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AlphaLISA detection kit for type B staphylococcal enterotoxin

A Staphylococcus aureus enterotoxin and detection kit technology, applied in the biological field, can solve the problems of goat antibody troubles, complex preparation of Fab segments, false positive immunological results, etc., and achieve rapid detection methods, good application prospects, and reduced impact Effect

Active Publication Date: 2018-11-23
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the immunological identification of the toxin in the culture supernatant of Staphylococcus aureus, there is also protein A in the culture supernatant of Staphylococcus aureus, which can bind to the Fc region of various mammalian IgG antibodies, leading to immunological results false positive
The current solution to this problem is to modify the antibody to remove its Fc segment, but the preparation of the Fab segment is relatively complicated; or use goat antibodies, but the preparation of goat antibodies is more troublesome than mouse monoclonal antibodies

Method used

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  • AlphaLISA detection kit for type B staphylococcal enterotoxin
  • AlphaLISA detection kit for type B staphylococcal enterotoxin
  • AlphaLISA detection kit for type B staphylococcal enterotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the impact of protein A on the detection of SEB by ELISA and AlphaLISA

[0046] 1. Protein A is used as the sample to be tested

[0047] 1. ELISA detection

[0048] (1) Add 100 μL of SEB capture antibody at a concentration of 1 μg / mL to a 96-well plate, and coat at 4°C overnight.

[0049] (2) Discard the coating solution, and block with 200 μL of PBST (PBS+10% Tween) containing 10% FBS.

[0050] (3) Discard the blocking solution, and add protein A solutions with concentrations of 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL and 100 μg / mL respectively (protein A was purchased from Sigma Company, product number is P6031; protein A The solvent of the solution was PBST) 100 μL, incubated at 37°C for 30 minutes, discarded the supernatant, and washed 3 times with PBST.

[0051] (4) Add 1:2000 biotin-labeled SEB detection antibody, incubate at 37°C for 30 minutes, discard the supernatant, and wash 3 times with PBST.

[0052] (5) Add 1:8000 diluted HRP-labeled str...

Embodiment 2

[0066] Embodiment 2, ELISA and AlphaLISA to the detection of SEB in S. aureus culture supernatant

[0067] 1. Detection of SEB-producing strains by qPCR and R-Biopharm RIDASCREEN

[0068] Select 10 strains of Staphylococcus aureus (S-6, 1169, 3-4, AT, SC2, 2-1, 3-3, SC1, A8 and SC3), and use RT-qPCR and R-Biopharm RIDASCREEN to detect 10 strains respectively SEB-producing and non-SEB-producing strains in Staphylococcus aureus.

[0069] 1. Detection of SEB-producing strains by R-Biopharm RIDASCREEN

[0070] Ten strains of Staphylococcus aureus were cultured in the broth medium for 24 hours, centrifuged at 12000rpm for 10min, the supernatant was taken, sterilized by filtration with a 0.22μm filter membrane, and SEB in the supernatant was detected by SEB enzyme-linked method. The SEB enzyme-linked method was operated according to the instructions of R-Biopharm RIDASCREEN (R-Biopharm AG, Darmstadt, Germany).

[0071] R-Biopharm RIDASCREEN test results are as follows: figure 2...

Embodiment 3

[0088] Example 3, Sensitivity and specificity detection of the AlphaLISA detection kit of Staphylococcus aureus type B enterotoxin

[0089] 1. Sensitivity detection

[0090] SEB solutions of different concentrations were used as samples to be tested, and SEB was detected according to the AlphaLISA detection method in Step 1-2 of Example 1. The SEB solution concentrations of the 4 parameter curves are 0.03ng / mL, 0.05ng / mL, 0.10ng / mL, 0.19ng / mL, 0.39ng / mL, 0.78ng / mL, 1.56ng / mL, 3.13ng / mL, 6.25 ng / mL, 12.5ng / mL, 25ng / mL, 50ng / mL, 100ng / mL, 200ng / mL and 300ng / mL; linear curve SEB solution concentration is 0.03ng / mL, 0.05ng / mL, 0.10ng / mL, 0.19ng / mL, 0.39ng / mL, 0.78ng / mL, 1.56ng / mL, 3.13ng / mL, 6.25ng / mL, 12.5ng / mL, and 25ng / mL.

[0091] The result is as image 3 shown. The results showed that the established AlphaLISA detection system had high sensitivity to SEB detection, the detection limit could reach 25pg / mL, and the linear range was from 25pg / mL to 25ng / mL.

[0092] 2. Spe...

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Abstract

The invention discloses an AlphaLISA detection kit for type B staphylococcal enterotoxin (SEB). The AlphaLISA detection kit provided by the invention comprises an SEB capturing antibody labeled receptor microsphere, a biotin labeled SEB detection antibody and a streptavidin labeled donor microsphere. The invention further provides a detection method of the type B staphylococcal enterotoxin; the detection method of the type B staphylococcal enterotoxin, provided by the invention, is used for detecting the type B staphylococcal enterotoxin by adopting an AlphaLISA technology. An experiment proves that the detection method of the type B staphylococcal enterotoxin can be used for greatly reducing influences on protein A; the detection method provided by the invention is rapid, accurate and reliable and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an AlphaLISA detection kit for type B staphylococcus aureus enterotoxin. Background technique [0002] SEB (Staphylococcus aureus enterotoxin type B) is an important toxin secreted by Staphylococcus aureus into the supernatant. It is an important biological warfare agent and an important factor for food poisoning caused by Staphylococcus aureus contamination. SEB itself has no cytotoxicity, and its toxicity comes from its superantigen properties. The so-called superantigen (Superantigen, SAg) is a class of antigen molecules that can directly bind to MHC class II molecules without the treatment of antigen-presenting cells (APC), leading to the proliferation of T cells with specific Vβ segments. Its activation ability is 2 000-50 000 times that of ordinary antigens, so only a very low concentration (1-10 pg / mL) can stimulate T cell activation and proliferation, and release...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/531G01N33/6854
Inventor 律清宇江华姜永强刘鹏郑玉玲孔德聪赵俊清
Owner ACADEMY OF MILITARY MEDICAL SCI
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