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Enzyme-linked immune sorbent assay (ELISA) kit for detecting Shiga toxin II (StxII)

A technology in the kit and sequence listing is applied in the field of preparation of monoclonal antibodies S2D8 and S2C6, which can solve the problems of delaying the best time for treatment and being difficult to distinguish, and achieves a high-accuracy and easy-to-use method to curb the spread of the epidemic. Effect

Active Publication Date: 2015-05-27
JIANGSU BIG DIPPER ENVIRONMENTAL PROTECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinically, it is difficult to distinguish the early diarrhea caused by EHEC from the diarrhea caused by other pathogens. On the one hand, the arbitrary use of antibiotics kills a large number of bacteria, and the diarrhea symptoms of patients are relieved, resulting in the illusion of apparent "recovery", which makes Diagnosis by isolating pathogens mostly ends in negative results, which delays the best time for treatment. On the other hand, since Stx is encoded by the lambda phage gene integrated into the EHEC genome, the use of antibiotics can easily make the remaining bacteria enter the SOS stress state , the phage enters the lytic state from the static lysogenic state, produces a large amount of Stx toxin in the intestinal tract, and enters the blood circulation, causing the patient to develop HUS

Method used

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  • Enzyme-linked immune sorbent assay (ELISA) kit for detecting Shiga toxin II (StxII)
  • Enzyme-linked immune sorbent assay (ELISA) kit for detecting Shiga toxin II (StxII)
  • Enzyme-linked immune sorbent assay (ELISA) kit for detecting Shiga toxin II (StxII)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Construction of anti-StxII monoclonal antibody hybridoma cell line

[0042] 1. Materials

[0043] Fusion protein StxIIA-GST, protein G affinity chromatography column, fetal bovine serum: product of Beijing Yuanheng Shengma Institute of Biotechnology; serum-free RPMI 1640: product of Gibco company; OPTI-MEM medium, Freund's complete adjuvant and Freund's incomplete adjuvant, chromogenic reagent TMB: product of Sigma; SP2 / 0 cells: imported from ATCC; Balb / c and C57BL / 6 mice: provided by the Experimental Animal Center of Academy of Military Medical Sciences; other reagents are commercially available.

[0044] 2. Methods and Results

[0045] (1) Fusion protein StxIIA-GST immunized 5-week-old female Balb / c mice, 100 μg / mouse, for the first immunization, 100 μg antigen was mixed with an equal volume of Freund’s complete adjuvant, and injected intraperitoneally; after the third week, an equal amount of antigen was used Intraperitoneal immunization after mixing w...

Embodiment 2

[0052] Example 2 Application of Double Antibody Sandwich ELISA to Select the Best Antibody Pairing

[0053] A total of 5 monoclonal antibodies against the StxII A subunit were obtained through Hybridoma Technology Example 1: S2D8, S2C6, 4F5, 1G7 and 11H5, and the double antibody sandwich ELISA method was used to pair the above 5 antibodies with each other to select the best Combined to facilitate StxII toxin detection.

[0054] 1. Materials

[0055] NaHCO 3 / Na 2 CO 3 Buffer (pH9.6), Tween-20, bovine serum albumin, 96-well microtiter plate, activated horseradish peroxidase: Beijing Taitianhe Biotechnology Co., Ltd.; other reagents are commercially available.

[0056] 2. Method results

[0057] Monoclonal antibody coating. with 0.1M NaHCO 3 / Na 2 CO 3 Buffer (pH9.6) to dilute antibody concentration to 10 μg / ml, add to 96-well ELISA plate, 100 μl / well, coat overnight at 4°C, wash once with PBST (add 0.05% Tween-20 to PBS) , add 5% milk to block overnight at 4°C, wash o...

Embodiment 3

[0064] Identification of Heavy and Light Chain Isotypes of Example Three Monoclonal Antibodies S2D8 and S2C6

[0065] Use SouthernBiotech's SBA Cloning System / HRP Isotype Identification Kit (5300-05), dilute goat anti-mouse (H+L) antibody to 10 μg / ml with PBS (pH7.4), add to ELISA enzyme label plate, 100 μl per well, and incubated overnight at 4°C. Discard the coating solution, wash 3 times with PBS (PBST) containing 0.05% Tween20, add 200 μl of 1% BSA to each well, and block overnight at 4°C. Wash 3 times with PBST, add 100 μl hybridoma culture supernatant to each well of 12 wells, incubate in 37°C water bath for 1 hour, and wash 3 times with PBST. Dilute the HRP-labeled goat anti-mouse secondary antibody with PBS containing 1% BSA at 1:500, and its specificity is as follows: the heavy chain is IgG 1 , IgG 2a , IgG 2b , IgG 3 , the light chains are κ and λ, each specific secondary antibody was added to 2 wells, 100 μl per well, placed in a water bath at 37°C for 1 hour, ...

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Abstract

The invention discloses an enzyme-linked immune sorbent assay (ELISA) kit for detecting Shiga toxin II (StxII) and also discloses sequence information of monoclonal antibodies S2D8 and S2C6 in the ELISA kit and a preparation process of the monoclonal antibodies S2D8 and S2C6. The ELISA kit comprises the monoclonal antibody S2D8, the HRP-marked monoclonal antibody S2C6, a horse radish peroxidase substrate buffer solution, a protein standard product StxII (100 ug / ml, 0.1 ml), a negative control sample BSA and lotion (PBS+0.05%Tween20). The kit can sensitively detect the StxII content of samples, and the sensitivity can reach 4 ng / ml. The kit provided by the invention also can effectively identify subtype StxIIc and StxIIvha of the StxII.

Description

technical field [0001] The invention relates to an ELISA kit, in particular to an ELISA kit for detecting type II Shiga toxin (StxII), and also relates to the preparation of monoclonal antibodies S2D8 and S2C6 in the ELISA kit and the method for using the kit. Background technique [0002] Enterohemorrhagic E.coli (EHEC) infection is an important zoonotic disease, which is prevalent all over the world, including developed and developing countries, and its infection is fulminant, highly pathogenic and fatal It has become a global public health problem and poses a huge threat to human health. EHEC infection can cause diarrhea (Diarrhea), hemorrhagic colitis (Hemorrhagic Colitis, HC), can also cause hemolytic uremic syndrome (Hemolytic Uremic Syndrome, HUS), thrombotic thrombocytopenic purpura (Thromboric Thrombocytopenic Porpura, TTP) ) and other serious complications, especially HUS can cause irreversible functional damage to the kidneys of patients, and the mortality rate i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12
Inventor 焦永军曾晓燕郭喜玲史智扬汪华周明浩
Owner JIANGSU BIG DIPPER ENVIRONMENTAL PROTECTION CO LTD
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