Preparation And Identification Of Monoclonal Antibody Against Botulinum Toxin Type E

Botulinum neurotoxins(BoNT)is a very toxic protein neurotoxin produced by Clostridium botulinum,and it is currently the most toxic toxin known in nature among biological and chemical toxins.BoNT poisoning can block the release of neurotransmitters and cause flaccid muscle paralysis.In severe cases,it can cause death by asphyxiation.So far there is no effective chemical drug to treat BoNT poisoning.Horse-derived antitoxin is often used clinically.However,BoNT antitoxin has many defects and its clinical application is greatly restricted.It is necessary to find better therapeutic drugs to replace horse-derived antitoxin.The cloned antibody becomes the future star in the treatment of BoNT poisoning.There are 7 known serotypes of BoNT,of which types A,B,E,and F can cause human poisoning.Previous studies have mostly focused on types A and B.There are very few studies on BoNT/E,especially in monoclonal antibodies.There are fewer studies in this area,so no antibodies for the treatment of BoNT/E have been marketed so far,so screening of neutralizing antibodies against BoNT/E is of great significance.This study aims to screen and evaluate and identify mouse-derived neutralizing monoclonal antibodies with strong specificity and high affinity for BoNT/E through hybridoma technology.In order to prepare monoclonal antibodies to botulinum toxin,firstly construct recombinant antigens,first express and identify the antigens,then immunize animals with the recombinant antigens,screen monoclonal antibodies using hybridoma technology,and further evaluate the monoclonal antibodies.Firstly,synthesize the whole BoNT/E gene sequence and amplify the fragment BoNT/EHc,construct the recombinant plasmid pTIG-BoNT/E,pTIG-BoNT/EHc,and use the E.coli expression system to express the recombinant protein.The recombinant protein was purified using His Trap HP affinity chromatography column and PD10 desalting column.Gel-pro32 analysis of protein purity was>95%.After purification,the recombinant protein was identified by Western Blot for its antigenicity,indicating that the recombinant protein can be used as the required immune antigen and screening antigen for subsequent hybridoma antibody screening.A challenge test was performed on the BoNT/E recombinant protein to determine the virulence of the recombinant protein.Secondly,the recombinant protein BoNT/EHc was used as immunogen to immunize female BALB/c mice with routine immunization and rapid immunization respectively.indirect ELISA was used to measure the titer of mouse tail blood supernatant and screen positive cells.it was found that the titer of mouse tail blood with both immunization schemes could meet the needs of subsequent cell fusion.The average fusion rate and positive rate of routine immunization were 91.93%and 19.79%,respectively.Eight positive hybridoma cell lines(1A11,1F9,2D10,2D9,2E9,4E8,4E9,4E10)were screened.However,only three cells can complete the sequence of light and heavy chain variable regions.Therefore,three cells,1A11,1F9 and 4E8,were expanded and cultured,and ascites was prepared by allogeneic ascites induction method.The ascites was purified by Protein G column and PD 10 desalting column to prepare monoclonal antibodies.The purity of antibodies was over 95%by Gel-pro32 analysis.The antibody heavy chain subtypes are IgG2b,and the antibody light chain subtypes are κ.The purified three antibodies have high specificity for BoNT/EHc,but do not bind to BoNT of other serotypes,and all of them specifically bind to antigen BoNT/EHc with linear epitopes.The affinity constants of these three antibodies and antigen BoNT/EHc were determined by non-competitive ELISA.the results showed that the affinity constants of antibody 1A11 reached nmol/L level,which indicated that both antibodies and antigen had high affinity.The average fusion rate and positive rate of rapid immunization were 89.84%and 17.45%,respectively.Nine positive hybridoma cell lines(1D2,2B6,2D11,2D12,2F3,3B6,5F4,6C4,7B9)were finally screened.The light and heavy chain variable region sequences can be retrieved completely in 8 cells,but the heavy chain subtypes of 2B6,2D11 and 2D12 are IgG2b,and the heavy chain subtypes of 1D2,2F3,3B6,5F4,6C4 and 7B9 are IgM,2B6,2D11,2D 12,2F3,3B6,5F4,6C4 and 7B9.Purification and subsequent evaluation of the antibody whose heavy chain subtype is IgG2b.Therefore,2B6,2D11 and 2D12 cells were expanded and cultured,and then ascites was prepared by allogeneic ascites induction method.the ascites was purified by Protein G column and PD 10 desalting column to prepare monoclonal antibodies,and the purity of antibodies was>95%by Gel-pro32 analysis..The purified three antibodies have high specificity for BoNT/EHc,but do not bind to BoNT of other serotypes,and all of them specifically bind to antigen BoNT/EHc with linear epitopes.The affinity constants of these three antibodies and antigen BoNT/EHc were determined by non-competitive ELISA.the results showed that the affinity constants of antibody 2B6 reached nmol/L level,which indicated that both antibodies and antigen had high affinity.Then,the application of the selected BoNT/E monoclonal antibodies was evaluated,and the epitopes of the six antibodies obtained above were grouped by competitive ELISA.The results showed that the six antibodies could be divided into two groups,one was antibodies 1A11,1F9 and 4E8,and the other was antibodies 2D11 and 2D12.The neutralization titer of six strains of antibodies used alone was determined by mouse neutralization experiment.The results showed that the neutralization titer was low and there was no complete neutralization,but only some toxins could be bound.After further combining with antibodies with different epitopes,the neutralization titer of bivalent antibodies was determined.The results showed that the bivalent antibodies still did not have complete neutralization,but only some toxins could be neutralized,and the neutralization titer of bivalent antibodies improved little.Through the above studies have shown that BoNT/E and BoNT/EHc recombinant proteins can be expressed using the E.coli expression system,using BoNT/E recombinant antigens as immunogens,and hybridoma technology can be used to screen BoNT/E proteins with high affinity and high specificity.Cloned antibodies.There is no significant difference in the evaluation process of monoclonal antibodies obtained by the two immunization schemes,and rapid immunization may lead to the appearance of heavy chain subtype IgM of monoclonal antibodies.