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Staphylococcus aureus enterotoxin B resistant antibody, test paper and kit

A staphylococcus enteric and golden yellow technology, applied in the field of immunity, can solve the problems of inconvenient on-site operation, long time for processing and detection, etc.

Active Publication Date: 2020-12-04
北京弘进久安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing immunological detection methods mainly include immunoagglutination experiment, agarose diffusion method and enzyme-linked immunosorbent adsorption method, etc., but these sample pretreatment and detection take a long time and are not convenient for on-site operation

Method used

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  • Staphylococcus aureus enterotoxin B resistant antibody, test paper and kit
  • Staphylococcus aureus enterotoxin B resistant antibody, test paper and kit
  • Staphylococcus aureus enterotoxin B resistant antibody, test paper and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Expression and purification of SEB

[0034] The genomic DNA from the Staphylococcus aureus strain (ATCC accession number is BAA-1556) was amplified by PCR for the SEB gene, connected to the pGEX-6p-2 vector, transformed into TOP10 competent cells, and positive clones were picked and subjected to After DNA sequencing verified that the sequence was correct, it was transformed into BL21 competent cells. Positive clones were picked and induced to express GST-SEB protein overnight in Escherichia coli. Afterwards, the bacteria were collected, the GST-SEB protein was enriched with glutathione agarose 4B medium, digested with PreScission enzyme at 4°C overnight, the GST tag was removed, the SEB protein was collected, and stored at low temperature for later use. The amino acid sequence of SEB protein is shown in Table 1.

[0035] Table 3: Amino acid sequence of prokaryotic expressed SEB protein

[0036]

Embodiment 2

[0037] Embodiment 2: Preparation of SEB monoclonal antibody

[0038] The purified SEB antigen in Example 1 was used to immunize six Balb / c mice aged 8 to 12 weeks. After three times of immunization, blood was collected from the eyes of the mice to obtain mouse serum, and ELISA was used to detect SEB antibody drops in the mouse serum. Degree, antibody titer>10000.

[0039]The mouse splenocytes with antibody titer >10000 were fused with myeloma SP2 / 0 cells, and the fused cells were screened by HAT selection medium, and the fused cells were positively screened and subcloned by ELISA. Ascites was prepared from the positive monoclonal screened out, and the antibody was purified with Protein A / G antibody purification column. The ELISA titer of the purified antibody was >1:128000, and the purity was >90%.

[0040] The cell line numbers corresponding to the two monoclonal antibodies are 3F2G10 and 5A11G6, respectively. Example 3: Amplification and sequence determination of SEB monoc...

Embodiment 3

[0040] The cell line numbers corresponding to the two monoclonal antibodies are 3F2G10 and 5A11G6, respectively. Example 3: Amplification and sequence determination of SEB monoclonal antibody CDR region sequence

[0041] The hybridoma cell lines 3F2G10 and 5A11G6 were revived and cultured. After the cells grew to the logarithmic growth phase, the cell count was about 8×10 7 cells / ml, the cells were collected. According to the TaKaRa MiniBEST Universal RNA Extraction Kit instructions, extract the total RNA of hybridoma cells. According to the reaction system shown in Table 4, keep it at 65°C for 5 minutes, cool it rapidly on ice, and then place it at 42°C for 60 minutes according to the reaction system shown in Table 5. ; 15min at 70°C; 1min at 25°C for 1st-Strand cDNA synthesis reaction.

[0042] Table 4: 1st-Strand cDNA synthesis reaction mix

[0043] Reagent Usage amount Oligo dT Primer (50μM) 1μL dNTP Mixture (10mM each) 1μL template RNA <...

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Abstract

The invention discloses an antibody or an active fragment thereof specifically combined with staphylococcus aureus enterotoxin B, colloidal gold immunochromatographic detection test paper or a kit containing the antibody, and a preparation method and application thereof. The detection test paper or kit is convenient to produce, stable in effect, free of non-specific reaction, low in coating amountand beneficial to mass production. The detection method of the staphylococcus aureus enterotoxin B provided by the invention has the advantages of rapidness, sensitivity, simplicity, convenience, specificity and the like, and has wide application in food safety detection and monitoring.

Description

technical field [0001] The invention relates to the field of immunization, in particular to an anti-staphylococcus aureus enterotoxin B antibody, a test paper and a kit for detecting the staphylococcus aureus enterotoxin B. Background technique [0002] Staphylococcal enterotoxin (SE) is a kind of extracellular protein with related structure, similar virulence and different antigenicity. It is an important pathogen that causes food poisoning and nosocomial infection. Acute poisoning enterotoxins, more than 33 kinds of Staphylococcus aureus enterotoxins have been found so far, including SEA~SEE, SEI and enterotoxin-like serotypes G (enterotoxin-like serotypes G, selG), selH and selJ~selU, etc., among which SEA ~SEE is considered to be the main toxin causing poisoning, especially SEB is considered to be one of the most potent toxins due to its high toxicity and strong stability. [0003] Staphylococcal enterotoxin B (staphylococcal enterotoxin B, SEB) mainly exists in meat, m...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13G01N33/569G01N33/558G01N33/543
CPCC07K16/1271G01N33/56938G01N33/558G01N33/54346C07K2317/56C07K2317/565
Inventor 应天翼李岩松余涛许宏瑞崔贝贝段佳慧
Owner 北京弘进久安生物科技有限公司
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