Studies On Immunological Rapid Detection Technique Of Ractopamine And Sulfamethazine

Ractopamine(Rac) is a member of the phenethanolamine family of beta-adrenergic agonists(beta-agonists).This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species. Ractopamine also has a powerful effect of promoting pig growth with increased daily weight gain,increased carcass lean percentages,improved feed conversion,decreased accretion of body fat,shortened breeding period.For those purposes,Rac has been usually used as feed additive of pig to improve feed conversion efficiency and enhance lean meat rate.Illegal use of Rac has caused people acute food poisoning. The EU and China have been officially banned the use of ractopamine as growth promoting agents and import of ractopamine contaminated meat.Sulfamethazine is commonly incorporated into pig feed for growth promotion and for control of certain diseases.The sulfamethazine can be retained in tissues of animals that have eaten the medicated diet.The residues of sulfamethazine in pork constitutes a potential hazard for the consumer and may cause allergic reactions, interference in the intestinal flora,and generate resistant populations of bacteria, thereby rendering antibiotic treatment ineffective.Sulfamethazine is also a suspected carcinogen.Research has shown that high concentrations of sulfamethazine may cause thyroid tumors of mice.The US Food and Drug Administration(FDA) requires a 15-day withdrawal period for feed containing sulfamethazine and has set its maximum residue limits(MRLs) in tissues for human consumption as 100ng/g. Recently,ractopamine and sulfamethazine residues have become a great matter of public concern all over the world.The confirmatory methods for ractopamine and sulfamethazine have been established based on GC,LC and MS.These methods are highly sensitive,but require expensive instruments,complicated pretreatments and are also time-consuming.Therefore sample testing on a large scale is difficult.Easier,cheaper and faster detection methods are urgently needed to meet the requirement of large scale and on-site scanning of samples.The aim of this study was to develop simple, rapid,sensitive,specific and reliable screening methods for use in routine monitoring of ractopamine or sulfamethazine residues.In the present study,we focused on the the immunogenicity of BSA-SM2 and BSA-Rac,established nine hybridoma strains effectively secreting monoclonal antibodies(mAbs) against SM2 and Rac respectively.Four ELISA-kits and two rapid test strips for detecting SM2 and Rac residues have been developed and the properties of the kits and strips were confirmed by GC-MS and HPLC.Basis on analysis of molecular structure and immunogenicity of Rac,the active carboxyl group was introduced to phenyl of Rac by chemical modification.The carboxyl group introduced to phenyl of Rac was formed amide bond with amino-group of bovine serum albumin(BSA) or ovalbumin(OVA) to prepare BSA-Rac or OVA-Rac by the mixed anhydride method.Diazotization was used to conjugate SM2 to BSA to obtain BSA-SM2 artificial immunogen.The OVA-SM2 coating antigen was also obtained in the same way.The conjugation rates were measured as about 1:19 for BSA-SM2 and 1:20 for BSA-Rac respectively,by the methods of infrared,ultraviolet and SDS-PAGE.Immunization of BSA-SM2 and BSA-Rac indued specific antibodies in mice.Balb/c mice were immunized with BSA-SM2 and BSA-Rac for five times.The antibody titers both of SM2 and Rac were about to 6.4×10~3 as detected by indirect ELISA.The 50%inhibitive concentrations(IC50) of SM2 and Rac were 9.51 ng/mL and 7.11 ng/mL respectively.The monoclonal antibodies to SM2 and Rac are highly specific apart from that the SM2 antibody crossreact with sulfamerazine slightly,as analysized by methods of blocking ELISA,agar diffusion and colloidal gold blotting.Hybridoma cell lines were established by fusion of NS0 cells and splenocytes from immunized mice using PEG 1500 as the fusion reagent.Nine stable hybridoma cell lines were obtained secreting anti-Rac and anti-SM2 antibodies with high affinities.Four SM2 hybridoma cell lines and the antibodies they secrete were named as SM2H10,SM2E2,SM2A10 and SM2G5.The titers of the hybridoma supematants and ascites were from 3.2×10~2 to 6.4×10~2 and 6.4×10~4 to 3.2×10~5 respectively.The isotypes of SM2 mAbs were IgG2a/κ,IgG1/λ,IgG1/κ,and the affinity constants(Ka) were from 3.71×10~8 to 2.21×10~9 L/mol.The Rac hybridoma cell lines and mAbs were named as RacB5,RacC3,RacE6,RacH1 and RacF4.Titers of supernatants and ascites were from 3.2×10~2 to 6.4×10~2 and from 1.28×10~5 to 6.4×10~5 respectively.The isotypes of Rac mAbs were IgG2a/κ,IgG2b/λ,IgG1/κ,IgG2a/λand the affinity constants(Ka) were from 5.46×10~8 to 3.43×10~9 L/mol.Four ELISA kits(SM2-Kit A,Rac-Kit A,SM2-Kit B,Rac-Kit B) were developed with the mAbs to SM2 and Rac.The calibration curves of both SM2-Kit-A and Rac-Kit A for standard solution were typical sigmoid curves fitted to the four parameters logistic equation with the linear range of 1.0 to 128.0 ng/mL,and their detection limits were 1.0 ng/mL.The recoveries of drugs spiked in feed were 89.4% for the SM2 kit-A and 89.1%for the Rac kit-A.In pig urine samples the recoveries were 90.5%for the SM2 kit-A,91.8%for the Rac kit-A.The coefficient variation (CV) of inter-assay and intra-assay were below 15%.SM2 kit-A generally had a CR of 2.75%to Sulfamerazine.Rac kit A has no crossreactivity with all other copounds detected.The dilution buffer of standard samples had no effect on the detection results of SM2 kit-A and Rac kit-A.The validity of SM2 kit-A in 4℃was above six months. Both SM2 kit-A and Rac kit-A have better detection properties than SM2 kit-B and Rac kit-B.Based on colloidal gold labelled mAbs,rapid test strips were developed to detect SM2 or Rac residues.The calibration curves of SM2-strip and Rac-strip had typical sigmoid curves fitted to the four parameters logistic.The detection limits of both SM2 and Rac were 0.165 ng/mL and 0.154ng/ml on a BioDot-TSR3000 reader,3.0 ng/mL and 2.0ng/mL with the unaided eye.SM2-strip had a lightly CR with Sulfamerazine and no CR to other compounds for Rac-strip.The validity of SM2-strip or Rac-strip in 4℃was above twelve months.The confirmatory experiments of HPLC indicated that the difference between SM2-strip and HPLC was from 1.3%to 4.3%,SM2-kit and HPLC from2.5%to 4.6%. The confirmatory experiments of GC-MS showed that the difference between Rac-strip and GC-MS was from 1.0%to 4.7%,Rac-kit and GC-MS from1.1%to 4.0%.Statistical analysis using student's t-test did not show a significant difference between the the methods we developed and those confirmatory methods.The kits and strips are sensitive,specific,rapid,economical and easy to use.The detection assays could be accomplished within 40 minutes with kits and 10 minutes with strips.Based on the experiment results,the kits and strips are reliable methods for qualitative or semi-quantitative assays of SM2 and Rac.