Preparation And Identification Of Monoclonal Antibodies Against Botulinum Toxin F

Botulinum toxins are an extremely poisonous protein neurotoxin generated by Clostridium botulinum,It is presently one of the most dangerous toxins known in nature,both biologically and chemically.When BoNT infecting human,BoNT can cause flaccid muscle paralysis by blocking the release of neurotransmitters,which can lead to death by asphyxiation in severe cases.Whatsmore,the prevention,diagnosis and treatment of BoNT in the clinic have received so much attention from researchers.Until now,there is no effective chemical drug to treat BoNT poisoning when Horse-derived antitoxins are often used clinically.To replace it with antitoxins,monoclonal antibodies have emerged as a novel therapy option in the treatment of BoNT poisoning.Today,with the increasing development of antibody drugs,monoclonal antibodies have become a popular research field and development starting point for the diagnosis and even treatment of BoNT poisoning.Among the seven distinct serotypes of BoNT,4 of them:A,B,F,and F have been shown to cause human poisoning,however,past research has mostly concentrated on types A and B.According to this,BoNT/F seems to be receiving less attention,especially in the aspect of monoclonal antibodies.Furthermore,no antibodies against BoNT/F poisoning have been approved for commercial use.In light of this situation,preparing the neutralizing antibody of BoNT/F is of great significance.In conclusion,this study aims to prepare and evaluate murine neutralizing monoclonal antibodies with strong specificity and high affinity for BoNT/F,which will be accomplished by the use of hybridoma technology and phage display technology,respectively.In order to prepare botulinum toxin monoclonal antibodies,a recombinant antigen against the toxic domain of BoNT/F is first constructed.After the antigen is expressed and identified,the recombinant antigen is obtained by affinity chromatography.Then use different immunization methods to immunize the animals.Further use two technical means,hybridoma and phage display library technology,to screen murine monoclonal antibodies and human single-chain antibodies,respectively,for further antibody evaluation.Utilizing the plasmid pTIG-BoNT/F that has been constructed in the early stage of the laboratory to amplify the fragment FLHn,construct the recombinant plasmid pTIG-BoNT/FLHn,and express the recombinant protein through the E.coli expression system.The recombinant protein was purified and desalted using His Trap HP affinity chromatography column and PD 10 desalting column,respectively.The purity of the protein analyzed by GFl-pro32 was>95%.After purification,the recombinant protein was identified by Western Blot for its antigenicity,indicating that the recombinant protein can be used as an immune antigen and screening antigen for subsequent antibody preparation.Furthermore,the recombinant protein BoNT/FLHn was used as the immunogen to immunize female BALB/c mice with two immunization schemes:rapid immunization and conventional immunization,respectively,and the indirect ELISA method was used to determine the titer of the tail blood supernatant of the mice and screen positive cells.It was found that the mouse tail blood titers of the two immunization regimens can meet the needs of subsequent cell fusion.The fusion rates of rapid immunization and conventional immune cells were 18.9%and 95.42%,respectively,and the positive rates were 79.35%and 21.72%,respectively.A total of 10 positive hybridoma cell lines were screened（1F8,1G11,2D3,5B7,10F6,1D2,1D6,1F9,2C11,3E3）,including 5 rapid immunization and conventional immunization.The monoclonal hybridoma cell line was obtained by the limiting dilution method and the ascites was prepared by the in vivo induction method.At the same time,the titer of the ascites was tested after the preparation of the ascites was completed.Then,according to the different antibody subtypes,Protein G column and Protein L column IgM Purification column were used for purification,and PD 10 desalting column was used for monoclonal antibody desalting operation.After analyzing the purity of the antibody by GF1-pro32,it was evaluated in many aspects.There are three antibody heavy chain subtypes,and the light chains are all kappa type.Except 2D3 which is of IgG1 type,the rest are of IgM type in rapid immunization.In routine immunization,1D2 and 1F9 are IgG2b type,1D6 and 3E3 are IgG1 type,and 2C11 is IgM type.All the obtained antibodies were tested by Western Blot for linear conformation.Results 1D6 and 3E3 were linear conformations in routine immunization.Then,the application evaluation of the selected BoNT/F monoclonal antibodies was carried out.The specificity test results showed that the specificity of 1G11,2D3,and 1F9 monoclonal antibodies was relatively strong,and the 1D6 antibody had a strong tendency to specifically bind to immune recombinant antigens.Although 3E3 has a relatively high affinity index,it has a low specificity index for immune antigens.In addition,the biological activity index,namely the neutralization activity,was evaluated.Through the BoNT/F challenge experiment,it was found that the obtained murine monoclonal antibody had no neutralization effect.At the same time,the phage display library technology uses the existing human phage display library through ELISA solid-phase screening,and coats the purified recombinant antigen as a substrate.After three rounds of enrichment-washing-amplification,the affinity is obtained.Four human single-chain antibodies with strong indicators.The sequences of the CDR regions obtained are:F2-1-C5:LCDR3:QVWAG NRSKC,HCDR3:YWRFNGSFDP;F1-2-C6:LCDR3:AADNGLGL,HCDR3:YGVY RDFDD;F2-1-B5:LCDR3:QVWDDNSLDQ,HCDR3:HSVFRVPFAV;F2-1-G2:LCDR3:SAHELRI,HCDR3:HFGHRRPFAY.After plasmid construction,expression and purification,its affinity and specificity still need to be verified.The above research shows that the BoNT/FLHn recombinant protein can be expressed using the E.coli expression system,the expression product is used as an immunogen,and hybridoma technology can be used to screen BoNT/F monoclonal antibodies with high affinity and high specificity.There is no significant difference in the evaluation process of the monoclonal antibodies obtained by the two immunization schemes except for the affinity index,but the rapid immunization may increase the probability of the heavy chain subtype IgM of the monoclonal antibody,thereby increasing the difficulty of purifying the antibody.However,for the screening of neutralizing antibodies,the screening antigen prepared by the transmembrane domain of BoNT/F as a hybridoma technology antibody in the early stage of the laboratory has obtained antibodies with good neutralizing activity.It is speculated that the yield of neutralizing antibodies may be The choice of antigen domain is related.Phage display library can also obtain high-affinity single-chain antibodies.Different from hybridoma technology,human phage library can be used to directly obtain human antibody sequences,without the need for later humanization of murine antibodies to reduce monoclonal antibodies.In addition to the therapeutic effect,its own potential immunogenicity.Finally,a double-antibody sandwich ELISA method was established by combining 1D6 antibody with an affinity index of nmol/L screened by hybridoma technology and 1E2B antibody with Hc terminal domain.The linear detection range was 2.048-32.000 ng/mL,repeatability was good and had statistical significance.In conclusion,the rat access to the source antibody can be BoNT/F for the establislunent and application in the clinical diagnosis of poisoning kit provides reference data,the sequence of single-chain antibodies are to achieve targeted treatment or botulinum toxin poisoning to provide reference in research and feasibility,this study obtained antibodies for subsequent BoNT/F poisoning diagnosis and treatment of drug development preliminary experimental basis,It has practical application significance.