After Warren and Marshall successfully isolated Helicobacter pylori (Hp) from human gastric mucosa in 1983, the correlation of Hp with gastritis and peptic ulcer has aroused great interest. It is speculated that it may become one of the causative factors of such diseases. Many researchers are further exploring its pathogenic mechanism and looking for rapid and reliable detection methods from different angles. So far, methods for detecting Hp include tissue specimen culture, section staining, bacterial direct smear staining, rapid urease determination, 13C urea breath test and 14C urea breath test. Except for the urea breath test, all other methods need to be completed by gastroscopy. In view of the difficulty in obtaining materials, it is necessary to further search for immunology to detect Hp infection. Some people abroad have used enzyme-linked immunosorbent assay (ELISA) to detect the Hp antibody in the serum of clinical patients, and there are no domestic research reports on this. This article reports the experimental results of establishing an ELISA for detecting human anti-Hp antibodies and comparing the test results with bacterial culture and urease assays. Studies have shown that this method is specific, sensitive, suitable for a large number of specimens and timely judgment of treatment response The results are reported below.
1 Materials and methods
1.1 Materials
â‘ 40-well polystyrene micro-concave orifice plate: the product of Shanghai No. 3 Plastic Factory. â‘¡Enzyme-linked plate centrifugal basket: designed and manufactured by our laboratory according to the 40-well enzyme-linked plate. â‘¢ DG-â… enzyme-linked immunoassay detector: the product of Nanjing Huadong Electron Tube Factory. â‘£Reagent: peroxidase (HRP), R2 = 3.2, product of American Sigma company. O-Phenylenediamine (OPD): Shanghai Baihe Chemical Plant product, formulated according to literature. Coating liquid: 1.59g of sodium carbonate, 2.93g of sodium bicarbonate and 1000ml of distilled water are prepared. Washing solution: The above washing solution without Tween-20 is 100ml + 2% calf serum + 4% polyethylene glycol (PEG). Plate blocking solution: 5% calf serum. Glutaraldehyde fixing solution: 25% glutaraldehyde solution, 100ml, and Kochligh import sub-packing, when diluted to 0.25% glutaraldehyde fixing solution. Stop solution: 2NH2SO4. ⑤ Preparation of antigen: Hp standard strain (NCTC11638) of Royal Perth Hospital in Australia and Hp strain isolated from gastric mucosa biopsy specimens of patients undergoing gastroscopy were inoculated on heart and brain blood plate respectively, and the colonies were taken for biochemical and Serological identification (self-made rabbit anti-Hp antibody), then take a single colony and transfer it to another blood plate, continue to culture for 3d, after identification, it will be multiplied and cultured, after 3d, the moss is scraped off and placed in physiological saline Bacteria solution, fixed with formalin (0.1% ～ 0.3%), overnight at 4 ℃, centrifuged at 3000r / nin for 30min, precipitated 3 times, suspend the last precipitate in a small amount of PBS solution, and the turbidity tube showed no bacterial concentration , 3 × 109ml containing bacteria, frozen in the refrigerator, repeated freezing and thawing 3 times, after microscopic examination of the presence of sterile body, made soluble antigen, protein quantification using Lowry's method, stored at -20 ℃ for future use. â‘¥Clinical serum specimens: collected from gastroscopy patients, randomly selected 38 patients from August to October 1988 gastroscopy, venous blood was drawn 2ml, separated serum was stored at -20 ℃ for future use. ⑦ Negative control serum, measured by ELISA, and take the average OD value as a control. ⑧Preparation of enzyme-labeled goat anti-human IgG conjugate (SAHIgG-HRP): According to the periodate modification method, slightly improved labeling is made into enzyme-labeled antibody. That is HRP8.0ml, stirring at 22 ℃ for 20min. Add 6 drops of ethylene glycol and continue stirring for 5 min. Dialyze overnight at pH 4.2, 0.001 mol / L acetate buffer (adjust the pH with 2 mol HC1), change the water 4 times in the middle, add goat anti-human IgG (Shanghai Bioproducts Research) Product) 14mg, dropwise add pH, 1.0mol carbonate buffer to raise the pH to 9.0 ~ 9.5, stirring at room temperature for 2h, add 0.2ml of sodium borohydride (4mg / ml), set at 4 ℃ for 2h, pH 7 .2, 0.01molPB-0.4molNaCl buffer is dialysis equilibrated at 4 ℃. Change the water 4 times, collect the conjugate in the dialysis bag, salt out with 50% saturated ammonium sulfate solution to remove the free enzyme, measure the refined conjugate, after the E / P molar ratio is qualified, divide the conjugate into a vial at -20 ℃ Save for future use. ⑨Urease assay and Hp separation and culture method are carried out by reference.
1.2 Method
And a little improvement, namely: Antigen coated microtiter plate: After the antigen was diluted with the coating solution at 2 × 10 8 billion / ml (containing protein 8.6 μg), add 100 μ to each well, centrifuge the basket for 20 min and 2000 r / min, pour Remove the liquid in the well, then add 0.25% glutaraldehyde solution 50μl / well, fix at 4 ° C for 30min, pour off the glutaraldehyde, wash with washing solution 3 times (3min each time), add 5% calf serum solution, 200ml / The wells were sealed at 37 ° C for 30 min and the liquid in the wells was poured. Add 100ml of serum to be tested (1: 100 dilution) to each well. After incubating at 37 ℃ for 1h, wash 3 times as above, and make negative control wells and blank wells at the same time. Then add 100ml of freshly diluted enzyme-labeled goat anti-human IgG conjugate to each well and incubate at 37 ° C for half an hour. After adding 2NH2SO450μl to each well to stop the reaction, measure the OD value at 490nm in the enzyme-linked analyzer. The ratio of the OD value (P) of the specimen to the OD value (N) of the negative control (P / N) is obtained. If the P / N ratio is greater than or equal to 2.0, it is positive.
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