Preparation Of BoNT/A Antibody And Establishment Of Pharmacokinetic Detection Methods

Botulinumneurotoxin（BoNT）is a protein neurotoxin secreted by Gram-positive anaerobic Clostridium botulinum under suitable conditions.It is one of the most toxic bacterial toxins known,There are seven different types of A-G,among which BoNT/A is the most toxic and most common.Currently,There are no chemical drugs that can effectively treat botulinum toxin poisoning.horse serum is often used in clinical treatment.However,horse serum is prone to contamination,long preparation cycle,unstable yield,and as a heterologous substance,it is easy to trigger hypersensitivity Can’t be a standing medicine.Therefore,the development of monoclonal antibodies has become the main research direction of toxin poisoning treatment in recent years.The laboratory has carried out the research and development of botulinum toxin prevention and treatment drugs,and early screening has obtained more than 60 strains of monoclonal antibodies against BoNT/A.The neutralizing activity of BoNT/A can reach1000LD₅₀/mg when 1F11、2D2 and 6B3 are used together.And it was preliminarily determined that the epitopes corresponding to 1F11,2D2 and 6B3 and toxin were located on H_CC,H_CN,and LH_N,respectively.The purpose of this study is to prepare 1F11,2D2,6B3monoclonal antibodies on the basis of previous studies,and then to detect the affinity constant between the antibody and the BoNT/A domain and establish an ELISA method to detect antibody concentration for pharmacokinetic studies.At the first,the preparation of antigen protein.The antigen protein consists of two parts.The first part is the preparation for BoNT/A domain H_CC,H_CN,H_C and LH_N proteins.The pTIG-H_CC,pTIG-H_CN,pTIG-H_C,and pTIG-LH_N were transformed into E.coli BL21（DE3）,respectively.After induction by IPTG,recombinant proteins of H_CC,H_CN,H_C,LH_N were obtained.The recombinant protein was purified by affinity chromatography column and analyzed by Gelpro Analyzer software,and the purity was above 90%.The second part is the preparation of full-length BoNT/A protein.In this study,srtA enzyme ligation method was used to prepare full-length BoNT/A protein.srtA is a sort of sortase from Staphylococcus aureus that can link proteins containing LPETG and Gly-Gly-Gly-（G-for short）sequences.The G-H_C,LH_N-LPETG（Thrombin）and srtA genes were amplified by PCR.The PCR products were cloned into pTIG plasmids to construct pTIG-G-H_C and pTIG-LH_N-LPETG（Thrombin）recombinant expression vectors.After successful construction,they were transformed into E.coli BL21（DE3）to express recombinant proteins G-H_C,LH_N-LPETG（Thrombin）,and srtA.The protein purified by affinity chromatography column was analyzed by Gelpro software,and the purity was above 90%.The full-length BoNT/A was obtained by srtA ligation method,and the full-length BoNT/A was obtained by Thrombin digestion of the full-length BoNT/A.SDS-PAGE and Western Blot identification showed a clear band that was consistent with the expected protein size.The prepared BoNT/A was injected into mice by intraperitoneal injection to evaluate the virulence.The results showed that the prepared BoNT/A antigen was active and the virulence was further enhanced by Thrombin digestion.Secondly,antibodies were prepared,and 1F11,2D2,and 6B3 antibodies were prepared by HEK-293F cell expression system.The antibody light chain and heavy chain expression vectors constructed in the previous stage were transiently transfected into HEK-293F cells for expression.The purified antibodies were identified by SDS-PAGE and analyzed by Gelpro Analyzer software,and the purity was above 90%.Using BIAcore to detect the affinity constants of 1F11,2D2,and 6B3 antibodies,the results are as follows:the affinity constant between 1F11 and H_C is 1.378×10^-12,the affinity constant between 2D2 and H_C is 7.094×10^-11,and the affinity between 2D2 and H_CN The constant is 1.256×10^-8,and the affinity constant between 6B3 and LH_N is 5.615×10^-10.The last part is to establish an ELISA method for detecting antibody concentration for pharmacokinetic studies.To detect 1F11 antibody,H_CC was used as coating substrate to establish ELISA method;H_C was used as coating substrate to establish 2D2 antibody detection method;LH_N was used as coating substrate to establish ELISA method to detect 6B3 antibody.Determine the standard curve and linear range of each detection method and evaluate the recovery rate,coefficient of variation（CV%）,sensitivity,serum interference level and the effect of linear dilution.The results are as follows:the linear range of the 1F11 detection method is 0.5 ng/mL-6 ng/mL;the linear range of the 2D2 detection method is 3.5 ng/mL-25ng/mL;the linear range of the 6B3 detection method is 4 ng/mL-20 ng/mL.The recovery rate of the above detection methods is between 90%-110%,and the CV%is less than 15%.It has good precision,sensitivity,no interference of serum and linear dilution does not affect sample detection.In summary,in this study,various partial domain proteins of BoNT/A and antibodies of1F11,2D2,and 6B3 were prepared,and the affinity constant between the domain and antibody was detected by BIAcore;the full-length BoNT/A was prepared by enzyme ligation method;The antibody concentration detection method with good performance provides a basic tool for subsequent pharmacokinetic research.