Abstract
Antigen Used for Immunization
The recombinantly produced carboxy terminus of C. botulinum type C neurotoxin (rBoNTC Hc) was used as the immunizing antigen.
Method of Immunization
The mice were immunized with rBoNTC Hc for a total of seven injections. For the first two injections, the mice received 2 μg of antigen split between s.q. and i.p. in Freund's complete adjuvant. The third, fourth, and fifth injections were the same as the first two, except Freund's incomplete adjuvant was used. The sixth injection was 6 μg of antigen split between two i.m. sites in ImmunEasy adjuvant. The seventh and final injections were 3 μg of antigen/PBS into the spleen, and 3 μg of antigen in ImmunEasy injected i.m.
Parental Cell Line Used for Fusion
Mouse myeloma cell line Sp2/O-Ag14.
Selection and Cloning Procedure
Fusion supernatants were tested against rBoNTC Hc in initial screening and following two rounds of subcloning.
Heavy and Light Chains of Immunoglobulin
5D9-H9-A9 is in the IgG class; subtype undetermined.
Specificity
5D9-H9-A9 is specific to the heavy chain carboxy terminus of botulinum type C neurotoxin in ELISA. 5D9-H9-A9 will also bind to mosaic botulinum neurotoxin type D/c; the carboxy terminus on this molecule has almost identical amino acid sequence to botulinum type C toxin. It will not bind to mosaic botulinum neurotoxin type C/d or to any other botulinum neurotoxin type. 5D9-H9-A9 will not bind to botulinum neurotoxin type C in a Western blot due to the denatured state of the antigen.
Specific Antigen Identified
5D9-H9-A9 binds to the heavy chain carboxy terminus of botulinum type C neurotoxin or to the carboxy terminus of mosaic botulinum type D/c.