Abstract
Antigen Used for Immunization
Botulinum type C neurotoxin from strain NCTC 8264 was toxoided in 0.4% formalin. Prior to the initial and second immunizations, the toxoid was mixed with Ribi's Adjuvant System (Ribi ImmunoChemical, Hamilton, MT).
Method of Immunization
Initial immunization was with 20 μg total antigen, injected s.q. at two different sites (total injection volume for all immunizations was 0.2 mL). The second immunization was with 1 μg of the toxoid injected s.q. at two sites. The third and final immunizations were 1 μg of toxoid in phosphate-buffered saline injected s.q. at two sites.
Parental Cell Line Used for Fusion
Myeloma cell line P3X63Ag8.653, also known as ATCC CRL 1580.
Selection and Cloning Procedure
Fusion supernatants were tested against botulinum neurotoxin type C in initial screening and following two rounds of subcloning.
Heavy and Light Chains of Immunoglobulin
14G8 is in the IgG class; subtype undetermined.
Specificity
14G8 is specific to the light chain of botulinum type C/d neurotoxin in ELISA. It will not bind to any other botulinum neurotoxin type or to tetanus toxin. 14G8 will not bind to the botulinum neurotoxin type C in a Western blot due to the denatured state of the antigen.
Specific Antigen Identified
14G8 binds to the light chain of botulinum type C neurotoxin when the light chain is in native conformation.