Preparation Of The Generic Monoclonal Antibody For Phenothiazines And The Single Chain Fragment Viarable Antibogy For Amoxycillin

With the development of animal husbandry, the use of veterinary drugs and feed additives showes a rising trend year by year. For example, some feeds producers add phenothiazine drugs into feeds in order to increase the weight gaining, or use phenothiazines to the animals during transport to reduce the animal’s weight loss and mortality, or use penicillin drugs to treat various bacterial induced deseases. However, the use of these drugs in food producing animals can cause their residues in foods of animal origin that are dangerous to the consumers. The objective of this study is to prepare the monoclonal antibody for phenothiazine drugs and the single chain fragment viarable(Sc Fv) antibody for amoxicillin, which could provide the basis for the further preparation of the immunoassayes.During the experiments, 2-chlorophenothiazine(Ch PZ) was derivatized with bromoacetic acid to synthesize the hapten(Ch PZD) that was then conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to prepare the immunogen(Ch PZD-BSA) and the coating antigen(Ch PZD-OVA). The immunogen Ch PZD-BSA was used to immunize Balb/c mice, and the serum titer and specificity were measured by indirect ELISA. The mouse spleen cells with the high serum titer and specificity for chlorpromazine were fused with myeloma cells, and the hybridoma cells were screened by indirect ELISA method. After several times of screening, two positive hybridoma cell lines were obtained. The ascites were purified by saturated ammonium sulfate and Protein A affinity chromatography to obtain the monoclonal antibody that simultaneously recognized 5 phenothiazines(Chlorpromazine、Perphenazine、Promethazine、Acepromazine、 Triflupromazine).During the experiments, the total RNA from a hybridoma cell stain secreting the anti-amoxicillin mononlonal antibody were extracted, which was used to reverse transcrib the c DNA. Then the heavy chain gene fragment(VH) and light chain gene fragment(VL) were obtained by PCR method that were used to construct the Sc Fv gene fragment. The Sc Fv gene and expression plasmid PET-32 a were linked to obtain the recombinant expression vector that was transformed into expression competent strain BL21(DE3) cell to generate the anti-amoxicillin Sc Fv antibody with His tag.In this study, the monoclonal antibody for phenothiazines and the Sc Fv for amoxicillin were obtained, which should be valuble for the effective monitoring the residue of phenothiazines and amoxicillin in animal derived foods.