Determination of the specificity of CD45 and CD45R monoclonal antibodies through the use of transfected hamster cells producing individual porcine CD45 isoforms.
The exclusive presence of the tyrosine phosphatase, CD45, on the surface of hematopoietic cells coupled with the differential expression of its various isoforms has enabled the selection of lymphocyte subsets based on their reactivity with monoclonal antibodies (mAbs) specific for one or more CD45 species. As a prelude to defining the specificity of anti-porcine CD45 mAbs for this purpose, Chinese hamster ovary cells were transfected with constructs containing cDNAs encoding the extracellular and transmembrane domains of four pig CD45 isoforms. Cells expressing only one of the three predominant types (CD45RO, CD45RC, and CD45RAC) or the minor species (CD45RA) of porcine CD45 on their surface were sorted based on positive reactivity with the CD45 mAb K252.1E4. Initially, these CD45+ cells were used as a source of antigen when determining the specificities of nine mAbs, which had been identified during the First and Second International Swine CD Workshops as being reactive with porcine CD45. Later, cloned cell lines were established and phenotypically verified for the production of the correct CD45 transcript by RT-PCR. Binding of two more mAbs (74-9-3 and 10-14-1) in addition to the original mAb panel to these cell lines was assessed by using a cell ELISA in lieu of one-color flow cytometry. Despite differences in detection methodology, identical mAb binding results were obtained. As anticipated, CD45 mAbs K252.1E4, MAC 323, and 74-9-3 which recognize an epitope(s) in the common portion of porcine CD45, reacted with cells expressing any one of the four isoforms but not with the parental CHO cells. In contrast, none of the restricted (CD45R) mAbs bound to cells producing the CD45RO isoform which lacks any of the alternate extracellular regions. However, three of these mABs (6E3/7, FG2F9 and STH267) did react specifically with the CD45RA and CD45RAC isoforms, indicating their specificity for an epitope(s) encoded by the CD45 A exon. The other four CD45R mAbs (MAC326, 3a56, MIL5, and -a2) recognized the CD45RC isoform. Interestingly, only CD45R mAb MAC326 also bound to cells expressing the CD45RAC isoform, suggesting that the epitope(s) recognized by the other three may have arisen due to the juncture of the invariant 5' leader sequence with the CD45C exon. The eleventh mAb (10-14-1) was unique in that it did not react with any of the expressed CD45 isoforms. This inability coupled with the previously demonstrated recognition of a 240 kDa protein suggests that it may be specific for CD45RABC. Overall, this panel of CD45R mAbs should prove useful for obtaining functionally distinct subpopulations of B and T lymphocytes.
Schnitzlein
W M
WM
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801, USA.
Zuckermann
F A
FA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D016466:CHO Cells; D006224:Cricetinae; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000276:immunology; D007536:Isomerism; D017493:Leukocyte Common Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D013552:Swine / Q000276:immunology*; D014162:Transfection / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017493:Leukocyte Common Antigens