| The safety of animal-derived food, especially due to the veterinary drugs residues, have been paid more attention all over the world and become the main agents of economy disputes in the international trades recently. The Medroxyprogesterone Acetate(MPA) feed pollution accidents in 2002 gave a strong impact to the confidence of consumers on food safety again in European Union(EU), and influenced widely in the word.MPA is a kind of synthetic progesterone derivative drug, which was widely used in young women for contraception and treatment of a number of gynecological conditions, and combination hormone therapy for postmenopausal women. In order to improve fertility, MPA was also used in husbandry and animal breeding industry in some countries. MPA has been banned not to use in food-producing animals by any way and was prohibited to be detected from animals and animal-derived foods in many countries such as China, EU, etc, because its residues in food may lead to breast cancer,infertilitas, and other adverse reactions. It is necessary and important to strengthen the monitoring and inspection of MPA residues for the quality control of import-export products. In order to develop the immunoassay technology for the detection of MPA, the indirect competitive ELISA based on the monoclonal antibody(McAb) against MPA was studied in this paper, which may be summarized as follows:1. According to molecular structure of MPA, one intermolecular spacerarm with 4 carbon atoms was designed through oximation and acylation reactions, and MPA hemi-succinate (MPA-HS) was produced by the reactions, hydroxylamine hydrochloride and MPA, oximated MPA and succinic anhydride. then MPA-HS was coupled to protein carriers, bovine serum albumin(BSA) and ovalbulmin(OVA) by carbodiimide method, and the mixed acid anhydride method in different reaction conditions respectively. One artifical immunogen(MPA-BSA)and coating antigen(MPA-OVA) were selected through animal(Balb/c mice)-inoculation immunization test and ELISA, then were identified by full-wavelength ultraviolet scanner and SDS-PAGE. The results showed that the MPA-BSA and MPA-OVA were successfully synthesized, with good immunogenicity and reactiongenicity, and the conjugation molecular ratio of MPA with BSA and OVA were about 12:1 and 8:1 respectively. The two artificial antigens in this section supplied ideal materials for the preparation and identified of McAb against MPA.2. Through optimization of coating condition, incubation time, dilution of goat anti-mouse IgG-HRP(secondary antibody), reaction buffer system and other conditions, the reaction conditions of indirect ELISA and inderect competitive ELISA for the screening and identification of McAb against MPA were determined, by using MPA-OVA, anti-MPA antibody positive Balb/c serum, and the Balb/c serum immunized with normal saline(NS) in section 1 separately as coating antigen, positive control and negative control. The Balb/c mice immunized 3 times with MPA-BSA(in section 1) were immunized for the 4th times, and the immunized effects were measured by indirect ELISA and competitive ELISA, 10 days later using the best immunized Balb/c for strengthen immunization, and then the monoclonal antibodies(McAb) against MPA was studied by the classical McAb preparation method, including cell fusion with PEG1500, screening of fusion cells, cloning and sub-cloning, identifying the main characteristics of McAb against MPA. The results showed one hybridoma cell line named M68F9 was obtained, which grew stably and secreted high titer anti-MPA McAb, the titers of anti-MPA McAb determined by indirect ELISA in cell culture supernatants and ascites were more than 1:1500 and 1:1×107 respectively, the McAb against MPA belongs to IgG1 subclass determined by antigen-capture ELISA, the concentration of MPA that inhibits 50% of antigen-antibody binding(the value of IC50) was 2-4 ng/mL, the cross-reactions rates of megestrol acetate (MA), Clenbuterol, Rac and DES were 25%, <0.01%,<0.01% and <0.01% respectively by inderect competitive ELISA. Preparation of anti-MPA McAb with good sensitivity and specificity sets basis for developing MPA residues immunoassay analysis methods.3. To improve the stability of immunoassay analysis methods for MPA residues detection, Protein G Sepharose 4 Fast Flow-affinity chromatography was employed to purify the anti-MPA McAb in ascites, and the purified McAb proved to be with good purification and reactive ability identified by SDS-PAGE and indirect ELISA. Using artificial antigen MPA-BSA as coating antigen, 1% BSA PBS as blocking buffer, a indirect competitive inhibition ELISA(ciELISA) method for the quantitative determination of MPA was developed through the optimization of the dilution of coating antigen, anti-MPA McAb, and goat anti-mouse IgG-HRP(secondary antibody), reaction condition, and other components respectively. Then the sensitivity, specificity, precision and storage of this method were evaluated, and was compared with the commercial MPA EIA Kit(5131MPA1p[3]08.05) in the primary application test of spike samples with different concentration MPA and field samples. Results showed the IC50 of the calibration curves(R2=0.9925)of this ciELISA method was about 3.3914 ng/mL, the linear range was from 1 ng/mL to 80 ng/mL, and the limit of detection was 0.1-0.5 ng/mL; the cross-reactivity was 25% with MA, while the others were all <0.01%, and this method was with excellent precision, the intra-assay and inter-assay coefficient of variation were both <10%, the correlation parameters and the detection results of this methods are concordance with the MPA EIA Kit method. All these indicated the ciELISA method developed in this study was with practicability, and can meet the need of the detection of MPA.The McAb against MPA and ciELISA method successfully developed based on the McAb in this paper were the first reports in our country. It offered a good technique platform for developing MPA residues immunoassay analysis methods, and set basis for the production of MPA detection EIA kit with our own property. |
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