Preparation Of Monoclonal Antibody Against Sulfamethazine And Establishment Of Immunoassay Method For Sulfamethazine

Sulfamethazine （Sulfamethazine, SM2）, which belongs to thesulfonamides （Sulfonamides, SAs） is widely used.SM2has the typical structure ofamino benzene sulfonamide.SM2has a broad antimicrobial spectrum,quick effect andlow cost,they are used to prevent and cure animal diseases. Effectiveness of SM2ongrowth of animals, therefore,it is for long-term use as feed additive in animalgrowth.Excessive use of SM2may cause a large number of residues in animal derivedfood.It cause serious damage to human health,also attracted wide attention from allover the world.As a result, the European Union set the maximum value ofsulfonamides in milk and meat food,that is the total amount of sulfonamides must notexceed100ug/kg,the single of sulfonamides shall not exceed25ug/kg. SAS has beenprohibited from been used in feeds in Japanese.Since December of2002, SAS hasbeen set the maximum residue limits of100ug/kg used in all food animal muscle, fat,liver and kidney in bulletin no.235issued by the Ministry of Agriculture of China.China’s agriculture ministry announcement no.235document of sulfonamidesmaximum, and sulfadimidine was monitored as the focus of the veterinary drugresidue.There are so many methods for using to detect sulfamethazine at present,such asmicrobial method,physical and chemical method, and enzyme-linked immunosorbentassay （ELISA）., etc.In these methods, Microbial method are fast speed,but they arelack of sensitivity and specity. Physical and chemical method have the characteristicsof cumbersome sample pretreatment, need expensive instrument and professionaloperation,etc. Enzyme-linked immunoassays is sensitive,rapid,highthroughput,doesn’t need expensive equipment, more suitable for the screening ofa large number of samples at the local level.In present study, sulfamethazine was prepared and conjugated to carrier proteinusing diazotization. The UV scanning,SDS-PAGE and IR scanning were used forconfirming that the SM2had connected to the carrier protein. The coupling ratios of BSA and OVA were13:1and10.5:1, respectively. Two antibodies （MAbs） againstsulfamethazine were prerpared by using SM2-BSA artificial complete antigenimmune BALB/c mine. Through the identification, the titers in the cell culturesupernatant and ascites of3A12were1:5.0×10⁴and1:4.0×10⁵; the titers in the cellculture supernatant and ascites of4H4were1:2.5×10⁴and1:2.0×10⁵.To detect theaffinity constant, K （3A12）=4.9×10⁹L/moL, K （4H4）=8.3×10⁸L/moL,the both celllines had good affinity. The IC₅₀value of3A12and4H4were1.4μg/L and1.8μg/L,respectively.The conditions of ELISA experiment were optimized. The package concentrationof antigen was0.25mg/mL,and the working concentration of antibody was1:2000.The test step method was used one footwork instead of two footwork, makedthe reaction time shortened to20min, under the condition of the reaction, the bestvolume ratio between standard samples and the working concentration of antibodywas100μL:20μL.The dynamic working curves range from0.5-40.5μg/L for sulfamethazine wasobtained by optimizing the system, The equation was y=－0.283x+0.546，r2=0.993,the sensitivity and detection limit are1.4μg/L and0.1μg/L respectively.The average recovery of sulfamethazine added to urine, pork, milk, feed wascarried out by the preliminary established ELISA detection.The contrast test wascarried out with the instrument method and purchased kit. The results showed that theELISA detection method could meet the actual sample testing standards.