| Aflatoxin B1widely exists in food crops and feed, which is one of the strongest carcinogensfound so far. Its toxicity is much higher than that in cyanides, organic pesticides etc, whichthreatens life safety of humans and animals seriously. Most of aflatoxin B1detection methodsrely on expensive instruments, such as liquid chromatography, to carry out currently, which isnot conducive to field testing and requires professional staffs to operate these instruments, so thatit takes a long time and high cost of testing. Therefore, it is very significant to develop newdetection methods which is suitable to field testing and takes a short time and low cost.Polyclonal antibodies can not be obtained by immuning directly. because Aflatoxin B1is asmall molecular chemical. Firstly, I transformed AFB1with CMO in order to bring carboxyl tothe position of C3.2mg AFB1was putting into2mL Pyridinemethanol (V/V1:3) solution tomake it react at room temperature directly, which produces oxime compounds AFB1-O.Reactants were identified by thin layer chromatography, UV scanning method and massspectrometry, which leaded to85.7%utilization of AFB1and209,220,264,360nmwavelength of UV absorption. AFB1-O was transformed into AFB1-O-BSA and AFB1-O-OVA bycoupling with bovine serum albumin and egg albumin in the role of EDC or DCC.Polyclonal antibody serum was got by immuning mice with immunogen AFB1-O-BSA,which was purified by caprylic acid ammonium sulfate and dialysis method. It showed2bandswhen the purified serum went by SDS-PAGE electrophoresis. The serum titer was determined byindirect ELISA method, and titers of6mice were over6000. The titer of No3mouse was about10000, the antibody concentration was1.37mg/mL, standard curve equation of IC-ELISA wasy=-43.50x+99.37(R2=0.988), and IC50was12.59ng/mL.Colloidal gold was prepared by reducting HAuCl4with sodium citrate. Orthogonalexperiments were made on the conditions of putting2mL1%sodium citrate into50mL0.01%HAuCl4based on different reaction time pH and rotation speeds, which leaded to the bestreaction conditions: no acid and alkaline substance,100r/min rotation speed and10min reactiontime after boiling. Average diameter of colloidal gold particle was14.6nm under the electronmicroscope and the UV absorption wavelength was518.5nm. By computing, the particleconcentration was6.8×1010/cm3, the molar concentration was1.13×10-10mol/L, every colloidalgold particle consists of9.64×105gold atoms and the critical flocculation concentration was0.8g/mL. The testing strip mainly consists of a sample pad, a gold labeled pad, a NC membrane and aabsorbent paper. The optimum amount of labeled antibody and the optimum pH were defined byvisual method and photoelectric colorimetry. The optimum amount of labeled antibody was55.2μg/mL, and the optimum pH appeared after putting12μL0.1mol/L potassium carbonate into1.0mL colloidal gold. The UV absorption peak wavelength of prepared colloidal gold antibodycomplexes was528.5nm. The gold labeled pad was made by leaching method,1mg/mLAFB1-O-OVA was labeled on the detection line,1.2mg/mL goat anti-mouse secondary antibodywas labeled on the quality control line, and the sensitivity of assembled testing strip was250ng/mL. |
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