Treponema pallidum igm antibody biotin avidin ELISA kit and preparation method thereof
An ELISA technology for Treponema pallidum, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of low sensitivity and specificity of serological detection, inability to culture Treponema pallidum in vitro, clinical missed diagnosis and high rate of misdiagnosis , to improve the sensitivity and specificity, shorten the window period, and achieve the effect of accurate results
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[0057] The preparation method of described Treponema pallidum IgM antibody biotin avidin ELISA kit comprises the following steps:
[0058] 1) Extract the outer membrane proteins of Treponema pallidum Nichol and Reiter strains for two-dimensional electrophoresis, transfer them to nitrocellulose membranes, and perform immunoblotting experiments with syphilis serum to obtain characteristic immunoblotting spots, and take corresponding protein spots for mass spectrometry analysis . Find out the outer membrane proteins with high immunogenicity of Nichol strain and Reiter strain respectively, and find out the highly immunogenic outer membrane protein unique to Nichol strain but lacking in Reiter strain;
[0059] 2) Find the corresponding gene in the whole genome sequence of Treponema pallidum, design primers and construct the corresponding expression vector, study the effect of different induction conditions on the expression of the recombinant target protein in E. coli, obtain the b...
Embodiment 1
[0075] The preparation method of described Treponema pallidum IgM antibody biotin avidin ELISA kit comprises the following steps:
[0076] (1) Immunoproteomics study of outer membrane protein:
[0077] The outer membrane proteins of Treponema pallidum Nichol and Reiter strains were extracted for two-dimensional electrophoresis, then transferred to nitrocellulose membranes, and immunoblotting experiments were performed with syphilis serum to obtain characteristic immunoblotting spots, and corresponding protein spots were taken for mass spectrometry analysis. Find out the outer membrane proteins with higher immunogenicity of Nichol strain and Reiter strain respectively, and find out the outer membrane protein with high immunogenicity unique to Nichol strain but lacking in Reiter strain.
[0078] (2) Cloning and purification of candidate proteins
[0079] Find the corresponding gene in the whole genome sequence of Treponema pallidum, design primers and construct the correspondin...
Embodiment 2
[0105] The Treponema pallidum antibody in clinical specimens detected by the Treponema pallidum IgM antibody biotin avidin enzyme-linked immunoassay kit is given below:
[0106] (1) Specimen processing: Serum: 5 mL of venous blood, placed in a 37°C water bath for 30 minutes, centrifuged at 3000 g for 10 minutes, and the supernatant was used as a test sample for later use.
[0107] (2) Adding samples: add 100 uL of the sample and 50 uL of biotin-labeled anti-human IgM specific fragment μ chain monoclonal antibody to the reaction wells, and simultaneously serve as blank, negative and positive control wells. Incubate at 37°C for 1h.
[0108] (3) Washing: wash five times with washing buffer and then buckle dry.
[0109] (4) Enzyme addition: 100 μL of horseradish peroxidase-labeled avidin was added to each well, and incubated at 37° C. for 30 min.
[0110] (5) Washing: wash five times with washing buffer and then buckle dry.
[0111] (6) Color development: add 50 μL each of colo...
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