CN118440192B

CN118440192B - Anti-haptoglobin monoclonal antibody composition and application thereof

Info

Publication number: CN118440192B
Application number: CN202410700528.3A
Authority: CN
Inventors: 杨金奎; 陈兴; 杨芳远; 杨艳慧
Original assignee: Shijiazhuang Heya Biotechnology Co ltd; Beijing Diabetes Institute Beijing Diabetes Prevention And Control Office; Beijing Tongren Hospital
Current assignee: Shijiazhuang Heya Biotechnology Co ltd; Beijing Diabetes Institute Beijing Diabetes Prevention And Control Office; Beijing Tongren Hospital
Priority date: 2024-05-31
Filing date: 2024-05-31
Publication date: 2025-02-18
Anticipated expiration: 2044-05-31
Also published as: CN118440192A

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

Anti-haptoglobin monoclonal antibody composition and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to an anti-haptoglobin monoclonal antibody composition and application thereof.

Background

Haptoglobin (HP), also known as binding globin, is located on human chromosome 16q22.Hp molecules are tetramers composed of two beta chains and two alpha chains linked by disulfide bonds. Where the beta strand is identical, the polymorphism of Hp is mainly caused by genetic variation of the alpha strand. The alpha chain has two alleles Hp1 and Hp2, forming 3 distinct genotypes, hp1-1, hp2-1 and Hp2-2, respectively. The alpha chain has two types a1 and a2, wherein a1 has two subtypes a1S and a1F, the two subtypes differ by only 1 amino acid, the 54 th 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 internal repeats of 142 amino acids and has a relative molecular mass of about 16,000. The protein product Hp1-1 of the homozygous Hp1 allele is a monovalent entity with a relative molecular mass of 86,000. Hp2 allele products heterozygous (Hp 2-1) or homozygous (Hp 2-2) form multimers of multiple Hp molecules Hp2-1 is a variety of linear homodimers and multimers, hp2-2 forming a cyclic polymer.

Hp is an acidic glycoprotein in the serum alpha 2 globulin component, which is widely found in the blood and other body fluids of humans and many mammals. The concentration in human plasma circulation is 0.3-3 g/L. Hp was initially isolated as an acute phase response protein in the liver as an inflammatory response, the main site of synthesis being the liver, which was later found to be present in immune cells including neutrophils and monocytes. In addition, studies have demonstrated that renal proximal tubular epithelial cells synthesize and secrete Hp under stimulation by inflammatory response factors, and that white adipose tissue and other tissues (stomach, intestine, adrenal gland, muscle, brain, epididymis, esophagus, placenta, ovary, ureter, etc.) also have mRNA expression of Hp.

The most important function of Hp is to bind to and be cleared of free hemoglobin (Hb) in plasma, thereby preventing further redox reactions and corresponding tissue damage of the free Hb. In clinic, many reasons for the destruction of erythrocytes such as hemolysis, trauma and the like can cause the obvious rise of free Hb in blood, wherein the free Hb is a powerful oxidation protein, and the ferrous heme component can cause cell injury and tissue injury after being oxidized. The Hb binding site on each β chain of the Hp molecule can bind to free Hb in the plasma, forming a high affinity, irreversible Hp-Hb complex, which is then recognized by the scavenger receptor CD163 and eliminated in the cell, preventing damage to the tissue by free Hb. In addition, free Hb may pass through the glomerular filtration membrane, resulting in oxidative damage to the kidney, while Hp in the plasma may rapidly bind to and be cleared of free Hb, thereby preventing free Hb from filtering from the glomeruli, preventing tubular damage that Hb may cause. In addition, hp has long been recognized as a marker of inflammatory response, having antioxidant effects, which plays an important role in resisting infection, repairing damaged tissues, and maintaining stable internal environment. The serum content of the medicine is obviously increased in pathological states such as infection, wound, inflammatory reaction, tumor, myocardial infarction and the like. In inflammatory reactions and vascular injury, hp increases in serum, and sustained changes in blood flow also induce Hp expression in the arterial wall and are involved in vascular remodeling and repair by promoting fibroblast migration. Hp also plays an important role in inflammatory reactions after implantation, initiating the graft rejection process. In addition, hp has antioxidant activity, inhibits prostaglandin synthesis, inhibits bacteria, and participates in apoptosis and immune response. In many cases, hp upregulation is to address potentially deleterious conditions, which may often be early in the hint of disease or other pathological changes. Thus, it is of great significance for the detection of Hp.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide an anti-haptoglobin monoclonal antibody composition and application thereof, which can detect micro haptoglobin with high sensitivity and high specificity.

For this purpose, the invention provides the following technical scheme:

An antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH and VL comprising determinant complementary regions each consisting of CDR1, CDR2 and CDR3, the antibody comprising at least one of:

(1) Antibody HY2B4:

CDR1 of the VH has an amino acid sequence shown as SEQ ID NO.1 or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

CDR2 of the VH has an amino acid sequence shown as SEQ ID NO.2 or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

CDR3 of the VH has an amino acid sequence as shown in SEQ ID No.3, or has at least 90%, 95% or more than 99% similarity to the amino acid sequence;

CDR1 of the VL has an amino acid sequence as shown in SEQ ID No.4 or has at least 90%, 95% or more than 99% similarity to the amino acid sequence;

The CDR2 of the VL has an amino acid sequence of YAS, or has at least 90%, 95% or more than 99% similarity to the amino acid sequence;

CDR3 of the VL has an amino acid sequence as shown in SEQ ID No.5 or has at least 90%, 95% or more than 99% similarity to said amino acid sequence;

(2) Antibody HY6D7:

CDR1 of the VH has an amino acid sequence shown as SEQ ID NO.6 or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

CDR2 of said VH has an amino acid sequence as shown in SEQ ID NO.7 or has at least 90%, 95% or more than 99% similarity to said amino acid sequence;

The CDR3 of the VH has an amino acid sequence shown as SEQ ID NO.8 or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

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

The CDR2 of the VL has an amino acid sequence of GTN, or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

The CDR3 of VL has an amino acid sequence as shown in SEQ ID No.10, or has at least 90%, 95% or more than 99% similarity to the amino acid sequence.

Optionally, at least one of the following is included:

(1) The antibody is selected from any one of monoclonal antibodies mAb, fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments and single chain antibodies;

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

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

(4) The antibody is a murine antibody.

Optionally, at least one of the following is included:

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

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

the full-length amino acid sequence of the heavy chain of the antibody HY6D7 is shown as SEQ ID NO.15 or has at least 90%, 95% or more than 99% similarity with the amino acid sequence;

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

Alternatively, the antibody HY2B4 (mouse anti-human haptoglobin monoclonal cell strain) is prepared from a hybridoma cell strain with a preservation number of CGMCC No.45850 which is preserved in the China general microbiological culture Collection center, and the antibody HY6D7 (mouse anti-human haptoglobin monoclonal cell strain) is prepared from a hybridoma cell strain with a preservation number of CGMCC No.45851 which is preserved in the China general microbiological culture collection center.

The mouse hybridoma cell strain secretes and generates the antibody, and the preservation numbers are CGMCC NO.45850 and CGMCC NO.45851.

Any of the following biomaterials:

(1) The humanized antibody is obtained by performing humanized transformation on the antibody;

(2) A nucleic acid molecule encoding said antibody;

(3) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising the nucleic acid molecule of (2);

(4) The antibody conjugate is obtained by coupling the antibody with a marker or protein, wherein the marker is one or more selected from ELISA dye markers, enzyme markers, biotin markers, fluorescent dye markers, colloidal gold markers and radioactive markers;

(5) And a kit containing the antibody.

The antibody has the use as any one of the following:

(1) Use in the detection of haptoglobin or in the preparation of a product for detecting haptoglobin;

(2) For immunohistochemistry of haptoglobin-containing tissues;

(3) For detecting secreted haptoglobin in cell culture supernatants.

An ELISA kit for detecting micro 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.

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

And/or, the enzyme-labeled antibody is labeled by HRP or alkaline phosphatase;

And/or the working concentration of the enzyme-labeled antibody is that the enzyme-labeled antibody stock solution and the diluent are diluted according to the dilution ratio of 1:1000-8000, and the concentration of the enzyme-labeled antibody stock solution is 1.77mg/ml;

and/or, ELISA plates, standard solutions, substrate color solutions, stop solutions and/or antibody dilutions;

the ELISA plate is coated with the antibody.

Optionally, the preparation of the ELISA plate comprises the steps of coating, sealing, drying and/or sealing;

In the coating step, a coating antibody is added into an enzyme-labeled plate hole to carry out coating;

in the sealing step, the coated ELISA plate is washed, and sealing liquid is added into the holes for sealing;

In the drying step, washing the sealed ELISA plate and drying;

In the sealing step, the dried ELISA plate is subjected to sealing treatment;

optionally, the coating concentration of the coated antibody is 1-9ug/ml, preferably 3ug/ml;

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

and/or the blocking condition is 35-38 ℃ for 1-2 hours, preferably 37 ℃ for 2 hours.

A method for detecting micro-haptoglobin, which comprises the steps of carrying out qualitative or quantitative detection by using the ELISA kit for detecting the micro-haptoglobin:

The quantitative detection comprises the steps of adding a sample or standard substance solution to be detected into an ELISA plate coated with an antibody, setting blank holes at the same time, incubating, washing the plate, adding an ELISA antibody with working concentration, incubating, washing the plate, adding a substrate color development liquid, developing color, adding a stop liquid to terminate the color development reaction, and detecting an optical density value;

Qualitative detection, namely respectively adding a sample to be detected, a positive control solution and a negative control solution into an ELISA plate coated with an antibody, incubating, washing the plate, then adding the ELISA antibody with working concentration, incubating, washing the plate, adding a substrate color development solution, developing, then adding a stop solution to stop the color development reaction, and detecting an optical density value;

Optionally, the incubation condition of the sample or standard solution to be tested is 35 ℃ to 38 ℃ for 30 minutes to 1.5 hours, preferably 37 ℃ for 1 hour;

And/or the conditions for incubation of the enzyme-labeled antibody are 35-38 ℃ for 30 minutes to 1.5 hours, preferably 37 ℃ for 30 minutes;

and/or the sample to be tested is urine, serum, saliva or cerebrospinal fluid;

Optionally, adding a sample or standard solution to be detected into the ELISA plate coated with the coated antibody, wherein each hole is 100ul, incubating, washing the plate, then adding 100ul of the ELISA antibody with working concentration, incubating, washing the plate, adding 50ul of substrate chromogenic solution A and B, developing for 15 minutes at 37 ℃ in a dark place, then adding 50ul of stop solution, and stopping the chromogenic reaction.

The technical scheme of the invention has the following advantages:

1. The invention provides two antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH and the VL comprise determinant complementary regions, and the determinant complementary regions of the VH and the VL are respectively composed of CDR1, CDR2 and CDR3, (1) an antibody HY2B4, wherein the CDR1 of the VH has an amino acid sequence shown as SEQ ID NO.1 or has at least 90% of similarity with the amino acid sequence, the CDR2 of the VH has an amino acid sequence shown as SEQ ID NO.2 or has at least 90% of similarity with the amino acid sequence, the CDR3 of the VH has an amino acid sequence shown as SEQ ID NO.3 or has at least 90% of similarity with the amino acid sequence, the CDR1 of the VL has an amino acid sequence shown as SEQ ID NO.4 or has at least 90% of similarity with the amino acid sequence, the CDR2 of the CDR has an amino acid sequence of YAS or has at least 90% of similarity with the amino acid sequence shown as SEQ ID NO.1 or has at least 90% of similarity with the amino acid sequence shown as SEQ ID NO.2, the CDR3 has at least 90% of similarity with the amino acid sequence shown as SEQ ID NO.6 or has at least 90% of similarity with the amino acid sequence shown as SEQ ID NO.3 or has at least 90% of similarity with the amino acid sequence shown as SEQ ID NO.6 or has at least 90% of the amino acid sequence shown as SEQ ID NO.6, or has at least more than 90% of similarity with the amino acid sequence, the CDR2 of the VL has an amino acid sequence of GTN or has at least more than 90% of similarity with the amino acid sequence, the CDR3 of the VL has an amino acid sequence shown as SEQ ID NO.10 or has more than 90% of similarity with the amino acid sequence, the two antibodies HY2B4 and HY6D7 obtained by the invention can specifically bind with the haptoglobin, the paired combination of the two antibodies can detect the haptoglobin with the concentration as low as 1ng/ml, the detection sensitivity is high, and body fluid samples containing the haptoglobin such as urine, saliva, serum, cerebrospinal fluid and the like can be detected.

2. The ELISA kit for detecting the micro-haptoglobin provided by the invention comprises the antibody, wherein one of the antibody HY2B4 and the antibody HY6D7 is used as a coating antibody, and at least one of the antibodies is used as an enzyme-labeled antibody. Because the two antibodies HY2B4 and HY6D7 have the advantages, the kit for detecting the HP content by adopting the antibody to prepare the double-antibody sandwich ELISA has the advantages of wide detection linear range, low detection limit, high recovery rate, strong anti-interference capability and the like, and can accurately, highly sensitively and highly specifically detect the trace haptoglobin in the sample.

3. The ELISA kit for detecting the micro-haptoglobin provided by the invention has the advantages that the antibody HY6D7 is used as a coating antibody, the HY2B4 is used as an enzyme-labeled antibody, and the invention verifies that the HY6D7 is used as the coating antibody and the HY2B4 is used as the enzyme-labeled antibody and is used as the optimal combination of Hp content measurement.

4. A method for detecting micro-haptoglobin comprises the step of carrying out qualitative or quantitative detection by using the ELISA kit for detecting the micro-haptoglobin. The detection method is based on double antibody sandwich ELISA to detect HP, and the principle is that one of the antibodies is physically adsorbed on the solid phase surface, and the immunocompetence of the antibody is kept, and the antibody is combined with an antigen in a sample to be detected, and then enzyme-labeled second monoclonal antibody is added to combine with the combined antigen. A solid phase antibody-antigen-HRP-antibody complex is formed, which in turn catalyzes the development of the substrate. By selecting two antibodies HY2B4 and HY6D7 as coating antibodies and enzyme-labeled antibodies, trace haptoglobin in a sample can be accurately detected with high sensitivity and high specificity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is an electrophoretogram of affinity purified haptoglobin antigen according to example 1 of the present invention;

FIG. 2 is a standard graph in example 15 of the present invention;

fig. 3 is a standard graph in the comparative example.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1 preparation of antibodies HY2B4 and HY6D7

1, Antigen preparation:

According to the characteristic that haptoglobin and hemoglobin are specifically combined, NHS-activated agarose gel 1ml is selected to couple 20mg of hemoglobin to prepare an affinity purification column, and since HP2-1 phenotype haptoglobin contains a1, a2 and beta chains simultaneously, 10 cases of serum samples with the phenotype HP2-1 are selected to be screened through molecular biology technology, 20ml of serum samples containing natural HP2-1 phenotype haptoglobin are obtained through mixing, 5ml of natural HP2-1 phenotype haptoglobin with the concentration of 1.5mg/ml is obtained through column separation and purification, the purity and molecular weight of the haptoglobin are identified through SDS-polyacrylamide gel electrophoresis, referring to FIG. 1, the whole antigen protein molecular weight is larger than 100kD, a plurality of zones are shown, corresponding polymers corresponding to Hp2-1 phenotypes are shown, the dissociated proteins form 3 zones, alpha 1, alpha 2 and beta chains specific to the Hp2-1 phenotypes are consistent with the report of the prior art. Meanwhile, no impurity band is seen in electrophoresis, and the protein purity is more than or equal to 95%. As an immunogen for immunization of mice.

2 Preparation of antibodies:

(1) Immunization of animals

2 Male Balb/c mice of 8 weeks of age were selected and had a body weight of about 28 g. The specific immunization scheme is that Hp protein of 1mg/mL is fully and uniformly mixed with Freund's complete adjuvant with equal volume for primary basic immunization, and each mouse is injected with 0.2mL intraperitoneally. Two booster immunizations were performed on days 14 and 28 after the primary immunization, using equal volumes of Hp protein (1 mg/mL) and Freund's incomplete adjuvant, and injecting 0.2mL intraperitoneally. Day 42 direct spleen injections of 1mg/mL Hp protein 0.1mL.

On day 46 after primary immunization, mice with higher titers of indirect ELISA detection antibodies were selected, the eyeballs of the mice were removed to obtain blood and sacrificed by neck breakage, and spleens were isolated under aseptic conditions for cell fusion.

(2) Cell fusion

Spleen cells were collected by grinding the spleen of the mice, washed with GKN buffer, and resuspended in 10mL DMEM. Spleen cells and mouse Sp2/0 myeloma cells are uniformly mixed according to the ratio of 1.6:1, and then are centrifuged, supernatant is removed, and a centrifuge tube is lightly buckled to precipitate into paste. 50% PEG3500 1ml pre-warmed at 37℃was added dropwise with shaking, and after 1 minute the addition was completed, the mixture was further shaken for 1 minute. 2ml of serum-free DMEM pre-warmed at 37℃was added dropwise, after which the addition was completed within 2 minutes. The addition of serum-free DMEM 7ml was continued and completed within 2-3 minutes with shaking. The cell suspension was centrifuged to discard the supernatant, and the cells were homogenized by adding a pre-warmed HAT medium containing 20% bovine serum, and then added to a 96-well plate and cultured in a 5% co ₂, 37 ℃ cell incubator.

(3) Positive clone screening and subcloning

Hp protein was diluted to 3. Mu.g/mL with carbonate buffer, coated on 96-well ELISA plates, washed, blocked, and incubated with cell culture supernatants for detection with HRP-labeled rabbit anti-mouse IgG antibodies overnight at 4 ℃. Through 3 subcloning, two high-specificity and high-affinity antibodies HY2B4 and HY6D7 are finally obtained, and the two monoclonal antibodies obtained through Western blotting verification screening can specifically identify human haptoglobin. The two antibodies were as follows:

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

Antibody HY6D7 includes a heavy chain variable region VH and a light chain variable region VL, which include determinant complementary regions (CDR 1-CDR 3) and framework regions. The amino acid sequences of CDR1-CDR3 of the VH are shown as SEQ ID NO. 6-8, the amino acid sequences of CDR1 and CDR3 of the VL are shown as SEQ ID NO. 9-10, and the amino acid sequence of CDR2 of the VL is GTN. The full-length amino acid sequence of the heavy chain of the antibody HY6D7 is shown as SEQ ID NO.15, wherein 118 th to 447 th positions are constant regions, and the nucleotide sequence of the heavy chain of the antibody HY6D7 is shown as SEQ ID NO. 16. The full-length amino acid sequence of the light chain of the antibody HY6D7 is shown as SEQ ID NO.17, wherein 108 th to 215 th positions are constant regions, and the nucleotide sequence of the light chain of the antibody HY6D7 is shown as SEQ ID NO. 18.

(4) Preparation of ascites in mice and purification of antibodies

10 Days before hybridoma cell inoculation, mice were intraperitoneally injected with 0.5mL of liquid paraffin. Hybridoma cells in the logarithmic growth phase were collected, and the cell concentration was adjusted to 1X 10 ⁶ cells/mL, and 0.5mL was injected into each mouse. And after 7-9 days, after the abdomen of the mouse is raised, the ascites is collected by sacrifice, and the mice are frozen by centrifugation. Hybridoma cells producing the HY2B4 antibody (mouse anti-human haptoglobin monoclonal cell line) and hybridoma cells producing the HY6D4 antibody (mouse anti-human haptoglobin monoclonal cell line) are sent to China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation address of North Star West Lao No. 1,2 in Beijing, postal code 100101, and preservation numbers of CGMCC No.45850 and CGMCC No.45851, respectively, and a preservation date of 2024, 04 and 29.

Antibody purification affinity chromatography was performed using protein G-sepharose pre-loaded columns. The pre-packed column balances the volume of 5-10 column beds with the binding buffer solution, and after the ascites of the mice are centrifuged, the pre-packed column is diluted by 10 times of the binding buffer solution, and the pre-packed column is filtered and loaded with a 0.45um filter membrane. After balancing 5-10 column bed volumes by using a binding buffer solution, eluting by using an eluent, collecting each tube of an eluting peak, presetting 60-100 mu l of a neutralization buffer solution in a collecting tube, and regulating the PH of the tube to be neutral. The purified antibody protein was dialyzed against 0.02M PBS1000ml in three times to calculate the antibody concentrations as HY2B 4.8 mg/ml and HY6D 4.55 mg/ml, respectively.

EXAMPLE 2 HRP-labeled screening of antibody ELISA detection titers

Dissolving 5mg HRP in 0.5mL distilled water, adding 0.5mL of newly prepared 0.06mol/L NaIO ₄ solution, stirring at room temperature in a dark place for 20min, standing at 4 ℃ for half an hour, adding 0.5mL of 0.16M glycol aqueous solution, standing at room temperature for 30 min after uniform mixing, adding 1mL of the antibody solution prepared in example 1, mixing, placing into a dialysis bag, dialyzing at 4 ℃ in 0.05M carbonate buffer solution with pH of 9.6 for overnight, adding 0.2mL of 5mg/mL NaBH ₄ solution into the dialyzed solution, standing at 4 ℃ for 2 h, adding 500mL PBS with pH of 0.02M and pH7.4 for overnight at 4 ℃, and adding a proper amount of neutral glycerol for constant volume to 5mL after taking out for standby. The labeled antibodies were HRP-HY2B4 (1.2 mg/ml) and HRP-HY6D7 (1.4 mg/ml), respectively. ELISA detection of the titer of the enzyme-labeled antibody based on the double-antibody sandwich method comprises the following steps:

Purified haptoglobin (3 ug/ml) was coated on an ELISA plate, 100ul per well, at 4℃overnight, washed, blocked for 2 hours at 37℃by adding 200ul of PBS solution containing 1% BSA (wt/vol) per well, washed, and dried for use. Adding 100ul of enzyme-labeled antibody diluents with different dilution factors, incubating for 30min at 37 ℃, washing the plate 5 times by PBST, adding 50ul of substrate chromogenic solutions A and B (same as in example 3) into each hole, uniformly mixing, developing for 15min at 37 ℃ in a dark place, adding 50ul of stop solution (same as in example 3), stopping the chromogenic reaction, and reading the OD450nm detection value by an enzyme-labeled instrument

The titers of the enzyme-labeled antibodies detected with purified antigen are shown in table 1.

TABLE 1 ELISA detection titers for HRP-HY2B4, HRP-HY6D7 antibodies

Example 3 ELISA kit for detecting micro haptoglobin

The embodiment provides an ELISA kit for detecting micro haptoglobin, which comprises an ELISA plate and an enzyme-labeled antibody;

The ELISA plate is coated with an antibody HY6D7;

the enzyme-labeled antibody is HRP-HY2B4 in example 2, the working concentration is HRP-HY2B4 stock solution (concentration is 5.8 mg/ml), the dilution ratio is 1:2000, and the dilution is PBS.

Further, the kit also comprises a standard substance solution, a substrate chromogenic solution, a stop solution and an antibody diluent. The standard solution is prepared as a series of gradient concentration haptoglobin solutions, and the haptoglobin is diluted to 0,1, 5, 25, 50, 100 and 200ng/ml by 0.02M PH7.4 PBS dilution containing 1% BSA (bovine serum albumin).

The substrate color development solution comprises a color development agent A and a color development agent B, wherein the color development agent A is 4.2g of citric acid, 13.6g of sodium acetate and 0.5g of carbamide peroxide are dissolved in 100mL of deionized water, the volume is fixed to 1L, and the pH value is adjusted to 5.0. The color reagent B is that lg TMB color reagent is dissolved in 50mL dimethyl sulfoxide, 100mL formaldehyde is added after dissolution, 16.0g polyvinylpyrrolidone is added after dissolution, and deionized water is used for volume fixation to 1L. The stop solution was 2M H ₂SO₄.

The antibody dilution was 0.05m ph9.6 carbonate buffer.

The ELISA plate was prepared as follows:

The coated antibodies were diluted to 3ug/ml with 0.05M PH9.6 carbonate buffer, coated overnight at 100ul at 4℃per well, PBST plates washed 5 times, 1% BSA, PBS blocking solution was added separately, 200ul per well blocked at 37℃for 2 hours, plates washed 5 times, and dried at 33 ℃.

Example 4

This example differs from example 3 in that the coating concentration of the coated antibody is 1ug/ml. The dilution factor of the HRP-HY2B4 stock solution is 1:1000.

Example 5

This example differs from example 3 in that the coating concentration of the coated antibody was 9ug/ml. The dilution factor of the HRP-HY2B4 stock solution is 1:8000.

Example 6

This example differs from example 3 in that the blocking solution is a 3% BSA in PBS blocking for 3 hours at 37 ℃.

Example 7

This example differs from example 3 in that the blocking solution is a 5% nonfat dry milk in PBS, blocking conditions are 37℃for 3 hours.

Example 8

The present embodiment provides a method for detecting micro haptoglobin, using the kit of embodiment 3, comprising the steps of:

In ELISA plate coated with antibody, positive hole (P) is added with sample or standard solution to be detected, negative hole (N) is added with standard solution containing 0ng/ml haptoglobin, each hole is 100ul,37 ℃ is incubated for 1 hour, PBST (phosphate buffer) is washed 5 times, enzyme-labeled antibody diluent is added with 100ul (working concentration: dilution is 1:2000), 37 ℃ is incubated for 30 minutes, PBST is washed 5 times, each hole is added with substrate developing solution A and B for 50ul, mixing is carried out uniformly, 37 ℃ is shaded for 15 minutes, then 50ul of stop solution is added, the chromogenic reaction is stopped, OD450nm optical density value is read by an enzyme-labeled instrument, the optical density value of the sample to be detected is substituted into the standard curve according to the haptoglobin content of the standard solution and the optical density value corresponding to OD450nm, and the haptoglobin content of the sample to be detected is calculated.

Example 9

The difference between this example and example 8 is that the test sample or standard solution is incubated at 37℃for 30 minutes and the enzyme-labeled antibody dilution is incubated at 37℃for 1.5 hours using the kit of example 4.

Example 10

The difference between this example and example 8 is that the test sample or standard solution is incubated at 37℃for 1.5 hours and the enzyme-labeled antibody diluent is incubated at 37℃for 1 hour using the kit of example 5.

Example 11 optimal pairing combinations of coated antibody-enzyme-labeled antibody

This example examined the best pairing combination of coated antibody-enzyme-labeled antibody, comprising the following methods:

The procedure of example 3 was followed, except that the coated antibodies were HY6D7 and HY2B4, respectively, and the enzyme-labeled antibodies were HRP-HY2B4 and HRP-HY6D7, respectively.

The reagents obtained above and standard solutions containing different concentrations of haptoglobin were tested as in example 8. The lowest detection limit is measured, a standard curve is obtained, and the specificity (the specificity refers to the binding specificity of an antigenic determinant on an antigen molecule and an antibody molecule hypervariable region is determined by the complementarity of a space structure between the two molecules), and in the test, the antigen with the same concentration, different antibodies are paired and combined, and the higher the detection OD value is, the better the specificity is). The results are shown in the following table. The optimal combination of the Hp content measurement can be determined by taking HY6D7 as a coating antibody and taking HRP-HY2B4 as an enzyme-labeled antibody.

Table 2 summary of antibody pairing results

Coated antibodies	Enzyme-labeled antibody	Minimum detection limit	Specificity (specificity)	Linearity of
					HY6D7	HRP-HY6D7	Difference of difference	Difference of difference	Difference of difference
HY2B4	HRP-HY6D7	Poor quality	Poor quality	Poor quality
					HY6D7	HRP-HY2B4	Good (good)	Excellent (excellent)	Preferably, it is
HY2B4	HRP-HY2B4	Preferably, it is	Preferably, it is	Poor quality

EXAMPLE 12 optimal coated antibody concentration and optimal ELISA antibody dilution determination

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

And screening the optimal concentration by adopting a chessboard titration method. On ELISA plates with different coating antibody coating concentrations, a standard solution containing haptoglobin 50ng/ml is added to a positive well (P), a standard solution containing haptoglobin 0ng/ml is added to a negative well (N), each well is incubated for 1 hour at 100ul and 37 ℃, PBST wash plates are respectively added for 30 minutes at 1:1000, 1:2000, 1:4000 and 1:8000 dilutions of enzyme-labeled antibody HRP-HY2B4 and 37 ℃, PBST wash plates are respectively added for 5 times, each well is added with substrate color development liquid A and B respectively for 50ul, uniformly mixed, colored at 37 ℃ for 15 minutes in a dark state, then 50ul of stop solution is added to terminate the color development reaction, OD450nm detection values are read through an enzyme-labeled instrument, and OD450nm detection values of P/N=positive wells (P)/OD 450nm detection values of negative wells (N) are calculated. And calculating the P/N value of paired detection of different dilutions, wherein the concentration corresponding to the larger P/N value is the optimal working concentration of the coated antibody for detecting the haptoglobin and the enzyme-labeled antibody, and finally determining that the optimal coated concentration of the capture antibody is 3ug/ml and the optimal dilution multiple of the enzyme-labeled antibody is 1:2000 through calculation. The results are shown in Table 3.

TABLE 3 pairing analysis of coated antibodies at different concentrations and different dilutions of enzyme-labeled antibodies

Example 13 optimal closed condition optimization

The procedure of example 3 was followed, except that PBS blocking solutions containing 1% BSA, 3% BSA and 5% skimmed milk powder were used as blocking solutions, and blocking conditions were selected to block at 37℃for 1 hour and 2 hours, respectively.

On the different ELISA plates obtained, 50ng/ml calibrator was added to the positive wells (P), 0ng/ml calibrator was added to the negative wells (N), 100ul of each well was incubated at 37℃for 1 hour, PBST was washed 5 times, enzyme-labeled antibody diluted with PBS1:2000 was added, incubated at 37℃for 30 minutes, PBST was washed 5 times, each well was added with substrate color development solutions A and B, each 50ul was mixed, developed at 37℃in the dark for 15 minutes, then 50ul of stop solution was added, the color development reaction was terminated, OD450nm detection value was read by an enzyme-labeled instrument, and OD450nm detection value of P/N=positive wells (P) was calculated. The P/N values of the different blocking conditions corresponding to the detection are calculated, the blocking condition corresponding to the larger P/N value is the optimal blocking condition for detecting the haptoglobin, and the result is shown in the following table, and the data in the table show that the P/N values of the different types of blocking solutions for detecting the haptoglobin with the same concentration are obviously superior to those of the other two groups when 1% BSA is used as the blocking solution, and in addition, the blocking time is longer than 1 hour when the blocking time is used for 2 hours at 37 ℃, so that the blocking time is selected from 1% BSA37 ℃.

TABLE 4 detection and analysis of different confining liquids and different confining times

Example 14 optimal incubation condition optimization

HP was detected using the kit of example 3, 50ng/ml calibrator solution was added to the positive well (P), 0ng/ml calibrator solution for haptoglobin was added to the negative well (N), each well was incubated for 30 min, 1 hr, and 1.5 hr at 37℃respectively, PBST plate was washed 5 times, enzyme-labeled antibody diluted with PBS1:2000 was added, incubation was carried out for 30 min, 1 hr, and 1.5 hr at 37℃respectively, PBST plate was washed 5 times, each well was added with 50ul of substrate development solutions A and B respectively, mixed well, developed for 15 min at 37℃in the dark, and then 50ul stop solution was added to terminate the development reaction, and OD450nm detection values were read by an enzyme-labeled instrument. The P/N values of the detection corresponding to different incubation conditions are calculated, the incubation condition corresponding to the larger P/N value is the optimal incubation condition for detecting haptoglobin, and the result is shown in the following table, and the data in the table shows that the P/N values are obviously superior to those of other groups when the incubation time of the enzyme-labeled antibody is 30 minutes in different antigen incubation times and different enzyme-labeled antibody incubation times, the antigen incubation time is 1 hour, and the enzyme-labeled antibody incubation time is 30 minutes in the optimal antigen incubation time is 1 hour.

TABLE 5 detection assay for different antigen incubation times and different enzyme-labeled secondary antibody incubation times

Example 15 kit Performance verification

(1) Preparing calibration product and quality control product

The method takes approved multiple protein calibration products (national food and drug supervision (in) 2014, 3402493) as Hp detection calibration products, which are manufactured by SIEMENS company, batch number 083651, hp content of 0.930ng/ml, and expiration date of 2024.11.02. The international standard erm@470 can be traced. Antigen was diluted to 0,1, 5, 25, 50, 100, 200ng/ml using antigen dilution as calibrator and placed in calibrator tube. And diluting the antigen to 40 ng/ml and 120ng/ml by using the antigen diluent to serve as a quality control product, and placing the quality control product into a quality control product tube. Antigen dilutions were 0.02M PH7.4 PBS containing 1% BSA (bovine serum albumin).

(2) Accuracy:

using the kit of example 3, the detection method was performed as in example 8. Sample a of known concentration of high level haptoglobin (198 ng/mL) was added to sample B of low concentration, the volume ratio between sample a and sample B was 1:9, each repeated 3 times, averaged, and recovery calculated according to the following formula.

Wherein:

r is the recovery rate;

c, adding the average value of the detection concentration of the solution A into the solution B;

v ₀ -volume of liquid B;

v _S -volume of liquid a;

c ₀ -average value of B solution concentration;

C _S -concentration of A liquid.

The results are shown in the following table:

TABLE 6 recovery of experimental test results

(3) Linear range:

Using the kit of example 3, the detection method was performed as in example 8. The haptoglobin dilutions at different concentrations were used as standard, the results were linear between 1 and 200ng/mL, the lowest limit of detection was 1ng/mL, the results are shown in Table 7, and the standard curve is shown in FIG. 2.

TABLE 7HP detection Standard Curve

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

The optical density is plotted on the ordinate and the double logarithm on the abscissa of the concentration, as shown in fig. 1. Standard curve equation y= 0.53331x-0.78052, correlation coefficient R ² =0.998.

(4) Limit of detection:

Using the kit of example 3, the detection method was performed as in example 8. The sample diluent (the haptoglobin content is 0) is taken as a sample for continuous detection for 20 times, the average value M and the standard deviation SD are calculated according to the detection result, then M+2SD is substituted into the standard curve, the blank detection limit is calculated to be 0.046ng/mL, and the blank detection limit is calculated to be lower than the detection limit by 1ng/mL, and the result is shown in Table 8.

TABLE 8 repeated 20-time determination of OD450nm detection values with sample dilutions

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

(5) Coefficient of variation:

using the kit of example 3, the detection method was performed as in example 8. The concentration, average value (M) and Standard Deviation (SD) were calculated according to standard curves, and the Coefficient of Variation (CV) was calculated according to the formula cv=sd/mx100%, with the measured intra-batch coefficient of variation being 3.47% and 2.23%, respectively, and the average intra-batch coefficient of variation being 2.85%, with the results shown in table 9, using quality control at concentrations of 40ng/mL and 120ng/mL for 20 consecutive measurements.

Table 9 calculation of 20 repeated measurements (ng/mL) for 40ng/mL and 120ng/mL concentration standards

(6) Interference experiment

Using the kit of example 3, the detection method was performed as in example 8. The interference substances with different final concentrations are respectively added into quality control products with the concentration levels of 40ng/mL and 120ng/mL of haptoglobin, an unadditized group is taken as a control group, the relative deviation of the added group and the control group is less than 5% and is an acceptable standard of the interference substances, the maximum concentration levels (not exceeding the highest clinically visible concentration) of the different interference substances are studied, and the results of the interference resistance analysis of the detection kit are shown in Table 10. The results show that the concentrations of the interfering substances in the table below do not significantly interfere with the detection results.

TABLE 10 influence of different interfering substances on HP detection results

Comparative example

The procedure of example 3 was followed, except that the coated antibody was used to select mab 5C4 in patent document CN111323601, and the antibody used in the enzyme-labeled antibody was used to select mab 1A2 in patent document CN 111323601. Then, the test sensitivity was 5ng/mL and the result was linear in the linear range of 5-200ng/mL, the standard curve was shown in FIG. 3, and the result was shown in the table, according to the test method of the test kit of example 15.

TABLE 11HP detection Standard Curve

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

The optical density is plotted on the double log plot with the concentration on the abscissa and the optical density on the ordinate. Standard curve equation y= 0.75394x-1.25330, correlation coefficient R ² = 0.99916.

The blank detection limit is calculated to be 0.411ng/mL, and the result is shown in the table, wherein the blank detection limit is lower than the detection limit by 2 ng/mL.

TABLE 12 repeated 20-time measurement of OD450nm detection values with sample dilutions

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

In conclusion, compared with the comparative example, the kit provided by the invention selects better antibodies and antibody combinations, and the detection sensitivity is obviously improved.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope 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 haptoglobin, characterized in that it comprises the antibody according to any one of claims 1-2, 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 up 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.