Immunological detection of Helicobacter pylori

Helicobacter pylori immunological tests detect H. pylori infection by measuring Helicobacter pylori antibodies in serum, including passive hemagglutination assays, immunoblotting techniques, and enzyme-linked immunosorbent assays (ELISA). The patient took the cerebrospinal fluid and diluted the antigen with the 96-well V-type hemagglutination plate to dilute the JE antigen, so that each well contained 25 uL, and the serum to be tested was diluted 1:10 with each dilution. Add 25uL, add lyophilized Japanese brain monoclonal antibody to sensitize sheep red blood cells 25uL, and observe the results after standing at 37 °C for several hours. Basic Information Specialist classification: growth and development examination classification: blood examination Applicable gender: whether men and women apply fasting: fasting Tips: Keep an empty stomach from the exam. Normal value The result was negative. Clinical significance Abnormal results: 1. The serum agglutination titer of the test is 4 times or more than 4 times higher than the antigen control. The serum antigen titer during the recovery period was 4 or more times higher than the acute phase and was positive. 2. There is a special color reaction, which proves that a certain protein exists. 3. The ratio of the serum to be tested to the known negative serum (P/N) ≥ 2.1, and the OD value of the serum to be tested is ≥ 0.4, then it is judged as positive. Need to check the crowd: patients with peptic ulcer. Precautions Contraindications before inspection: Pay attention to protecting the brain; prepare various packaging liquids and configure the concentration. Taboo when checking: 1. The patient takes the cerebrospinal fluid to sit, so as not to hurt the head. 2. Monoclonal antibodies used to sensitize sheep red blood cells to be lyophilized before use. 3. The dilution, duration of action and temperature of the primary and secondary antibodies should be pre-experimented to determine the optimal conditions for different proteins. 4. The color developing solution must be freshly configured and finally added to H2O2. 5. DAB has the potential to cause cancer, so be careful when handling it. 6. Strictly control factors affecting the efficiency of labeling such as temperature, time, pH, enzyme and antibody amount. 7. When installing the column, make the column uniform, the cylinder surface is flat, and there are no bubbles or cracks. Inspection process First, passive blood coagulation The patient took the cerebrospinal fluid and diluted the antigen with the 96-well V-type hemagglutination plate to dilute the JE antigen, so that each well contained 25 uL, and the serum to be tested was diluted 1:10 with each dilution. Add 25 uL, and add 25 uL of sensitized sheep red blood cells by lyophilized Japanese brain monoclonal antibody, and observe the results after standing at 37 ° C for several hours. Second, immunoblotting technology (A) protein sample obtained: after bacterial induction of expression, the cells can be directly lysed by electrophoresis loading buffer, eukaryotic cells plus homogenization buffer, mechanical or ultrasonic room temperature homogenate 0.5-1min. It was then centrifuged at 13,000 g for 15 min at 4 °C. Take the supernatant as a sample. (2) Electrophoresis: An electrophoresis gel was prepared and subjected to SDS-PAGE. (3) Transfer: (semi-dry transfer) 1. After the electrophoresis is finished, cut the strip to the appropriate size and equilibrate with the membrane buffer, 5 min × 3 times. 2. Membrane treatment: The filter paper and NC membrane of the same size as the strip were pre-cut and immersed in the transfer buffer for 10 min. 3. Transfer film: The film transfer device is placed in order from bottom to top according to the order of anode carbon plate, 24 layers of filter paper, NC film, gel, 24 layers of filter paper and cathode carbon plate. The filter paper, gel and NC film are precisely aligned. The bubbles were removed in one step, and a weight of 500 g was applied thereto to dry the excess liquid on the carbon plate. Turn on the power, constant current 1mA/cm2, transfer 1.5hr. After the transfer is completed, the power is removed and the membrane is taken out, and the strip to be tested is cut for immunoblotting. The protein standard strips were stained, placed in the membrane staining solution for 50 s, and then decolorized in 50% methanol several times to a clear background, then washed with double distilled water, air-dried in two layers of filter paper, and left to color The results are compared. (4) Immune response: 1. Wash the membrane with 0.01 M PBS for 5 min x 3 times. 2. Add the coating solution and shake gently at room temperature for 2 hr. 3. Discard the solution and wash the membrane with 0.01 M PBS for 5 min × 3 times. 4. Add primary antibody (diluted with 0.01 M PBS in a suitable dilution ratio, the liquid must cover all of the membrane), and leave at 4 °C for more than 12 hr. In the negative control, the primary antibody was replaced with 1% BSA, and the remaining steps were the same as in the experimental group. 5. Discard the primary antibody and 1% BSA, and wash the membrane with 0.01 M PBS, 5 min × 4 times. 6. Add horseradish peroxidase-conjugated secondary antibody (diluted with 0.01 M PBS at the appropriate dilution ratio) and shake gently for 2 hr at room temperature. 7. Discard the secondary antibody and wash the membrane with 0.01 M PBS for 5 min × 4 times. 8. Add the coloring solution, avoid the light and develop color until the band appears. Put in the double distilled water to stop the reaction. Third, enzyme combined adsorption determination Commonly used ELISA methods include a double antibody sandwich method and an indirect method, the former for detecting macromolecular antigens and the latter for measuring specific antibodies. Indirect ELISA This method is mainly used to detect antibodies. The procedure for indirect ELISA is as follows. (1) Materials 1 coating liquid, washing liquid, heat preservation liquid, substrate liquid, and stopping liquid; 2DVH coated antigen, enzyme-labeled anti-antibody, negative and positive DVH reference serum; 3 ELISA detector, sampler, polystyrene microplate. (2) Method steps 1 plus antigen coating → 4 ° C overnight, washed three times, throw dry; 2 plus serum to be tested → 37 ° C for 2 hours, washed three times, throw dry; 3 plus enzyme labeled antibody → 37 ° C for 2 hours, washed three times, throw dry; 4 add substrate solution → 37 ° C for 30 minutes, add stop solution; 5 The OD value was measured by an ELISA detector, and the P/N ratio was calculated. 2. Double antibody sandwich ELISA This method is mainly used to detect macromolecular antigens. 1 plus antibody coating → 4 ° C overnight, washed three times, throw dry; 2 plus the antigen to be tested → 37 ° C for 30 minutes, washed three times, throw dry; 3 plus enzyme labeled antibody → 37 ° C for 30 minutes, washed three times, throw dry; 4 add substrate solution → 37 ° C for 15 minutes, add stop solution; 5 The OD value was measured by an ELISA tester. Not suitable for the crowd Not suitable for checking the crowd: none. Adverse reactions and risks No related complications or hazards.