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Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody

A monoclonal antibody, Vibrio hemolyticus technology, applied in the field of bacterial monitoring and immunological analysis, can solve the problems of complex procedures, low detection rate, difficulty in providing relevant reagents, etc. technologically novel effects

Inactive Publication Date: 2012-09-19
SHANGHAI OCEAN UNIV +1
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Conventional isolation and culture operations are cumbersome, the cycle is long, and the detection rate is low; although the PCR method has the characteristics of specificity and sensitivity, its procedures are complicated and require sophisticated instruments and equipment, which is not conducive to popularization and application; in terms of immunology applications, it is used in The method of Vibrio parahaemolyticus mainly detects Vibrio parahaemolyticus with polyclonal antibody detection and dot ELISA (Dou Yong, 2006; Zang Hongmei, 2006), all of which use Vibrio parahaemolyticus cells as antigens to immunize animals to obtain antiserum, To obtain the detection effect of Vibrio parahaemolyticus
This method also has its limitations, specifically reflected in the fact that polyclonal antibodies are affected by various factors such as different strains, immunization doses, experimental animals, immunization time, immune serum storage time, etc., it is difficult to provide standardized related reagents, while monoclonal antibodies The antibody has strong specificity, good homogeneity, high effect, stability and cheapness, and has obvious advantages over immunization methods such as polyclonal antibody technology

Method used

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  • Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody
  • Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody
  • Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the establishment of mouse-derived hybridoma cell line

[0048] (1) Preparation of immune antigen TDH

[0049] Using multiple chromatography methods, glucose, Na 2 HPO 3 And tryptone and NaCl are the TDH in the bacterial culture fluid of main component to extract and purify, as immune antigen of the present invention;

[0050] (2) Mice immunization and serum titer determination

[0051] Select 6-8 week-old female mice (BalB / c mice), immunize with the antigen obtained in step (1), and use the indirect ELISA method to measure the antibody titer in the mouse serum;

[0052] (3) Cell fusion and culture

[0053] Culture mouse myeloma cells SP2 / 0, mix with splenocytes of immunized mice, carry out cell fusion under the mediation of polyethylene glycol (PEG), carry out in 15% FCS selection medium containing HAT at 37°C, 5%CO 2 to cultivate;

[0054] (4) Screening and cloning of hybridoma cells

[0055] Using indirect ELISA method to screen the positive hy...

Embodiment 2

[0058] Embodiment 2, preparation of anti-Vibrio parahaemolyticus thermostable hemolytic toxin (TDH) monoclonal antibody

[0059] (1) Preparation of immune antigen TDH

[0060] ① Production of TDH crude protein

[0061] The pathogenic Vibrio parahaemolyticus standard strain ATCC33846 (preserved by the American Type Culture Collection) was inoculated in the culture medium, and each liter of the culture medium contained 5g glucose, 5gNa 2 HPO 3 , 10g tryptone and 30gNaCl, pH6.5-6.8; at 37°C, 180 rpm, shaker culture for 15h, after centrifugation to remove bacteria, add 351g ammonium sulfate per liter of supernatant, salt out, centrifuge, put The precipitate was dissolved in 0.01mol / L phosphate buffer solution with pH 7.0, dialyzed overnight, and finally the crude protein of TDH was obtained;

[0062] ②Purification of crude protein

[0063] A DEAE-cellulose chromatography

[0064] The previously obtained crude protein was purified by DEAE-cellulose chromatography (2.6×60).

...

Embodiment 3

[0094] Example 3, Identification of Monoclonal Antibody Characteristics

[0095] ①Determination of antibody affinity

[0096]ELISA sandwich method is used to determine the concentration of mAb in the hybridoma cell culture supernatant: get 1: 3000 diluted rabbit anti-mouse IgG antibody (Sigma Chemical Co. the same below) of appropriate concentration to coat the microtiter plate, 0.1mL / well, overnight at 4°C. After washing, block with blocking solution for 2h at 37°C. Add the supernatant of hybridoma cells to be tested and pure mouse IgG (SigmaChemical Co., the same below) diluted serially, 0.1 mL / well, and incubate at 37°C for 1 h. After washing, add 1:3000 dilution of horseradish peroxidase HRP-labeled rabbit anti-mouse IgG antibody (Sigma Chemical Co., the same below), 0.1 mL / well, and incubate at 37°C for 1 h. After washing, add substrate solution ( OPD, Sigma Chemical Co.), 0.1 mL / well, and develop color at room temperature for 20 min in the dark. After terminating the...

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Abstract

The invention provides a murine hybridoma cell line, a monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and secreted by the hybridoma cell line and a preparation method of the monoclonal antibody. The monoclonal antibody resistant to the thermostable direct hemolysin of the vibrio parahaemolyticus for mice is stably secreted by one hybridoma cell line T6D4 obtained by means of B lymphocyte hybridoma technique after TDH (thermostable direct hemolysin) of the vibrio parahaemolyticus is extracted and purified and the BalB / c mice are immunized, and is used for rapid detection of pathogenic vibrio parahaemolyticus. The antibody is IgG2a secreted by one stable murine hybridoma cell line and is capable of generating idiosyncratic reaction to TDH.

Description

technical field [0001] The invention belongs to the technical field of bacterial monitoring and immunological analysis, and specifically relates to a mouse-derived hybridoma cell line, an anti-vibrio parahaemolyticus heat-resistant hemolytic toxin monoclonal antibody secreted by the cell line, and a preparation method thereof. Background technique [0002] Thermostable direct hemolysin (TDH) is a hemolytic toxin produced by Vibro parahaemolyticus (VP). It is a transit dimeric protein secreted by VP, without sugar or lipid. The molecular mass of TDH is about 46,000. TDH is a pore-forming toxin, which has toxic effects on various cultured cells and cells, and has potential cardiotoxic effects. The most studied is its hemolytic effect. TDH can hemolyze red blood cells (RBC) of humans, mice, and rabbits, but does not hemolyze RBCs of horses and sheep. [0003] Vibrio parahaemolyticus is a halophilic bacterium belonging to the genus Vibrio. It exists in seawater, fish and shel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12C12R1/91
Inventor 杨靖亚徐莉张建赵勇陆晓帆晁若瑜
Owner SHANGHAI OCEAN UNIV
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