A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody (12-09) raised against Eimeria acervulina sporozoites. The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gtl 1, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products. The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E. coli
Resident (R), Brewer's thioglycollate broth (TG) and C. parvum (CP)-elicited murine peritoneal cell populations were separated into subpopulations by centrifugation on discontinuous Ficoll gradients consisting of layers of 4, 6, 8 and 10% Ficoll. The resulting subpopulations were shown to be distinct on the basis of cell size, ectoenzyme phenotypes and antitumor activity. The cell size distributions were analyzed by means of a Coulter channelyzer and software developed for a computer. The 4-6% Ficoll interface fraction comprised the smallest macrophages, with most cells in this subpopulation appearing to range in cell volume from 150-275 microns 3. The largest sized macrophages were found in the 10%-pellet fraction, with most cells appearing to range in cell volume from 300-600 microns 3. The ectoenzyme phenotypes of the R, TG and CP unseparated macrophage populations were significantly different. Moreover, the ectoenzyme phenotypes of the smallest, (4-6% Ficoll interface) subpopulation in the R and CP macrophages differed from the other subpopulations. Reduction in alkaline phosphodiesterase 1 (APD-1) ectoenzyme activity (as compared with unseparated R macrophages) appeared to be a marker for acquisition of antitumor activity. The small CP macrophages (4-6% Ficoll interface) showed no antitumor activity while the unseparated CP macrophages and all other CP macrophages subpopulations exhibited antitumor activity. The CP macrophage unseparated population and the subpopulations with antitumor activity expressed reduced ADP-1 activity. Conversely, the R or TG macrophage unseparated populations and their subpopulations, along with the CP macrophage 4-6% subpopulation, lacked antitumor activity and failed to show a change in ADP-1 ectoenzyme activity.
Protein conjugates of salinomycin were prepared and utilized to produce murine monoclonal antibodies (MAbs) in the BALB/c-P3X63.Ag 8.653 fusion system. Several antibodies were cloned, stabilized, and evaluated for cross-reactivity to other ionophorous antibiotics. Selected antibodies were used to develop quantitative assays for salinomycin by means of an enzyme-linked immunosorbent assay (ELISA). These assays were sensitive to 50 ng of salinomycin.
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