Development Of The Immunoassay Method For Rapid Determination Of E.coli In Pharmaceutical Wastewater

World experts and scholars trying to find a rapid method to detect E.coli, now has developed molecular biological detection method, enzyme substrate method and immunological method, etc.This research sieved E.coli from pharmaceutical waste water, and using it manufactured immunogen, then prepared polyclonal escherichia coli antibodies, and established the indirect competitive enzyme-linked immunosorbent adsorption method, and based on this we developed indirect ELISA kit, and the specific results are as follows:1. The research sieved E.coli from pharmaceutical waste water, and using it manufactured immunogen. Using the E.coli immunogen injecting New Zealand big white rabbit, we obtained polyclonal escherichia coli antibodies, after purification antibody protein content is 2.50 mg/mL, the titer of all bacteria antibody is more than 25600.2.Establish and optimize the E.coli indirect ELISA method: the best technological conditions of the concentration of antigen is 200 times, the best diluted concentration of antibody and enzymes standard antibody respectively 12800 times and 2000 times, 0.5%BSA is the best closed liquid. Closed 2h as the best closed time, Choose 37℃temperature 60min, contain 4% PEG diluent as the best of antigen and an antioxidant reaction conditions, Choose 37℃temperature 60min, contain 4% PEG diluent as best antibody and HRP-IgG reaction conditions.3. Prepared the E.coli gradient solution, the concentration is 1012cfu/mL-10cfu/mL, with the optimized indirect competitive ELISA determination, the results showed good linear relationship between the 103cfu/mL-109cfu/mL, showed that the detection limit for ELISA was 103cfu/mL. Using the indirect competitive ELISA and MPN method to detect E.coli in pharmaceutical wastewater, the result showed that the indirect competitive ELISA method was consistent with MPN method, but the indirect ELISA can detect dead bacteria, so the MPN method have big assessment.4. This study we prepared indirect competitive ELISA kit. Optimized the kit coating time, the experimental results show that compared coating under 37℃for one hour and 4℃overnight, samples'OD value were similar; The research does the methodological evaluation of the kits, including: specificity, repeatability and sensitivity. Detected the E.coli respectively (screening), E.coli, Yeast, Providencia bacteria (Rettgeri), Alkali coli (Alcaligenes Faecalis) samples for testing, the results showed that the kits has strong specificity; Using five different samples made repeated experiments, only part of the samples'coefficient of variation were more than 10%, indicating that this kit has good repeatability, The detect limit of E.coli was 103cfu/mL.