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

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 native forms of challenging protein targets. 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 native forms of challenging protein targets. Monoclonal antibodies that bind the native form of a protein are indispensable for the development of sensitive immunoassays, production of therapeutic antibodies and for studying protein interaction networks by affinity purification-mass spectrometry (1Weiner L.M. Surana R. Wang S. Monoclonal antibodies: versatile platforms for cancer immunotherapy.Nat. Rev. Immunol. 2010; 10: 317-327Crossref PubMed Scopus (904) Google Scholar, 2Lambert J.P. Ivosev G. Couzens A.L. Larsen B. Taipale M. Lin Z.Y. Zhong Q. Lindquist S. Vidal M. Aebersold R. Pawson T. Bonner R. Tate S. Gingras A.C. Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.Nat. Methods. 2013; 10: 1239-1245Crossref PubMed Scopus (206) Google Scholar). Large-scale purification of native proteins from biological samples may be challenging, so recombinant proteins or protein fragments are often used for antibody production. Antibodies produced against short peptides, protein fragments, or even full length recombinant proteins, however, may not bind the native protein conformation present in biological fluids, thus limiting the utility of antibodies. Rapid screening of antibody-producing hybridoma clones for native protein binders requires highly specific and sensitive assays, performed under nondenaturing conditions. Here, we report the capability of an immunocapture-SRM assay to facilitate fast screening of hybridoma cultures for monoclonal antibodies that recognize the native conformation of testis-expressed sequence 101 (TEX101) 1The abbreviations used are:TEX101testis-expressed sequence 101 proteinABCAmmonium bicarbonateBCABicinchoninic acidBMGYBuffered complex glycerol mediumBMMYBuffered complex methanolDFPDiflunisal phosphateDOCDeoxycholateDTTDithiothreitolELISAEnzyme-linked immunosorbent assayFcconstant region of immunoglobulinFWHMFull width at half maximumGPIglycosylphosphatidylinositolHATHypoxanthine-aminopterin-thymidine mediumHRPHorseradish peroxidaseKLKKallikreinMSMass spectrometryNOANon-obstructive azoospermiaOAObstructive azoospermiaPBSPhosphate-buffered salinePSAProstate-specific antigenPVPost-vasectomySPseminal plasmaSRMSelected reaction monitoringTBSTris-buffered saline. 1The abbreviations used are:TEX101testis-expressed sequence 101 proteinABCAmmonium bicarbonateBCABicinchoninic acidBMGYBuffered complex glycerol mediumBMMYBuffered complex methanolDFPDiflunisal phosphateDOCDeoxycholateDTTDithiothreitolELISAEnzyme-linked immunosorbent assayFcconstant region of immunoglobulinFWHMFull width at half maximumGPIglycosylphosphatidylinositolHATHypoxanthine-aminopterin-thymidine mediumHRPHorseradish peroxidaseKLKKallikreinMSMass spectrometryNOANon-obstructive azoospermiaOAObstructive azoospermiaPBSPhosphate-buffered salinePSAProstate-specific antigenPVPost-vasectomySPseminal plasmaSRMSelected reaction monitoringTBSTris-buffered saline. protein in biological fluids. testis-expressed sequence 101 protein Ammonium bicarbonate Bicinchoninic acid Buffered complex glycerol medium Buffered complex methanol Diflunisal phosphate Deoxycholate Dithiothreitol Enzyme-linked immunosorbent assay constant region of immunoglobulin Full width at half maximum glycosylphosphatidylinositol Hypoxanthine-aminopterin-thymidine medium Horseradish peroxidase Kallikrein Mass spectrometry Non-obstructive azoospermia Obstructive azoospermia Phosphate-buffered saline Prostate-specific antigen Post-vasectomy seminal plasma Selected reaction monitoring Tris-buffered saline. testis-expressed sequence 101 protein Ammonium bicarbonate Bicinchoninic acid Buffered complex glycerol medium Buffered complex methanol Diflunisal phosphate Deoxycholate Dithiothreitol Enzyme-linked immunosorbent assay constant region of immunoglobulin Full width at half maximum glycosylphosphatidylinositol Hypoxanthine-aminopterin-thymidine medium Horseradish peroxidase Kallikrein Mass spectrometry Non-obstructive azoospermia Obstructive azoospermia Phosphate-buffered saline Prostate-specific antigen Post-vasectomy seminal plasma Selected reaction monitoring Tris-buffered saline. Recently, we discovered, verified, and validated two proteins, testis-specific protein TEX101 and epididymis-specific protein ECM1, as biomarkers for the differential diagnosis of azoospermia (3Batruch I. Lecker I. Kagedan D. Smith C.R. Mullen B.J. Grober E. Lo K.C. Diamandis E.P. Jarvi K.A. Proteomic analysis of seminal plasma from normal volunteers and postvasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system.J. Proteome Res. 2011; 10: 941-953Crossref PubMed Scopus (130) Google Scholar, 4Drabovich A.P. Jarvi K. Diamandis E.P. Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay.Mol. Cell. Proteomics. 2011; 10 (M110 004127)Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Combination of TEX101 and ECM1 proteins measured in seminal plasma could differentiate between normal spermatogenesis, obstructive azoospermia (OA), and nonobstructive azoospermia (NOA) with very high diagnostic sensitivity and specificity. TEX101 levels in seminal plasma also facilitated classification of NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome (5Drabovich A.P. Dimitromanolakis A. Saraon P. Soosaipillai A. Batruch I. Mullen B. Jarvi K. Diamandis E.P. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.Sci. Transl. Med. 2013; 5: 212ra160Crossref PubMed Scopus (115) Google Scholar). A clinical laboratory test for TEX101 in seminal plasma may confirm the success of vasectomy or vasovasostomy, eliminate diagnostic testicular biopsies, and predict the success of sperm cell retrieval for assisted reproduction. Human TEX101 is a membrane GPI-anchored protein encoded by the TEX101 gene, located in the 19q13.31 region of chromosome 19. According to the Human Protein Atlas, TEX101 expression is restricted to testicular tissue and male germ cells, with no evidence of expression in any other human tissue or cell type (6Djureinovic D. Fagerberg L. Hallstrom B. Danielsson A. Lindskog C. Uhlen M. Ponten F. The human testis-specific proteome defined by transcriptomics and antibody-based profiling.Mol. Hum. Reprod. 2014; 20: 476-488Crossref PubMed Scopus (125) Google Scholar). Investigation of the function of mouse TEX101 demonstrated its direct role in fertilization (7Fujihara Y. Okabe M. Ikawa M. GPI-anchored protein complex, LY6K/TEX101, is required for sperm migration into the oviduct and male fertility in mice.Biol. Reprod. 2014; 90: 60Crossref PubMed Scopus (63) Google Scholar, 8Fujihara Y. Tokuhiro K. Muro Y. Kondoh G. Araki Y. Ikawa M. Okabe M. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 8111-8116Crossref PubMed Scopus (106) Google Scholar, 9Li W. Guo X.J. Teng F. Hou X.J. Lv Z. Zhou S.Y. Bi Y. Wan H.F. Feng C.J. Yuan Y. Zhao X.Y. Wang L. Sha J.H. Zhou Q. Tex101 is essential for male fertility by affecting sperm migration into the oviduct in mice.J. Mol. Cell. Biol. 2013; 5: 345-347Crossref PubMed Scopus (22) Google Scholar). We initially measured TEX101 levels in seminal plasma by mass spectrometry-based selected reaction monitoring (SRM) and immuno-SRM assays, with limits of detection of 120 and 5 ng/ml, respectively (4Drabovich A.P. Jarvi K. Diamandis E.P. Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay.Mol. Cell. Proteomics. 2011; 10 (M110 004127)Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5Drabovich A.P. Dimitromanolakis A. Saraon P. Soosaipillai A. Batruch I. Mullen B. Jarvi K. Diamandis E.P. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.Sci. Transl. Med. 2013; 5: 212ra160Crossref PubMed Scopus (115) Google Scholar). However, because of the ultra-wide range of TEX101 concentrations in seminal plasma of infertile and healthy men (0.5 ng/ml to 50,000 ng/ml) and theoretically zero levels for some azoospermic patients, a sensitive TEX101 immunoassay is required to develop a clinical laboratory test. In addition to immunoassay, monoclonal antibodies against native TEX101 would allow investigating its interactome and revealing its functional role in spermatogenesis and male fertility. Because TEX101 may emerge as a novel biomarker of male infertility, in this work we focused on the development of an ELISA for sensitive measurement of TEX101 in seminal plasma and serum. Our initial efforts to develop a TEX101 immunoassay using commercially antibodies not We that antibodies the form of TEX101 and for and not for the analysis of native TEX101 in seminal Here, we the production of mouse monoclonal antibodies against native TEX101, screening of antibody-producing clones by the immunocapture and development of a sensitive ELISA and measurement of TEX101 in seminal plasma and serum A Expression used for production of recombinant on the TEX101 sequence a of and for of the for the form of TEX101 present in seminal plasma facilitated the of for into the as as the of a for protein purification TEX101 human used as a performed in a 20 reaction with of of which and in the of the and of on an The for by of for 10 for 20 and for 20 with a at for of the into the expression using and and of the two fragments The sequence of the confirmed by to of cells, the TEX101 with to the of the in P. The into the and by A selected from the to the clones in the complex glycerol medium the culture the cell in the complex methanol to an of and at with TEX101 production with 10 methanol over culture TEX101 and the initially by in an with a membrane by A antibody and a mouse monoclonal antibody used to TEX101 production by The recombinant TEX101 from culture supernatants by affinity for recombinant proteins with used to TEX101 from culture to the In the affinity with of to and with of in to application on the affinity the recombinant protein by to a between and protein with affinity which with TEX101 with in at The of TEX101 in with by using antibody The and the mass of TEX101 by with The TEX101 protein by the acid assay and by as Smith C.R. Soosaipillai A. Diamandis E.P. Differential of from cancer or the Cell. Proteomics. Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In from using and in 5 of A of to and to a a 40 of a that with 5 of acid in used to the from the a using The coupled to an mass and a used with a of and of A used to samples and a full from in the mass of by of the in the mass The and with of and not protein identification and used to of used to on with Protein performed against the human using the mass mass of and of of of from of the at of of and of the The TEX101 protein by of recombinant TEX101 protein with the The at for and TEX101 to with obtained from the for by with of recombinant TEX101 with with of antigen in performed at an of of antigen in saline 10 mouse and with using selected using with serum culture supernatants for the of and antibodies by using the immunoassay with antibody in with 20 in 10 and of in for at of hybridoma and the in two and for at of of and of antibodies on of the a of and for at with of on measured with the at with a of in culture clones for reaction with the using an immunoassay TEX101 protein on in and of in for at by of hybridoma and the and for at and of antibody in on the of and for at with of on measured with the at with a of with of antibody in with 10 culture supernatants at two in in to the and for at with A protein mouse antibody used as a for the with and of seminal from a normal in and of at with with and with of TEX101 with a and and of this to and for at 5 of and samples for 40 in the at by addition of 5 of of in and of with the addition of and of as coupled to a mass using a a unique TEX101 with the with used for and the used for of TEX101 in as a to the The the to of the of the of the for with the and used to and protein concentrations in We confirmed of immunocapture-SRM screening by plasma samples in and and to performed at for with using a and a in in 20 in for at to a culture supernatants on and for at A antibody used as a the membrane in by the addition of or in in and at for The membrane with and of The membrane into an and and using sensitive 10 and in medium and using a protein to the supernatants with the 10 and on the The with and antibodies with acid at ELISA with of mouse monoclonal antibody in with 20 in 20 TEX101 and samples for into and for with with the A mouse monoclonal antibody in a normal mouse normal and 10 in of antibody of and for and in the for 20 at with by a Diflunisal phosphate in in on the and for 10 at with the and on and for measured with the as Diamandis E.P. immunoassay with PubMed Scopus Google Scholar). pre- and seminal samples as as male and female serum samples, obtained and to at for in and at for at to plasma from and The seminal plasma at seminal plasma samples and to the TEX101 in the using a to the with a used as the for the absolute of TEX101 by of the to into 10 of seminal plasma proteomic and In of the seminal plasma and of seminal with of the and with and measured by with two used to TEX101 concentrations in the seminal plasma of 20 of the and at for the seminal plasma assay by of with and The for at and with assay normal mouse normal and 10 in of the in a from ng/ml to ng/ml) and on ELISA plasma samples from with the by at with assay on the for the serum assay by in a female serum and of this with The for at and with assay normal mouse normal and 10 in of the in a from ng/ml to ng/ml) and on ELISA samples from with by at to with assay on the a using the of TEX101 and analysis and using from The for TEX101 protein to into a P. expression and TEX101 by affinity from the culture two forms of TEX101 protein and as by and mass spectrometry forms produced because of the of recombinant proteins in P. expression in a PubMed Scopus Google Scholar). of recombinant TEX101 with their to the recombinant TEX101 protein used as an to mouse monoclonal antibodies. The of with in the of hybridoma with antibodies with recombinant antibodies for reaction with the native TEX101 protein in seminal Immunocapture-SRM revealed that produced antibodies that could bind to the native form of TEX101 present in seminal plasma however, of for seminal plasma TEX101 of the also by by the hybridoma in the and using protein mouse monoclonal antibodies and TEX101 epitopes used to develop an antibodies could the native form of TEX101 in seminal we a of that would ELISA and thus allow for TEX101 concentrations in the of seminal plasma with at or with at as the We that a of TEX101 is present in seminal plasma in the is to other proteins, into as or into membrane fragments of that the of plasma of the are in the form of B. L.M. of to plasma membrane from a novel for monitoring membrane and Reprod. PubMed Scopus Google Scholar). are by at B. L.M. of to plasma membrane from a novel for monitoring membrane and Reprod. PubMed Scopus Google and thus are present in seminal plasma samples by of at of membrane and of proteins requires high as with at S. G. A. T. P. T. S. A. sperm plasma membrane and PubMed Scopus Google Scholar). of seminal plasma with at or at may to the of TEX101 from for ELISA by seminal plasma samples obtained from healthy fertile The of TEX101 protein in the by a The and measured in of seminal with full for and two for that of the of the TEX101 protein as the assay for TEX101 measurements in seminal the of for diagnosis of male infertility. monoclonal antibodies and in the The TEX101 protein present in seminal plasma used to the Because of detection of the immunoassay seminal plasma not high we immunosorbent assay of PubMed Scopus Google Scholar, C. M. M. R. A immunosorbent assay for the of membrane and 2010; PubMed Scopus Google Scholar, M. T. M. T. M. proteins as in Immunol. Methods. PubMed Scopus Google Scholar, S. P. cell ELISA to the of cell in GPI-anchored protein Methods. 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Full Text Full Text PDF PubMed Scopus (63) Google Scholar, Diamandis E.P. immunoassay with PubMed Scopus Google Scholar, expression in a PubMed Scopus Google Scholar). a the of at and a of ELISA and sensitivity of TEX101 in seminal plasma The of of the immunoassay, measured with as The of detection as with a range from to ng/ml of seminal plasma measured over in to in and in two According to and for the range of TEX101 concentrations and the of at the TEX101 in this to the pg/ml). of TEX101 by measurements of TEX101 concentrations in seminal plasma at and for a of TEX101 in the seminal plasma measured in the seminal plasma at TEX101 from to ng/ml and TEX101 measured in of pre- and seminal plasma TEX101 concentrations in samples from to TEX101 in samples the ng/ml) on TEX101 in the systemic we to allow for TEX101 measurements in male and female serum TEX101 by of seminal plasma in female serum. of serum with at to be with at A for the TEX101 assay in female serum is in of the immunoassay measured with female serum samples as The as with a range from to ng/ml in and in two over for the range of TEX101 concentrations and of at the pg/ml). of TEX101 from serum by seminal plasma TEX101 into female serum According to TEX101 as by the measured with the Measurement of serum samples from healthy and healthy revealed undetectable levels of TEX101 in the systemic circulation 40 pg/ml). The of TEX101 in serum be by its high tissue specificity and the of in healthy rapid in and mass monoclonal antibodies indispensable for the analysis of proteins in biological fluids. antibody-based are the of for clinical laboratory and biomarker because of their high sensitivity and high and A.P. E. Diamandis E.P. an for protein biomarker 2014; Google Scholar). of the of ELISA development is the of antibodies that bind the native form of We an immunoassay for TEX101 because of the of this for diagnosis of male infertility (5Drabovich A.P. Dimitromanolakis A. Saraon P. Soosaipillai A. Batruch I. Mullen B. Jarvi K. Diamandis E.P. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.Sci. Transl. Med. 2013; 5: 212ra160Crossref PubMed Scopus (115) Google Scholar). Our initial efforts to an immunoassay using commercially antibodies not assays, and immuno-SRM revealed that antibodies the form of TEX101, could not bind the native form present in seminal a mouse antibody from could the native form of TEX101, as revealed by immuno-SRM (5Drabovich A.P. Dimitromanolakis A. Saraon P. Soosaipillai A. Batruch I. Mullen B. Jarvi K. Diamandis E.P. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.Sci. Transl. Med. 2013; 5: 212ra160Crossref PubMed Scopus (115) Google Scholar). to its and high as a for not for ELISA we with production of TEX101 monoclonal antibodies. native we to TEX101 from seminal and of seminal plasma from fertile with TEX101 by and mass spectrometry. TEX101 from the initial of to of however, not for mouse We also to TEX101 in E. however, expression levels and of TEX101 because of protein Expression in the P. and the recombinant TEX101 used for mouse Because of TEX101 in P. however, the produced antibodies recombinant TEX101, not the TEX101 in seminal as confirmed by ELISA and immuno-SRM The form of recombinant TEX101 a and led to of hybridoma by immunocapture-SRM assay revealed that produced antibodies against the native form of In to the sensitivity of the immunoassay, we a of with at and or at for seminal with the TEX101 protein present in a of seminal plasma samples of We also using the recombinant TEX101 in matched the with at TEX101 also from seminal plasma samples and and female serum samples and with the TEX101 screening of antibody clones by under or by Mass and antibody production the rapid screening of hybridoma clones S. L. A for the identification and of monoclonal antibodies from PubMed Scopus Google Scholar). spectrometry detection demonstrated by proposed a for the screening of hybridoma supernatants for antibodies Zhao L. Feng L. L. screening of monoclonal antibodies for using a and reaction Immunol. Methods. 2010; PubMed Scopus Google Scholar). the of immunocapture by detection to hybridoma clones the native form of proteins present in biological not purification using antibodies is a of of to be rapid screening is are in the using a cell supernatants produced by mouse antibodies against TEX101 are with antibodies and with seminal plasma the native form of by identification of hybridoma that antibodies against the native form of Our be to develop antibodies against a of proteins with function A.P. Saraon P. Jarvi K. 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Full Text Full Text PDF PubMed Scopus Google with According to the of immunocapture or over ELISA are with their high specificity and its for multiplex thus to be a to We that ng/ml or even for TEX101 be by assay with a as as of seminal plasma from the clinical In with a high samples a multiplex assay for TEX101 and ECM1 may ELISA for differential diagnosis of we that immunocapture-SRM screening facilitate the development of monoclonal antibodies and immunoassays against the native forms of challenging protein as testis-specific clinical the TEX101 ELISA may emerge as a novel noninvasive clinical laboratory test for the differential diagnosis of male infertility. protein antibodies allow of the human TEX101 interactome and its functional role in and male infertility. We Dimitromanolakis for with with

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