Preparation method of Treponema pallidum total antibody biotin avidin ELISA kit
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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[0052] The preparation method of the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit comprises the following steps:
[0053] 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 protein 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; find out in the whole genome sequence of Treponema pallidum Corresponding to the gene, design primers and construct corresponding expression vectors, according to the influence of different induction conditions on the expression of the recombinant target protein in E. co...
Embodiment 1
[0066] The Treponema pallidum total antibody biotin avidin ELISA kit of the present invention consists of an outer packaging box 1, a concentrated washing liquid bottle 2, a negative control bottle 3, a positive control bottle 4, and a biotin-labeled recombinant antigen bottle 5 A bottle of horseradish peroxidase-labeled avidin 6, a bottle of TMB chromogenic solution A 7 , a bottle of chromogenic solution B 8 , a bottle of reaction termination solution 9 , and a microwell plate 10 coated with recombinant antigen.
[0067] The preparation method of the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit comprises the following steps:
[0068] 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 ma...
Embodiment 2
[0092] The following shows the Treponema pallidum antibody in the clinical specimens of patients detected by the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit:
[0093] (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.
[0094] (2) Adding samples: add 100uL of the sample and 50uL of biotin-labeled recombinant antigen to the reaction wells, and use them as blank, negative and positive control wells at the same time. Incubate at 37°C for 1h.
[0095] (3) Washing: Wash 5 times with washing buffer and then buckle dry.
[0096] (4) Enzyme addition: Add 100 μL of avidin-labeled horseradish peroxidase solution to each well, and incubate at 37° C. for 30 min.
[0097] (5) Washing: wash 5 times with washing buffer and then buckle dry.
[0098] (6) Color development: 50 μL each of TMB color development solut...
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