Although immunoassays are an indispensable tool for scientific research, antibody specificity has been recognized as a major challenge to the rigor and reproducibility of research findings. A 2016 proposal published by the International Working Group for Antibody Validation identified five pillars of antibody validation.¹ Among these is genetic validation, in which “The expression of the target protein is eliminated or significantly reduced by genome editing or RNA interference.”

Y chromosome-encoded genes present unique opportunities and challenges to validate antibodies on this genetic principle. Fortunately, readily available female-derived cells and tissues can serve as a target-negative source material, which is far more convenient than typical sources of genetic validation, which require knockout or knockdown approaches to a target gene. However, an additional challenge for the specificity of these antibodies is that many Y chromosome proteins have “gametologs,” or highly homologous genes encoded on the X chromosome. As gametologs can share over 90% amino acid identity, these protein targets present unique specificity challenges. However, this obstacle has not impeded commercial antibody suppliers who market hundreds of antibodies with purported specificity for Y chromosome-encoded genes.

We performed an analysis of the extent to which Y chromosome gene-targeted commercial antibodies recognize female-derived materials using data provided in their marketing materials (a detailed methodology is provided in the Supporting Information). Table 1 lists 65 antibodies purporting to target a Y chromosome-encoded gene with company-supplied marketing demonstrating immunoreactivity in female-derived tissues. Product page URLs are provided in Supporting Information: Table S1. For one example, an antibody targeting sex-determining region chromosome Y marketed by MyBioSource (catalog # MBS8513980) presents validation data in HeLa cells, which is a cervical cancer cell line with no Y chromosomes.²

Among these antibodies, frequently used female-derived cell lines were HeLa, 30/65 (46%), HEK293T, female human embryonic kidney cells used in 14 (22%), and MCF-7 breast cancer cells used in 7 (11%). One antibody, a rabbit polyclonal raised against the “N terminus” of DEAD-box helicase 3 Y-linked (DDX3Y) (LS Biosciences, catalog # LS-C355991) presented positive immunohistochemistry in human breast cancer tissue. While not definitively a Y-chromosome absent tissue, we included this as a likely female-positive tissue, based on the prevalence of breast cancer in females compared to males being roughly 99-to-1 in the United States.³ Among 65 antibodies, we noted just two that had disclaimers warning that the antibody may cross-react with homologous X chromosome-encoded proteins.

This survey provides evidence of widespread off-target antigen recognition in commercial antibodies purporting to recognize Y chromosome-encoded proteins. Some important caveats should be noted. First, many antibodies provided no primary data on female tissues. For example, 20/30 (67%) of DDX3Y antibodies provided no data in female tissues. Therefore, it seems likely that the 65 antibodies listed in Table 1 are a significant underrepresentation of Y chromosome-targeted antibodies lacking specificity. Second, this analysis assumes that the identities of the listed cell types provided in marketing materials are accurate and not subject to cell line contamination, conceivably with Y chromosome-containing cells. Cell line purity and identity are a major challenge in biomedical research. Third, in the case of human tissues such as endometrium and breast cancer, it is conceivable that positive immunoreactivity from Y chromosome-encoded proteins could represent true staining of microchimerism, in which an allogeneic cell population resides within a host. We think this is unlikely because even in the extreme case where every Y chromosome-containing cell expresses the antigen, one would expect a true positive staining pattern to be restricted to the small number of allogenic cells as in other in situ hybridization staining analyses of microchimeric tissues,⁴ rather than widespread staining as is reported in antibody marketing materials. Finally, published marketing materials are not independently validated, and typically lack important information such as the number of experimental replicates.

Thus, while the survey findings are sufficient to suggest that these antibodies are not valid, even were they to pass a genetic screen, additional testing may be required to confirm their validity.

The broader implications of an overall lack of protein-based methodologies mean that identifying the roles of sex chromosome-encoded genes in phenotypes and pathologies that vary with sex chromosome number is far more challenging. As antibodies often play a central role in defining molecular mechanisms of proteins, future studies on the mechanistic contributions of sex chromosome-encoded proteins in health and disease demand more accurate molecular tools. More broadly, these observations should instill caution in researchers to carefully confirm the specificity of immunoassays, particularly for highly related protein targets not just on sex chromosomes, but autosomal gene families as well.

In summary, many commercial antibodies targeting Y chromosome-encoded proteins are not validated for use in sex-specific applications. Researchers using these tools are encouraged to validate their reagents in tissues lacking a Y chromosome and should be cautious when interpreting findings regarding antigens encoded by the Y chromosome. Material from samples lacking the Y chromosome should be used as a negative control to confirm antibody specificity. Ideally, these validation studies should be supplemented with other gene function-specific approaches including genetic knockout and transgenic overexpression. We also urge commercial antibody suppliers to provide better warning to consumers about the lack of validated specificity among Y chromosome-targeted antibodies.

AUTHOR CONTRIBUTIONS

Bradley D. Gelfand: Conceptualization (lead); data curation (lead); formal analysis (equal); funding acquisition (lead); investigation (equal); methodology (equal); project administration (lead); supervision (lead); writing—original draft (lead); writing—review and editing (equal). Dionne A. Argyle: Formal analysis (equal); investigation (equal); writing—review and editing (equal). Joseph J. Olivieri: Formal analysis (equal); investigation (equal); writing—review and editing (equal). Jayakrishna Ambati: Conceptualization (equal); writing—review and editing (equal). All authors have read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no relevant conflicts with the present work.

ETHICS STATEMENT

Not applicable.