Rapid Immunoassay For Detecting Polypeptide Antibiotics?phenothiazines?new "Clenbuterol" And Other Veterinary Drugs

In recent years,veterinary medicine has been extremely widely used and increased production and efficiency of food animals with the rapid development of animal husbandry.However,their residues in animal-derived food have become an increasingly serious global public health problem.Improper use of veterinary drugs will cause the residues in animal edible tissues,eggs and dairy products.It not only affects international trade and causes economic losses,but more importantly,it could bring potential risks to consumer health.In order to meet the needs of detecting and controlling veterinary drug residues in food animals,simple,inexpensive,sensitive,fast and high-throughput monitoring and analysis methods will be established urgently.This study mainly focused on the peptide antibiotics bacitracin zinc(BAC)and polymyxin(POL),the pleuromutilin antibiotics tiamulin(TIA)and vonimirin(VAL),the antiparasitic drugs nitroxinil(NIT),closantel(CLT)and rafoxanide(RAF),non-steroidal anti-inflammatory drugs diclofenac(DIC)and carprofen(CAR),antihistamines cyproheptadine(CPH)and phenothiazines(PZs)to design and synthesize artificial antigens and prepare high-sensitive monoclonal antibodies.Based on these,the corresponding immunological detection methods were established.(1)According to the structural characteristic of BAC,the artificial antigen BAC-BSA was designed and synthesized,and two anti-BAC monoclonal antibodies 6D2-G10 and 3F2-F11 were obtained.Both of the antibody subtypes of them were IgGl.However,6D2-G10 exhibited higher sensitivity,and IC50 value was 0.59 ng/mL.The affinity constant of 6D2-G10 was 6.69×109 L/mol,and 6D2-G10 had almost no cross-reaction to other structural analogs.Based on 6D2-G10,we established a colloidal gold immunoassay strip for rapid monitoring BAC residues in milk,the limit of detection by the naked eye(cutoff value)was 25 ng/mL.Take a reading by a strip reader,the standard inhibition curve of BAC was established.The IC50 was 3.16 ng/mL.The limit of detection was 0.82 ng/mL.The average intra-assay and inter-assay recovery rates were from 84.3%to 96.0%,the coefficients of variation(CVs)were from 4.9%to 9.1%.(2)The artificial antigen POL-E-BSA was designed and synthesized,and a monoclonal antibody 4H11-C3 that can recognize POL-E and POL-B was obtained.The antibody belonged to IgGl subtype and the affinity constant was 3.2×109 L/mol.Based on 4H11-C3,we established an ic-ELISA method to detect POL-E and POL-B residues in milk samples.Most of the matrix effect interference could be reduced by 4-fold dilution of milk.The IC50 for POL-E was 0.21 ng/mL.The linear detection range was from 0.03 ng/mL to 1.44 ng/mL,and the limit of detection was 0.02 ng/mL.The IC50 for POL-B was 0.40 ng/mL,the linear detection range was from 0.06 ng/mL to 2.65 ng/mL,and the limit of detection was 0.03 ng/mL.The average intra-assay and inter-assay recovery rates were from 89.3%to 103.4%,and the CVs were from 3.9%to 6.9%.(3)According to the common three-ring structure of VAL and TIA,the artificial immunogen VAL-BSA was successfully synthesized and a highly sensitive monoclonal antibody 1B7-G10 recognizing both VAL and TIA was obtained.The antibody belonged to IgG1 subtype and the affinity constant was 1.28×109 L/mol,and there was no cross-reaction to other structural analogs RET,TYL,AVE,IVE and DOR.The IC50 values of VAL and TIA tested by ic-ELISA were 0.99 ng/mL and 0.39 ng/mL,respectively.Based on 1B7-G10,a colloidal gold test strip was developed for the qualitative and quantitative detection of VAL and TIA in porcine liver,the cutoff values were 50 ng/g and 25 ng/g,respectively.The IC50 values calculated from the standard curves were 6.06 ng/g and 3.45 ng/g,the limits of detection were 0.96 ng/g and 0.29 ng/g,the average recovery rates were from 80.7%to 102.0%,the CVs were from 4.6%to 10.8%.(4)According to the structural characteristics of NIT,CLT and RAF,the artificial immunogens NIT-BSA and CLT-BSA were designed and synthesized,and an anti-NIT monoclonal antibody cell line 8G7-C6 and a cell line 1E3-A10 that could recognize CLT and RAF simultaneously were obtained.Both the monoclonal antibodies were IgGl subtype,with affinity constants of 2.93×109 L/mol and 8.90×109 L/mol,respectively.The IC50 of 8G7-C6 detected by ic-ELISA was 0.96 ng/mL,and the antibody had no cross-reactivity to other structural analogues.The IC50 of 1E3-A10 against CLT was 0.28 ng/mL,and the IC50 against RAF was 0.39 ng/mL.Based on 8G7-C6,we successfully established a colloidal gold test strip for the detection of NIT residues in milk.The cutoff value was 50 ng/mL,the IC50 calculated from the standard curve was 5.71 ng/mL,and the limit of detection was 1.14 ng/mL.The average recovery rates in milk were 84.6%-106.9%,and the CVs were from 4.8%to 9.9%.Based on 1E3-A10,we established the ic-ELISA method to detect CLT and RAF in milk.The IC50 values were 0.28 ng/mL and 0.33 ng/mL,the limits of detection were 0.03 ng/mL and 0.05 ng/mL,and the linear detection range were from 0.05 ng/mL to 1.63 ng/mL and 0.08 ng/mL to 1.38 ng/mL.(5)According to the structural characteristics of DIC and CAR,the immunogens DIC-BSA and CAR-BSA were designed and synthesized by the active ester method.One anti-DIC monoclonal antibody 1E5-B12 and one anti-CAR monoclonal antibody 1F5-C8 were successfully obtained,and both the two antibodies were IgGl subtype,the affinity constants were 1.72×109 L/mol and 6.19×109 L/mol,respectively.According to ic-ELISA,the IC50 of 1E5-B12 was 3.14 ng/mL,and the IC50 of 1F5-C8 was 0.10 ng/mL.Based on 1E5-B12,we established a colloidal gold test strip method to detect DIC in milk.The cutoff value was 125 ng/mL.Detected by a strip reader,the IC50 was calculated as 8.54 ng/g and the limit of detection was 1.40 ng/g.The average recovery rates of DIC added to milk were from 78.4%to 104.3%,and the CVs were from 4.7%to 9.5%.Based on 1F5-C8,we established a colloidal gold test strip to detect CAR in bovine muscle,with a cutoff value of 12.5 ng/g.The IC50 was calculated as 1.74 ng/g,and the limit of detection was 0.28 ng/g.The average recovery rates were from 79.5%to 90.6%,and the CVs were from 5.9%to 8.8%?(6)The hydroxylated CPH(COH)was modified by bromoacetic acid substitution method to introduce carboxyl group,and the active ester method was used to couple it with BSA to prepare the immunogen CPH-BSA.A broad-spectrum high-sensitivity monoclonal antibody 10D3-C3 belonging to IgG1 subtype was obtained.The affinity constant was 7.35×109 L/mol.The antibody can simultaneously recognize CPH and 6 PZs(APZ,FPZ,CPZ,PPZ,PTZ,and TDZ).The IC50 values against CPH and PZs were from 0.03 ng/mL to 0.76 ng/mL by ic-ELISA.Based on 10D3-C3,we developed a colloidal gold test strip for detecting CPH and PZs in feed.The cutoff values were from 5 ng/g to 100 ng/g,the calculated IC50 values were from 0.57 ng/g to 7.75 ng/g,and the limits of detection were from 0.10 ng/g to 1.35 ng/g.The average recovery rates of CPH and PZs added to the feed were from 79.0%to 103.4%,and CVs were from 4.5%to 12.7%.