Accession Number:

ADA220848

Title:

Quarterly Report for January-April 1990 on Contract N00014-89-J-3073 (Massachusetts General Hospital)

Descriptive Note:

Rept. no. 3

Corporate Author:

MASSACHUSETTS GENERAL HOSPITAL BOSTON

Personal Author(s):

Report Date:

1990-04-12

Pagination or Media Count:

2.0

Abstract:

Our first approach to the purification of the LPS binding and neutralizing factors is to affinity purify the substances 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 0111B4 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 gml. 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.

Subject Categories:

  • Biochemistry
  • Medicine and Medical Research
  • Microbiology

Distribution Statement:

APPROVED FOR PUBLIC RELEASE