CN117025546A

CN117025546A - Hybridoma cell strain, anti-GST monoclonal antibody and application thereof

Info

Publication number: CN117025546A
Application number: CN202311053346.3A
Authority: CN
Inventors: 刘万建; 刘洋; 李文; 邵建华; 李萍萍; 曾景斌; 杨帆
Original assignee: Qingdao Hightop Biotech Co ltd; Qingdao Shuojing Biotechnology Co ltd
Current assignee: Qingdao Hightop Biotech Co ltd; Qingdao Shuojing Biotechnology Co ltd
Priority date: 2023-08-21
Filing date: 2023-08-21
Publication date: 2023-11-10

Abstract

The invention discloses a hybridoma cell strain, an anti-GST monoclonal antibody and application thereof, and relates to the technical field of immunodetection. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) NO: C2021187. according to the invention, a hybridoma cell strain capable of secreting anti-GST monoclonal antibody is screened out by a hybridoma cell technology; the anti-GST monoclonal antibody secreted by the hybridoma cell strain has the characteristics of high affinity, good specificity and high sensitivity.

Description

Hybridoma cell strain, anti-GST monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a hybridoma cell strain, an anti-GST monoclonal antibody and application thereof.

Background

Glutathione thiol transferase (GST) is an enzyme that is widely present in both prokaryotes and eukaryotes, primarily involved in detoxification of cells and bodies; the GST protein has a size of 26KD and a relatively large molecular weight, can be combined with the C end or the N end of the protein, and is applied to the expression of various fusion proteins.

GST proteins are commonly used as tag proteins in reagent assays, and GST tags have the following advantages over other tag proteins: the application range is wide, and the expression can be carried out in different hosts; in the expression process, the solubility of the expressed protein can be improved; does not affect the antigenicity and biological activity of the expressed protein; the stability of the protein is improved; GST labels can be removed by different proteases according to the use requirement of the expressed protein, so that the removal is convenient; GST tag has high specificity, and is convenient and mild in purification.

The in vitro diagnosis kit using double antigen sandwich method is based on antigen-antibody specific binding, and uses two antigens containing different antibody binding sites as coating and labeling respectively to prepare detection kit, detect antibody in sample, and perform serological screening; in the labeling process, especially for antigens with smaller molecular weight, the ratio of the antigen to the marker can influence the detection sensitivity and the specificity, and when the marker is too much, the detection signal can be improved, but the surface of the antigen can be combined with too much marker to cover the antigen structure, so that the antigen and the antibody are prevented from being combined, the detection sensitivity and the specificity are reduced, and the accuracy of a detection result is influenced.

At present, in order to reduce the influence of the label on the antigen structure, a specific GST (GST) label antibody can be used as a labeling raw material, and then is specifically combined with a corresponding GST label protein on the antigen to jointly form the labeling raw material of the kit, so that the sensitivity and the specificity of the kit are improved.

Based on the application of the GST protein, the inventor considers how to screen a hybridoma cell strain capable of secreting the anti-GST monoclonal antibody by a hybridoma cell technology, and based on the screened hybridoma cell strain, the anti-GST monoclonal antibody secreted by the hybridoma cell strain is subjected to sequencing of the full-length sequence of the variable region, so that the stable anti-GST monoclonal antibody can be conveniently produced in a subsequent reverse direction by means of genetic engineering, and the technical problem which is needed to be solved by the person skilled in the art is solved.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Disclosure of Invention

Aiming at the technical problems, the embodiment of the invention provides a hybridoma cell strain, an anti-GST monoclonal antibody and application thereof, so as to solve the problems in the background technology.

The invention provides the following technical scheme:

a hybridoma cell line, designated as hybridoma cell line 62H9, was deposited at China Center for Type Culture Collection (CCTCC) at 8.12 of 2021, accession number: the university of Wuhan in China is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021187.

an anti-GST monoclonal antibody secreted by the hybridoma cell strain described above.

Preferably, the anti-GST monoclonal antibody can be specifically combined with GST tag protein, and the amino acid sequence of the GST tag protein is shown as SEQ ID NO. 1.

Preferably, the subtype of the anti-GST monoclonal antibody is IgG1.

An anti-GST monoclonal antibody, the light chain variable region of which comprises the amino acid sequence set forth in SEQ ID NO:4, as set forth in SEQ ID NO:5 and CDR2 as set forth in SEQ ID NO: CDR3 shown in fig. 6;

the heavy chain variable region of the anti-GST monoclonal antibody comprises a sequence as shown in SEQ ID NO:7, as set forth in SEQ ID NO:8 and CDR2 as set forth in SEQ ID NO: CDR3 as shown in 9.

An anti-GST monoclonal antibody, the light chain variable region of which comprises the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); the heavy chain variable region of the anti-GST monoclonal antibody comprises the amino acid sequence of SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

The recombinant expression vector is formed by recombining the expression vector and the encoding gene of the anti-GST monoclonal antibody.

A recombinant engineering bacterium obtained by transforming the recombinant expression vector according to claim 7 into a host bacterium.

A test kit comprising an anti-GST monoclonal antibody as described above.

Preferably, the anti-GST monoclonal antibody is used as a tag protein in a detection kit.

The hybridoma cell strain, the anti-GST monoclonal antibody and the application thereof provided by the embodiment of the invention have the following beneficial effects:

1. according to the invention, a hybridoma cell strain capable of secreting anti-GST monoclonal antibody is screened out by a hybridoma cell technology;

2. the anti-GST monoclonal antibody secreted by the hybridoma cell strain has the characteristics of high affinity, good specificity and high sensitivity;

3. the invention can sequence the whole length of the variable region of the anti-GST monoclonal antibody, can stably produce the anti-GST monoclonal antibody through genetic engineering in the later period, and can not cause the performance reduction of the secreted anti-GST monoclonal antibody due to the occurrence of genetic mutation after a plurality of subcultures of the selected hybridoma cell strain.

Drawings

FIG. 1 is a graph showing the results of detection of secretion stability of hybridoma cell lines selected according to the present invention;

FIG. 2 shows the results of detection of affinity of anti-GST monoclonal antibodies.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person skilled in the art without making any inventive effort are within the scope of the present invention.

Example 1: preparation of hybridoma cell strain secreting anti-GST monoclonal antibody

1. Recombinant antigen preparation

Analyzing protein sequences by utilizing NCBI and GenBank databases, and preparing four kinds of tag proteins, namely GST tag proteins, trx tag proteins, sumo tag proteins and MBP tag proteins, and four kinds of recombinant antigens containing GST tag proteins, namely Ag3-GST, ag6-GST, ag7-GST and Ag10-GST through the processes of PCR identification, enzyme digestion, vector construction, plasmid construction, transformation and expression, identification and seed preservation and protein purification; wherein, the amino acid sequence of the GST tag protein is shown as SEQ ID NO:1 is shown as follows:

SEQ ID NO：1

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK

2. immunization of mice

Immunizing female balb/c mice of 6-8 weeks old with GST tag protein; GST tag protein is mixed with Freund's complete adjuvant with equal volume according to the dosage of 75 mug/dose, the emulsification is complete, and the mice are immunized subcutaneously at the back; three weeks later, the immunization is enhanced, GST tag is mixed with an equal volume of incomplete Freund's adjuvant according to a dosage of 35 mug/mouse, the emulsification is complete, and the mice are immunized subcutaneously at multiple points on the back; after 1 week, mice were subjected to tail-breaking blood sampling, and serum titers were detected. Serum titer reaches more than 10 ten thousand, prepare and fuse; mice were immunized by intraperitoneal injection 3 days prior to fusion, without adjuvant, at a dose of 100 μg/mouse.

3. Cell fusion

During fusion, spleen of an immunized mouse is taken, dispersed cells are ground, after centrifugation, B lymphocytes of the mouse are collected, and are mixed according to the proportion of 7:1 (B lymphocytes: SP 2/0), and are centrifuged, cells are washed by serum-free DMEM culture medium, and no fetal bovine serum is ensured in the mixed cells; and (3) carrying out cell fusion by using PEG, stopping fusion by using a DMEN medium after full fusion, and finally, suspending the fused cells by using a HAT medium, placing the fused cells in a 96-well cell culture plate, and placing the 96-well cell culture plate into a carbon dioxide incubator for culture.

3. Hybridoma cell screening and subcloning

Culture supernatants were assayed by indirect ELISA on day 7 after fusion by total exchange of HAT medium.

The specific detection method comprises the following steps:

coating: GST tagged protein was diluted to 1. Mu.g/mL with CBS buffer (pH 9.6), 100. Mu.L/well coated in ELISA plates, coated overnight at 4 ℃. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate; closing: blocking the ELISA plate with PBST buffer containing 3% BSA, 200. Mu.L/well, and incubating at 37℃for 2h; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

and (3) detection: adding cell supernatant to be detected into an ELISA plate, wherein 100 mu L/hole is obtained, simultaneously taking mouse immune serum as positive control, taking HAT culture medium as blank control, and incubating at 37 ℃ for 1h; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse antibody was diluted 2-fold with PBST buffer, added to the elisa plate, 100 μl/well, incubated for 1h at 37 ℃; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 15min; stopping with 2M sulfuric acid stopping solution, 50 μl/well, and detecting at 450nm wavelength;

high selection potency (OD ₄₅₀ Subcloning the cells of 2.0) or more, and taking the subclones by a multiple dilution method200-500 cells, transferred to A1 wells of a new 96-well cell culture plate, and subcloned by 2-fold dilutions in the longitudinal and transverse directions, respectively; detecting culture supernatant by indirect ELISA after 7-9 days, selecting single positive hole of cell mass, and continuing subcloning until the positive rate of 96-well plate reaches 100%; repeating the subcloning for 2 times, wherein the positive rate is 100%, ending the subcloning, and ensuring the stability of the hybridoma cell strain.

4. Hybridoma cell line preservation

Selecting hybridoma cell strains with stable growth, coating Trx tag protein, sumo tag protein, GST tag protein and MBP tag protein by using the indirect ELISA method, verifying the specificity and titer of cell supernatant, and performing cell primary screening; as shown in Table 1, cell line 1 having high specificity, high titer and good linearity was selected for the expansion culture, and when the cell density reached 80%, the cells were collected and frozen with a cell frozen solution.

The cell is named as hybridoma cell strain 62H9 and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2021187.

TABLE 1 preliminary cell screening results

5. Hybridoma secretion stability assay

Subculturing the hybridoma cell strain 62H9, co-culturing for 30 generations, collecting culture supernatants at 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 generations respectively, detecting GST tag protein by using an indirect ELISA method, and analyzing the stability of cell secretion antibodies; the detection results are shown in fig. 1: the cell passage 30 times showed higher uniformity of secreted antibody titers.

Example 2: preparation of anti-GST monoclonal antibodies

1. Ascites preparation

Balb/c female mice with the age of about 12 weeks are selected, each mouse is injected with 500 mu L of liquid paraffin in the abdominal cavity, hybridoma cells are injected in the abdominal cavity after one week, and the dosage is 10 ⁶ Individual/individual; after 5 days, continue to observeAfter the abdomen of the mice swells obviously, collecting ascites, centrifuging at 12000rpm/min, removing impurities, and storing in a refrigerator at-80 ℃.

2. Antibody purification

Taking the ascites out of the refrigerator, centrifuging after melting, and filtering with a 0.22 mu m filter membrane; purification is performed with reference to the Boglycoprotein-A column specification; the purified antibodies were collected, dialyzed against 20mM PB, collected after 48h, filtered through a 0.22 μm filter, and sub-packaged for storage in a-80℃refrigerator for subsequent detection and validation.

The specific purification method for antibody purification is as follows:

balance: taking a Protein-A chromatographic column, and balancing the column by using a 10-time volume of loading buffer solution;

loading: adding the treated ascites into a chromatographic column, and collecting fluid passing through a centrifuge tube; after all the samples pass through the column, the flow-through liquid is sampled again, meanwhile, the flow-through liquid is collected, and the flow-through liquid is marked (in the process of sampling, the flow speed is controlled to be a little slower, so that the samples are fully combined with the purification column);

balance: after the sample loading is finished, balancing the column by using a sample loading buffer solution with the volume of 10 times of the column, detecting outlet liquid by using a coomassie brilliant blue reagent, and balancing completely when the concentration of liquid protein is lower than 1 mg/mL;

eluting: 30 μl of neutralization buffer was added to the 600 μl tube in advance, and elution was performed with 10 column volumes of elution buffer; during elution, 400uL of eluent is collected in each tube and is evenly mixed in time; detecting the concentration of the antibody in the eluent (note the node at which collection starts and ends) with coomassie brilliant blue reagent, and marking;

balance: balancing by using a loading buffer solution with the volume of 10 times of the column volume, detecting the PH of the outlet liquid by using PH test paper, and ending balancing when the PH of the outlet liquid is consistent with the PH of the loading buffer solution; the column was rinsed with 20% ethanol and stored in 20% ethanol and stored at 4 ℃.

Example 3: monoclonal antibody variable region sequencing

1. Cell culture and RNA extraction

Taking out the cells from the liquid nitrogen tank, resuscitating and expanding the cells for culture, and collecting the cultured cells; RNA from the above cells was extracted according to the instructions of the RNA extraction kit from Thermo company.

2. Reverse transcription

The above-mentioned extracted RNA was immediately reverse transcribed to prepare cDNA by referring to the kit instructions of Thermo company.

PCR amplification and recovery

The variable region universal primer of the IgG subtype mouse monoclonal antibody is used, the cDNA is used as a template to carry out PCR amplification on the variable regions of the light chain and the heavy chain, the amplified products are identified by DNA gel, and the PCR products are recovered by gel cutting of a kit of TIANGEN company.

4. And (3) carrying out vector construction and expression identification on the light chain PCR products and the heavy chain PCR products, selecting positive clone strains, and sending the positive clone strains to a sequencing company for sequencing.

The sequence of the light chain variable region (VL) of the anti-GST monoclonal antibody is determined by sequencing as shown in seq id NO:2, the sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:3 is shown in the figure;

by database analysis of the above sequences, the light chain contains the following complementarity determining regions: CDR1 sequences are set forth in SEQ ID NO:4, the CDR2 sequence is as shown in SEQ ID NO:5, the CDR3 sequence is as shown in SEQ ID NO:6 is shown in the figure; the heavy chain contains the following complementarity determining regions: CDR1 sequences are set forth in SEQ ID NO:7, the CDR2 sequence is as shown in SEQ ID NO:8, the CDR3 sequence is as shown in SEQ ID NO: shown at 9.

SEQ ID NO：2

DIVLTQSPTSLAVSLGQRATISCRASQGVSASSYSYIHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNVHPVEEEDTATYYCQHSWEIPLTFGGGTKLEIK

SEQ ID NO：3

EVQLQESGPELVKPGASVRMSCKASGYTFTSYFMHWVKQKSGQGLEWIGYINPYHDGTNYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYSCARSQRLHWYFDVWGAGTTVTVSS

SEQ ID NO：4

QGVSASSYSY

SEQ ID NO：5

YAS

SEQ ID NO：6

QHSWEIPLT

SEQ ID NO：7

GYTFTSYF

SEQ ID NO：8

INPYHDGT

SEQ ID NO：9

ARSQRLHWYFDV

Example 4: monoclonal antibody identification

1. Antibody affinity assay

The titer and the affinity of the antibody are detected by adopting an indirect ELISA method, and the antibody disclosed by the invention has good linearity and the affinity is about 20 ng/ml; the detection result is shown in figure 1;

it should be noted that, the patent application number is CN201310706852.8, the patent name is hybridoma cell capable of secreting anti-GST monoclonal antibody, monoclonal antibody and applied patent, and anti-GST monoclonal antibody is also prepared; however, the anti-GST monoclonal antibody is not sequenced over the entire length of the variable region, i.e., the structure of the anti-GST monoclonal antibody cannot be known, and stable production of the anti-GST monoclonal antibody by genetic engineering is not possible at the later stage;

meanwhile, when the antigen concentration of the anti-GST monoclonal antibody prepared by the patent is 25ng/ml, the corresponding OD is 0.6; when the antigen concentration of the invention is 20ng/ml, the corresponding OD is more than 1.0; as can be seen from comparative analysis, the affinity of the anti-GST monoclonal antibody prepared by the invention is far greater than that of the anti-GST monoclonal antibody prepared by the above patent.

Specific detection methods for antibody affinity assays are as follows:

coating: GST tag protein was diluted to 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL with CBS buffer (pH 9.6), and a blank control was set, 100. Mu.L/well was coated in an ELISA plate, and the coating was carried out overnight at 4 ℃. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

closing: the ELISA plate was blocked with 3% BSA in PBST buffer, 200. Mu.L/well, and incubated at 37℃for 2h. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

and (3) detection: the antibody to be detected was diluted to 1. Mu.g/mL, added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 1h. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse antibody was diluted 2-fold with PBST buffer, added to the elisa plate, 100 μl/well, and incubated for 1h at 37 ℃. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 15min; the reaction was stopped with 2M sulfuric acid stop solution, 50. Mu.L/well, and the reaction was detected at a wavelength of 450 nm.

2. Antibody subtype detection

The subtype of the antibody of the invention was IgG1, detected according to the instructions using the SBA subtype detection kit from Southern Biotech; the test results are shown in Table 2.

TABLE 2 antibody subtype detection results

The specific detection method comprises the following steps:

coating: diluting the coated antigen in the kit into 5 mug/mL with CBS buffer, coating 100 mug/hole into an ELISA plate, and coating at 4 ℃ overnight; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

closing: blocking the ELISA plate with 2% glycine-containing PBST buffer, 200. Mu.L/well, and incubating at 37deg.C for 2h; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

and (3) detection: adding the antibody to be detected into an ELISA plate (8 detection is carried out on each antibody), and incubating for 1h at 37 ℃ with 100 mu L/hole; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse antibody (comprising IgA-HRP, igM-HRP, igG1-HRP, igG2a-HRP, igG2b-HRP, igG3-HRP, kappa-HRP, lambda-HRP) was diluted 500-fold with PBST buffer, added to the elisa plate, 100 μl/well, incubated for 1h at 37 ℃; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

3. Antibody specific detection

Coating 4 different tag proteins (Trx tag protein, sumo tag protein, GST tag protein and MBP tag protein) and 4 recombinant proteins containing GST tags (Ag 3-GST, ag6-GST, ag7-GST and Ag 10-GST) by adopting an indirect ELISA method, and performing 3 dilution gradients on the coated proteins and blank control to detect the specificity of the anti-GST monoclonal antibody; the antibody does not react with other tag proteins, and has good reaction and high specificity with recombinant proteins containing GST tags; the test results are shown in Table 3.

TABLE 3 antibody specificity detection results

The specific detection method comprises the following steps:

coating: diluting 8 coating proteins with CBS buffer solution, wherein the concentration is respectively 0.5 mug/mL, 0.1 mug/mL and 0.02 mug/mL, the blank control coating PBST,100 mug/hole coating into an ELISA plate, and coating at 4 ℃ overnight; washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

closing: the ELISA plate was blocked with 2% glycine in PBST buffer, 200. Mu.L/well, and incubated at 37℃for 2h. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

and (3) detection: the antibody to be detected was added to the ELISA plate at 100. Mu.L/well and incubated at 37℃for 1h. Washing the plate with PBST buffer solution for 5 times, and drying the ELISA plate;

Example 5: application of anti-GST monoclonal antibody in-vitro diagnosis kit

Taking syphilis detection as an example, the anti-GST monoclonal antibody can be prepared into a colloidal gold test strip, and can also be used in detection kits of other products, wherein the types of the kits comprise a colloidal gold method, an enzyme-linked immunosorbent assay method and the like, and the detection products can be syphilis, hepatitis C and the like, but are not limited to the above-mentioned kits and products.

1. Preparation of colloidal gold test strip

1) Adding an anti-GST monoclonal antibody into a colloidal gold solution, uniformly mixing, standing for 10min, adding a blocking solution (2% BSA and 0.5% trehalose) for blocking, centrifuging to remove the supernatant, re-suspending with a colloid Jin Xishi solution, adding a syphilis recombinant antigen (mark) containing a GST tag, fully reflecting, centrifuging to remove the supernatant, re-suspending with a colloid Jin Xishi solution, uniformly mixing, uniformly spreading on glass fibers, and drying and preserving to obtain a gold-labeled pad of a test strip; meanwhile, directly marking the recombinant antigens (marks) of the syphilis containing GST labels on colloidal gold to prepare gold-labeled pads;

2) The recombinant antigen (coating) of syphilis and the goat anti-mouse polyclonal antibody are diluted to the working concentration of 1mg/ml by a coating buffer (20 mM PBS, PH7.4), and are fully and uniformly mixed to prepare a detection line coating liquid and a quality control line coating liquid. Starting a film drawing instrument, setting film drawing parameters, uniformly spraying a detection line coating liquid and a quality control line coating liquid on an NC film, and drying and storing at a distance of 5-7mm to obtain a detection line and a quality control line of the test strip;

3) And fixing the sample pad, the gold mark pad, the NC film (the detection line is close to the sample pad end, the quality control line is close to the water absorbing paper end) and the water absorbing paper on a PVC board in sequence to prepare the colloidal gold test strip. Cutting into test strips with proper sizes by a strip cutting machine for standby.

2. Test strip performance detection

435 cases of positive serum samples and 5000 cases of negative samples of syphilis are detected by using the two test strips, and meanwhile, a treponema pallidum antibody detection kit of the Kang Hua organism of Shandong is used as a control. The test strip for indirectly labeling the anti-GST tag protein antibody has the detection sensitivity of 100%, the specificity of 99.82%, and the result is superior to that of the test strip for directly labeling the antigen (the sensitivity is 94.02%, and the specificity is 97.10%); meanwhile, compared with a Kang Hua kit, the test strip provided by the invention has the advantages of sensitivity and specificity; the test results are shown in Table 4.

Table 4 test strip Performance test results

The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.

Claims

1. A hybridoma cell strain, which is characterized by being preserved in China center for type culture collection (cctccc) NO: C2021187.

2. an anti-GST monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. An anti-GST monoclonal antibody according to claim 2, wherein the anti-GST monoclonal antibody specifically binds to a GST-tag protein having the amino acid sequence shown in SEQ ID No. 1.

4. The anti-GST monoclonal antibody of claim 2, wherein said subtype of anti-GST monoclonal antibody is IgG1.

5. An anti-GST monoclonal antibody, wherein the light chain variable region of said anti-GST monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:4, as set forth in SEQ ID NO:5 and CDR2 as set forth in SEQ ID NO: CDR3 shown in fig. 6;

6. An anti-GST monoclonal antibody, wherein the light chain variable region of said anti-GST monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); the heavy chain variable region of the anti-GST monoclonal antibody comprises the amino acid sequence of SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

7. A recombinant expression vector, which is characterized by being formed by recombining an expression vector and the encoding gene of the anti-GST monoclonal antibody according to claim 5 or 6.

8. A recombinant engineering bacterium, which is formed by transferring the recombinant expression vector according to claim 7 into a host bacterium.

9. A test kit comprising an anti-GST monoclonal antibody according to any one of claims 2-6.

10. The test kit of claim 9, wherein the anti-GST monoclonal antibody is used as a tag protein in the test kit.