Listeria monocytogenes monoclonal antibody hybridoma cell strain and application thereof
A technology for Listeria monocytogenes and hybridoma cell lines, which is applied in the directions of antibacterial immunoglobulins, biochemical equipment and methods, instruments, etc., can solve the problems of high detection cost, dependence, and high requirements for operators
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Embodiment 1
[0018] Example 1 Preparation of Monoclonal Cell Line A
[0019] 1. Synthesis of complete antigen: Dissolve 1mg of SMCC in DMF and add it to 5mg of BSA solution dissolved in coupling buffer (0.1M PB buffer, containing 0.15M NaCl). Millipore, cutoff 3000) was washed and centrifuged 3 times, and the activated BSA was resuspended with an appropriate amount of coupling buffer. Weigh 3.75 mg of peptide D, dissolve it with an appropriate amount of coupling buffer, and slowly add it to the activated BSA solution. After stirring at room temperature for 2 hours, dialyze at 4°C for three days, and store in separate packages at -20°C.
[0020] 2. Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. The complete antigen of peptide D (1 mg / mL) was mixed with an equal volume of Freund's adjuvant (Freundadjuvant, Sigma), and BALB / c mice were immunized by subcutaneous injection. Freund's complete adjuvant was used for the first immunization, and Freund's i...
Embodiment 2
[0027] Example 2 Preparation and Identification of Monoclonal Antibody
[0028] Get 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 For monoclonal cell line A, the ascites was collected from the seventh day, and the ascites was purified by octanoic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.
[0029] The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody purified from ascitic fluid, and its subtype was IgG1.
[0030] The subtype identification of the monoclonal antibody of monoclonal cell line A is shown in Table 1.
[0031] Table 1 Subtype identification of the monoclonal antibody of the monoclonal cell line A
[0032] IgG1 1.856 IgG2a 0.169 IgG2b 0.168 IgG3 0.049 IgM 0.055 IgA 0.056
Embodiment 3
[0033] Example 3 Application of Monoclonal Antibody
[0034] The monoclonal antibody prepared by monoclonal cell line A through in vivo ascites was applied to the detection of Listeria monocytogenes, and the specific steps were as follows:
[0035] (1) 0.5 μg / mL PepD-SPDP-OVA diluted with 0.01M carbonate buffer (CBS) was used as the original coating material to coat a 96-well ELISA plate, 100 μL per well, and after coating at 37°C for 2 h, Wash the plate three times with PBST washing solution, 250 μL per well each time, 3 min each time, and pat dry;
[0036] (2) Block with CBS containing 0.01% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry;
[0037](3) Prepare 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9 μg / L standard solutions of Listeria monocytogenes P60 protein (recombinant protein, purchased from AdipoGen) in phosphate buffered saline (PBS). Add the standard solution and ...
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