Quarterly Report for January-April 1990 on Contract N00014-89-J-3073 (Massachusetts General Hospital)
Abstract
Our first approach to the purification of the LPS binding and neutralizing factor(s) is to affinity purify the substance(s) using anti-LPS immunoaffinity columns. Over the last several months we have incubated tritiated LPS prepared from S. typhimurium in normal and tolerant sera and passed the mixture over an anti-LPS column filled with affinity-purified rabbit anti-O- polysaccharide IgG. Approximately 50% of the radioactivity was recovered in the eluted fraction. SDS-PAGE analysis of this material showed no difference between normal and tolerant serum, with and without added LPS. There was, however, considerable non-specific binding. We prepared a new column by coupling 17 mg of murine monoclonal IgG directed to the O-polysaccharide of E. coli 0111:B4 to 5 mls of GNBr-activated sepharose 4B. Our second approach involves classical protein purification techniques to progressively purify the LPS binding factors. We have completed the packing and calibration of a sepharose 6B-C1 molecular sizing column. We purified the lipoproteins from normal and tolerant serum by ultracentrifugation in KBr at a density of 1.21 g/ml. We have refined our Tumor Necrosis Factor (TNF) inhibition assay and compared our pools of normal and tolerant rabbit sera for the ability to decrease the production of TNF by the RAW 264.7 cell line.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 12, 1990
- Accession Number
- ADA220848
Entities
People
- H. S. Warren
Organizations
- Massachusetts General Hospital