Development Of Gentamicin ELISA Kit And Preliminary Establishment Of Nanobody Mass Spectrum Screening Platform

Gentamicin（GM）is an aminoglycoside antibiotic,which is widely used in veterinary clinic because of its good antibacterial effect.Consequently,gentamicin residues may exist in food and raw materials,with ototoxicity,nephrotoxicity and other potential hazards.Therefore,it is urgent to develop a rapid,sensitive and accurate method to detect gentamicin residues in order to monitor and ensure the safety of animal-derived food.The immunological assay is characterized by convenience of operation,high specificity and sensitivity,in which monoclonal antibody and nanobody are the main immune components.Based on the characteristics of convenient operation,high specificity and sensitivity of immunological analysis method,this study used anti-GM monoclonal antibody to prepare GM indirect competitive ELISA kit,and realized the detection of GM in actual samples.The main antibodies used in enzyme-linked immunosorbent assay are monoclonal antibodies and nanobodies,among which nanobodies have the advantages of easy transformation and easy expression,and have great development potential in the field of small molecule immunoassay.In this study,anti-GM nanobodies were prepared by using this advantage.In order to improve the screening efficiency of nanobodies,a mass spectrum screening platform for nanobodies was initially established based on the advantages of high sensitivity and high accuracy by ultra-high performance liquid chromatography tandem mass spectrometry.Specific research results are as follows:1.Development and application of gentamicin ELISA kit.Based on anti-GM monoclonal antibody,an indirect competitive ELISA method for GM detection was successfully established and the ELISA kit was prepared.The IC₅₀of detection method was 0.724 ng/m L.The evaluation of the standard curve of indirect competitive ELISA method shows that the proposed method has high sensitivity and accuracy.It is proved that the ELISA kit developed in this study is feasible to detect gentamicin residue in actual food through the spiking experiment.2.Construction and panning of phage display library for anti GM nanobody.We immunized alpaca with GM-BSA complete antigen,isolated PBMC from peripheral blood of alpaca after the sixth immunization.Then we constructed a 3.8×10⁹ cfu original library of anti-GM nanobody,and obtained a phage display library with a titer of 1.56×10¹³ cfu/m L by M13K07 rescue.Using GM-OVA as panning antigen,14positive phages were detected by Phage-ELISA after four rounds of immobilization and panning,and one specific binding phage was obtained by indirect competition with Phage-ELISA.By constructing recombinant vector p ET-25b-148,the vector was transferred into Rosetta（DE3）receptor cells,expressed by prokaryotes and purified to obtain 148 protein.The standard curve of indirect competitive ELISA was established.The IC₅₀of detection method was 11.168μg/m L.3.Preliminary establishment of mass spectrum screening platform for nanobodies.Alpaca serum was purified by GM affinity purification column and then separated by Protein A and Protein G affinity chromatography column to obtain Ig G2 and Ig G3.Subsequently,we set up a UPLC-MS/MS method,and the feasibility and accuracy of the method were verified with the standard of Escherichia coli trypsin hydrolysis.The samples Ig G2 and Ig G3 were hydrolyzed with trypsin and analyzed by UPLC-MS/MS.The data set contained 27931 fragment ions.The data processing software identified140 peptide segments of the sample based on the personalized database,which were matched to 14 proteins.Seven of the protein sequences were consistent with those of the nanobodies panned from the phage library.