SENSITIVE ASSAY FOR MEASUREMENT OF ANTIBODIES TO CLOSTRIDIUM-BOTULINUM NEUROTOXIN-A, NEUROTOXIN-B, AND NEUROTOXIN-E - USE OF HAPTEN-LABELED-ANTIBODY ELUTION TO ISOLATE SPECIFIC COMPLEXES

The measurement of chicken and human antibodies to Clostridium botulin um neurotoxins A, B, and E was accomplished by affinity isolation of c omplexes containing these antibodies. By this approach, a mixture of t oxin with the test antibody, fluoresceinated antibody, and enzyme (Rus sell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase. This complex is then bound to a matr ix containing antifluorescein antibody. All components not bound to th e matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix. The eluted complex is then bound to a matrix w hich specifically binds the test antibody (anti-chicken immunoglobulin Y [IgY] or anti-human IgG), and the bound complex is measured by usin g the enzyme label. Using this approach, we were able to measure as li ttle as 1 ng of specific antibody per mi from affinity-isolated, monos pecific chicken antibody preparations and to measure antibody specific ally from IgY fractions of monospecific chicken antibody preparations. Human antibodies from subjects immunized with pentavalent toroid prep arations were detectable at dilutions as great as 24,300-fold, and und iluted serum from most control subjects showed no measurable antibody. Antibody was also measured in 65 subjects who were receiving preparat ions of neurotoxin A (BOTOX) for the treatment of spastic disorders. E ighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A. The tw o patients having antibody to toxin A were refractory to treatment wit h this toxin. This approach of isolation of hapten-labeled immune comp lexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concent rations in serum.