Method capable of detecting four kinds of type I diabetes autoimmune antibodies simultaneously
An autoimmune antibody and diabetes technology, applied in the field of medical testing, can solve problems such as cross-reaction, false-positive reaction, tissue sample processing is not fast enough, inactivation of degradable enzymes and salts, and impact on results, etc., to achieve stable, reliable and accurate measurement values High efficiency, quick and easy method
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[0049] The method of the present invention capable of simultaneously detecting four kinds of type I diabetes autoimmune antibodies comprises the following steps:
[0050] 1. Construction of the fusion vector of luciferase gene and multi-antigen gene
[0051] (1) Select the target sequences of luciferase gene and multi-antigen gene (GAD65 antigen gene sequence, IA-2a antigen gene sequence, IA-2b antigen gene sequence and insulin antigen gene sequence), and perform PCR amplification. Reaction condition program: Denaturation at 90°C to 95°C for 5 to 10 minutes; then denaturation at 93°C to 98°C for 45 to 60 seconds, annealing at 45°C to 50°C for 30 to 45 seconds, extension at 65°C to 70°C for 50 to 60 seconds, and 25 to 30 cycles; finally, extend at 70°C to 75°C for 10 to 15 minutes, and keep warm at 0°C to 4°C.
[0052] Introduce a restriction endonuclease at the kpnl site (GGTACC) to obtain the target fragment and identify it by gel electrophoresis;
[0053] Polyantigens are ...
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