Glycomapping the fine specificity of monoclonal and polyclonal Lewis antibodies with type‐specific Lewis kodecytes and function‐spacer‐lipid constructs printed on paper

BACKGROUND Lewis serologic reagents frequently give inaccurate phenotyping results. Furthermore these serologic reagents are often used in nonserologic assays such as inhibition and immunohistochemistry. In both scenarios knowledge of the fine specificity and cross-reactivity of these reagents will improve the quality of results obtained. STUDY DESIGN AND METHODS A range of contemporary and historical workshop and developmental Lewis reagents including mouse monoclonal (MoAb) and human and goat polyclonal (PoAb) reagents were evaluated. All were evaluated both against Lewis kodecytes expressing only single Lea, Leb, ALeb, BLeb, Lex, Ley, ALey, or BLey antigens and against the same antigens inkjet printed on a paper-based microplate and analyzed by enzyme immunoassay. Nine clinical samples were also evaluated. A kodecyte antigen dilution sensitivity assay was used to establish the ratio of Leb antigen between group A1/A2 and O RBCs. RESULTS A continuum of cross-reactivity from Lex through to H was observed with MoAbs. All PoAb and few MoAb anti-Lea samples and reagents cross-reacted to some degree with Leb antigen. Some PoAb and MoAb anti-Leb did not cross-react with Lea. All polyclonal goat anti-Leb reagents showed substantial activity against ALeb and BLeb, while no MoAb reagent had this activity. A1 RBCs had less than half the Leb antigen of A2/O RBCs. CONCLUSIONS Substantial cross-reactivity of both MoAbs and PoAbs with related antigens highlights the risks of using serologic reagents in nonserologic assays or against synthetic antigens. The lack of ALeb activity in anti-Leb MoAbs explains their poor performance against blood group A1 Le(a–b+) phenotypes.