Preparation method of human prostate immunodominance antigen and application method of human prostate immunodominance antigen
A technology of immunodominant antigen and application method, which is applied in the preparation of human prostate immunodominant antigen and the application field of detection of human prostate autoantibodies, can solve the problem that it is difficult to confirm the existence of prostatitis patients, the lack of specificity in the diagnosis of prostatitis, and the lack of specificity in the diagnosis of prostatitis. Waiting for questions
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Embodiment 1
[0025] Preparation of human prostatic tissue protein purification solution: human benign prostatic hyperplasia tissue was provided by the Department of Urology, Nanjing General Hospital of Nanjing Military Region, from 5 patients with benign prostatic hyperplasia. This study was approved by the Hospital Ethics Committee and informed consent was obtained from the patients. Place the acquired prostate tissue in ice PBS (0.01mmol / L, pH 7.4), trim off excess tissue such as fat and bladder, wash 5 times, cut up the prostate tissue, and add 0.5% TritonX-100 containing physiological Saline, homogenate in ice bath, centrifuge at 12000r / min at 4°C for 30min, remove the adipose tissue in the upper layer, keep the supernatant as human prostate tissue protein purification solution, measure its total protein concentration with an automatic biochemical analyzer, and store in separate packages (-80°C).
Embodiment 2
[0027] Preparation of antiserum against human prostate tissue antigen: the experimental rabbit protocol was approved by the Animal Committee and the Ethics Committee. Experimental rabbit is through back intradermal and subcutaneous multi-point injection of the human prostate tissue protein purification solution (protein concentration is 0.5mg / ml) that obtains in the embodiment 1 of Freund's complete adjuvant emulsification, every experimental rabbit 1ml; Control rabbit injection Physiological saline emulsified with complete Freund's adjuvant, 1ml each. The rabbits were injected again on the 10th, 20th, and 30th day, and the rabbits were killed 7 days after the last injection, and all the blood of the rabbits was collected, and the serum was separated by centrifugation, and the serum was frozen at -80°C for future use. The success of preparing antiserum in immunized rabbits was evaluated by detecting the titer of specific antibodies contained in the rabbit serum by ELISA method...
Embodiment 3
[0029]Detection of antibody titer in experimental rabbit serum: Anti-prostate tissue protein antibody titer in experimental rabbit serum was detected by conventional ELISA method. Coat a 96-well ELISA plate with human prostatic protein homogenate, 50ng protein per well. Serum to be tested was diluted in a gradient of 1:10, 1:100, 1:1000, 1:10000, 1:100000. HRP-labeled goat anti-rabbit IgG polyclonal antibody was used as the secondary antibody (diluted 1:1000), developed with TMB chromogenic solution for 10 min, and the reaction was terminated with 2 mol / L sulfuric acid. The absorbance value of the experimental rabbit serum / absorbance value of the control rabbit serum ≥ 2.1 was used as the positive judgment standard. The results showed that the antibody titer of the experimental rabbit serum was greater than 1:10000.
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