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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

Active Publication Date: 2014-12-03
无锡迪腾敏生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, molecular biology methods rely on detection instruments and have high requirements for operators, so the detection cost is relatively high

Method used

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  • Listeria monocytogenes monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a Listeria monocytogenes monoclonal antibody hybridoma cell strain and an application thereof and belongs to the technical field of food safety immunological detection. According to the invention, a complete antigen of a specific polypeptide of Listeria monocytogenes is uniformly mixed with an equal volume of Freund adjuvant (Sigma), BALB / c mice are immunized by subcutaneous injection, after the BALB / c mice are immunized for four times, spleen cells of immunized mice are fused with mice myeloma cells of immunized mice by virtue of a PEG method, an indirect ELISA screening and three times of subcloning are carried out to obtain a monoclonal cell strain A. The monoclonal antibody secreted by virtue of the monoclonal cell strain A has cross reaction with different Listeria monocytogenes culture supernatants and no cross reaction with listeria except Listeria monocytogenes, E. coliO 157, Salmonella and Campylobacter jejuni and thus the Listeria monocytogenes monoclonal antibody hybridoma cell strain can be used in the specific detection of Listeria monocytogenes in a food.

Description

technical field [0001] The present invention relates to a Listeria monocytogenes monoclonal antibody hybridoma cell line and its application, in particular to the monoclonal cell line A and the application of the Listeria monocytogenes monoclonal antibody it produces, which belongs to food safety immunology detection technology field. Background technique [0002] Listeria monocytogenes (L.monocytohenes) is a Gram-positive bacterium that widely exists in the environment and is an important zoonotic pathogen in the world. Listeria monocytogenes is a type of Listeria. Other Listeria include L.iuanuii, L.innocua, L.welshimeri ), L.seeligeri, L.grayi, L.murrayi, but L.monocytogenes is the only Listeria that causes human disease bacteria. [0003] The susceptible population of Listeria monocytogenes is mainly children, the elderly, pregnant women, and patients with chronic diseases with poor immunity. After infection, the main manifestations are sepsis, meningitis, and mononuc...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569C12R1/91
Inventor 马伟胥传来王文彬匡华徐丽广刘丽强宋珊珊吴晓玲
Owner 无锡迪腾敏生物科技有限公司
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