Preparation And Immunoassay Application Of ?-lactam Antibiotic Receptors And Polypeptide Antibiotic Antibodies

China is a big country in livestock and poultry breeding,and also a big country in antiobitic production and use.The excessiove use and abuse of antibiotics lead to serious residues in animal-derived food,which can human cause allergic reactions,dysbacteriosis,antibiotic resistance and so on.Antibitic resistance is a global public problem facing the world in the 21^st century.In order to prevent and control the generation and spread of drug-resistant genes,many countries and relevant institutions have successively issued“restrict antibitics,ban antibiotics”orders.Therefore,continuous monitoring of antibiotic residues in food-borne foods is of great significance.The test strip technology based on nanomaterial biomarkers has the advantages of high sensitivity,simple operation,quick analysis,low cost and so on,and therefore is particularly suitable for rapid screening of massive samples.In this study,?-lactam antibotics and polypeptide antibiotics were chosen as the research objects.Based on receptor of lactam antibitocs and antibodies of polypeptide antibiotics,multi-residues test strips were respectively developed for lactam antibitocs and polypeptide antibiotics.The receptors PBP2x?Thr52-ASP750?of Streptococcus pneumoniae R6,the Bla R-CTD?Met346-Arg601?of Bacillus licentius ATCC14580 and its mutant Bla R-CTD?I188K/S19C/G24C?,and the mutant Bla R-CTD?Gly331-Gln585,N439V?of Staphylococcus aureus NRS128 were expressed in E.coli by fusion a 6×His tag at the N-terminus,which were respectively denoted as PBP2x',Bla R-CTD?14580?,Bla R-CTD-M?14580?,and Bla R-CTD-M?NRS128?.With Amp-BSA as the antigen and HRP-labelled anti-6×His tag antibody as the secondary antibody,four receptors were characterized.As a result,receptor Bla R-CTD-M?14580?exhibited the highest affinity and stability.By optimizing the induced expression conditions,the soluble expression level of Bla R-CTD-M?14580?was up to 8 mg/100 m L at 0.2 m M IPTG and 20°C for 18 h.Anti-Bla R-CTD-M?14580?monoclonal antibody?m Ab 6B11?was produced by immunizing mice,with an affinity constant?Ka?of 4.5×10¹⁰ L/mol.Then,recptor Bla R-CTD-M?14580?was directly labelled by 35 nm gold nanoparticles?GNPs?or indirectly labelled by GNPs with m Ab 6B11 as a linker to respectively prepare a binary or ternary marker.Respectively based on the two markers,two colloidal gold chromatographic strips were established for the detection of?-lactam antibotics residues in milk and chicken samples.The test strips based on binary marker could detect 18?-lactams except penicillin G,amoxicillin,cefalexin,cefalexin and cefazolin in milk samples and 21?-lactams except cefazolin,cefazolin and cefazolin in chicken samples.The test strips based on ternary markers could detect 33?-lactams including the above-mentioned drugs in milk samples,with the visual detection limit?v LOD?or cut-off values less than or equal to the maximum residue limits?MRLs?set by the European Union.Complete antigens of colistin?COL?,polymyxin B?PMB?and bacitracin?Baci?were synthesized using protein crosslinking agents including glutaraldehyde,4-maleimide butylate-N-succinimide,carbodiimide and N-hydroxysuccinimide,and then m Abs were prepared by immunizing mice.For COL specific m Ab?1C10?,the half inhibition concentration(IC₅₀)and Ka values were 2.67 ng/m L and 1.9×10⁹L/mol.For PMB specific m Ab?2A2?,the IC₅₀ and Ka values were 15.26 ng/m L and 8.95×10⁸L/mol,respectively.For Baci specific m Ab?4B11?,the IC₅₀ and Ka values were 18.3 ng/m L and 2.6×10⁸L/mol,respectively.For COL and PMB broad-spectrum m Ab?2A6?,the Ka value was 4.04×10¹⁰L/mol,the IC₅₀ for COL and PMB were 4.44 ng/m L and 4.2 ng/m L,respectively.Fluorescent microspheres were applied to label Baci and COL specific antibodies,respectively.Then a fluorescence immunochromatographic asay?FICA?was established for simultaneously testiong Baci and COL residues in milk samples.The cut-off values of Baci and COL were 100 and 50 ng/m L,respectively;the quantitative detection limits?q LOD?were7.85 and 1.89 ng/m L,respectively;and the detection linear ranges were 7.85–28.42 ng/m L and 1.89–5.81 ng/m L,respectively.The linear ranges were narrow,but the FICA can be used for simultaneously qualitative analysis of COL and Baci.In addition,a colloidal gold immunochromatographic assay?GICA?for the combined detection of COL and PMB in milk was established based on the broad-spectrum m Ab?2A6?labelled by GNPs.The cut-off values of COL and PMB were 50 and 100 ng/m L,respectively;the q LOD were 0.53 and 0.94ng/m L,respectively;and the linear ranges were 0.53–61.48 ng/m L and 0.94–480.93 ng/m L,respectively.With wide linear ranges,the GICA can be applied for the combined quantitative detection of COL and PMB.