The Use of a Resin Bound Monoclonal Antibody in the Purification of Various Components in Anthrax Vaccines.
Abstract
Lethal factor from B. anthracis (Vollum 1B strain) has been purified 1130 fold by immunosorbent chromatography using a mouse anti-lethal factor monoclonal antibody Sepharose-4B column. The antibody and was eluted with buffer containing 4 M NaSCN with 77% recovery of the immunological activity. Pre-elution with 4 M NaCl was found to be effective in eluting non-biospecifically bound proteins. Migration of lethal factor in SDS-polyacrylamide gel electrophoresis indicated a single component with a molecular weight of 82,000. The effect of a number of dissociation buffers on the antigen-antibody complex has been investigated. Sodium thiocyanate (4M) in 0.05 M sodium phosphate buffer, pH 7, was the most effective eluting solution causing complete dissociation of the antigen-antibody complex while 4 M NaI and 4 M NaCl caused 93% and 15% dissociation respectively. The antigen-antibody complex was found to undergo a reversible dissociation at moderately high pH values. The ionizable group (s) responsible for this dissociation exhibited a pK sub a value of 9.90. Purified lethal factor exhibited a significant decrease in immunological activity upon freeze-thawing with 52% loss in potency observed after three freeze-thaw cycles.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 1985
- Accession Number
- ADA177579
Entities
People
- Gary J. Calton
- Joseph W. Burnett
Organizations
- University of Maryland School of Medicine