Production Of Monoclonal Antibody Against Forchlorfenuron And Its Application In Immunoassay

Forchlorfenuron,a synthetic phenylurea-derived cytokinin,is widely used as a plant growth regulator,mainly to increase berry size in fruits such as kiwifruit and grapevine.The widespread use of agrochemicals can result in violative residues in the human food supply,and residue levels for forchlorfenuron in different fruits and vegetables were clearly defined by China government department.Currently,forchlorfenuron residue analysis mainly depends on instrument-based detection methods to ensure the effectiveness of the monitoring system in food risk management.The instrument was stable and the results were reliable and these instrumental detection methods may be limited by the need of expensive and complex instruments,well-trained personnel,and high operation costs.Immunoassay based on the specific binding reaction between antigen and antibody has become a mature technology for trace analysis of biological and environmental samples,which has advantages over the common chromatographic analysis methods including easy performed with labor,high throughput,and quick results in experimental operation.For this,it is crucial to produce antibody with high sensitivity,specificity and strong stability.The subject of this work,therefore,aims at screening monoclonal antibodies that recognize forchlorfenuron.Magnetic-bead ELISA（MB-ELISA）based monoclonal antibody for detection of forchlorfenuron was also established and recombinant antibody（single-chain antibody）was developed to address the disadvantage of high cost and time-consuming in preparation.The present work sheds new light on rapid forchlorfenuron residue detection and the detailed research contents is as follows.（1）Preparation of monoclonal antibodies to forchlorfenuronIn order to prepare highly sensitive monoclonal antibodies against forchlorfenuron.The heterologous coating was used for screening,CPPU-OVA was selected as the immunogen,and the hybridoma supernatants were screened as well as ELISA experimental analysis coated with CPPU-BSA.Through three rounds of screening and subcloning,two hybridoma cell strains stably secreting m Ab against CPPU were generated,named as 14G1 and 16B1 respectively and the sensitivity of antibody produced in ascites by the two hybridoma cell lines was compared by indirect competitive ELISA,and finally 14G1 hybridoma cell line was used for the production of monoclonal antibodies.Monoclonal antibodies produced by hybridoma clones（14G1）were of Ig G1 subtype and the affinity constant was 4.74×10¹⁰L/mol after the ascites was purified by caprylic acid-ammonium sulfate method,which was a high-affinity antibody.After the optimization of the conditions of antigen coating concentration and working buffer related parameters,the sensitivity of the final optimized antibody was 0.0253 ng/m L with good specificity and linear range(IC₂₀-IC₈₀)of 0.005-0.231 ng/m L,which prepared monoclonal antibodies provide important biological material for the construction of forchlorfenuron immunoassays.（2）Development of magnetic bead ELISA method for the detection of forchlorfenuron and comparison of ELISA,MB-ELISAand HPLC methods for the spiked recovery of forchlorfenuron in kiwifruit and grape samplesMagnetic nanobeads-based immunomagnetic separation was used to increase the sensitivity of detection method and a direct competition ELISA reaction system was constructed using complex of magnetic bead streptavidin coupled to biotin forchlorfenuron antigen as the coated antigen and HRP conjugated anti-forchlorfenuron monoclonal antibody as the primary antibody.After the optimization of the relevant parameters,the IC₅₀of MB-ELISA was 0.0061 ng/m L,and the assay was completed within 35min,which greatly shortened the analysis time and did not cross-react with other analogues.The results of the spiked recoveries of conventional ELISA,MB-ELISA and HPLC for actual forchlorfenuron samples of kiwifruit and grapes showed that the conventional ELISA and MB-ELISA matrix were diluted at 200 and 100 times,respectively.The recoveries of spiked samples were 99.0-117.4%,86.0%-120.0%and 82.0%-116.5%,respectively.All intra-assay coefficients of variation were less than 14.3%and verify the practicality and accuracy of the three methods for the detection of forchlorfenuron residues in kiwifruit and grape samples,which can be used for high-throμghput screening of forchlorfenuron residues in fruits.（3）Preparation of anti-forchlorfenuron single chain antibody and preliminary investigations into the mechanisms of molecular recognitionProduction of single chain antibody can provide effective alternative to conventional antibody expression methods.The m RNA was extracted from 14G1 hybridoma cells and reverse transcribed into c DNA,which was used as a template to amplify the heavy chain variable region（VH）and light chain variable region（VL）of the antibody,and the two gene fragments were assembled into a single chain antibody gene（sc Fv）by a linker peptide,and the sc Fv was inserted into p ET32m and p ET32m-MBP vectors and recombinant protein in vitro expression was achieved in E.coli BL21.The protein was purified by Ni column and dialyzed,and the sc Fv-ELISA curve was established with an IC₅₀of 0.13 ng/m L for forchlorfenuron and still retained the binding ability of the original monoclonal antibody,which can be used as an alternative method for forchlorfenuron monoclonal antibody preparation,but it was found that the fusion protein（MBP-sc Fv）lost its ability to bind small molecules of forchlorfenuron.After homology modeling,molecular docking and other computational simulations,it was verified that Asn30（CDR1）,Trp32（CDR1）and Ser99（CDR3）in sc Fv were involved in hydrogen bonding interactions and information and theoretical basis for antibody evolution and immune binding mechanism were provided.Taken together,highly sensitive and specific monoclonal antibodies and single-chain antibodies against forchlorfenuron have been produced;magnetic bead ELISAwas constructed based on monoclonal antibodies to achieve rapid and high-sensitivity analysis and detection;ELISA and MB-ELISA analytical methods can meet the detection of actual samples in this paper.