Validation of LILR antibody specificities and development of LILRA3-specific antibodies

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) constitute a family of 11 structurally related receptors predominantly expressed on immune cells. Despite their functional diversity, LILRs show a high degree of sequence homology within their extracellular domains, with amino acid sequence identities ranging from approximately 50% to nearly 100%. This molecular similarity poses a significant challenge for antibody-based detection, often resulting in cross-reactivity and misinterpretation of expression profiles. Nevertheless, a comprehensive assessment of antibody specificity across the entire LILR family has not been previously conducted. In this study, we performed a rigorous validation of commercially available antibodies to evaluate their specificity and cross-reactivity against all LILR family members. Using flow cytometry with LILR-transfected K562 cells, we reliably identified specific antibodies for LILRA2, LILRA4, LILRA5, LILRB1, LILRB2, and LILRB4. Notably, more than half of the commercial antibodies showed cross-reactivity. Given the lack of a reliable antibody for LILRA3, the only known soluble member of the family, we generated novel anti-LILRA3 monoclonal antibodies in mice. Three hybridoma clones exhibiting high specificity for LILRA3 were successfully isolated and validated. Using these antibodies, we established a sensitive sandwich ELISA, which successfully detected LILRA3 in individuals carrying functional alleles, while no protein was detected in those with the 6.7-kb deletion or premature termination codons. Moreover, serum concentrations of LILRA3 were substantially higher than previously reported. These findings not only provide essential tools for accurate detection of LILRA3 but also underscore the importance of rigorous antibody validation in LILR-related research.