Preparation of Monoclonal Antibodies against Type A Botulinum Neuro-toxin and Application of Chemiluminescent Enzyme Immunoassay Tech-nology
Monoclonal antibodies against type A botulinum toxin were prepared by extracting monoclonal antibody genes from hybri-doma cells of type A botulinum toxin and expressing and purifying them in mammalian systems.In order to establish a rapid detection method for type A botulinum toxin using chemiluminescence enzyme immunoassay(CLEIA)technology,hybridoma cell sequences were detected using rapid amplification of cDNA ends(RACE)technology,plasmids containing monoclonal antibody genes were used as templates for subcloning,and expression plasmids for light and heavy chains were constructed and transferred to the JM108 clone strain.The transfected plasmids were extracted using a plasmid extraction kit,and then transfected into mammalian cell CHO for expression.Finally,the antibodies were purified by affinity chromatography using a Protein G column.Then using the double anti-body sandwich principle,the optimal detection scheme for the CLEIA method was optimized,a detection method for type A botulinum toxin CLEIA was established and the detection limit,specificity,and durability of this analytical method were verified one by one.This study prepared monoclonal antibodies against type A botulinum toxin and established a rapid detection method for type A botuli-num toxin with a detection limit of 0.156 ng/mL;Type A botulinum toxin has no cross reactivity with other types and its detection limit is not affected in different solvent media,demonstrating good specificity and durability.This method has been applied to prove 100 U balance force® best redissolution detection effect of type A botulinum toxin for injection is achieved under 0.02 mol/L,pH=7.8 phosphate buffer conditions.The monoclonal antibody against type A botulinum toxin prepared in this study and the established CLEIA detection method for type A botulinum toxin have good practicality,providing another approach and method for clinical detec-tion and quality control analysis of type A botulinum toxin.
type A botulinum neurotoxinmonoclonal antibody against type A botulinum toxin neurotoxinCLEIA technologyRACE technologydouble antibody sandwich method
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