Study On The Residue Determination Of Sulfamethoxydiazine By ELISA

Sulfonamides are now used widely as chemical antimicrobials and additives which are often added in animal feeds for animal growth stimulation and for prevention and treatment'of animals diseases. Owing to their characteristics of cheapness,convenience, stability, effectiveness in some animal diseases, sulfonamides are applied in stock; raising industry more and more often. But. sulfonamides will accumulate in animal tissues if they are not used properly and the residues in edible tissues will be potentially harmful to food hygiene and to human health. Therefore, their residues in the edible tissues of food-producing animals such as meat, milk and egg et al. are paid much more attention increasingly. It is stipulated in our country that the total residues of sulfanonamides should not exceed 300ng /ml while in EU the total residues should not exceed 100μg/kg. At present, the chemical methods for detecting sulfonamides residues are mainly as follows: high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and gas chromatography (GC) et al.. Though all these methods are very sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-taking, costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological methods is gradually applied to detecting sulfonamides residues. Sulfamethoxydiazine (SMD) is often used as an antimicrobial and an additive for domestic animals and fowls. It is necessary to establish ELISA analysis protocol for determing SMD' s residues. So, this research is launched to develop the pilot study on the establishment of this ELISA protocol with anticipation to obtain a convenient, speedful and sensitive method for the detection of the residue of SMD. This research is eager to supply some practical and useful data for further study on SMD residue analysis.Bovine serum albumin (BSA) and ovalbumin (OVA) were used as two protein carriers respectively to couple with semiantigen SMD by glutaraldehyde method in this paper. The complete antigens SMD-BSA and SMD-OVA were thus prepared and acted as immunoantigen and coating antigen respectively in ELISA protocol. Selected a few of immunal programs to immunize four healthy experimental rabbits to observe and obtain a suitable, best program of producing antiserum. Determined the property of this antiserum with two-direction agar diffusion test and it showed that antiserum was special to SMD. And the titer of antiserum was 1 : 2560 by ELISA test. Meanwhile ,some ELISA tests were conducted to identify the antiserum with OVA, SMD-OVA, BSA,SMD-BSA coating the microplates separately and the results indicated that the OD of the holes coated with OVA was very low while the OD of the holes coated with SMD-OVA was much high. It was clearly showed that this antiserum was sure to contain the antibody of SMD and this result was up to the need of request of practical application.Indirect competitive ELISA (icELISA) was established with this antiserum. The suitable working condition of icELISA was obtained by several grid tests which were used to calculate the suitable concentration and dilution of the coating antigen, enzyme labelled IgG and antiserum relatively. The most appropriate concentration and dilution of them was 50μg/mL, 1 : 500 and 1 : 100 correspondingly. The standard curve of icELISA was also established and the curve indicated that the lowest detection limit was 63μg/L which was under the demanded detection limit of 100μg/kg (EU) and 300 μg/kg (domestic). The curve had a favorable linearity relation within the concentration range of 10-2000μg/L. However the curve had a rather unfavorable linearity relation at the concentration exceeding over 2000 μg/L.The recovery ratio was favorable to accord with anticipation. One was 94.7% with no background and the other was 82.6% with added background beforehand. On the basis of the established protocol, two hens 20 months old were taken as practical test samples. One h...