Screening Of Microcystin Alpha Type Anti-idiotypic Nanobodies And The Establishment Of A Novel Immunoassay Method

Microcystin（MC）is a toxic secondary metabolite produced by cyanobacteria.279microcystins had been reported,and MC-LR is one of the most toxic and acutely harmful cyanobacterial toxins known.In this paper,anα-type MC-LR anti-idiotypic phage nanobody was isolated from a camel-derived nanobody library by using an anti-MC-LR monoclonal antibody as the target molecule,and a novel ELISA assay based onα-type anti-idiotypic phage nanobody was established with an improved sensitivity of the assay and low usage amount of anti-MC-LR monoclonal antibody.The results are as follows:（1）Preparation and identification of monoclonal antibodies against MC-LR.The lyophilized anti-MC-LR hybridoma cells were resuscitated and cultured.The ascites were purified by Hi Trap Protein G HP column,and the purification effect was identified by SDS-PAGE.The performance of the purified monoclonal antibody was determined.It was found that the antibody titer was 1:2560,000,and the optimal working concentration of the coated antigen MC-LR-BSA and purified MC-LR monoclonal antibody were 2μg/mL and6.25 ng/mL,respectively.Under the optimal working concentration,the calculated values of the linear response range(IC₂₀-IC₈₀)were 7.0-80.5 ng/mL for MC-LR standards.The IC₅₀and IC₁₀were 13.7 ng/mL and 4.5 ng/mL respectively.The minimum detection limit did not meet the national limit of 1 ng/mL for microcystin in drinking water.（2）The screening and identification of anti-idiotypic antibodies for MC-LR.After three rounds of"binding-washing-eluting-amplification"panning in the camel-derived nanobody library,anti-idiotypic antibodies were isolated by using anti-MC-LR monoclonal antibody as the target molecule.Four positive clones（B5,D11,F7,E8）were successfully screened,among which F7 and E8 had the highest binding activity.Three positive clones with different amino acid sequences were finally confirmed by PCR and DNA sequencing.The three anti-idiotypic nanobodies could bind to anti-MC-LR monoclonal antibodies without affecting the binding of monoclonal antibody to MC-LR,and had no inhibitory effect.Therefore the three anti-idiotypic antibodies were identified as Ab2α.The E8 positive clone with the strongest binding signal was selected for the following experiments.（3）Establishment of a novel immunoassay method for microcystins based onα-type anti-idiotypic phage nanobody.Firstly,the incubation time was optimized for the indirect competition immunoassay method established in the second chapter.The results showed that3h was the optimal incubation time,and the IC₅₀of the detection method was increased by2.97 times.Subsequently,a new immunoassay for microcystin based onα-type anti-idiotypic phage nanobody was established by using the signal amplification effect of the phage capsid and non-competitive inhibitory properties of Ab2αantibody.Under optimal optimization conditions,the linear response range of the optimized ELISA were 1.2-6.9 ng/mL for MC-LR standards.The IC₅₀and IC₁₀were 2.8 ng/mL.and 0.8 ng/mL respectively.As compared with IC₁₀of traditional ELISA,the optimized phage ELISA showed 5.6-fold improved sensitivity.The working concentration of MC-LR monoclonal antibody is reduced from 6.25ng/mL to 2.86 ng/mL,which saved the amount of monoclonal antibody.Soluble expression of E8 was performed,and the IC₅₀of the novel ELISA assay based on soluble E8 was 14.8ng/mL,which did not have the effect of improving sensitivity.It was inferred that the signal amplification effect of the phage capsid improved the sensitivity of the assay.MC-RR,MC-YR and MC-LA were then tested for cross-reactivity.The corresponding cross-reaction rates were 99.2%,107.0%and 48.0%,respectively.Finally,the spiked recovery test for MC-LR was performed on blank tap water samples,and the spiked recoveries of the novel ELISA and HPLC methods were between 89.3-99.3%and 97.1-114.3%respectively,with R²of 0.9535.Good correlation was observed between the two methods.In this paper,the new application ofα-type anti-idiotypic phage nanobodies in improving the sensitivity of immunoassay were explored.A new immunoassay method for microcystins was established,which has the potential for rapid screening and detection of microcystins in drinking water.