Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility

Abstract

BackgroundTEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men.MethodsMass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples.ResultsWe demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 μg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia.ConclusionsWe demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-017-0817-5) contains supplementary material, which is available to authorized users.

Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies

“…1B), and were subsequently expanded in serum-free media and purified using protein G columns. We showed previously that the commercial polyclonal antibody ab69522 could capture native TEX101 (28,35). Immunocapture-SRM results revealed that our monoclonal anti-TEX101 antibodies possessed higher affinity for native TEX101 than ab69522 (Fig.…”

“…In this study, we focused on the testis-specific protein TEX101, which we previously discovered and validated as a biomarker of male infertility (27)(28)(29)(30)(31). Similarly to other testisspecific proteins involved in PPIs, TEX101 is exclusively expressed on the surface of testicular germ cells (32) and was suggested to be a cell-surface chaperone involved in trafficking and maturation of numerous cell surface proteins essential for fertilization in mice (7,33).…”

Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

“…http://dx.doi.org/10.1101/120873 doi: bioRxiv preprint first posted online Mar. 26, 2017; Our work resulted in first-of-a-kind SP-based biomarkers for the differential diagnosis of male infertility [23][24][25] . Success with male infertility biomarkers motivated us to apply a similar strategy to prostate cancer biomarkers.…”

“…We also found that unlike mouse antibody, sheep antibody could barely capture rhTGM4. We then coated ELISA plates with 300 ng/well of either sheep or mouse antibodies, incubated with either rhTGM4 (0, 5, 20 and 100 ng/mL) or endogenous TGM4 in SP (0, 5, 20 and 100 ng/mL) and detected with biotinylated sheep or mouse antibodies and standard time-resolved fluorescence ELISA format 25 .…”

“…Finally, the developing solution (1 M Tris-HCl, 0.4 M NaOH, 2 mM TbCl 3 and 3 mM EDTA) was added and mixed for 1 min. Time-resolved fluorescence was measured with the Wallac EnVision 2103 Multilabel Reader (Perkin Elmer), as previously described 25 . All SP and blood serum samples were measured in duplicates.…”