Screening And Preparation Of Monoclonal Antibody Against Sulfaguanidine

Sulfaguanidine (SG)belongs to the family of sulfa drugs, which is commonly used in the animal industry to treat enteritis and bacillary dysentery. As a favorable entero-antiseptic, sulfaguanidine has wide applications in fishery. However, SG is toxic, as it can cause kidney damage, blood abnormalities, and reduce the growth rate of mammalian species. So it is necessary to established rapid method to detect sulfonamides residues in aquatic animals.At present, the most common method to detect SG residues is chromatography, such as high performance liquid chromatography (HPLC) and high performance liquid chromatography coupled to a mass spectrometer (HPLC-MS). However, these methods are time-consuming, labor-intensive and require expensive equipments that are available only in laboratories, and therefore, are not suitable for convenient market monitoring. Enzyme-linked immunosorbent assay (ELISA) is demonstrated as a simple, rapid, cost-effective and highly sensitive method in quantitative detection of sulfonamide residue. In order to establish the ELISA assay method, it is necessary to get the monoclonal antibodies against sulfaguanidine residues.In this study, Hapten sulfaguanidine (SG) was coupled with carrier protein bovine serum albumin (BSA) to form a full antigen SG-BSA by diazotization and glutaraldehyde methods. Ovalbumin (OVA) was used as protein carriers to couple with Hapten SG by glutaraldehyde method. Then, the immunogen and the coating antigen were identified with SDS-PAGE, UV-scanning and Infrared spectrometry (IRS), and purified by dialysis and gel exclusion chromatography.The conjugated ratio of SG to BSA in artificial antigen was 8.5 (using diazotization method) and 6.0 (glutaraldehyde method), and the conjugated ratio of SG to OVA in coating antigen was 4.0 (glutaraldehyde method) by UV-visible spectrophotometer. The coupling was successful according to the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), UV-scanning and Infrared spectrometry (IRS). BALB/c mice were immunized with the antigen (SG-BSA), and the titers of antiserum were tested to be 1:12800 by indirect ELISA.The fresh spleen cells of immunized mice were used for fusion with myeloma cells. The confluent cell clones emerged in seven days after confusion. When the confluent cells have occupied 70% of a well bottom at thirteen days, positive cells were determined using indirect ELISA. Subclone the positive cells by limiting dilution for three times to get monoclonal cell clone. The efficiency of confluent cells was 67.9%, the rate of positive clone was 1.1%. At last, we got one positive hybridoma cells named 4D11.The immunigen and coating antigen were obtained by diazotization method and glutaraldehyde method, and the specific monoclonal antibodies against SG were prepared with cell fusion technic, which laid a foundation of getting a detective kit of SG residues.