Beta-fodrin antigen epitope polypeptide, and its screening method and use
An antigenic epitope, lining protein technology, applied in the direction of peptide sources, animal/human peptides, material testing products, etc., can solve the problem of lack of antigen sources and specific diagnostic methods, etc.
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Embodiment 1
[0041] Example 1 Screening and identification of β-fodrin epitope polypeptide
[0042] The aforementioned research work has proved that the β-fodrin fusion protein purified by genetic engineering can specifically bind to the anti-β-fodrin antibody in the serum of patients with Sjogren's syndrome, and then it can be purified by specific methods. Detected (Masataka K, Tetsuroh O, Yoko - O, Junichi K, and Yutaka K, Autoantibodies to the Amino-Terminal Fragment of β-Fodrin Expressed in Glandular Epithelial Cells in Patients with Sjogren's Syndrome. The Journal of Immunology, 2001, 167: 5449-5456.). In the experiment of the present invention, the inventor selected the amino acid sequence corresponding to the ORF nucleic acid sequence of the full-length β-fodrin gene as the object, and adopted five international standard methods for predicting antigenic epitopes, including Standard, Karplus, Emini, Amphiphi, and Pellequer. Comprehensive analysis of the above-mentioned 2365aa length...
Embodiment 2
[0066]Example 2 Detection of β-fodrin epitope polypeptide on anti-β-fodrin epitope polypeptide IgG antibody in patient serum and its value evaluation
[0067] In the experiment of the present invention, for the amino acid characteristics of the synthetic polypeptide and its reaction with the antibody, after a series of adjustments of the pH value and the concentration of the reactant ion, we determined that the self-prepared PBS with pH 9.6 was used as the coating buffer; Corresponding experiments were carried out on the concentration of the coating antigen, and the β-fodrin epitope polypeptide SEQ ID NO.1 was serially diluted and reacted. The results showed that the peptide was coated with the self-prepared PBS coating buffer at pH 9.6 above Dilute to 0.5μg / ml, add 100μl to each well, incubate at 37°C for 4 hours, then perform serum antibody detection, the effect is the best; in addition, with regard to the problem of preventing non-specific reactions, based on the inventor’s ...
Embodiment 3
[0114] Example 3 Application of β-fodrin epitope polypeptide as an antigen in the preparation of clinical Sjogren's syndrome diagnostic kit
[0115] The present inventors used the sandwich method solid-phase enzyme-linked immunosorbent assay (ELISA), coated the β-fodrin epitope polypeptide antigen on a microwell plate, and prepared β-fodrin epitope polypeptide IgG The kit for clinical diagnosis is used for the quantitative detection of anti-β-fodrin epitope polypeptide IgG antibody, which can complete the detection of 96 people in total. The specific content is as follows:
[0116] 1. Kit composition materials
[0117] One detachable microwell plate (1×8) with 12 strips coated with highly purified β-fodrin epitope polypeptide;
[0118] β-fodrin epitope polypeptide IgG standard: 0 / 6.3 / 12.5 / 25 / 50 / 100U / ml, 1 bottle each, 1.5ml per bottle;
[0119] 1 bottle of quality control serum (including IgG and negative quality control), 1.5ml;
[0120] 1 bottle of concentrated sample buff...
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