Isolation and Immunochemical Characterization of Soluble Anaplasma Marginale Antigen (Rickettsia, Hemoparasite, Disease, Elisa)

238 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.The present work was designed to characterize and isolate a soluble A. marginale (noncaudal Florida isolate) antigen derived from culture supernatants and infected erythrocytes. Two immunoassays, double immunodiffusion and enzyme-linked immunosorbent assay were adapted for monitoring the presence of soluble Anaplasma antigen using bovine immune sera. A soluble antigen was detected in the supernatant of short-term cultures which had a reaction of identity with a soluble Anaplasma antigen from infected bovine erythrocytes. Gel filtration in Sephadex G-200 and anion--exchange chromatography in DEAE cellulose were used to isolate this antigen from infected erythrocytes. A fraction with antigenic activity was eluted in the ascending phase of the hemoglobin peak by gel filtration chromatography. Furthermore, two fractions eluted with 0.5 M and 1.0 M NaCl in the anion-exchange chromatography showed antigenic activity. The soluble Anaplasma antigen was highly anionic as demonstrated by its fast mobility in immunoelectrophoresis, crossed immunoelectrophoresis and non-denaturating polyacrylamide gel electrophoresis with electrotransfer blotting analysis. According to gel filtration sieve chromatography the antigen was located primarily in the region of 90-100 kd molecular size. When the antigen previously isolated by anion-exchange chromatography was re-chromatographied by gel filtration and its elution volume compared with those of the protein markers, a molecular size of 88 kd was estimated for the soluble Anaplasma antigen reported in this study. This antigen was thermolabile and sensitive to enzymatic degradation by protease but resistant to amylase and lysozyme. Soluble Anaplasma antigen contained in crude and purified preparations were able to induce precipitating antibodies in experimental animals only after challenge exposure with virulent organisms.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD