Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

Abstract

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein-a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre-and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against na-

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

“…We developed a first-of-a-kind TEX101 ELISA (35) and demonstrated its clinical utility in large cohorts of fertile, sub-fertile and infertile individuals (28). Since interactions between testis-specific proteins are particularly important, in the present study we first focused on elucidation of the human TEX101 interactome in testicular tissues, spermatozoa and SP.…”

“…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.…”

“…Screening for clones producing antibodies against native TEX101 and DPEP3 was performed according to our established protocol (35). A commercial mouse polyclonal anti-TEX101 antibody (ab69522; Abcam, Cambridge, MA) was used as a positive control for anti-TEX101 antibody secreting clone screening.…”

“…1A). The purified recombinant TEX101 was quantified by an in-house TEX101 ELISA, as previously described (35).…”

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

“…Since prostatic proteins are more concentrated in semen than in serum or urine, PCa biomarkers might be more easily identified and quantified in SP by mass spectrometry. We previously discovered male infertility biomarkers in SP and developed a simple 2-biomarker algorithm for the differential diagnosis of male infertility 23,24,58 . Our success with male infertility biomarkers motivated us to search for prostate cancer biomarkers in SP.…”