Kit for combined detection of 6 diabetic antibodies
A technology of autoantibodies and diabetes, applied in the biological field, to achieve the effect of ensuring accuracy, simple operation, and improving detection efficiency
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Embodiment 1
[0048] The preparation of embodiment 1 kit
[0049] Extraction and purification of 6 islet cell protein antigens including IA, GAD, IA-2, CPH, ZnT8 and ICA:
[0050] Screen pancreatic tissues from pigs, cattle, rats and humans, and select pancreatic tissues containing 6 islet cell protein antigens;
[0051] Extract 6 kinds of islet cell protein antigens: crush the pancreatic tissue with an ultrasonic cell breaker, put it in a high-speed refrigerated centrifuge, collect the supernatant, put it in a dialysis bag for dialysis, concentrate it in polyethylene glycol, and measure DM with an ultraviolet spectrophotometer protein antigen content. The key problem it mainly solves is that in the process of extracting and purifying DM antigen, its antigenic components will be degraded and lost.
[0052] Electrophoresis: The six antigens of islet cells were properly diluted with sample buffer, and SDS-polyacrylamide gel gradient discontinuous electrophoresis was used to separate various...
Embodiment 2
[0063] Embodiment 2 is based on the detection method of the kit provided by the invention
[0064] Serum or whole blood to be tested is 10 μl. Fresh serum or whole blood is suitable for the specimen to be tested. Serum can generally be collected from veins and centrifuged after coagulation. Point-of-care testing can be performed by finger pricking and whole blood testing. If the collected serum or whole blood is not tested on the same day, it should be stored at 4°C; if it exceeds seven days, it should be stored below -20°C.
[0065] Preparation of washing solution: the concentrated washing solution stored at 2-8°C will crystallize, dilute the whole bottle (25mL) of concentrated washing solution to 250mL with distilled water or pure water, put it into the reagent bottle, and indicate the preparation on the label Time, stored at 2-8°C, valid for 3 months, if there is floc or mildew, it should be discarded;
[0066] Use tweezers to put the required amount of blotting membrane ...
Embodiment 3
[0075] Comparison of several detection methods of embodiment 3
[0076] 45 cases of T1DM patients were newly diagnosed patients from the Department of Secretology and Pediatrics, all of which met the 1999 diagnostic criteria of the World Health Organization (WHO). Aged 5-48 years old, average age (25±12 years old), including 27 males and 18 females, healthy control group was 50 cases who came to our hospital for physical examination, aged 15-41 years old, average age (24±5 years old) , including 24 males and 26 females, all the controls had normal fasting blood sugar, no family history of diabetes and autoimmune disease history.
[0077]All tested samples were fasting venous blood collection, separated serum, aliquoted and stored at -20°C for testing. The IAA radioimmunoassay kit was purchased from the Beijing Institute of Biotechnology, and the SN-697 fully automatic dual-probe radioimmuno r-counter was produced by the Shanghai Atomic Energy Research Institute Rihuan Instrum...
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Abstract
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