CN116239684B

CN116239684B - Rabbit monoclonal antibody aiming at human calreticulin, and preparation method and application thereof

Info

Publication number: CN116239684B
Application number: CN202310127574.4A
Authority: CN
Inventors: 王亚群; 胡文娟; 郭夏阳; 孙倩; 张莹; 李智; 徐若; 吴海
Original assignee: Wuhan Abclonal Inc
Current assignee: Wuhan Abclonal Inc
Priority date: 2023-02-12
Filing date: 2023-02-12
Publication date: 2023-10-20
Anticipated expiration: 2043-02-12
Also published as: CN116239684A

Abstract

The invention provides a rabbit monoclonal antibody aiming at human calreticulin, a preparation method and application thereof. The method specifically provides a high-affinity rabbit monoclonal antibody pair 2G2 and 3B3 for human calreticulin, and utilizes the rabbit monoclonal antibody pair to develop a double-antibody sandwich ELISA detection method, and has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, and the detection sensitivity reaches 1pg/mL. The double-antibody sandwich ELISA detection can stably detect trace human calreticulin in serum and cell culture medium samples, and has important significance in clinical diagnosis and scientific research application.

Description

Rabbit monoclonal antibody aiming at human calreticulin, and preparation method and application thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a rabbit monoclonal antibody aiming at human calreticulin, and a preparation method and application thereof.

Background

Calreticulin (CALR) is a soluble Ca retained in the endoplasmic reticulum ²⁺ Binding proteins, present in most eukaryotic cells. CALR acts as a key regulator of intracellular free calcium levels to maintain Ca ²⁺ Steady state effects. CALR can also act as a lectin-type chaperone in the endoplasmic reticulum, binding to oligosaccharide residues on newly synthesized glycoproteins to ensure proper folding.

In recent years, research has also found that: CALR not only resides in the endoplasmic reticulum, but also localizes to the cell membrane and is secreted outside the cell, performing its specific function. CALR is expressed on the surface of tumor cells, apoptotic cells, some drug-treated cells (e.g., anthracyclines) in higher amounts than normal cells, and CALR on the surface of these cells can bind to CD91/LRP1, a receptor on the surface of some innate immune cells, such as macrophages, dendritic cells, etc., to enhance the immune response of the immune system to these cells. In patients with rheumatoid arthritis, CALR on the cell surface can bind to HLA-DR beta on the leukocyte surface, and stimulate the production of protein autoantibodies. Studies have also shown that: the NDomain of CALR can be secreted extracellularly, called angiostatin, and can inhibit the formation of microvessels, playing an important role in tumor formation. CALR is therefore an important drug target for the treatment of tumors, rheumatic diseases and other autoimmune diseases. By measuring CALR levels in human body fluids or specific cell culture supernatants, diagnostic assessments can be made of their associated diseases and prognosis.

At present, almost all CALR ELISA detection kits on the market use mouse anti-human CALR monoclonal antibodies, and the affinity and the specificity of the detection kit are generally low. For example, patent document CN113960311a discloses a kit for rapidly detecting calreticulin, a pancreatic cancer marker, and a method thereof, wherein the preparation method of the murine monoclonal antibody is dependent on hybridoma cells, and the preparation process is relatively complex and the batch-to-batch difference is large.

Therefore, the development of a detection method with high affinity for human calreticulin protein has very important significance.

Disclosure of Invention

Based on the above, it is necessary to provide a rabbit monoclonal antibody against human calreticulin, a preparation method and application thereof, and in particular to provide a rabbit monoclonal antibody with high affinity to 2G2 and 3B3 and human calreticulin, and a double antibody sandwich ELISA detection method established by using the high-affinity rabbit monoclonal antibody has high sensitivity and high specificity.

The invention adopts the following technical scheme:

the invention provides a rabbit monoclonal antibody pair aiming at human calreticulin, wherein the rabbit monoclonal antibody pair is rabbit monoclonal antibody 2G2 and rabbit monoclonal antibody 3B3.

Wherein the full-length sequence of the light chain of the rabbit monoclonal antibody 2G2 is shown as SEQ ID NO.1, the sequence of the light chain variable region is shown as SEQ ID NO.2, and the sequences of the light chain complementarity determining regions are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively; the full-length sequence of the heavy chain is shown as SEQ ID NO.6, the sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the sequences of the heavy chain complementarity determining regions are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

The full-length sequence of the rabbit monoclonal antibody 3B3 is shown as SEQ ID NO.11, the sequence of the light chain variable region is shown as SEQ ID NO.12, and the sequences of the light chain complementarity determining regions are shown as SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 respectively; the full-length sequence of the heavy chain is shown as SEQ ID NO.16, the sequence of the heavy chain variable region is shown as SEQ ID NO.17, and the sequences of the heavy chain complementarity determining regions are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.

The invention also provides a preparation method of the rabbit monoclonal antibody aiming at the human calreticulin, which comprises the following steps: loading heavy chain genes and light chain genes of rabbit monoclonal antibodies aiming at human calreticulin on expression vectors respectively, and transfecting 293F cells; culturing to obtain a supernatant containing rabbit monoclonal antibody pairs 2G2 and 3B 3; purifying to obtain the final product.

The invention also provides application of the rabbit monoclonal antibody aiming at the human calreticulin in preparation of a reagent or a kit for detecting the human calreticulin. The reagent or the kit is used for enzyme-linked immunosorbent assay of human calreticulin, wherein the rabbit monoclonal antibody 2G2 is used as a capture antibody, and the rabbit monoclonal antibody 3B3 is used as a labeled antibody.

Specifically, the invention can also provide a reagent or a kit for detecting human calreticulin, which comprises rabbit monoclonal antibodies 2G2 and 3B3 aiming at the human calreticulin. The human calreticulin is at least one of recombinant human calreticulin and human calreticulin secreted by cells.

Compared with the prior art, the invention has the core advantages that:

(1) The invention is based on B cell marking and sorting technology, and can directly enrich and separate B lymphocyte capable of recognizing human CALR protein from the immunized rabbit spleen, thereby greatly improving the antigen specificity B cell screening efficiency. And the isolated B lymphocytes are cultured in a single cell form, so that the monoclonal antibody is directly obtained, and the tedious steps of multiple subcloning in the hybridoma technology are omitted.

(2) The monoclonal antibody prepared by the invention is a recombinant antibody. After positive clones are selected from spleen, antibody VH/VL genes are directly amplified from B cells, and recombinant expression vectors are constructed, and the obtained antibody has the characteristics of high stability, good specificity, small batch-to-batch difference and the like.

(3) The rabbit monoclonal antibodies prepared by the invention have different antigen binding sites for 2G2 and 3B3. The double-antibody sandwich method ELISA kit developed by the antibody has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like.

Drawings

FIG. 1 is a diagram of alignment of VH and VL sequences of rabbit monoclonal antibodies to 2G2 and 3B3.

FIG. 2 is a light and heavy chain expression vector map of an antibody.

Fig. 3 is a graph of affinity assays for rabbit monoclonal antibodies to 2G2 and 3B3 and Human CALR.

FIG. 4 is a chart of epitope testing of rabbit monoclonal antibody pairs 2G2 and 3B3 with recombinant Human CALR.

Fig. 5 is a standard graph of a method for detecting Human CALR based on a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) established for 2G2 and 3B3 by a rabbit monoclonal antibody.

Detailed Description

The technical conception of the invention is to provide rabbit monoclonal antibodies against human calreticulin, including rabbit monoclonal antibody 2G2 and rabbit monoclonal antibody 3B3. The double-antibody sandwich ELISA detection method developed by utilizing the rabbit monoclonal antibody to 2G2 and 3B3 has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like.

The sequence of the specific selected rabbit monoclonal antibody 2G2 is as follows:

the full length sequence of the light chain is as follows:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSTSAAVGGTVTINCQASESVYGNNRLAWYQQKPGQPPKRLIYDASTLASGVPSRFSGSGSGKQFTLTMSGVQCDDAATYYCIGHFYSGINSISFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:1)。

the sequence of the light chain variable region is as follows:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSTSAAVGGTVTINCQASESVYGNNRLAWYQQKPGQPPKRLIYDASTLASGVPSRFSGSGSGKQFTLTMSGVQCDDAATYYCIGHFYSGINSISFGGGTEVVVK(SEQ ID NO:2)。

the sequence of the light chain complementarity determining region LCDR1 is:

ESVYGNNRLAW(SEQ ID NO:3)。

the sequence of the light chain complementarity determining region LCDR2 is:

LIYDASTLASGV(SEQ ID NO:4)。

the sequence of the light chain complementarity determining region LCDR3 is:

IGHFYSGINSISF(SEQ ID NO:5)。

the full length sequence of the heavy chain is as follows:

METGLRWLLLVAVLKGVQCQEQLEESGGGLVQPEGSLTLTCTASGFSINNNYWICWVRQAPGKGLEWVGCISIADNSNTYYATWAKGRVTISKTSSTTVTLQMTSLTAADTATYFCARGPAVISFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:6)。

the sequence of the heavy chain variable region is as follows:

METGLRWLLLVAVLKGVQCQEQLEESGGGLVQPEGSLTLTCTASGFSINNNYWICWVRQAPGKGLEWVGCISIADNSNTYYATWAKGRVTISKTSSTTVTLQMTSLTAADTATYFCARGPAVISFNLWGPGTLVTVSS(SEQ ID NO:7)。

the sequence of heavy chain complementarity determining region LCDR1 is:

FSINNNYWIC(SEQ ID NO:8)。

the sequence of heavy chain complementarity determining region LCDR2 is:

WVGCISIADNSNTYYATWAK(SEQ ID NO:9)。

the sequence of heavy chain complementarity determining region LCDR3 is:

YFCARGPAVISFNL(SEQ ID NO:10)。

the sequence of the specific selected rabbit monoclonal antibody 3B3 is as follows:

the full length sequence of the light chain is as follows:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSTSAAVGGTVTINCQASESVYNNNRLAWYQQKPGQPPKRLIYDASKLASGVPSRFKGSGSGKQFTLTMSGVQCDDAATYYCIGHFYTSINSVTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:11)。

the sequence of the light chain variable region is as follows:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSTSAAVGGTVTINCQASESVYNNNRLAWYQQKPGQPPKRLIYDASKLASGVPSRFKGSGSGKQFTLTMSGVQCDDAATYYCIGHFYTSINSVTFGGGTEVVVK(SEQ ID NO:12)。

the sequence of the light chain complementarity determining region LCDR1 is:

ESVYNNNRLAW(SEQ ID NO:13)。

the sequence of the light chain complementarity determining region LCDR2 is:

LIYDASKLASGV(SEQ ID NO:14)。

the sequence of the light chain complementarity determining region LCDR3 is:

IGHFYTSINSVTF(SEQ ID NO:15)。

the full length sequence of the heavy chain is as follows:

METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSSYWICWVRQAPGKGLEWIGCISIDDNSNTYYASWAKGRVTISKTSSTTVTLQMTSLTAADTATYFCARGPGIIFYNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:16)。

the sequence of the heavy chain variable region is as follows:

METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSSYWICWVRQAPGKGLEWIGCISIDDNSNTYYASWAKGRVTISKTSSTTVTLQMTSLTAADTATYFCARGPGIIFYNLWGPGTLVTVSS(SEQ ID NO:17)。

the sequence of heavy chain complementarity determining region LCDR1 is:

FSFSSSYWIC(SEQ ID NO:18)。

the sequence of heavy chain complementarity determining region LCDR2 is:

WIGCISIDDNSNTYYASWAK(SEQ ID NO:19)。

the sequence of heavy chain complementarity determining region LCDR3 is:

YFCARGPGIIFYNL(SEQ ID NO:20)。

the present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention. In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

Example 1

The present example provides a method for preparing recombinant human CALR fusion protein by in vitro expression comprising the steps of:

downloading the full-length sequence (NP_ 004334.1) of the human CALR gene from NCBI, calling the sequence corresponding to 18-203 aa, designing a primer and introducing enzyme cutting sites EcoRI and XhoI, wherein the primer sequence is as follows:

F:GAATTCGAGCCTGCCGTCTACTTCAAG；

R:CTCGAGCAGGAAGTCCCAATCGTCTTC。

PCR amplification was performed using the above primers with human cDNA as a template, and the amplification system and procedure were as follows:

the reaction system:

the amplification procedure was:

98℃，30s；35×(98℃，10s+55℃，30s+72℃，30s)；72℃，5min。

after agarose gel electrophoresis, selecting a target fragment for gel cutting and recycling to obtain a target PCR product; respectively carrying out double enzyme digestion on a target PCR product and a vector pGEX-4T-1, and carrying out gel electrophoresis and gel cutting recovery to obtain a DNA fragment with a sticky end; connecting the digested DNA fragment and the vector through T4 ligase; the ligated target fragments were transformed into competent E.coli and cultured overnight on LB plates. Picking single spots on a flat plate, shaking the flat plate in an LB culture medium for culturing overnight, taking bacterial liquid for PCR verification, taking corresponding bacterial liquid for sequencing after the bacterial liquid is qualified for verification, and preserving seeds after the sequencing is correct.

Performing amplification culture on qualified bacterial liquid overnight, and centrifuging to obtain a supernatant; the culture supernatant was purified by immobilized metal ion affinity chromatography (IMAC) and verified that the purity of the recombinant human CALR fusion protein was above 90%.

Example 2

The present example provides a method for preparing a rabbit monoclonal antibody directed against human calreticulin, comprising the steps of:

the recombinant human CALR fusion protein prepared in example 1 was used as an immunogen to immunize New Zealand white rabbits. Each white rabbit is immunized by 200 mug, the immunogen is mixed with the complete Freund's adjuvant with the same quantity to prepare an emulsifying agent for the first time, 100 mug immunogen is mixed with the incomplete Freund's adjuvant with the same quantity to prepare the emulsifying agent at intervals of 3 weeks, the subcutaneous multi-point injection is carried out on the abdomen and the back, the serum titer is measured by ELISA method after the immunization is enhanced twice, the rabbit with high serum titer is taken, the subcutaneous multi-point injection is carried out for one time by 200 mug immunogen, and the spleen is taken after three days.

The spleens of rabbits with high serum titers were placed in RMPI basal medium containing 100U/mL penicillin and 100. Mu.g/mL streptomycin, chopped with a surgical blade and transferred to a 100 μm cell screen for milling. The resulting cell suspension was filtered to remove large cell clusters and tissue envelopes, centrifuged at 400g for 5min, and the supernatant was removed to retain spleen cell clusters. Spleen cell clusters were resuspended in hypotonic solution and after lysis of erythrocytes, centrifuged again at 400g for 5min, and spleen cells were retained. Spleen cells were resuspended in RMPI basal medium of 100U/mL penicillin and 100. Mu.g/mL streptomycin, centrifuged at 400g for 5min, and the resulting spleen cells were resuspended in complete medium (RMPI basal medium containing 10% fetal calf serum, 100U/mL penicillin and 100. Mu.g/mLl streptomycin) for further use.

B lymphocyte separation is then carried out from the obtained spleen cells, and the method is described in patent document 201910125091.4, namely, a method for efficiently separating single antigen-specific B lymphocytes from the spleen cells, so as to obtain a B cell supernatant.

The cultured B cell supernatant was used for identifying positive clones by antigen-coated ELISA, which comprises the following specific steps: coating a 96-well plate overnight with a recombinant human CALR fusion protein; adding the cultured B cell supernatant, and incubating for 1h at 37 ℃; after washing the plate 3 times, adding the Goat anti Rabbit IgG secondary antibody marked by HRP, and incubating for 1h at 37 ℃; after washing the plate 3 times, 100 mu LTMB of color development solution is added and incubated for 15min at 37 ℃; adding stop solution, and detecting OD by enzyme-labeled instrument _450nm And OD (optical density) _630nm Absorbance at wavelength, in OD value>More than 0.7 was identified as positive clones. 8 positive clones were identified by the above method, with clone numbers 1B11, 2D12, 2G2, 3B3, 4E3, 4G3, 5C11, 7H2. And finally, screening by a subsequent test to obtain 2G2 and 3B3 with excellent pairing effect.

Positive cloned cells were collected, lysed using lysate, RNA was extracted and reverse transcribed into cDNA. The light and heavy chain variable region genes (VH and VL) of the naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones by PCR and sequenced to determine the sequence, sequence alignment of the 2G2 and 3B3 antibodies is shown in fig. 1.

Wherein, the gene sequence of the 2G2 antibody is as follows:

the 2G2-FL light chain gene sequence is:

ATGGACACGAGGGCCCCCACTCAGCTGCTGGGCCTGCTCCTGCTCTGGTTGCCCGGTGCAACATTCGCCCAAGTTCTGACGCAGACTCCCAGTTCAACATCAGCAGCCGTTGGGGGTACCGTGACCATCAACTGTCAGGCGAGTGAAAGCGTGTATGGCAATAATAGATTGGCATGGTACCAACAAAAGCCTGGACAGCCGCCAAAGCGGTTGATCTACGATGCTAGCACGCTGGCCTCTGGAGTGCCTTCCAGATTTAGTGGATCCGGTTCCGGCAAGCAATTCACATTGACGATGTCTGGAGTGCAGTGCGATGATGCAGCTACATACTATTGTATTGGTCACTTTTACTCAGGGATAAACAGCATTTCTTTCGGAGGCGGGACTGAGGTCGTGGTCAAAGGTGACCCGGTGGCACCCACAGTCCTGATTTTTCCCCCAGCCGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG。

the gene sequence of the 2G2-VL light chain variable region is as follows:

GGCCTGCTCCTGCTCTGGTTGCCCGGTGCAACATTCGCCCAAGTTCTGACGCAGACTCCCAGTTCAACATCAGCAGCCGTTGGGGGTACCGTGACCATCAACTGTCAGGCGAGTGAAAGCGTGTATGGCAATAATAGATTGGCATGGTACCAACAAAAGCCTGGACAGCCGCCAAAGCGGTTGATCTACGATGCTAGCACGCTGGCCTCTGGAGTGCCTTCCAGATTTAGTGGATCCGGTTCCGGCAAGCAATTCACATTGACGATGTCTGGAGTGCAGTGCGATGATGCAGCTACATACTATTGTATTGGTCACTTTTACTCAGGGATAAACAGCATTTCTTTCGGAGGCGGGACTGAGGTCGTGGTCAAAGGTGACCCGGTGGCACCCACAGTCCTGATTTTTCCCCCAGCC。

the gene sequence of the 2G2-FH heavy chain is as follows:

ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTTGCAGTTCTGAAGGGGGTCCAATGCCAAGAACAGCTCGAGGAAAGCGGTGGGGGGTTGGTGCAGCCTGAGGGGAGCCTGACCCTGACGTGTACGGCAAGTGGTTTCTCTATCAATAATAACTACTGGATCTGTTGGGTGAGACAAGCCCCTGGGAAGGGCCTCGAATGGGTTGGTTGTATCTCTATTGCCGACAATTCTAATACATACTATGCAACATGGGCAAAAGGCCGCGTCACAATCTCTAAGACCAGTAGCACTACAGTCACCCTGCAAATGACCTCTTTGACAGCAGCCGATACTGCAACATATTTTTGTGCCAGGGGACCTGCGGTCATTTCTTTTAATCTGTGGGGACCGGGGACGCTGGTGACCGTTTCCAGCGGGCAACCAAAGGCGCCATCAGTCTTTCCTCTCGCGCCTTGCTGTGGCGACACTCCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAA。

the gene sequence of the 2G2-VH heavy chain variable region is:

GTTGCAGTTCTGAAGGGGGTCCAATGCCAAGAACAGCTCGAGGAAAGCGGTGGGGGGTTGGTGCAGCCTGAGGGGAGCCTGACCCTGACGTGTACGGCAAGTGGTTTCTCTATCAATAATAACTACTGGATCTGTTGGGTGAGACAAGCCCCTGGGAAGGGCCTCGAATGGGTTGGTTGTATCTCTATTGCCGACAATTCTAATACATACTATGCAACATGGGCAAAAGGCCGCGTCACAATCTCTAAGACCAGTAGCACTACAGTCACCCTGCAAATGACCTCTTTGACAGCAGCCGATACTGCAACATATTTTTGTGCCAGGGGACCTGCGGTCATTTCTTTTAATCTGTGGGGACCGGGGACGCTGGTGACCGTTTCCAGCGGGCAACCAAAGGCGCCATCAGTCTTTCCTCTCGCGCCTTGCTGTGGCGACACT。

wherein, the gene sequence of the 3B3 antibody is as follows:

the light chain gene sequence of 3B3-FL is:

ATGGACACGAGGGCCCCCACTCAGCTGCTGGGACTCTTGCTCCTCTGGCTGCCAGGCGCCACATTTGCACAGGTTCTCACACAAACACCGAGCAGCACGTCTGCGGCCGTGGGGGGCACGGTGACGATAAACTGCCAGGCGTCTGAAAGCGTTTACAATAACAACCGCCTCGCATGGTATCAACAGAAACCCGGCCAGCCTCCTAAACGCCTCATTTATGACGCGAGTAAACTGGCCTCTGGGGTTCCCTCAAGATTCAAGGGTTCTGGTAGTGGAAAGCAGTTCACCTTGACGATGAGTGGTGTCCAATGCGACGACGCAGCAACCTACTACTGCATTGGGCACTTTTATACCAGCATCAACAGCGTTACTTTTGGTGGCGGGACCGAAGTTGTTGTTAAAGGAGATCCCGTTGCGCCTACAGTCTTGATCTTTCCTCCGGCCGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG。

the light chain variable region gene sequence of 3B3-VL is:

GGACTCTTGCTCCTCTGGCTGCCAGGCGCCACATTTGCACAGGTTCTCACACAAACACCGAGCAGCACGTCTGCGGCCGTGGGGGGCACGGTGACGATAAACTGCCAGGCGTCTGAAAGCGTTTACAATAACAACCGCCTCGCATGGTATCAACAGAAACCCGGCCAGCCTCCTAAACGCCTCATTTATGACGCGAGTAAACTGGCCTCTGGGGTTCCCTCAAGATTCAAGGGTTCTGGTAGTGGAAAGCAGTTCACCTTGACGATGAGTGGTGTCCAATGCGACGACGCAGCAACCTACTACTGCATTGGGCACTTTTATACCAGCATCAACAGCGTTACTTTTGGTGGCGGGACCGAAGTTGTTGTTAAAGGAGATCCCGTTGCGCCTACAGTCTTGATCTTTCCTCCGGCC。

the heavy chain gene sequence of 3B3-FH is:

ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCAGTTTTGAAAGGAGTCCAATGCCAGAGTCTGGAAGAGAGTGGTGGTGGCTTGGTCCAACCTGAGGGGAGCTTGACATTGACATGCACCGCGTCCGGCTTTAGTTTCTCTAGTTCTTATTGGATCTGTTGGGTTCGGCAAGCACCGGGGAAGGGGCTGGAGTGGATTGGGTGTATAAGCATTGACGATAACAGTAATACTTACTACGCGTCTTGGGCTAAAGGCCGCGTCACCATTAGCAAAACATCTTCCACGACTGTTACGCTCCAGATGACCTCACTGACAGCCGCTGACACTGCGACTTACTTTTGCGCGAGAGGCCCTGGCATCATATTCTATAACTTGTGGGGTCCAGGGACCCTGGTTACGGTTTCCTCTGGACAACCAAAAGCGCCCAGTGTTTTTCCCTTGGCTCCTTGTTGTGGGGATACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAA。

the heavy chain variable region gene sequence of 3B3-VH is:

GTCGCAGTTTTGAAAGGAGTCCAATGCCAGAGTCTGGAAGAGAGTGGTGGTGGCTTGGTCCAACCTGAGGGGAGCTTGACATTGACATGCACCGCGTCCGGCTTTAGTTTCTCTAGTTCTTATTGGATCTGTTGGGTTCGGCAAGCACCGGGGAAGGGGCTGGAGTGGATTGGGTGTATAAGCATTGACGATAACAGTAATACTTACTACGCGTCTTGGGCTAAAGGCCGCGTCACCATTAGCAAAACATCTTCCACGACTGTTACGCTCCAGATGACCTCACTGACAGCCGCTGACACTGCGACTTACTTTTGCGCGAGAGGCCCTGGCATCATATTCTATAACTTGTGGGGTCCAGGGACCCTGGTTACGGTTTCCTCTGGACAACCAAAAGCGCCCAGTGTTTTTCCCTTGGCTCCTTGTTGTGGGGATACA。

the rabbit monoclonal antibodies 2G2 and 3B3 heavy and light chain genes were further loaded onto the expression vector pBR322, respectively, using the mammalian expression vector shown in fig. 2. Wherein pBR322Ori and f1Ori are replication initiation sites in E.coli, ampcilin is a plasmid resistance gene, CMV Promoter is a transcription Promoter, SV 40 PAterminal is a tailing signal, heavy chain constant (FIG. 2 a) and Light chain constant (FIG. 2 b) are rabbit heavy chain constant region and light chain constant region sequences, respectively.

Mammalian expression plasmids containing rabbit heavy chain constant region and light chain constant region sequences are subjected to conventional linearization treatment by using NheI and XbaI restriction enzymes respectively, heavy chain variable region genes and light chain variable region genes are respectively connected to corresponding mammalian expression vectors by adopting a homologous recombination mode, and the sequences are determined by sequencing, and the sequencing work is completed by Jin Kairui biotechnology limited company.

Transfecting the plasmid into 293F cells; collecting culture supernatant after transfection for 72-96 hours; purifying recombinant rabbit monoclonal antibodies 2G2 and 3B3 recognizing human CALR protein by affinity chromatography, split charging after antibody identification, and preserving at-20deg.C for use.

Example 3

This example was conducted for antibody affinity test and antigen recognition epitope test against the rabbit monoclonal antibodies 2G2 and 3B3 obtained by preparation.

The affinity of the rabbit monoclonal antibodies 2G2 and 3B3 prepared in example 2 was precisely determined by using Biacore 3000 biomolecular interaction analyzer from GE company, and the test results are shown in FIG. 3.

Affinity of rabbit monoclonal antibody 2G2 for recombinant human CALR protein (prepared in example 1) was 4.88×10 by affinity curve fitting and calculation ^-9 Affinity of the rabbit monoclonal antibody 3B3 for recombinant human CALR protein (prepared in example 1) 4.34×10 ^-9 M。

The antigen recognition epitopes of the rabbit monoclonal antibodies 2G2 and 3B3 prepared in example 2 were identified using a Gator biomolecular interaction analyzer from Probe Life, and the test results are shown in FIG. 4.

The concentrations of the rabbit anti-human CALR monoclonal antibodies 2G2 and 3B3 used were 500nM and 300nM, respectively, and the concentration of recombinant human CALR protein (prepared in example 1) was 100nM. As can be seen from the analysis of the pairing data between the two antibodies, after the rabbit monoclonal antibody 2G2 is combined with the recombinant human CALR protein, the rabbit monoclonal antibody 3B3 can still be combined with the recombinant human CALR protein, and the fact that the rabbit monoclonal antibodies 2G2 and 3B3 are combined at different positions on the surface of the CALR protein is proved, and the rabbit monoclonal antibodies do not interfere with each other.

Example 4

The embodiment establishes a double-antibody sandwich method ELISA detection method based on rabbit monoclonal antibodies 2G2 and 3B3, and comprises the following steps:

1) Coating: the rabbit monoclonal antibody 2G2 (capture antibody, prepared in example 2) was diluted to 2. Mu.g/mL with 1 XPBS buffer, vortexed, added to 96-well microwell plates at 100. Mu.L/well, covered with cover plate membrane, and incubated at 4deg.C for 16-20 h in a refrigerator.

2) Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.

3) Closing: adding a blocking solution (5% BSA) into the plate holes at a concentration of 200 mu L/well, covering a cover plate film, blocking for 2 hours at 37 ℃, discarding the blocking solution after blocking, drying the ELISA plate, drying in a drying oven at 37 ℃ for 0.5-2 hours, and taking out for later use.

4) Protein adding: human CALR (prepared in example 1) was diluted with a diluent (1% bovine serum albumin, 0.05% tween-20 in phosphate buffer) at a dilution concentration of: 200. 100, 50, 25, 12.5, 6.25, 3.12, 0ng/mL, then added to the ELISA plate in sequence at 100. Mu.L/well, covered with cover plate membrane and incubated for 2h at 37 ℃.

5) Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.

6) Adding a detection antibody: the biotin-labeled rabbit monoclonal antibody 3B3 (detection antibody) was diluted to 0.083. Mu.g/mL, and then added to the ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 1 hour.

7) Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.

8) Adding SA-HRP: 100 XSA-HRP concentrate was diluted 100 times, and then added to an ELISA plate in order of 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.

9) Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.

10 Adding TMB color development liquid: TMB color development solution is added into an ELISA plate in sequence at 100 mu L/well, a cover plate film is covered, and incubation is carried out for 15min at 37 ℃.

11 After incubation was completed, the ELISA plate was removed, 50. Mu.L of oxalic acid stop solution was added to each well, and absorbance at 450nm and 630nm, OD was measured _450nm Subtracting OD _630nm The corrected absorbance value.

The linear curve drawn by the statistical result is shown in fig. 5.

The test results show that the double-antibody sandwich ELISA detection method for the needle human calreticulin is established based on the rabbit monoclonal antibody pair 2G2 and 3B3, and has the advantages of high specificity, high detection sensitivity and the like.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rabbit monoclonal antibody directed against human calreticulin, wherein the rabbit monoclonal antibody is a first rabbit monoclonal antibody or a second rabbit monoclonal antibody;

the sequences of the light chain complementarity determining regions 1-3 of the first rabbit monoclonal antibody are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the sequences of the heavy chain complementarity determining regions 1-3 are respectively shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10;

the sequences of the light chain complementarity determining regions 1-3 of the second rabbit monoclonal antibody are respectively shown as SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, and the sequences of the heavy chain complementarity determining regions 1-3 are respectively shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20.

2. The rabbit monoclonal antibody directed against human calreticulin of claim 1, wherein the sequence of the light chain variable region of the first rabbit monoclonal antibody is shown in SEQ ID No. 2; and/or

The sequence of the heavy chain variable region of the first rabbit monoclonal antibody is shown as SEQ ID NO. 7.

3. The rabbit monoclonal antibody directed against human calreticulin of claim 2, wherein the full length sequence of the light chain of the first rabbit monoclonal antibody is shown in SEQ ID No. 1; and/or

The full-length sequence of the heavy chain of the first rabbit monoclonal antibody is shown as SEQ ID NO. 6.

4. The rabbit monoclonal antibody directed against human calreticulin of claim 1, wherein the sequence of the light chain variable region of the second rabbit monoclonal antibody is shown in SEQ ID No. 12; and/or

The sequence of the heavy chain variable region of the second rabbit monoclonal antibody is shown as SEQ ID NO. 17.

5. The rabbit monoclonal antibody directed against human calreticulin of claim 4, wherein the full length sequence of the light chain of the second rabbit monoclonal antibody is shown in SEQ ID No. 11; and/or

The full-length sequence of the heavy chain of the second rabbit monoclonal antibody is shown as SEQ ID NO. 16.

6. Use of a rabbit monoclonal antibody directed against human calreticulin according to any one of claims 1 to 5 for the preparation of a reagent or kit for the detection of human calreticulin.

7. The use according to claim 6, wherein the reagent or kit is used for enzyme-linked immunosorbent assay of human calreticulin, the first rabbit monoclonal antibody being used as a capture antibody and the second rabbit monoclonal antibody being used as a marker antibody.

8. A reagent or kit for detecting human calreticulin comprising a first rabbit monoclonal antibody and a second rabbit monoclonal antibody directed against human calreticulin according to any one of claims 1 to 5.

9. The reagent or kit for detecting human calreticulin according to claim 8, wherein the human calreticulin is at least one selected from recombinant human calreticulin and human calreticulin secreted by cells.

10. A method of preparing a rabbit monoclonal antibody to human calreticulin according to any one of claims 1 to 5, comprising the steps of:

loading the heavy chain gene and the light chain gene of the rabbit monoclonal antibody against human calreticulin according to any one of claims 1 to 5 on expression vectors, respectively, and transfecting 293F cells;

culturing to obtain a supernatant containing the first rabbit monoclonal antibody and the second rabbit monoclonal antibody;

purifying to obtain the final product.