CN113817062A

CN113817062A - Rabbit monoclonal antibody against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) and application thereof

Info

Publication number: CN113817062A
Application number: CN202110841196.7A
Authority: CN
Inventors: 扈晓敏; W·付; 柳孟姣; 钮倩; 李婷; 任琪; 张杨
Original assignee: WUXI ORIGENE BIO-TECHNOLOGY CO LTD
Current assignee: WUXI ORIGENE BIO-TECHNOLOGY CO LTD
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2021-12-21
Anticipated expiration: 2041-07-26
Also published as: CN113817062B

Abstract

The invention relates to the technical field of biology, and discloses rabbit anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) monoclonal antibodies OTIR5C10 and OTIR13E1, wherein the antibodies are generated by sorting specific B cells, culturing, screening and recombining molecular clones. The immunogen of the rabbit monoclonal antibody is HSD17B13 full-length protein expressed by eukaryotic cell HEK293T, the 2 rabbit monoclonal antibodies can recognize different antigenic determinants on the surface of HSD17B13 protein, and the amino acid sequence of the OTIR5C10 antibody light chain (VL) is shown as SEQ ID NO. 1; the amino acid sequence of the heavy chain (VH) of the OTIR5C10 antibody is shown in SEQ ID No. 2. The amino acid sequence of the OTIR13E1 light chain (VL) is shown as SEQ ID NO. 11; the amino acid sequence of the heavy chain (VH) of OTIR13E1 is shown as SEQ ID No. 12. The anti-HSD 17B13 rabbit monoclonal antibody can be applied to the preparation of various immunodetection kits for detecting HSD17B13, including but not limited to the preparation of double-antibody sandwich ELISA or chemiluminescence method kits, provides an auxiliary means for the diagnosis of diseases related to HSD17B13, and provides a basis for the preparation of next engineering antibodies.

Description

Rabbit monoclonal antibody against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibody and application thereof in immunodetection.

Background

17 β -hydroxysteroid dehydrogenases (HSD17Bs) are a class of enzymes that catalyze the conversion between 17-keto-and 17-hydroxysteroids. To date, 15 HSD17B members have been identified, most of which are involved in regulating the biological activity of sex hormones, including HSD17B1, HSD17B2, HSD17B3, HSD17B5 and HSD17B6, and others are also involved in fatty acid metabolism, cholesterol biosynthesis and bile acid production in vivo. The human HSD17B13 gene is located on chromosome 4 (4q22.1), and its expression is highly restricted to liver, particularly hepatocytes, but not to other cell types of liver. The human HSD17B13 gene encodes a 300 amino acid protein, which is localized on lipid droplets and is a novel liver-specific Lipid Droplet (LD) -related protein.

HSD17B13 is a hepatic retinol dehydrogenase associated with histological features of non-alcoholic fatty liver disease. In non-alcoholic fatty liver patients, HSD17B13 expression was significantly upregulated, and its overexpression in the liver promoted lipid accumulation in the liver. HSD17B13 plays an important role as a liver-specific LD protein in regulating liver lipid homeostasis and lipid metabolism, and may be a novel traction target for the treatment of nonalcoholic fatty liver disease (NAFLD) and related liver diseases.

Chinese patent has no item about the preparation of HSD17B13 rabbit monoclonal antibody, application publication No. CN 110199032A relates to HSD17B13 protein variant and its use, and does not mention mouse monoclonal antibody or rabbit monoclonal antibody and antibody CDR sequence. Application publication No. CN 103520724A describes a new use of HSD17B13 protein or an inhibitor of its coding gene, and does not relate to a murine monoclonal antibody or a rabbit monoclonal antibody and an antibody CDR sequence. The international patent application utilizes HSD17B13 or HSD17B13+ monoclonal antibody as a keyword, and does not find patents about monoclonal antibodies having high affinity and specific binding with HSD17B13, and does not find patents about detection of an immunoassay kit prepared by using anti-HSD 17B13 rabbit monoclonal antibodies OTIR5C10 and OTIR13E 1.

Disclosure of Invention

The invention aims to provide anti-HSD 17B13 rabbit monoclonal antibodies OTIR5C10 and OTIR13E1 with good specificity and high affinity and application thereof in an immunodetection kit prepared by a double-antibody sandwich ELISA or a chemiluminescence method, provides auxiliary diagnosis for a disease related to HSD17B13, namely nonalcoholic fatty liver disease (NAFLD), and also provides a basis for the preparation of a next engineering antibody.

The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR5C10 or rabbit monoclonal antibody OTIR13E 1.

The rabbit monoclonal antibody OTIR5C10 and the rabbit monoclonal antibody OTIR13E1 take full-length HSD17B13 protein expressed by eukaryotic cell HEK293T as immunogen. The preparation method comprises the steps of carrying out immune injection on a New Zealand white rabbit, taking peripheral blood mononuclear cells (PMBCs) of the immune rabbit, sorting specific B cells, culturing, screening and obtaining the immune rabbit by utilizing a molecular cloning recombination technology.

The rabbit monoclonal antibody OTIR5C10 has a light chain variable region (VL) of 109aa and an amino acid sequence shown in SEQ ID NO. 1; the heavy chain (VH) of the antibody contains 115aa, and the amino acid sequence of the heavy chain (VH) is shown as SEQ ID No. 2.

The rabbit monoclonal antibody OTIR5C10, wherein the VL region comprises 3 antigenic determinants: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 27aa-34aa, 52aa-54aa and 91aa-99 aa. The amino acid sequences are respectively shown in SEQ ID No. 3-5.

The rabbit monoclonal antibody OTIR5C10, wherein the VH region comprises 3 antigenic determinants: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 25aa-32aa, 50aa-56aa and 93aa-104 aa. The amino acid residue sequences are respectively shown in SEQ ID No. 6-8.

The rabbit monoclonal antibody OTIR13E1 has a light chain variable region (VL) containing 108aa and an amino acid sequence shown in SEQ ID NO. 11; the heavy chain (VH) of the antibody contains 116aa, and the amino acid sequence of the heavy chain (VH) is shown as SEQ ID No. 12.

The rabbit monoclonal antibody OTIR13E1, wherein the VL region comprises 3 antigenic determinants: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 27aa-32aa, 50aa-52aa and 89aa-98 aa. The amino acid sequences are respectively shown in SEQ ID No. 13-15.

The rabbit monoclonal antibody OTIR13E1, wherein the VH region comprises 3 antigenic determinants: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 25aa-32aa, 50aa-56aa and 93aa-105 aa. The amino acid residue sequences are respectively shown in SEQ ID No. 16-18.

The rabbit monoclonal antibody comprises a rabbit monoclonal antibody OTIR5C10 or a rabbit monoclonal antibody OTIR13E1, can be combined with HSD17B13 in a high specificity manner, and can be prepared into various immunoassay kits for detecting HSD17B13 by methods known by a person skilled in the art. In particular to an immunoassay kit prepared by double-antibody sandwich ELISA or a chemiluminescence method. The double-antibody sandwich ELISA detects the HSD17B13 standard substance, and the detection sensitivity can reach 300 pg/ml; the plate-type chemiluminescence detection HSD17B13 standard substance has the detection sensitivity reaching 78 pg/ml.

Drawings

FIG. 1 is an electrophoretogram of full-length amplification products of heavy and light chains of rabbit monoclonal antibodies OTIR5C10 and OTIR13E1, M is a DNA molecular weight Marker;

FIG. 2 is a diagram showing the results of Westernblot detection of a rabbit monoclonal antibody OTIR5C10 specifically recognizing the complete HSD17B13(Full length HSD17B13, HSD17B13-FL) protein;

FIG. 3 is a diagram showing the results of Westernblot detection of a rabbit monoclonal antibody OTIR13E1 specifically recognizing the complete protein HSD17B13(Full length HSD17B13, HSD17B 13-FL);

FIG. 4 is a standard curve for detecting HSD17B13 protein by a double antibody sandwich ELISA method;

FIG. 5 shows plate-type chemiluminescence detection of HSD17B13 protein.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not noted in the following examples are generally performed according to conventional conditions, as described in laboratory manuals, or according to conditions recommended by the manufacturer.

EXAMPLE 1 preparation of Rabbit monoclonal antibody against HSD17B13

Firstly, preparation of HSD17B13 recombinant protein

HSD17B13 gene NM-178135.5 (NP-835236.2) encoding HSD17B13 full length 300 amino acids (aa) was selected from Genebank. Plasmid RC213132 (containing HSD17B13 ORF 900bp) obtained from Aorutong bioscience, Inc. in USA is used as a template, two primers are designed and are respectively introduced into enzyme cutting sites SgfI and MluI, and the two primers are cloned into an expression vector pCMV6 to establish the HSD17B13 recombinant expression plasmid. The gene is transfected into HEK293T cells by a technical method known by a person skilled in the art, cell lysis is carried out after 48 hours of transfection, lysate in all culture dishes is collected, centrifugation is carried out at 4 ℃, purification is carried out by a DDK affinity chromatography column, and the purity is identified by SDS-PAGE electrophoresis. After electrophoresis, a target protein band with the molecular weight of about 34kDa is observed on the gel, and the purity is more than 85 percent, thereby meeting the purity requirement of the monoclonal antibody.

Second, animal immunization

The purified HSD17B13 recombinant protein is emulsified by complete Freund's adjuvant, and is used for immunizing New Zealand white rabbits of about 2kg by subcutaneous injection, wherein the immunization dose is 500 mu g/rabbit, the second immunization is carried out after two weeks, and the emulsification is carried out by incomplete Freund's adjuvant, and the immunization dose is 250 mu g/rabbit. After twice immunization, tail blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; according to the standard that the OD450 is more than 1.0 when the ELISA titer is 128000, whether PBMCs are collected or immunization is continued is judged according to the result, and the rabbit with the highest antibody titer is selected for collecting the PBMCs.

Thirdly, PBMCs are separated, specific B cells are sorted, cloning and recombining are carried out, a rabbit is fixed on an operating table in a supine mode, the hair of a heart part is removed, the skin is disinfected by alcohol, the most obvious part of the heart beat is punctured by a 50ml syringe, blood flows into the syringe after the needle penetrates the heart, the needle is quickly pulled out after the required blood volume is obtained, whole blood in the syringe is transferred into a sterile 50ml tube, the whole blood is uniformly mixed with equal amount of PBS and then slowly added above lymphocyte separation liquid drop by drop, the mixture is centrifuged for 30 minutes at the room temperature of 400 Xg, and after centrifugation, the liquid level is divided into four layers from top to bottom: and (3) carefully sucking the mononuclear cell layer, washing with PBS to remove a platelet and lymphocyte separation solution to obtain the rabbit PBMCs. And continuously sorting antigen-specific B cells from the rabbit PBMCs for culture, and screening positive clones from the cultured B cell supernatant by using an ELISA plate coated with the antigen. And (3) collecting and cracking positively cloned cells, extracting RNA (ribonucleic acid) and carrying out reverse transcription on the RNA to obtain cDNA (complementary deoxyribonucleic acid), amplifying a naturally paired light-heavy chain full-length sequence of the rabbit monoclonal antibody from the cDNA corresponding to the positive cloning, constructing a rabbit monoclonal antibody expression vector by a cloning and recombination method, and determining the sequence by sequencing. The results of the amplified full-length PCR products are shown in FIG. 1.

Fourthly, preparation and purification of the monoclonal antibody in order to obtain a plurality of rabbit monoclonal antibodies for identifying the human HSD17B13 protein, heavy chain and light chain genes of the rabbit monoclonal antibody are loaded on an expression vector, and plasmids are transfected into HEK293 cells; the rabbit monoclonal antibody recognizing human HSD17B13 protein contained in the culture supernatant was obtained after 120-144 hours of transfection. Collecting cell suspension, centrifuging, collecting supernatant, and purifying antibody by affinity chromatography. And (5) determining the concentration of the purified monoclonal antibody by using a BCA method, subpackaging and freeze-drying.

Example 2 identification of anti-HSD 17B13 Rabbit monoclonal antibodies OTIR5C10 or OTIR13E1

First, specificity identification of rabbit monoclonal antibody

Westernblot (WB) was used for detection. Protein samples such as HSD11B1, HSD17B2, HSD17B3, HSD17B4, HSD17B6, HSD17B7, HSD17B8, HSD17B10, HSD17B11, HSD17B13 and the like are subjected to SDS-PAGE, the loading amount of the protein is 20ng, and WB detection is performed after membrane transfer.

The results showed that the rabbit monoclonal antibodies OTIR5C10 or OTIR13E1 specifically recognized HSD17B13 protein only, but not other 9 family members, indicating that the anti-HSD 17B13 rabbit monoclonal antibodies OTIR5C10 or OTIR13E1 of the present invention have high specificity for HSD17B13 protein. The results are shown in FIGS. 2 and 3.

Second, potency of Rabbit monoclonal antibody

The rabbit monoclonal antibodies OTIR5C10 or OTIR13E1 are diluted by adopting a double-proportion dilution method, the antibody titer is measured by indirect ELISA, and the result shows that the rabbit monoclonal antibodies OTIR5C10 or OTI related to the inventionThe R13E1 titer was 6.6X 10⁶。

Antibody pairing

In order to select the optimal combination of the coating antibody and the detection antibody, a checkerboard combination was used to pair the obtained multiple HSD17B13 rabbit monoclonal antibodies with each other. The method comprises the following basic steps: the enzyme label plate is coated with a plurality of monoclonal antibodies respectively and stays overnight at 4 ℃. Taking out the enzyme label plate on the next day, washing the enzyme label plate by PBST once, blocking the enzyme label plate by 1 percent BSA solution at 37 ℃ for 2 hours, and washing the enzyme label plate by PBST 3 times; adding 100 μ l HSD17B13 protein to each well at 20, 5, 1 and 0ng/ml, respectively, and incubating at 37 deg.C for 1 hr; after incubation, taking out the enzyme label plate, washing by PBST for 3 times, respectively adding the biotin-labeled polyclonal antibody as a detection antibody, and incubating for 1 hour at 37 ℃; after the incubation, the microplate was removed, washed 3 times with PBST, added with HRP-labeled Avidin, and incubated at 37 ℃ for 0.5 hour. PBST was washed 5 times, TMB substrate was added, and color development was carried out at 37 ℃ for 10 min. After being taken out, the stop solution is added, and OD450 reading is measured on a microplate reader. Based on the OD of the sample and the background of the negative control, the most ideal rabbit monoclonal antibody pair was selected, and the results of the paired selection are shown in Table 1. The antibody OTIR5C10 is used as a coating antibody, and the antibody OTIR13E1 is optimal as a detection antibody. Anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibodies OTIR5C10 and OTIR13E1 recognize different epitopes on the surface of HSD17B13 protein.

TABLE 1 screening results of antibody pairing experiments

Example 3 analysis of the genes and amino acid sequences of the variable regions of the rabbit monoclonal antibodies OTIR5C10 or OTIR13E1

Recombinant plasmids of OTIR5C10 or OTIR13E1 antibodies are respectively used as DNA templates, light chain variable region and heavy chain variable region sequencing primers are designed according to vector sequences at the 5' ends of light chains and heavy chains on the templates, and sequencing is carried out by adopting a sequencer ABI 3730. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibodies OTIR5C10 and OTIR13E1 were obtained by sequencing.

The nucleotide sequences of the light chain and the heavy chain are subjected to sequencing result data analysis by using the Internet and IMGT/V-QUEST analysis software on http:// www.imgt.org respectively to obtain the light chain amino acid sequence of the rabbit monoclonal antibody OTIR5C10 shown as SEQ ID No.1 and the heavy chain amino acid sequence shown as SEQ ID No. 2. The total length of VL is 109 amino acids, the number of amino acids in the 4 domains of FR is 26, 17, 36 and 10 respectively, the number of amino acids in the 3 domains of CDR is 8, 3 and 9 respectively, the regions of CDR1, CDR2 and CDR3 are 27aa-34aa, 52aa-54aa and 91aa-99aa respectively, and the amino acid sequences are QSVYRNQ, GAS and GGGHSTGVE respectively.

Through analysis, the whole length of the rabbit monoclonal antibody OTIR5C10 VH is 115 amino acids, the number of 4 domain amino acids of FR is 24, 17, 35 and 11 respectively, the number of 3 domain amino acids of CDR is 8, 7 and 12 respectively, CDR1, CDR2 and CDR3 are 25aa-32aa, 50aa-56aa and 93aa-104aa respectively, and the amino acid sequences thereof are SLSGGFTNA, ISSRGNT and ARDPTYTNDRWCL respectively.

Through analysis, the light chain amino acid sequence of the rabbit monoclonal antibody OTIR13E1 is shown as SEQ ID NO.11, and the heavy chain amino acid sequence is shown as SEQ ID NO. 12. The total length of VL is 108 amino acids, the number of amino acids in 4 domains of FR is 26, 17, 36 and 10 respectively, the number of amino acids in3 domains of CDR is 6, 3 and 10 respectively, the regions of CDR1, CDR2 and CDR3 are 27aa-32aa, 50aa-52aa and 89aa-98aa respectively, and the amino acid sequences are EKIYSF, LAS and QQTYTYENGD respectively.

By analysis, the whole length of VH of the rabbit monoclonal antibody OTIR13E1 is 116 amino acids, the number of amino acids of 4 domains of FR is 24, 17, 36 and 11 respectively, the number of amino acids of 3 domains of CDR is 8, 7 and 13 respectively, CDR1, CDR2 and CDR3 are 25aa-32aa, 50aa-56aa and 93aa-105aa respectively, and the amino acid sequences thereof are SLSGTYASGST, ARDFPSGPDTGY respectively.

Example 4 anti-HSD 17B13 Rabbit monoclonal antibodies OTIR5C10 or OTIR13E1 for preparing a double antibody sandwich ELISA test kit

The detection technology based on the ELISA double-antibody sandwich method principle known by the technicians in the field is adopted to manufacture the HSD17B13 detection kit, so that auxiliary diagnosis is provided for the disease non-alcoholic fatty liver disease (NAFLD) related to the HSD17B13, and a foundation is also provided for the preparation of the next engineering antibody.

Kit composition

1. Panel coated with OTIR5C10 antibody: diluting the antibody to 5 μ g/ml with PBS buffer solution, coating on a microplate, incubating at 4 deg.C overnight with 100 μ l per well, washing with PBST for 3 times, and spin-drying; adding PBS containing 1% BSA, 5% sucrose and 0.05% Proclin300 for blocking, reacting at 37 deg.C for 2 hr, discarding blocking solution in the hole, and spin-drying; and (3) putting the coated plate in a 37 ℃ oven for 2h to finish coating, sealing the coated plate by using an aluminum foil bag, and storing the coated plate at 4 ℃ for later use.

2. Reagent preparation

1. Biotin conjugate formulation EZ-Link from Thermo Scientific^TMSulfo-NHS-LC-Biotin labeled anti-HSD 17B13 rabbit monoclonal antibody OTIR13E1, titrating the working concentration to prepare a concentrated solution with the ratio of 1:20, diluting the concentrated solution with PBS containing 1% BSA, 5% glycerol and 0.05% Proclin300, and filtering and sterilizing the concentrated solution.

2. The wash buffer was conventional PBST pH7.4 containing 0.05% Proclin300 and was prepared as a 20-fold concentrate.

HRP-labeled Avidin was formulated as a 1:20 concentrate in PBS containing 1% BSA, 5% glycerol, and 0.05% Proclin300, and sterilized by filtration.

4. Substrate solution (color developing solution) of enzyme single component TMB (purchased from market).

5. Sample dilutions were filter sterilized with 1% BSA, 0.05% Proclin300 in PBS.

6. The stop solution was added to 110ml of distilled water with 10ml of HCl (36-38%), and the mixing was carried out slowly to prepare 1N HCl.

7. The HSD17B13 recombinant protein standard is diluted to 20 mu g/ml by PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin300, and then the sample is filtered, sterilized and aseptically packaged.

Secondly, detecting the operation key points

1. In order to ensure the accuracy of the detection result, the standard substance and the sample are both provided with double-hole measurement. A standard curve is required for each test.

2. If the content of the substance to be detected in the specimen is too high, the specimen is firstly diluted by using a specimen diluent so as to enable the specimen to conform to the detection range of the kit, and finally, the specimen is multiplied by the corresponding dilution times during calculation.

3. Sample adding: when the sample is added, please use a disposable clean suction head to avoid cross contamination. The sample adding should be gentle as far as possible, avoid bubbling, add the sample in the bottom of the ELISA plate hole, surely don't add the sample along the pore wall.

4. And (3) incubation: in order to prevent the sample from evaporating or polluting, the enzyme label plate must be covered with a plate paste in the incubation process, and the enzyme label plate should be prevented from being in a dry state in the experiment process. The temperature of the incubator should be observed whether to be constant at 37 ℃ at any time during the incubation process, and the temperature should be adjusted in time. In the incubation process, the incubator is not easy to open too many times so as not to influence the temperature balance.

5. Washing: the washing process is very important and inadequate washing is prone to false positives.

6. Color development: to ensure the accuracy of the experimental results, the stop solution should be added as soon as the reaction time of the substrate is up. The reaction time can be controlled by observing the color development at intervals after the addition of the substrate solution (e.g., at intervals of 10 minutes). When the obvious gradient blue color of the front 3-4 holes and the unobvious color development of the back 3-4 holes of the standard product are visible by naked eyes, the stop solution is added to stop the reaction, and the blue color is changed into yellow color immediately. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution.

7. The substrate solution should be pale blue or colorless and must be discarded if the color becomes too dark. The substrate solution is easily contaminated and should be properly preserved in the dark.

Thirdly, determining standard curves of different dilutions of standard HSD17B13 protein

The prepared double-antibody sandwich ELISA detection kit for detecting HSD17B13 protein is taken out from a 4-degree refrigerator and is balanced to room temperature. Diluting the standard substance with PBS from 20 μ g/ml to 5ng/ml, preparing into series of 7 concentration standard substances of 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, adding 100 μ l standard substance into each well according to the above detection method, incubating, discarding liquid, adding biotin-labeled detection antibody working solution, incubating, discarding liquid in the well, and adding HRP-labeled Avidin working solutionIncubation is carried out as a liquid, liquid in the holes is removed, a substrate solution is added into each hole after spin-drying, a termination solution is added into each hole after light shielding and color development, the optical density OD value of each hole is measured by an enzyme-labeling instrument within 5 minutes after the reaction is terminated in order at the wavelength of 450nm to form a standard curve, and the result is shown in table 2. HSD17B13 assay standard curves were generated according to Table 2 and are shown in FIG. 4. R of the standard curve²0.9934, the sample concentration calculation formula is derived: y is 0.0045x + 0.1622.

TABLE 2 detection of HSD17B13 Standard protein by double antibody Sandwich ELISA

Coated antibodies	5C10
		ng/ml	OD450nm
5	1.67
		2.5	0.925
1.25	0.507
		0.625	0.286
0.3125	0.22
		0.15625	0.158
0	0.092
		Detection of antibodies	13E1

Example 5: application of rabbit monoclonal antibody OTIR5C10 or OTIR13E1 resisting HSD17B13 protein in plate-type chemiluminescence immunoassay

The plate-type chemiluminescence immunoassay technology based on the principle of a double-antibody sandwich method, which is known by the technicians in the field, is adopted to manufacture the HSD17B13 protein detection kit for the clinical aided diagnosis of diseases related to the HSD17B13 protein.

Detection principle and method

A plate-type chemiluminescence immunoassay technology based on the principle of a double-antibody sandwich method is adopted. And (2) internally wrapping a rabbit monoclonal antibody OTIR5C10 of the anti-HSD 17B13 protein in a light-tight white micropore plate, placing the sealed coating plate on a Cosimei full-automatic luminescence chemiluminescence apparatus, setting an apparatus program, and simultaneously incubating a standard substance or a sample to be tested and the rabbit monoclonal antibody OTIR13E1 marked by biotin to form an antibody-antigen-antibody compound. Washing to remove unbound substances, adding Horse Radish Peroxidase (HRP) -labeled Avidin (Avidin), washing to remove unbound substances, adding Beijing Kejieke Zhongshi chemiluminescent substrate, and reacting for 10min to display chemiluminescence value by a Cosmei full-automatic chemiluminescence apparatus. The reading result is judged to be positive according to the signal to noise ratio of more than 2.0. The magnitude of the luminescence reflects the amount of enzyme-labeled antibody bound, which is proportional to the concentration of HSD17B13 protein in the sample. And drawing a standard curve according to the measured luminescent value of the standard product, wherein the concentration value of the HSD17B13 protein in the sample to be measured can be obtained from the standard curve.

Secondly, a plate-type chemiluminescence detection kit for detecting HSD17B13 protein

1. Panel coated with OTIR5C10 antibody: the same as in example 4.

2. Reagent preparation

1. The biotin conjugate was formulated as in example 4.

2. The wash buffer was the same as in example 4.

4. Chemiluminescence color development liquid A and B are purchased from Beijing Kezhongzhong Biotechnology Co.

5. Sample dilutions were filter sterilized with 1% BSA, 0.05% Proclin300 in PBST.

6. The HSD17B13 full-length protein standard is diluted to 5 mu g/ml by PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin300, and then sterilized by filtration and aseptically packaged.

Third, detecting HSD17B13 full-length protein

The HSD17B13 full-length protein was subjected to gradient dilution to 0pg/ml, 78pg/ml, 156pg/ml, 312pg/ml, 625pg/ml, 1260pg/ml, 2500pg/ml and 5000pg/ml, respectively, the OTIR5C10 antibody was used as a coating antibody and the OTIR13E1 antibody was used as a detection antibody, and the detection results of the 8 gradient-diluted antigens were shown in Table 3. According to the judgment that the signal-to-noise ratio is more than 2.0, the rabbit monoclonal antibody is used for a plate-type chemiluminescence immunoassay reagent, the lowest sensitivity is more than 78pg/ml, and the linear range is 78-5000 pg/ml. HSD17B13 protein detection Standard Curve was generated according to Table 3, as shown in FIG. 5

TABLE 3 detection of HSD17B13 full-length protein by plate-type chemiluminescence method

Sequence listing

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Claims

1. A rabbit monoclonal antibody to human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) characterized by: the rabbit monoclonal antibody is rabbit monoclonal antibody OTIR5C10 or rabbit monoclonal antibody OTIR13E 1.

2. The rabbit monoclonal antibody OTIR5C10 and the rabbit monoclonal antibody OTIR13E1 of anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) according to claim 1, wherein: the rabbit monoclonal antibody OTIR5C10 and the rabbit monoclonal antibody OTIR13E1 are obtained by taking full-length HSD17B13 protein expressed by eukaryotic cells HEK293T as immunogen, taking immune rabbit peripheral blood mononuclear cells (PMBCs) through immune injection of New Zealand white rabbits, sorting specific B cells, culturing, screening and utilizing molecular cloning recombination technology.

3. The rabbit monoclonal antibody OTIR5C10 against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) according to claim 1, wherein the light chain variable region (VL) comprises 109aa, and the amino acid sequence thereof is shown in SEQ ID NO. 1; the heavy chain (VH) of the antibody contains 115aa, and the amino acid sequence of the heavy chain (VH) is shown as SEQ ID No. 2.

4. The anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibody OTIR5C10 of claim 1, wherein VL region comprises 3 epitopes: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 27aa-34aa, 52aa-54aa and 91aa-99 aa. The amino acid sequences are respectively shown in SEQ ID No. 3-5.

5. The anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibody OTIR5C10 of claim 1, wherein VH region comprises 3 epitopes: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 25aa-32aa, 50aa-56aa and 93aa-104 aa. The amino acid residue sequences are respectively shown in SEQ ID No. 6-8.

6. The rabbit monoclonal antibody OTIR13E1 against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) according to claim 1, wherein the light chain variable region (VL) comprises 108aa, and the amino acid sequence thereof is represented by SEQ ID NO. 11; the heavy chain (VH) of the antibody contains 116aa, and the amino acid sequence of the heavy chain (VH) is shown as SEQ ID No. 12.

7. The anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibody OTIR13E1 of claim 1, wherein VL region comprises 3 epitopes: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 27aa-32aa, 50aa-52aa and 89aa-98 aa. The amino acid sequences are respectively shown in SEQ ID No. 13-15.

8. The anti-human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) rabbit monoclonal antibody OTIR13E1 of claim 1, wherein VH region comprises 3 epitopes: CDR1, CDR2 and CDR3, the regions of the antigenic determinant being respectively 25aa-32aa, 50aa-56aa and 93aa-105 aa. The amino acid residue sequences are respectively shown in SEQ ID No. 16-18.

9. The rabbit monoclonal antibodies OTIR5C10 and OTIR13E1 against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) as claimed in claim 1 can recognize different epitopes on the surface of human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) protein.

10. The use of the rabbit monoclonal antibody against human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) according to claim 1 for the preparation of human hydroxysteroid 17-beta dehydrogenase 13(HSD17B13) immunoassays, including but not limited to the double antibody sandwich ELISA and the chemiluminescent immunoassay kit.