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hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum, and monoclonal antibody and application thereof

A technology of Salmonella pullorum and hybridoma cell lines, which is applied in the direction of analysis materials, immunoglobulins, anti-bacterial immunoglobulins, etc., can solve the problems that there is no strong specificity and low-cost detection of Salmonella pullorum, and achieve The effects of shortened detection time, high titer, and easy operation

Active Publication Date: 2019-03-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no simple, rapid, specific and low-cost effective method for the detection of Salmonella pullorum

Method used

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  • hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum, and monoclonal antibody and application thereof
  • hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum, and monoclonal antibody and application thereof
  • hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum, and monoclonal antibody and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the acquisition of hybridoma cell line SP-4G6

[0045] The deposit number is CCTCC NO: C2018145.

[0046] 1. Animal immunization

[0047] The specific immunization procedure is as follows: for the first immunization, the purified protein from tapping was injected intraperitoneally, and an appropriate amount of rubber foam was used as an adjuvant to mix with 100 μg of recombinant rHis-SP-IpaJ protein to immunize the mice. Foam was used as an adjuvant and mixed with 100 μg protein for the second immunization. After an interval of 2 weeks, 100 μg purified protein without adjuvant was injected intraperitoneally for the third immunization. Orbital blood was collected 7 days after the second immunization to determine the serum antibody titer, and the titer was selected. Taller mice undergo cell fusion.

[0048] 2. Cell Fusion

[0049] The specific steps are as follows: 3 days after the tail vein booster immunization, a small amount of blood was collected, and...

Embodiment 2

[0058] Embodiment 2: the preparation of Salmonella pullorum IpaJ monoclonal antibody

[0059] 1. Ascites Preparation

[0060] The method of inducing ascites in vivo was carried out according to the conventional method. Healthy BALB / c mice aged 10-12 weeks were intraperitoneally injected with liquid paraffin 0.3-0.5mL / mouse, and after 7-10 days, hybridoma cells SP-1C3 and SP- 1F3, SP-2D9, SP-3B5, SP-3D8, SP-4D5, SP-4G6 and SP-5D1, 5×10 5 After 10 days, the ascites was collected, the precipitate was removed by centrifugation, and the supernatant was collected, and the antibody titer was determined by indirect ELISA, aliquoted, and stored at -70°C. Hybridoma cell lines SP-1C3, SP-1F3, SP-2D9, SP-3B5, SP-3D8, SP-4D5, SP-4G6 and SP-5D1.

[0061] 2. Labeling for Antibody Purification

[0062] The prepared SP-3D8, SP-4G6 and SP-5D1 ascites were purified using Protein G affinity chromatography.

[0063] The purified SP-3D8, SP-4G6 and SP-5D1 mAbs were labeled with horseradish per...

Embodiment 3

[0064] Embodiment 3: monoclonal antibody characteristic detection

[0065] 3. Identification of mAb subclasses

[0066] According to the instructions of the monoclonal antibody subclass kit of Sigma Company, the antigen-mediated brief ELISA method was used. Add 100 μL / well of the cell culture supernatant to the coated ELISA plate, wash at 37°C for 1 hour, and wash 3 times with PBST for 5 minutes each time; add 1:1000 diluted goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, 50 μL / well of IgM subclass antibody, 0.5 h at 37°C, add two wells of each subclass to each monoclonal antibody, wash 3 times with PBST for 5 min each time; add 50 μL / well of rabbit anti-sheep enzyme-labeled secondary antibody diluted 1:5000, Wash at 37°C for 15 minutes with PBST for 3 times; add 50 μL / well of chromogenic solution tetramethylbenzidine (TMB) solution, develop color at 37°C for 10 minutes in the dark, 2M H 2 SO 4 50 μL / well was used to terminate the reaction, and the subtype antibody added to thos...

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Abstract

The invention provides a hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum, and a monoclonal antibody and application thereof. The hybridoma cell strain capable of secreting IpaJ monoclonal antibody of salmonella pullorum is preserved in CCTCC. The monoclonal antibody secreted by the hybridoma cell strain has the advantages of high valence, good specificityand high in affinity to natural antigens. A competitive ELISA detection method for the IpaJ monoclonal antibody of salmonella pullorum has relatively good sensitivity and specificity and is used forevaluating the immune state of a chicken body and researching related infectious diseases.

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting Salmonella pullorum IpaJ monoclonal antibody, the monoclonal antibody secreted by it and the application in the field of immune detection. Background technique [0002] Salmonella pullorum is an important host-specific Salmonella, which can cause pullorum in chickens and cause serious economic losses to the poultry industry. The characteristic of the pathogen is that the mortality rate is the highest in chicks, and although the infected adult chickens do not show obvious clinical symptoms, they may show weight loss, decreased egg production, dysentery, reproductive system damage and deformity, etc., and the bacteria Long-term persistence in recovered hosts. So far, there is no simple, rapid, specific and low-cost effective method for detecting Salmonella pullorum. [0003] Enzyme-linked Immunosorbent Assay (ELISA) technology has high sensitivity and wide application, and has become one of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569
CPCG01N33/56916G01N33/577C07K16/1235
Inventor 焦新安李求春尹克全朱悦徐黎娟王鑫李扬
Owner YANGZHOU UNIV
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