| Microcystins?MCs?belong to a type of small biotoxins which are produced by cyanobacteria,and they are detrimental to waterbody,soil environment,agricultural products and food pollution.Until now,more than 200 MCs have been reported.Among them,the MC-LR isoform is the most toxic with most ubiquity.Also,it has been classified as a possible human carcinogen?Class 2B?for human health by International Agency for Research on Cancer?IARC?,and its minimum residue limit?MRL?was set at a guideline value of 1.0?g/L in drinking water from World Health Organization?WHO?.It has been a hot topic to explore the research of a rapid and high sensitive detection mothod for MCs monitoring.The present doctoral degree study was based on phage display antibody library,antigen-specific antibodies high throughput screening and characteristic modification techniques,and then developing the high sensitivity binding activity of MC-LR genetically engineered antibodies?GEAbs?by multiple ways.In final,the obtained GEAbs were used to establish corresponding immunoassay methods for MC-LR rapid and high sensitivity determination.The specific contents of the study are as follows.Screening and application of MC-LR alpaca nanobody.The MC-LR was conjugated to keyhole limpet haemocyanin?KLH?and bovine serum albumin?BSA?,respectively,then the conjugates were used as coated antigens for enrichment?coated MC-LR-KLH?and screening?coated MC-LR-BSA?of MC-LR phage nanobodies from an alpaca phage display nanobody library.After four rounds of enrichment and biopanning,total 20 positive monoclonal phage display nanobodies were obtained from the library.The most three positive nanobodies genes,ANAb12,ANAb9 and ANAb7were cloned into p ET26b vector,then the nanobodies proteins were expressed in E.coli BL21 respectively.The purified nanobodies,ANAb12,ANAb9 and ANAb7 were used to establish the indirect competitive ELISA?IC-ELISA?for MC-LR,and their limits of detection(LOD-IC10)were 0.06,0.08 and 0.12?g/L,respectively.All of them showed strong cross-reactivity?CRs?of 82.7%?116.9%for MC-RR,MC-YR and MC-WR.It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy and stability,their recoveries were 84.0%?106.5%and coefficient variations?CVs?were 3.4%?10.6%.MC-LR immunized rabbit phage display antibody library construction and its sc Fv screening and application.In this study,MC-LR-KLH conjugate was used as an immunogen to induce rabbit to express anti-MC-LR p Abs,after five rounds of immunization,its serum titer reached to 1:2500000.The total RNA of immunized rabbit spleen was reverse transcribed as c DNA,and then they were used as the template for amplifying VH and VL genes fragments with specifically designed primers,respectively.The VH and VL genes fragments were spliced as the whole sc Fv genes,then the sc Fv genes were cloned into p IT2 phagemid vectors,which were used for transforming into E.coli TG1 electro-competent cells.A MC-LR-immunized rabbit phage display sc Fv library with its capacity of 3.26×109 cfu/m L was constructed successfully.Using MC-LR-BSA conjugate as coat antigen,after four rounds of enrichment and biopanning,total 18 positive monoclonal phage display sc Fvs were obtained from the MC-LR-immunized rabbit phage display sc Fv library.The most positive phage display sc Fv?Rsc Fv3?was transformed into E.coli HB2151 to make sc Fv proteins by soluble expression.The purified anti-MC-LR p Abs and Rsc Fv3-sc Fv proteins based IC-ELISAs were established for MC-LR and its analogues,and the results showed they all had high sensitivity for MC-LR with LOD-IC10 of 0.03 and 0.05?g/L,and had strong CRs of 56%?136%for MC-RR,MC-WR and MC-YR,respectively.The recovery rate were 86.8%?106%with coefficient variation were 1.8%?10.8%for Rsc Fv3-sc Fv based IC-ELISA to detect MC-LR spiked in drinking bottled water,tap water and lake water samples,which indicated the IC-ELISA had good accuracy and stability.MC-LR immunized mouse phage display antibody library construction and its sc Fv screening and application.In this study,MC-LR-BSA conjugate was used as an immunogen to induce mouse to express anti-MC-LR antibodies,after five rounds of immunization,its serum titer reached to 1:3600000.The total RNA of immunized mouse spleen was reverse transcribed as c DNA,and then they were used as the template for amplifying VH and VL genes fragments with specifically designed primers,respectively.The VH and VL genes fragments were spliced as the whole sc Fv genes,then the sc Fv genes were cloned into p IT2 phagemid vectors,which were used for transforming into E.coli TG1 electro-competent cells.A MC-LR-immunized mouse phage display sc Fv library with its capacity of 8.67×107 cfu/m L was constructed successfully.Using MC-LR-KLH conjugate as coat antigen,after four rounds of enrichment and biopanning,total 16 positive monoclonal phage display sc Fvs were obtained from the MC-LR-immunized mouse phage display sc Fv library.The most positive phage display sc Fv?Msc Fv7?was transformed into E.coli HB2151 to make sc Fv proteins by soluble expression.The purified anti-MC-LR Msc Fv7-sc Fv proteins based IC-ELISA was established for MC-LR and its analogues,and the results showed that it had high sensitivity for MC-LR with LOD-IC10 of 0.044?g/L,and had strong CRs of 85.9%and 93.1%for MC-RR and MC-YR,respectively.The recovery rates were 81.2%?106.3%with coefficient variation were 2.62%?10.22%for Msc Fv7-sc Fv based IC-ELISA to detect MC-LR spiked in drinking bottled water,tap water and lake water samples,which indicated that the IC-ELISA had good accuracy and stability.MC-LR heterologous GEAb recombinant and its activity modification and application.In this study,a new anti-MC-LR AVHH-MVH recombinant was constructed based on anti-MC-LR ANAb12-VHH and anti-MC-LR Msc Fv7-sc Fv templates by chain shuffling technology.The binding activity and thermal stability of anti-MC-LR AVHH-MVH recombinant for MC-LR were improved more or less compared with anti-MC-LR ANAb12-VHH and anti-MC-LR Msc Fv7-sc Fv by ELISA detection analysis.A capacity of 8.99×105 cfu/m L of phage display AVHH-MVHmutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3region with specifically designed primers based on AVHH-MVH template.Using MC-LR-KLH conjugate as coat antigen,after three rounds of enrichment and biopanning,total 15 positive monoclonal phage display AVHH-MVH mutants were obtained from the phage display AVHH-MVH mutagenesis library.Among them,total five positive mutants have higher antigen-binding activity compared with the native AVHH-MVHtemplate for MC-LR.The most positive anti-MC-LR AVHH-MVH mutant 3 gene was cloned into p ET25b vector,and then it was transformed into E.coli Rosetta to make AVHH-MVH mutant 3 proteins by soluble expression.The purified anti-MC-LR AVHH-MVH mutant 3 proteins based IC-ELISA was established for MC-LR,and the result showed that it had ultrasensitive binding activity for MC-LR with LOD-IC10 of 0.0075?g/L,which was 5.86-fold higher than anti-MC-LR ANAb12-VHH and 8-fold higher than anti-MC-LR Msc Fv7-sc Fv.In addition,the anti-AVHH-MVH mutant 3 based IC-ELISA shows strong CRs of 90.2%and 122.5%for MC-RR and MC-YR.The recovery rates were 82.68%?99.25%with coefficient variation were 2.64%?10.85%for anti-AVHH-MVH mutant 3 based IC-ELISA to detect MC-LR spiked in drinking bottled water,tap water and lake water samples,which indicated that the IC-ELISA had good accuracy,stability and repeatability.In summary,the present doctoral degree study had successfully obtained anti-MC-LR GEAbs reach up to 70.And it contians alpaca nanobodies,rabbit sc Fvs,mouse sc Fvs and heterologous AVHH-MVHrecombinants,which depending on different design screening paths from commercial natural antibody library,self-constructed MC-LR immuned rabbit/mouse antibody library and site-mutated antibody library based on a high quality GEAb template,which means the study reserves abundant anti-MC-LR GEAbs resources for MC-LR immunological determination research.Further more,all the established IC-ELISAs based on representative highly antigen-binding activity of anti-MC-LR GEAbs showed good LOD-IC10 values,which were far lower than the MRL?the guideline value is 1.0?g/L in drinking water from WHO?.Finally,all these established IC-ELISAs?with good accuracy and stability?are expected to provide accurate risk assessment and safety warning in drinking water,soil environment and even agricultural products and food. |
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