Hepatitis C virus envelope antigen ELISA kit and detecting method
A technology of hepatitis C virus and envelope antigen, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of low magnification, false positive, high cost, etc.
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Embodiment 1
[0181] Configuration of Hepatitis C virus envelope antigen kit:
[0182] a. 12 peptides labeled with biotin and the like that can specifically bind to the HCV E2 protein;
[0183] b. The polypeptide is a sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4;
[0184] c. Rabbit polyclonal antibody against E2;
[0185] d. horseradish peroxidase-labeled streptavidin;
[0186] E. horseradish peroxidase substrate;
[0187] f. Microplate plate (CellStar@, purchased from Wuhan Dafeng Biotechnology Co., Ltd.), microplate reader;
[0188] g. E2 protein as a standard positive well;
[0189] h. Enzyme-linked immunoassay instrument (purchased from Nanjing Huadong Electronics Group Medical Equipment Co., Ltd.).
[0190] The steps of a polypeptide PF2A, PE2B, PE2C, PE2D are as follows:
[0191] 1. The steps for preparing E2-GST fusion protein are as follows:
[0192] A. Pick the Escherichia coli containing pGEX-KG-E2 (see reference Li, et al., Engineering of N-glycosylation o...
Embodiment 2
[0317] The steps of surface plasmon resonance (SPR) detection of affinity between polypeptide and E2 protein are as follows:
[0318] A. Let the test instrument Biocore (USA) pass through a period of HBS buffer until the baseline is stable.
[0319] B. Inject 40 μl of amino coupling reagent (containing 0.02M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS) ), to activate the carboxyl group on the CM5 sensor chip.
[0320] C. Dilute the E2 protein to 5 mg / ml with an acetate buffer solution with a pH value of 5.0, and inject 40 μl of the solution.
[0321] D. On-machine detection, the flow rate is 20 μl / min.
[0322] E. Dilute the polypeptides PE2A, PE2B, PE2C, and PE2D to different concentrations, and react with the E2 protein on the CM5 sensor chip at 2 μl / min for 7 minutes.
[0323] F. Inject 10 μl of HCl regeneration solution at a flow rate of 2 μl / min to restore the baseline, and collect response signals in real time.
[0324] G. Us...
Embodiment 3
[0328] The steps for flow cytometry to detect the combination of polypeptides and target molecules are as follows:
[0329] A. Culture human liver cancer cells Huh7.5 (see reference Zhong, et al., Robust hepatitis C virus infection in vitro, 2005, PNAS, 102: 9294-9299) and DC-SIGN + - NIH3T3 (fibroblasts from mice with DC-Sign molecules on their surface) cells (see reference Wu, et al., Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGNinteractions with ICAM-3 do not promote human immunodeficiency virustype I transmission. J. virus. 2002, 76: 5905-5914). The culture conditions were all DMEM medium (purchased by Gibco) with 10% fetal bovine serum, cultured at 37° C. in an incubator containing 5% carbon dioxide.
[0330] B. Mix the polypeptides PE2A, PE2B, and PE2D with the E2-GST protein respectively (GST protein is added to the control tube), the total volume is 50 μl, the concentration of the polypeptide is 500 μM, and the concentration of E2-GST protein ...
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