CN117821395A - Hybridoma DF2 of HCV core antigen core, monoclonal antibody and application - Google Patents
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Abstract
Hybridoma DF2 of HCV core antigen core and monoclonal antibody and application thereof. The invention discloses screening and application of hybridoma cells and monoclonal antibodies for detecting an HCV core antigen, wherein the preservation number of the hybridoma cells is GDMCC No:63836 the preservation date is 2023, 09 and 26, the preservation unit is the Guangdong province microorganism strain preservation center, and the hybridoma cell can secrete monoclonal antibody DF2 and is used for detecting HCV core antigen core, and has the advantages of good specificity and high sensitivity, so that the hybridoma cell can be used as a detection reagent for detecting the HCV core antigen core.
Description
Technical Field
The invention relates to the detection field, in particular to a hybridoma DF2 of an HCV core antigen core, monoclonal antibodies secreted by the hybridoma and application of the monoclonal antibodies in preparation of detection reagents.
Background
Hepatitis C (hepatitis C) is a liver disease caused by Hepatitis C Virus (HCV) infection, and is generally susceptible to population, 80% of patients with acute hepatitis C are converted into chronic infection, and 10-20% of patients with chronic hepatitis C are developed into liver cirrhosis and liver cancer. At present, polyethylene glycol Interferon (IFN) and ribavirin are mainly adopted for treating hepatitis C, and although new medicines such as semepivir (simeprevir), sofosbuvir (sofosbuvir) and the like are marketed, the treatment effect is obviously improved, but the cost is higher, and certain toxic and side effects are caused, so that the further popularization and the use of the medicines are limited. Therefore, developing new HCV detection technology is important for early and timely diagnosis and treatment of HCV infected persons, improving safety of blood products, and fundamentally eliminating spread of HCV.
Diagnosis of HCV infection is an important component of the work of prevention and control of hepatitis c, and establishing a sensitive and practical detection method is particularly important for monitoring, diagnosis or blood screening to control the prevalence of HCV. There are many methods for detecting HCV, and generally, two general categories are antibody detection and virus detection, including nucleic acid detection (nucleic acid testing, NAT) and core antigen (core) detection.
anti-HCV antibody detection methods are the earliest established laboratory diagnostic methods and are currently widely used for clinical diagnosis and screening of asymptomatic populations. According to the appearance time of the antibody to be detected and different antigens are used, the enzyme-linked immunosorbent assay (enzyme-linked immunosorbnent assay, ELISA) is used for detecting the HCV antibody in serum, the technology is changed for 4 times, the generation 1 to 3 is a HCV antigen coated microplate, the enzyme-labeled mouse anti-human IgG monoclonal antibody is used for assisting in detection, the generation 4 detection technology is used for detecting by using the HCV NS3/NS4 double-antigen sandwich ELISA, and the sensitivity and the detection time are improved to a certain extent. The anti-HCV antibody detection is an indirect serological technology, which has a long window period, namely, the anti-HCV antibody detection can not timely generate enough specific antibody in an infected person within 2 months after HCV infection, and the blood at the moment has infectivity, although the anti-HCV antibody detection is economical, convenient and widely applied. In addition, HCV antibody detection does not distinguish between acute, chronic and past infections in patients, nor does it allow for monitoring of clinical efficacy.
Nucleic acid amplification detection technology (nucleic acid testing, NAT) is currently the means of definitive diagnosis of HCV infection, and in 1999 the european union began to use NAT for HCV screening of medical plasma, and NAT was applied successively to blood screening and clinical sample detection in developed countries such as the united states and japan. By means of nucleic acid amplification technology, NAT detection has the advantages of high specificity, high sensitivity and the like, and the detection window period is shortened greatly by about 10-14 days. However, NAT detection is suitable for the active infection phase of HCV, transient, locally controlled viral replication of the immune response is temporarily undetectable, and about 15% -20% of HCV antibody positive patients are diagnosed as recovered due to failure to detect virus. In addition, viral RNA is very unstable in the sample, which means that the blood sample must be immediately tested or flash frozen and stored at-20℃or-80 ℃.
The HCV core antigen core has been particularly focused and rapidly developed in recent years as an index for diagnosing early infection of hepatitis C. HCV core is located at the N-terminus of the polyprotein precursor, consists of 191 amino acids, and has a molecular weight of about 21 kDa. Compared with other structural proteins and non-structural proteins, the core protein has conserved amino acid sequences among different HCV strains, has no glycosylation site after processing and maturation, has strong antigenicity, and is a good marker protein for HCV infection detection. In addition, the HCV core serum conversion time is 2-8 weeks before the HCV antibody, and is basically consistent with the HCV RNA serum conversion time, so that the HCV core serum conversion time is an important diagnostic target for the early infection of HCV. The existing clinical HCV antigen detection methods mainly adopt ELISA, electrochemiluminescence methods and the like, the detection methods are complex in operation, high in reagent cost and limited in sensitivity improvement degree, and popularization and development of HCV core detection are restricted, so that development of sensitive and specific HCV core rapid detection means becomes urgent.
The colloidal gold immunochromatography is an economical and rapid detection method with wide application fields. Because the operation is convenient and the special equipment and the site are not needed, the method is the method which is most suitable for popularization and use in basic medical institutions and home self-test at present. However, the current immune binding colloidal gold chromatography method has certain defects such as low sensitivity to samples, insufficient quality control capability, excessive variation coefficients among batches, in-batch and different reagents, and the like. Therefore, the sensitivity and the specificity of the anti-monoclonal antibody are improved, and the method is an important means for compensating the defects of the immune binding colloidal gold chromatography.
Disclosure of Invention
In order to solve the problems, the invention adopts a hybridoma monoclonal antibody preparation technology to obtain a hybridoma cell strain which specifically secretes the HCV core antigen monoclonal antibody, and a high-specificity and high-sensitivity anti-HCV core antibody pair is screened from the hybridoma cell strain. Based on the screened HCV core monoclonal antibody pair, a high-sensitivity HCV core antigen detection test strip is developed by adopting a mode of combining a colloidal gold labeling technology and an immune layer technology, so that the aim of early diagnosing HCV infection is fulfilled. The test paper is based on a double-antibody sandwich method, a specific HCV core monoclonal antibody is coated and fixed on an NC film to be used as a detection line T, a goat anti-mouse IgG is coated and fixed on the NC film to be used as a quality control line C, a colloidal gold labeled HCV core monoclonal antibody is used as a detection reagent to be adsorbed on a binding pad, after a sample to be tested is added on a sample pad at one end of a test strip, the sample to be tested moves forwards through capillary action, the colloidal gold labeled reagent on the binding pad is dissolved and reacts with each other to form an antigen-antibody-colloidal gold compound, the compound moves to an area where a capture antibody is fixed on the NC film, the compound is specifically combined with the fixed antibody to be trapped and gathered on a detection belt to form a macroscopic red band, and whether the HCV core is positive or not is judged. The whole detection process only needs 5-10 minutes, and the result interpretation is convenient and accurate. The HCV core antigen detection test paper can effectively detect the early infection of HCV, has simple operation, low cost and convenient and accurate result interpretation, is suitable for popularization and application in medical institutions at all levels, and is particularly suitable for primary screening detection of blood donors, rapid bedside detection and home self-detection.
Accordingly, it is an object of the present invention to provide a hybridoma DF2 that detects HCV core antigen; the second object of the present invention is to provide a monoclonal antibody DF2 secreted by the hybridoma DF2; the third purpose of the invention is to provide the application of the monoclonal antibody DF2 in preparing test paper for detecting HCV core antigen; the fourth object of the present invention is to provide a test strip containing the monoclonal antibody DF2.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. a hybridoma cell DF2 that detects HCV core antigen, said hybridoma cell DF2 deposited under the accession number GDMCC No:63836, the date of deposit is 2023, 09, 26, and the collection unit is the collection of microorganism strains from Guangdong province.
2. The monoclonal antibody DF2 secreted by the hybridoma DF2.
3. The monoclonal antibody DF2 is applied to the preparation of a detection reagent for detecting HCV core antigen.
In the invention, the detection principle of the detection reagent is preferably a colloidal gold method, an immunofluorescence chromatography method, an enzyme-linked immunosorbent assay method or a chemiluminescence method.
4. And a detection reagent containing the monoclonal antibody DF2.
In the invention, the detection principle of the detection reagent is preferably a colloidal gold method.
Preferably, the detection reagent comprises a colloidal gold test strip, wherein the colloidal gold test strip comprises a sample pad, a colloidal gold pad and a nitrocellulose membrane coated with a detection T line and a quality control C line, and absorption pads are mutually connected and adhered to a substrate plate; the detection T line is coated with an antibody DF2, the colloidal gold pad is combined with an antibody GE1, and the antibody GE1 is secreted by hybridoma cells GE1.
Preferably, the detection reagent comprises a colloidal gold test strip, wherein the colloidal gold test strip comprises a sample pad, a colloidal gold pad and a nitrocellulose membrane coated with a detection T line and a quality control C line, and absorption pads are mutually connected and adhered to a substrate plate; the detection T line is coated with an antibody DF2, the colloidal gold pad is marked with an antibody GE1, and the antibody GE1 is secreted by hybridoma cells GE1.
Preferably, the quality control C line is coated with l.5mg/mL goat anti-mouse IgG; the concentration of the monoclonal antibody coating on the T line is 1.0mg/mL; the colloidal gold-labeled antibody was bound to 50. Mu.g of antibody in 1mL of colloidal gold.
Preferably, the colloidal gold particles on the colloidal gold pad are prepared by a trisodium citrate reduction method.
Preferably, the quality control C line coating goat anti-mouse IgG is preferable in the invention, and the colloidal gold particles on the colloidal gold pad are prepared by adopting a trisodium citrate reduction method.
Preferably, the quality control C line is coated with goat anti-mouse IgG.
The invention has the beneficial effects that: the invention discloses a hybridoma DF2 for detecting HCV core antigen, and also discloses a monoclonal antibody secreted by the hybridoma DF2, wherein the secreted antibody can be used for preparing a polypeptide with a preservation number of GDMCC No: the GE1 antibody secreted by the 63837 hybridoma cell strain is paired, and has the advantages of good specificity and high sensitivity for detecting the HCV core antigen, so that the reagent for detecting the HCV core antigen can be prepared, and a reagent strip for detecting the HCV core antigen can be developed with the paired antibody, thereby providing a new tool for detecting the HCV core antigen.
The invention is subsidized by the natural and scientific fund project of Chongqing city, and the project name is as follows: high-sensitivity rapid detection research on early infection of hepatitis C based on quantum dot labeling technology, and project number: cstc2021jcyj-msxmX0198.
Preservation of biological materials
The BABL/C mouse hybridoma cell HCV core antigen monoclonal antibody hybridoma cell strain DF2 is preserved in the Guangdong province microorganism strain collection center, and is located at the Guangdong province academy of sciences of Guangzhou Kogyo No. 59 building No. 5, guangdong province, and the preservation number is GDMCC No:63836, with a date of preservation of 2023, month 09, 26, and a classification of BABL/C mouse hybridoma cells.
The BABL/C mouse hybridoma cell HCV core antigen monoclonal antibody hybridoma cell strain GE1 is deposited in the Guangdong province microorganism strain collection center, and is positioned at the Guangdong province academy of sciences of Guangzhou Kogyo No. 59 building No. 5, guangdong province, and the deposit number is GDMCC No:63837, with a date of preservation of 2023, month 09, 26, and a classification of BABL/C mouse hybridoma cells.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a graph showing the results of qualitative detection using the color intensity of the signal generated by colloidal gold in example 2.
FIG. 2 is a graph showing the results of specific detection experiments on 89 HCV negative sera using the color shade of the signal generated by colloidal gold in example 2.
Fig. 3 is a schematic diagram of an assembly structure of an immune colloidal gold test paper (1, sample pad; 2, substrate board; 3, colloidal gold pad; 4, T line; 5, C line; 6, cellulose film; 7, absorption pad).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
The invention provides a hybridoma cell pair, wherein the hybridoma cell pair comprises a first hybridoma cell strain and a second hybridoma cell strain which are independently stored; the first hybridoma cell line has a deposit number of GDMCC No:63836; the second hybridoma cell line has a deposit number of GDMCC No:63837.
the invention also provides a monoclonal antibody pair comprising a first monoclonal antibody and a second monoclonal antibody stored independently; the first monoclonal antibody is produced by a first hybridoma cell line; the second monoclonal antibody is produced by a second hybridoma cell line; the first hybridoma cell line has a deposit number of GDMCC No:63836; the second hybridoma cell line has a deposit number of GDMCC No:63837.
the invention also provides a monoclonal antibody as described above and the use of a monoclonal antibody pair as described above in the detection of HCV core antigen.
In still another aspect, the present invention further provides an immune colloidal gold test strip, which includes a water absorption pad, a base film, a gold-labeled pad and a sample pad sequentially connected; the base film is provided with a C line and a T line, the C line is coated with an anti-mouse IgG antibody, the T line is coated with a first monoclonal antibody, the gold-labeled pad contains a second monoclonal antibody marked by colloidal gold, and the first monoclonal antibody and the second monoclonal antibody form a monoclonal antibody pair. Wherein the monoclonal antibody pair is a monoclonal antibody pair as described above.
Wherein the first monoclonal antibody is produced by a first hybridoma cell line; the second monoclonal antibody is produced by a second hybridoma cell line; the first hybridoma cell line has a deposit number of GDMCC No:63836; the second hybridoma cell line has a deposit number of GDMCC No:63837.
the present invention will be described in further detail with reference to examples. In the examples below, the reagents used are all commercially available.
Example 1 preparation of HCV core antigen
The full-length HCV core expression primers for the prokaryotic expression vector pQE30 were designed based on the HCV-1b subtype gene sequence number provided by NCBI (see Table 1). The full-length gene sequence of the HCV core protein is cloned into a pQE30 expression vector by adopting a molecular cloning technology, and is subjected to fusion expression, and induction expression conditions such as bacterial density, induction temperature, induction time, IPGT concentration and the like are optimized. Purifying the expressed antigen by adopting an affinity chromatography method to obtain recombinant protein with optimal antigenicity, and comparing antigenicity by ELISA and Western Blot analysis. Primer synthesis and sequencing were all completed by Shanghai Biotechnology service Co., ltd, and the specific primer sequences used are shown in Table 1.
TABLE 1 primer sequences for HCV core protein amplification
| Name of the name | Primer sequences | Introduction of cleavage sites |
| PF1 | 5’-AGTGGATCCATGAGCACGAATCCTAAACC-3’ | BamH I |
| PR2 | 5’-CCGGGTACCCAGTCCGTTCTCTCTCGTC-3’ | Kpn I |
Extracting HCV RNA of HCV subtype 1b patient, using reverse transcription product as amplification template, using sequences shown as PF1 and PR2 as primers to make PCR reaction, and adopting the following PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 30sec,30 cycles; additional extension was performed at 72℃for 5min. Amplified products were collected, then digested with BamH I and Kpn I, and the desired fragment was recovered, then ligated by T4 DNA ligase into BamH I and Kpn I restriction endonuclease digested expression vector pQE30, and after overnight ligation at 4℃the ligation products were transformed into competent cells of E.coli (E.coli) JM109, and plated on LB plates with Amp (50 mg/mL) for selection of white colonies for recombinant transformation. Plasmid extraction and enzyme digestion identification are carried out and then are sent to Shanghai Biotechnology engineering service Limited company for sequencing identification. The correctly sequenced pQE30-core plasmid was transformed into competent cells of E.coli (E.coli) SG13009, screened on plates containing 50. Mu.g/mL Km antibiotic and plasmid restriction identified.
SG13009 with pQE30-core transferred thereto was inoculated into 200mL of liquid LB medium containing kanamycin (final concentration: 50. Mu.g/mL) and ampicillin (final concentration: 100. Mu.g/mL), and shake-cultured at 37℃for 2 hours at 200 rpm. When the OD600 reaches between 0.6 and 0.8, IPTG with a final concentration of 1.0mM is added, the temperature is 30 ℃, the rpm is 200, the shaking table is used for induction culture overnight, and after 4 ℃, bacteria are collected by centrifugation for 15min at 7,000Xg. To the collected cells, 2ml of NTA-0 buffer and a final concentration of 1mM PMSF were added, and this step was ice-bath. Adding TritonX-100 to obtain final concentration of 0.05%, mixing, and ice-bathing for 15min. Cells were sonicated in an ice bath, centrifuged at 13,000Xg for 30min at 4℃and the supernatant was taken. Taking a small amount of supernatant as a crude extract, performing SDS-PAGE electrophoresis detection, adding 50% (w/v) glycerol into the rest supernatant, mixing uniformly, and preserving at-20 ℃ for later use.
HCV core antigen was purified from the supernatant, and the N-terminus of the fusion protein contained His-Tag, so that affinity chromatography purification was performed using Ni-NTA-coupled NTA resin. Adding the crude extract into chromatographic column, controlling flow rate, repeating the sample application to increase purification efficiency, and collecting effluent for SDS-PAGE. The column was then eluted with NTA eluents containing different imidazole gradients (10 mM,50mM,100mM,150mM,200mM,500 mM) and the eluents were collected in fractions. The protein elution was maximum when the imidazole concentration in the eluate was 200 mM. The purity of the collected proteins was checked by polyacrylamide gel electrophoresis, and the purified proteins were dialyzed against a dialysis solution (20 mM Hepes, pH7.5, 1mM DTT, 200mM KCl and 50% glycerol) and split-packed and stored at-80 ℃.
EXAMPLE 2 preparation and identification of HCV core antigen monoclonal antibodies
1. Immunization of BALB/c mice
Will be 6-8W + The BALB/c female mice of (1) were immunized in three groups, i.e., 50. Mu.g/mouse, 100. Mu.g/mouse, 150. Mu.g/mouse, 3 times in total, with the core antigen (i.e., the HCV core purified antigen obtained in example 1) injection. Taking ocular blood as negative control before primary immunization, adding equal volume of Freund's complete adjuvant for full emulsification during primary immunization, and performing subcutaneous multipoint injection; immunization was performed once a week later, and subcutaneous multiple injections of the same dose of recombinant antigen plus an equal volume of incomplete Freund's adjuvant were performed. The antibody titer of the serum of the mice was determined by indirect ELISA about 10 days after the end of the last immunization; three days prior to fusion the highest antibody titers (1:10) 5 Above) mice were boosted by intravenous injection (25 μg) for three daysCell fusion.
2. Cell fusion
A. Feeder cell preparation: the day before cell fusion, feeder cells were prepared as follows:
(1) Will be 8W + Healthy male BALB/c mice are immersed in 75% ethanol for 2-3min after neck-pulling and killing;
(2) Move into the super clean bench, fix on the dissecting plate in supine position, peel off the abdominal skin, expose the peritoneum.
(3) Gently pulling up the peritoneum by using hemostats, injecting 10mL of preheated 1640 culture medium into the abdominal cavity by using a syringe, gently kneading the abdominal cavity by using a cotton ball for 1-2min, sucking out cell suspension, and placing into a centrifuge tube;
(4) And (3) centrifuging: 1000rpm,5min, discarding the supernatant;
(5) Mixing cells with 10mL of HAT medium containing serum, counting cells, and adjusting cell density to 2×10 5 /mL；
(6) The cell suspension was added to a 96-well cell culture plate at 100. Mu.L/well to give a cell density of 2X 10 4 Holes;
(7) Placing at 37deg.C, 5% CO 2 Incubator culture for the next day fusion experiment.
B. Preparation of myeloma cell SP2/0
(1) Myeloma cells SP2/0 were resuscitated 5-7 days prior to fusion.
(2) The SP2/0 cells in logarithmic growth phase are harvested before fusion, centrifuged at 1000rpm for 5min, washed 3 times with 1640 medium and the supernatant discarded;
(3) The cells were resuspended in 1640 medium, 100 μl of the cell suspension was stained with 0.2% trypan blue, cell counted, cell viability >95% was required, and cell density was adjusted for use.
C. Preparation of spleen cells
(1) BALB/c mice immunized with an impact 3 days ago were exsector bled and ocular blood was collected as a positive control. Killing the broken neck, and soaking in 75% ethanol for 2min;
(2) Moving into an ultra clean bench, cutting off the abdominal skin and peritoneum, taking out the spleen, flushing the spleen with 1640 culture medium, and removing the fat and connective tissue;
(3) The spleen was placed on a 200 mesh screen, gently ground with a syringe core, rinsed with culture medium, the spleen cell suspension was collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded;
(4) The cells were resuspended in 1640 medium, centrifuged at 1000rpm for 5min, washed 2 times and the supernatant discarded;
(5) Resuspension was performed with 10mL of incomplete medium. 100 μl of the cell suspension was stained with 0.2% trypan blue, cell viability >95% was required, and spleen cells were counted and the remaining cells were conditioned for use.
D. Cell fusion and culture
(1) Uniformly mixing spleen cells and SP2/0 myeloma cells in a 50mL centrifuge tube according to the ratio of 5:1, centrifuging at 1000rpm for 5min, discarding supernatant, flicking the bottom of the centrifuge tube with an index finger, and loosening cell sediment;
(2) While the centrifuge tube was gently shaken, 4000 1mL of 50% PEG pre-warmed at 50℃was added dropwise with a 1mL pipette, and completed in 1 min;
(3) Adding 1mL of 1640 culture medium preheated at 37 ℃ and finishing within 1 min;
(4) 10mL of 1640 culture medium preheated at 37 ℃ is added, and the completion is completed within 5min;
(5) Centrifuging at 800rpm for 8min; resuspend cell pellet with 100mL HAT medium;
(6) The cell suspension was transferred to feeder cells-inoculated cell culture plates at 100. Mu.L/well while leaving 2 wells with unfused SP2/0 cells as a control. All plates were placed at 37℃with 5% CO 2 Culturing in incubator;
(7) After 6 days of fusion, the HAT culture solution is replaced by adopting a half-amount solution replacement mode. Half liquid change is carried out every 3-5 days later; after 2 weeks, HT medium was half-changed and after 3-4 weeks, the complete medium was changed for maintenance.
E. ELISA screening of Positive hybridoma cells
(1) When the fused cells grow to 1/4 of the bottom area of the culture hole (about 14-18 days of culture), the supernatant is taken and used for detecting specific reaction and cross reaction by an indirect ELISA method, and hybridoma cells are screened. Recombinant protein HCV core is a coating antigen, and the coating concentration is 4 mug/mL of conventional coating ELISA plate.100. Mu.L/well of cell culture supernatant was added to the coated ELISA plate, and the immunized mouse serum diluted with PBS1:50 was used as a positive control, and the SP2/0 cell well culture supernatant was used as a negative control. Cell supernatants were incubated with coated ELISA plates for 30min at 37℃and after extensive washing, HRP-labeled goat anti-mouse IgG antibody (1:10000 dilution) was added to 100. Mu.L/well, incubated for 30min at 37℃and secondary antibody was discarded. After washing the plate fully, adding 100 mu L TMB into each hole for color development for 15min, and adding 1N H 2 SO 4 Stop reaction at 50. Mu.L/well, determine OD 450 Values.
(3) ELISA screening to obtain 33 cell lines secreting HCV core monoclonal antibodies; the monoclonal antibodies secreted by the 33 cell lines of HCV core monoclonal antibodies all had positive responses to the recombinant HCV core antigen. Selecting 10 cells with strongest positive reaction for subcloning culture, directly enlarging culture of other cell strains, freezing and storing, and producing small amount of ascites.
F. Cloning culture by limiting dilution method
(1) The day prior to cloning feeder cells were prepared as described above; inoculating 100 mu L/well of HT culture solution into 96-well culture plate;
(2) Blowing and mixing cells to be cloned uniformly by using a pipette, and diluting the cells to be cloned to the density of 1 cell/hole by using HT selective culture solution containing 20% serum; cell plates added to existing feeder cells and placed in 5% CO 2 Culturing in an incubator at 37 ℃;
(3) On day 3 of culture, cell monoclonal growth wells were observed and recorded under an inverted microscope; culturing for about 1 week, sucking 100. Mu.L of the supernatant of the cell culture medium which has turned yellow, and detecting by the ELISA method;
(4) Subcloning is generally carried out for 2-3 times until the ELISA detection result of the culture hole supernatant of the last time of growth of only one cell colony is positive;
(5) Transferring the positive Kong Zajiao tumor cells into a 24-hole culture plate for expansion culture, detecting hybridoma culture supernatant by ELISA when the cell number is enough, detecting the cell strain still positive for further expansion culture, and freezing.
3. Preparation of monoclonal antibodies
A. Preparation of ascites
(1) Intraperitoneal injection of liquid paraffin, 0.5 mL/mouse, into 10-week-old BALB/c mice;
(2) After one week, the abdominal cavity was inoculated with positive hybridoma cells cultured to log phase diluted with PBS, 5×10 per mouse 5 /mL hybridoma cells;
(3) After 5 days, when the abdomen of the mice is obviously swelled, collecting ascites by a 12-gauge injection needle, and collecting every 3 days until the mice die;
(4) Centrifuging the ascites at 7500rpm for 10min; taking supernatant, subpackaging, and storing in a refrigerator at-80deg.C.
B. Purification of monoclonal antibodies (protein A affinity chromatography)
(1) Balance: adding a 10 column volume of Equilibration Buffer equilibrated protein A column;
(2) Loading: loading the ascites to make the ascites slowly flow through the gel bed; the collected effluent may be, if necessary, passed to the column again;
(3) Leaching: adding Equilibration Buffer with 10 times of column volume, collecting penetrating liquid according to 4-5 mL/tube until OD 280 <0.1；
(4) Eluting: eluting with 5 column volumes of Elutation Buffer (50mM glycine,0.5M NaCl,pH 2.3), adding neutralization Buffer (20 mM phosphate Buffer, pH 7.7) into the tube at 500 μl/tube, and collecting eluate at 1.5 mL/tube until OD 280 <0.1；
(5) The column was washed and equilibrated with 5 column volumes of Equilibration Buffer.
C. Monoclonal antibody pairing screening
The obtained 33 purified monoclonal antibodies are combined pairwise to obtain (33 multiplied by 33) pair combinations, two antibodies in each pair of combinations are respectively coated with nitrocellulose membranes and labeled colloidal gold, colloidal gold test strips are prepared, HCV core recombinant antigen detection is subjected to sensitivity screening, HCV NS3 and HCV NS4 are subjected to specificity screening, and finally pairing monoclonal antibodies only aiming at HCV core high specificity and high sensitivity are screened.
D. Gold label and membrane preparation of monoclonal antibody
Preparing colloidal gold particles: colloidal gold particles are prepared by adopting a trisodium citrate reduction method. The specific method comprises the following steps: chloroauric acid was prepared as a 0.01% aqueous solution with ultrapure water, 100mL was mixed with 1.2mL of a 1% aqueous solution of trisodium citrate, and heated to continuous boiling for 5min. After cooling, the solution was recovered to the original volume with ultrapure water to prepare colloidal gold particles having a particle diameter of about 50 nm.
Determining the optimal stabilizing amount of the colloidal gold-labeled antibody: the amount of antibody required to label 1mL of colloidal gold was 50. Mu.g as determined by classical MEY method.
Coating of nitrocellulose membrane (NC): c line: l.5mg/mL goat anti-mouse IgG (0.0 l dilution in PBS buffer pH 7.2); t line: 1.0mg/mL purified mab (0.0 l diluted in PBS buffer pH 7.2). Spraying the mixture on a nitrocellulose membrane at a speed of 30mm/s by using a working system of XYZ3050 of BIODOT company to form a detection T line and a quality control C line which are parallel to each other, and drying at 37 ℃.
E. Assembly of immune colloidal gold test paper
The nitrocellulose membrane 6, the absorption pad 7, the colloidal gold pad 3 and the sample pad 1 coated with the C line 5 and the T line 4 are sequentially adhered to the PVC coated substrate board 2 which is not water-absorbing, and an immune colloidal gold test paper is assembled as shown in figure 3.
F. Paired screening of monoclonal antibodies
33 monoclonal antibodies, a total of 33×33 (1089) matched monoclonal antibodies are obtained, and 1089 colloidal gold test strips are formed. When in paired screening, serum of healthy people is taken as a negative sample, 1 mug/mL of recombinant HCV core antigen is taken as a positive sample, and each test strip is tested. 10 strains of the monoclonal antibody, 14 pairs of which react positively against recombinant HCV core antigen and react negatively against serum of healthy people, were selected as candidate monoclonal antibodies. For the candidate 14 pairs of pairing monoclonal antibodies, recombinant HCV core antigen, serum of an HCV early infection patient, HCV negative serum and HBV, HIV, TP positive serum are further screened and detected. And selecting the paired monoclonal antibody with the detection sensitivity of 1ng/mL to the recombinant HCV core antigen, the strongest serum response to the patient with the early HCV infection and no cross reaction to other samples as the paired monoclonal antibody for preparing the double-antibody sandwich method detection kit. And finally obtaining the monoclonal antibody DF2 coated on the T line through experiments, wherein the second monoclonal antibody GE1 is combined with colloidal gold.
G. Pairing antibody optimization
(1) Concentration of coated antibody DF2
Diluting the screened coated antibody DF2 to 1.0mg/mL;1.5mg/mL;2.0mg/mL,2.5mg/mL, coated on NC membrane, and dried for standby.
(2) Labeling conditions for labeling antibody GE1 and concentration of colloidal gold antibody-conjugated solution
The selected labeled antibody GE1 was further refined in labeling conditions. And (3) after the best labeling conditions are selected, preparing a colloidal gold antibody coupling solution again, and regulating the solution to different concentrations to coat the colloidal gold pads.
(3) The prepared colloidal gold pad is matched with films with different concentrations in a pairwise crossing way, and the optimal concentration ratio of the coating to the colloidal gold pad is found. The experimental results are shown in table 2:
table 2, GE1 different labelling conditions and DF2 different coating conditions paired screening
Pairing results showed that coating concentrations above 2.0mg/mL would have false positive appearance. Preferably, the marking condition K is 3, and the A values 10 and 15 are the same, and the T line is stronger when the A value is 15, so K3A15 is selected. The difference in detection sensitivity and specificity of different colloidal gold pad sizes under the same labeling conditions were further compared at a coating concentration of 1mg/mL and 1.5 mg/mL. The pairing results of the coating concentration and the different sizes of the colloidal gold pad are shown in table 3.
Table 3, DF2 different coating conditions and GE1 different gold pad width pairing screening
When the size of the colloidal gold pad exceeds 12mm, the sensitivity is enhanced, but the reaction time is prolonged, and the colloidal gold pad has false positive results, so that the colloidal gold pad is not selected. The lowest detection sensitivity of the paired antibodies is 100pg/mL, so that the paired antibodies with the lowest detection sensitivity of 100pg/mL can be achieved by selecting the paired antibodies with the lowest coating concentration and proper colloidal gold concentration, the paired antibodies with the lowest detection sensitivity of 100pg/mL are prepared by adopting DF2 coating concentration of 1mg/mL and GE1 colloidal gold pad 50d size of about 10mm, and the final dosage is determined according to the detection sensitivity of 100 pg/mL.
H. Identification of monoclonal antibody subclasses
The monoclonal antibodies of each strain were subjected to subclass identification by using an immunoglobulin standard subclass identification kit (Sigma Co.), and the specific test method is as follows:
(1) Sheep anti-mouse IgG (IgM, igA, igG) diluted with 1:1000 fold PBS respectively 1 、IgG 2a 、IgG 2b And IgG 3 ) The ELISA plate was coated, 100. Mu.L/well, and left at 37℃for 1h.
(2) The liquid in the ELISA plate was discarded and washed 3 times with PBST.
(3) Adding PBS diluted purified monoclonal antibody (antibody concentration 2-5 mug/ml) into 100 mug/hole, and incubating for 1h at room temperature; PBST was washed 3 times.
(4) HRP-labeled goat anti-mouse IgG antibody (1:10000 dilution) was added to 100. Mu.L/well and incubated for 30min at room temperature.
(5) After washing the plate sufficiently, 100. Mu.L TMB was added to each well and developed for 15min, followed by 1N H 2 SO 4 Stop reaction at 50. Mu.L/well, determine OD 450 Values.
The results of the identification of the monoclonal antibody subclasses are shown in Table 4.
TABLE 4 subclasses of monoclonal antibodies
| Antibodies to | DF2 | GE1 |
| Antibody subclasses | IgG2a | IgG1 |
I. Paired antibody sensitivity evaluation:
paired monoclonal antibodies screened in this experiment: the detection sensitivity of the first monoclonal antibody DF2 and the second monoclonal antibody GE1 on the recombinant HCV core antigen reaches 100pg/mL; 25-fold dilutions were detected for serum detection of HCV early infection (fig. 1 and table 5).
TABLE 5 sensitivity evaluation results
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J. Paired antibody specificity evaluation
Paired monoclonal antibodies screened in this experiment: a specific detection experiment was performed on 80 HCV-negative serum samples with a first monoclonal antibody DF2 and a second monoclonal antibody GE1. The detection results are shown in fig. 2 and table 6.
TABLE 6 specificity evaluation results
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The paired monoclonal antibody DF2-GE1 detects that 80 serum samples which are not positive to HCV are negative results, and have no cross reaction with HBV, HIV, TP positive samples.
The hybridoma cell strain producing DF2 monoclonal antibody is preserved, and the preservation number is GDMCC No:63836 the preservation date is 2023, 09 and 26 days, the preservation unit is Guangdong province microorganism strain preservation center, the address is 59 th floor 5 th Guangdong province academy of sciences of building 5 in Guangzhou, and the classification is named as anti-HCV core monoclonal antibody hybridoma cell strain DF2; the hybridoma cell strain producing the GE1 monoclonal antibody is preserved, and the preservation number is GDMCC No:63837 the preservation date is 2023, 09 and 26 days, the preservation unit is Guangdong province microorganism strain preservation center, the address is 59 th floor 5 th Guangdong province academy of sciences of building 5 of Guangzhou, and the classification is named as anti-HCV core monoclonal antibody hybridoma cell strain GE1.
According to the invention, a pair of monoclonal antibodies are screened, the detection sensitivity of the immune colloidal gold test paper to the recombinant HCV core antigen can reach 100pg/mL, the immune colloidal gold test paper can be detected by diluting an HCV core positive serum sample by 25 times, and the immune colloidal gold test paper does not show cross reaction to 80 HCV negative serum samples and has higher sensitivity and specificity.
The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. The hybridoma DF2 for detecting the HCV core antigen core is characterized in that: the hybridoma cell DF2 accession number is GDMCC No:63836, the date of deposit is 2023, 09, 26, and the collection unit is the collection of microorganism strains from Guangdong province.
2. The monoclonal antibody DF2 secreted by the hybridoma cell DF2 of claim 1.
3. Use of the monoclonal antibody DF2 of claim 2 for preparing a detection reagent for detecting HCV core antigen.
4. A use according to claim 3, characterized in that: the detection principle of the detection reagent is a colloidal gold method, an immunofluorescence chromatography method, an enzyme-linked immunosorbent assay method or a chemiluminescence method.
5. A detection reagent comprising the monoclonal antibody DF2 of claim 2.
6. The detection reagent according to claim 5, wherein: the detection principle of the detection reagent is a colloidal gold method.
7. The detection reagent according to claim 6, wherein: the detection reagent comprises a colloidal gold test strip, wherein the colloidal gold test strip comprises a sample pad, a colloidal gold pad and a nitrocellulose membrane coated with a detection T line and a quality control C line, and absorption pads are mutually connected and adhered to a substrate plate; the detection T line is coated with an antibody DF2, the colloidal gold pad is marked with an antibody GE1, and the antibody GE1 is secreted by hybridoma cells GE1.
8. The detection reagent according to claim 7, wherein: the quality control C line is coated with 1.5mg/mL goat anti-mouse IgG; the concentration of the monoclonal antibody coating on the T line is 1.0mg/mL; the colloidal gold-labeled antibody was bound to 50. Mu.g of antibody in 1mL of colloidal gold.
9. The detection reagent according to claim 7, wherein: the colloidal gold particles on the colloidal gold pad are prepared by adopting a trisodium citrate reduction method.
10. The detection reagent according to claim 9, wherein: and the quality control C line is coated with goat anti-mouse IgG.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311553283.8A CN117821395A (en) | 2023-11-21 | 2023-11-21 | Hybridoma DF2 of HCV core antigen core, monoclonal antibody and application |
Applications Claiming Priority (1)
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