Research And Application Of High-throughput Immunoassay Technology For Antibiotics In Water

Antibiotics has been applied in the used fields of clinical diagnosis,agriculture and aquaculture as drugs aimed at bacterial growth inhabitation or sterilization.Because of releasing into the environment of large amounts of antibiotics,it do great harm to the ecological system and human health,lead to the emergence of resistant bacteria.Currently,antibiotics in water are usually detected using physical and chemical methods,such as High Performance Liquid Chromatography(HPLC).Although these methods have high sensitivity and accuracy,they require complicated pre-treatment processes and high detection cost with low throughput.Thus,high-throughput and rapid screening method for antibiotics detection in water is urgently needed.Based on the requirement of high-throughput and rapid screening method for antibiotic detection in water,this paper aims to establish an indirect competitive ELISA immunoassay for sulfa antibiotics detection by optimizing the detection conditions according to the principle of antigen-antibody-specific immune response.Furthermore,the matrix effect in the real water samples were studied,and an antibody buffer system was developed to mitigate or even eliminate such matrix interferences.Carry out the actual water sample spike detection research,conduct a systematic.Then,the performance of this method was evaluated systematically(detection limit,precision,accuracy)by the spiking experiments in real water.A standardized applicability protocol for this indirect competition ELISA immunoassay for antibiotics detection was proposed,which provides effective technical support for the actual detection of antibiotic contamination.The main conclusions of this paper are as follows:(1)An indirect competitive ELISA assay for sulfa antibiotics was initially established.By optimizing the conditions of coating antigen,primary antibody and secondary antibody concentration,blocking time and reaction time,the concentration of sulfa-coated antigen is 5?g/mL,and the dilution ratio of primary antibody is 1:2000.The concentration of the pyrimidine-coated antigen is 3 ?g/mL for SM2,the dilution factor of the primary antibody is 1:4000.The dilution factor of the secondary antibody is 1:700.The blocking time is at least 80 min,and the reaction time is 60 min or more.The standard curve of sulfa antibiotics indirect competitive ELISA is established.The detection limit of sulfa antibiotics is 2.64?g/L and the quantitative detection interval is 7.07?244.86?g/L by Logistic equation fitting analysis.The detection limit is 7.03?g/L,and the quantitative detection interval is 23.26?367.07?g/L for the SM2 by Logistic equation fitting analysis.Cross reactivity of sulfa antibiotics with other major classes of antibiotics and several common sulfonamide-like antibiotics such as SM2 were analyzed.Results show that sulfa antibiotics had no cross reactivity with tetracyclines,quinolones,and linco-like antigens.The cross-reaction rate of the SM2 antibody with the structural analogs such as sulfonamide p-methoxypyrimidine and sulfaquinoxaline is very low.(2)The effect and elimination of common matrix in indirect competitive ELISA were studied.Results indicate that when the pH is 6.0-8.0,the mass concentration of heavy metals is less than 0.32mg/L,the concentration of calcium and magnesium ions is less than 400mg/L,and the percentage of methanol is less than 20%,the matrix effect on the standard curves is little;when the mass concentration is 6mg/mL EDTA?8mg/mL BSA and 6×PBS are used as antibody dilutions,the buffering effect of the matrix for pH(5-9),heavy metals(27 heavy metals mixed concentration<1.25mg/L),hardness(calcium and magnesium ion concentration<800mg/mL)and methanol(volume ratio<40%)is better;The buffer system is used for indirect competitive ELISA detection with large matrix influence,and the buffering effect is better.(3)A standardization scheme for sulfonamide indirect competition ELISA detection is proposed,including collection,transportation and preservation of water samples,reagents and instruments required for main tests,standardization of experimental operations,data processing,and presentation and screening layout schemes.By three parallel spikes of three different concentrations of sulfa antibiotics and SM2,the detection limit of sulfa antibiotics indirect competition ELISA is 2.34pqg/L,and the detection limit of SM2 is determined.6.82?g/L.The relative standard deviation of sulfa antibiotics and SM2 is less than 20%,indicating that the indirect competition ELISA precision is good.The calculated average recovery of sulfa antibiotics and SM2 is within 100±20%,the detection accuracy of indirect competition ELISA is very good.