detection and analysis of different antigen incubation times and different enzyme-labeled secondary antibody incubation times

[0141]

[0142] Example 15 Kit Performance Verification

[0143] (1) Preparation of calibrators and quality control products

[0144] The approved multi-protein calibrator [National Food and Drug Administration (Import) No. 2014 3402493] was used as the Hp detection calibrator, produced by SIEMENS; batch number: 083651; Hp content: 0.930ng / ml; validity period until 2024.11.02. It can be traced back to the international standard ERM@-470. Use antigen diluent to dilute the antigen to 0, 1, 5, 25, 50, 100, 200ng / ml as a calibrator and place it in a calibrator tube. Use antigen diluent to dilute the antigen to 40, 120ng / ml as a quality control product and place it in a quality control tube. The antigen diluent is 0.02M PH7.4 PBS containing 1% BSA (bovine serum albumin).

[0145] (2) Accuracy:

[0146] Using the kit of Example 3, the detection method was implemented according to Example 8. Sample A with a known concentration of high-level haptoglobin (198 ng / mL) was added to sample B with a low concentration, and the volume ratio between sample A and sample B was 1:9. The test was repeated 3 times, and the average value was taken. The recovery rate was calculated according to the following formula.

[0147]

[0148] Where:

[0149] R——recovery rate;

[0150] C——the average value of the detected concentration after adding liquid A to liquid B;

[0151] V0——volume of liquid B;

[0152] V S ——Volume of liquid A;

[0153] C0——average value of concentration of liquid B;

[0154] C S ——Concentration of liquid A.

[0155] The results are shown in the table below:

[0156] Table 6 Recovery test results

[0157]

[0158] (3) Linear range:

[0159] The kit of Example 3 was used and the detection method was implemented according to Example 8. Different concentrations of haptoglobin dilutions were used as standard products. The results were linear in the range of 1-200 ng / mL, with a minimum detection limit of 1 ng / mL. The results are shown in Table 7 and the standard curve is shown in Figure 2 .

[0160] Table 7 HP detection standard curve

[0161] HP content (ng / mL) 1 5 25 50 100 200 OD1 0.180 0.382 0.849 1.287 2.054 2.879 OD2 0.164 0.390 0.883 1.305 1.954 2.901 Average OD value 0.172 0.386 0.866 1.296 2.004 2.890

[0162] With concentration as the horizontal axis and optical density as the vertical axis, a double logarithmic plot is made, such as Figure 1 . Standard curve equation: y = 0.53331x-0.78052, correlation coefficient R 2 =0.998.

[0163] (4) Detection limit:

[0164] Using the kit of Example 3, the detection method was implemented according to Example 8. The sample dilution (haptoglobin content was 0) was used as the sample for continuous detection 20 times, and the average value M and standard deviation SD of the detection results were calculated. Then M+2SD was substituted into the standard curve, and the blank detection limit was calculated to be 0.046 ng / mL, which was lower than the detection limit of 1 ng / mL. The results are shown in Table 8.

[0165] Table 8 OD450nm detection values ​​were measured 20 times with sample dilution

[0166] 0.026 0.022 0.031 0.019 0.013 0.028 0.018 0.020 0.025 0.014 0.022 0.016 0.023 0.030 0.024 0.019 0.013 0.025 0.017 0.025

[0167] (5) Coefficient of variation:

[0168] Using the kit of Example 3, the detection method was implemented according to Example 8. The quality control products with concentrations of 40 ng / mL and 120 ng / ml were used for continuous detection 20 times, and the concentration value, mean value (M) and standard deviation (SD) were calculated according to the standard curve, and the coefficient of variation (CV) was calculated according to the formula CV=SD / M×100%. The intra-batch coefficients of variation were 3.47% and 2.23%, respectively, and the average intra-batch coefficient of variation was 2.85%. The results are shown in Table 9.

[0169] Table 9 Calculation results of 20 repeated measurements of 40 ng / mL and 120 ng / mL concentration standards (ng / mL)

[0170]

[0171]

[0172] (6) Interference experiment

[0173] Using the kit of Example 3, the detection method was implemented according to Example 8. Different final concentrations of interfering substances were added to the quality control products with haptoglobin concentrations of 40 ng / mL and 120 ng / mL, respectively, and the non-added group was used as the control group. The relative deviation between the added group and the control group was less than 5% as the acceptable standard for the interfering substance. The maximum concentration levels of different interfering substances (not exceeding the highest concentration seen clinically) were studied, and the anti-interference ability of the detection kit was analyzed. The results are shown in Table 10. The results show that the above-mentioned interfering substances do not significantly interfere with the detection results at the concentrations in the table below.

[0174] Table 10 Effects of different interfering substances on HP detection results

[0175]

[0176] Comparative Example

[0177] The method in Example 3 was followed, except that the coated antibody was the monoclonal antibody 5C4 in the patent document CN111323601, and the antibody used in the enzyme-labeled antibody was the monoclonal antibody 1A2 in the patent document CN111323601. Then, the kit performance verification method in Example 15 was used for verification, and the detection sensitivity was 5 ng / ml, and the linear range was 5-200 ng / mL, and the standard curve was shown in FIG. Figure 3 , the results are shown in the table.

[0178] Table 11 HP detection standard curve

[0179] HP content (ng / mL) 5 25 50 100 200 OD1 0.182 0.623 1.159 1.864 3.045 OD2 0.196 0.631 0.909 1.914 2.907 Average OD value 0.189 0.627 1.034 1.889 2.976

[0180] The concentration is used as the horizontal axis and the optical density is used as the vertical axis. The standard curve equation is: y = 0.75394x-1.25330, and the correlation coefficient is R 2 =0.99916.

[0181] The blank detection limit was calculated to be 0.411 ng / mL, which was 2 ng / mL lower than the detection limit. The results are shown in the table.

[0182] Table 12 OD450nm detection values ​​were measured 20 times with sample dilution

[0183] 0.023 0.011 0.010 0.011 0.001 0.018 0.009 0.000 0.002 0.020 0.002 0.004 0.032 0.014 0.007 0.015 0.023 0.007 0.000 0.003

[0184] In summary, after comparison, the kit of the present invention selects better antibodies and antibody combinations, and the detection sensitivity is significantly improved compared with the control group.

[0185] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An anti-haptoglobin antibody, characterized in that The antibody is antibody HY2B4 or antibody HY6D7: The full-length amino acid sequence of the heavy chain of the antibody HY2B4 is shown in SEQ ID NO.11; The full-length amino acid sequence of the light chain of the antibody HY2B4 is shown in SEQ ID NO.13; The full-length amino acid sequence of the heavy chain of the antibody HY6D7 is shown in SEQ ID NO.15; The full-length amino acid sequence of the light chain of the antibody HY6D7 is shown in SEQ ID NO.

17.

2. The antibody according to claim 1, characterized in that The antibody HY2B4 is prepared from a hybridoma cell line deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO. 45850, and the antibody HY6D7 is prepared from a hybridoma cell line deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO. 45851.

3. A mouse hybridoma cell line, which secretes and produces the antibody according to any one of claims 1 to 2, and has a deposit number of CGMCC NO. 45850 or CGMCC NO. 45851.

4. Any of the following biological materials: (1) A humanized antibody is obtained by humanizing the antibody according to any one of claims 1 to 2; (2) A nucleic acid molecule encoding the antibody according to any one of claims 1 to 2; (3) An expression cassette, recombinant vector or recombinant cell containing the nucleic acid molecule described in (2); (4) Antibody conjugates, obtained by coupling the antibody according to any one of claims 1 to 2 with a marker or protein; the marker is selected from one or more of ELISA dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling; (5) A kit comprising the antibody according to any one of claims 1 to 2.

5. Use of the antibody according to any one of claims 1 to 2 in the preparation of a product for detecting haptoglobin.

6. An ELISA kit for detecting trace amounts of haptoglobin, characterized in that: The method comprises the antibody according to any one of claims 1 to 2, wherein the antibody HY6D7 is used as a coating antibody, and the antibody HY2B4 is used as an enzyme-labeled antibody.

7. The ELISA kit for detecting trace haptoglobin according to claim 6, characterized in that: The enzyme-labeled antibody is labeled with HRP or alkaline phosphatase; And / or, the working concentration of the enzyme-labeled antibody is that the enzyme-labeled antibody stock solution is diluted with the diluent at a dilution ratio of 1:1000-8000, and the concentration of the enzyme-labeled antibody stock solution is 1.77 mg / ml; And / or, further comprising an ELISA plate, a standard solution, a substrate color developing solution, a stop solution and / or an antibody diluent; The ELISA plate is antibody coated.

8. The ELISA kit for detecting trace haptoglobin according to claim 7, characterized in that: The preparation of the ELISA plate comprises the following steps: coating, blocking, drying and / or sealing; In the coating step, the coating antibody is added to the wells of the ELISA plate for coating; In the blocking step, the coated ELISA plate is washed, and a blocking solution is added to the wells for blocking; In the drying step, the blocked ELISA plate is washed and dried; In the sealing step, the dried ELISA plate is sealed.

9. The ELISA kit for detecting trace haptoglobin according to claim 8, characterized in that: The coating concentration of the coating antibody is 1-9 ug / ml; And / or, the blocking solution is a PBS solution containing 1-5 w / v % BSA or 5 w / v % skim milk powder; And / or, the sealing condition is 35°C-38°C for 1-2 hours.

10. The ELISA kit for detecting trace haptoglobin according to claim 9, characterized in that: The coating concentration of the coating antibody is 3ug / ml; And / or, the blocking solution is a PBS solution containing 1 w / v% BSA And / or, the blocking condition is blocking at 37° C. for 2 hours.

11. Use of the ELISA kit according to any one of claims 6 to 10 in preparing a product for qualitative or quantitative detection of trace haptoglobin: Quantitative detection: add the sample to be tested or the standard solution to the ELISA plate coated with the antibody, set a blank well at the same time, incubate, wash the plate, then add the enzyme-labeled antibody at the working concentration, then incubate, wash the plate, add the substrate color developing solution, color, then add the stop solution to stop the color development reaction, and detect the optical density value; draw a standard curve based on the haptoglobin content of the standard solution and the corresponding optical density value of the test, substitute the luminescence value of the sample to be tested into the standard curve, and calculate the haptoglobin content of the sample to be tested; Qualitative detection: Add the sample to be tested, positive control solution, and negative control solution to the ELISA plate coated with the antibody, incubate, wash the plate, then add the enzyme-labeled antibody at the working concentration, then incubate, wash the plate, add the substrate color development solution, develop the color, then add the stop solution to stop the color development reaction, and detect the optical density value.

12. The use according to claim 11, characterized in that The incubation condition of the test sample or standard solution is 35°C-38°C for 30 minutes-1.5 hours; And / or, the incubation condition of the enzyme-labeled antibody is 35° C.-38° C. for 30 minutes-1.5 hours; And / or, the sample to be tested is urine, serum, saliva or cerebrospinal fluid.

13. The use according to claim 12, characterized in that Add the sample or standard solution to each well of the antibody-coated ELISA plate, 100ul, incubate, wash the plate, then add 100ul of the working concentration of enzyme-labeled antibody to each well, then incubate, wash the plate, add 50ul of substrate color development solution A and B to each well, color at 37℃ in the dark for 15 minutes, then add 50ul of stop solution to each well to terminate the color reaction.

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