STRUCTURAL CHARACTERIZATION OF DEVELOPMENTALLY EXPRESSED ANTIGENIC MARKERS ON CHICKEN ERYTHROCYTES USING MONOCLONAL-ANTIBODIES

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity were used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The antigenic changes which occur on the avian erythrocyte membrane with organismic maturation were partially accounted for by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent MW. On 2-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displayed microheterogeneity, consisting of 8-9 closely spaced spots with an isoelectric midpoint of .apprx. 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however an antigen with similar but perhaps not completely identical structure can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent MW, but this antigen had a more basic isoelectric point. The adult antigen was polymorphic. Slightly different patterns were obtained on 2-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It had a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower MW near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisera suggest that these antigens may be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrated that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments were found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.