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

BACKGROUND: TEX101 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. METHODS: Mass 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. RESULTS: We 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. CONCLUSIONS: We 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 MATERIAL: The online version of this article (doi:10.1186/s12916-017-0817-5) contains supplementary material, which is available to authorized users

Development of new analytical methods for the determination of thiols in food and biological samples using the hyphenation of liquid chromatography and flow automated systems

In summary, the experimental data of the present Dissertation lead to the following:The first study reports the very first application of ethyl propiolate (EP) as anadvantageous pre-column derivatization reagent for the determination of cysteine,glutathione and N-acetylcysteine by liquid chromatography. These thiols werederivatized online under stopped-flow conditions in a sequential injection systemcoupled to liquid chromatography. The reaction was rapidly allowed to proceed understopped-flow conditions in the loop of the LC setup. The formed derivatives wereseparated isocratically using a monolithic stationary phase and were detectedspectrophotometrically at 285 nm. Critical parameters that affected the online precolumnderivatization reaction (e.g. the reaction time and the amount concentration ofEP) and the separation (e.g. pH and the composition of the mobile phase) wereinvestigated. The developed analytical scheme offers a total analysis time of less than10 min. The limits of detection were in the range of 0.24–0.40 × 10-3 mol L-1and thelinearity achieved were up to 200 × 10-3 mol L-1for all analytes. The proposed methodwas satisfactorily applied to the analysis of the selected thiols – that are oftenemployed as antibrowning agents – in fresh fruit samples.The second study reports a new High Performance liquid chromatographic (HPLC)method for the determination of the anti-hypertension drug captopril in human urine.After its separation from the sample matrix in a reversed phase HPLC column,captopril reacts with the thiol-selective reagent ethyl-propiolate (EP) in a postcolumnconfiguration (PCD) and the formed thioacrylate derivative is detectedspectrophotometrically at 285 nm. Automated 4-fold preconcentration of the analyteprior to analysis was achieved by an on - line solid phase extraction (SPE) step usinga Sequential Injection (SI) manifold. The Oasis HLB SPE cartridges offeredquantitative recoveries and effective sample cleaning by applying a simple SPEprotocol. The limits of detection and quantitation were 10 μg L-1and 35 μg L-1respectively. The percent recoveries for the analysis of human urine samples rangedamong 90 – 96 % and 95 – 104 % using aqueous and matrix matched calibrationcurves respectively. The features of the developed method are as follows: i) it is moresensitive and simple compared to the only previously reported PCD method; ii) thesample preparation and analysis steps are fully automated by coupling SI and HPLC; iii) the Oasis HLB SPE cartridges offer quantitative recoveries of the analyte from thebiological matrix, preconcentration potentials and adequate capacity and stability; iv)EP is an excellent reagent for captopril in terms of sensitivity and rapid reaction underflow conditions; (v) the method is capable of determining total captopril since theexcess of the reducing reagent does not interfere in the PCD mode.Finally the last work reports one of the very first applications of on-line post-columnderivatization to hydrophilic interaction chromatography, offering a viable tool for thedetermination of low molecular weight thiolic compounds. More specifically itproposes a new method for the determination of Glutathione and Cysteine in yeastsamples. The analytes were separated isocratically from the sample matrix byHydrophilic Interaction Chromatography (HILIC) and they were detectedspectrophotometrically at 285 nm following on-line post-column derivatization (PCD)by the thiol-selective reagent methyl-propiolate in alkaline medium. The method takesadvantage of the ability of HILIC to retain polar analytes and the well-establishedpotentials of post-column derivatization to the analysis of complicated matriceswithout matrix effects. The main figures of merit included linearity in the range of 5 –200.0 × 10-6 mol L-1and a limit of detection 0.6 × 10-6 mol L-1for both compounds.The absence of matrix effect allowed the determination of cysteine and glutathione invarious yeast samples. Glutathione was present in most of the samples at levelsranging among 0.9 – 3.1 mg g-1, whereas Cysteine was determined in only one sampleat significantly lower concentration. In terms of accuracy, the percent recoveriesranged among 91.2 to 105.6 % for glutathione and 91.6 to 106.9 % for cysteine.Συνοψίζοντας, τα πειραματικά δεδομένα της Διδακτορικής Διατριβής προκύπτουν ταπαρακάτω:1) Στην πρώτη πειραματική εργασία προτείνεται μια νέα φασματοφωτομετρικήμέθοδος ταυτόχρονου προσδιορισμού τριών θειολών, της κυστεΐνης, τηςγλουταθειόνης και της Ν-ακελυτοκυστεΐνης σε φρούτα. Η ανάπτυξη της μεθόδουπραγματοποιήθηκε με σύζευξη δύο επιμέρους αναλυτικών τεχνικών, της Τεχνικήςτων Διαδοχικών Εγχύσεων και της Υγρής Χρωματογραφίας Υψηλής Απόδοσης.Χαρακτηριστικό της μεθόδου είναι η πλήρης αυτοματοποίηση του συζευγμένουσυστήματος και η δυνατότητα ανεξάρτητης λειτουργίας και ελέγχου των επιμέρουςδιατάξεων από ηλεκτρονικό υπολογιστή. Στην προτεινόμενη μέθοδο γίνεται χρήσηγια πρώτη φορά των άλκυλο εστέρων του προπιολικού οξέως για τηνπαραγωγοποίηση θειολών, πριν τον διαχωρισμό τους σε χρωματογραφική στήλη. Μετην Τεχνική των Διαδοχικών Εγχύσεων επιτυγχάνεται η εν-ροή προσθήκη τωνδειγμάτων και του αντιδραστηρίου παραγωγοποίησης, ενώ με την ΥγρήΧρωματογραφία πραγματοποιείται ο διαχωρισμός των σχηματιζόμενων παραγώγωνμε τη χρήση μονολιθικής χρωματογραφικής στήλης αντίστροφης φάσης υπόισοκρατικές συνθήκες έκλουσης. Η εν ροή αντίδραση των τριών θειολών με τααντιδραστήρια παραγωγοποίησης μεθυλικός εστέρας του προπιολικού οξέος καιαιθυλικός εστέρας του προπιολικού οξέος πραγματοποιείται σε καναλικό σύστημα, τοοποίο παράλληλα χρησιμεύει για τη σύζευξη των δυο οργανολογικών διατάξεων. Ηχρήση μονολιθικής στήλης επιτρέπει την ταχεία έκλουση των παραγώγων και οδηγείσε υψηλούς ρυθμούς δειγματοληψίας. Η ανίχνευση πραγματοποιείταιφασματοφωτομετρικά στα 285 nm. Η προτεινόμενη μέθοδος επικυρώθηκε ως προς τηγραμμικότητα (έως 200 × 10-6 mol L-1), τα όρια ανίχνευσης (0,24 – 0,40 μmol L-1),ποσοτικού προσδιορισμού, την ακρίβεια, την εκλεκτικότητα και εφαρμόστηκε μεεπιτυχία για τον προσδιορισμό των τριών θειολών και την παρακολούθηση τουρυθμού κατανάλωσής στους σε φρούτα.2) Στη δεύτερη πειραματική εργασία προτείνεται μια νέα χρωματογραφική μέθοδοςγια τον προσδιορισμό της καπτοπρίλης σε βιολογικά δείγματα. Η αναλυτικήδιαδικασία χωρίζεται σε τρία μέρη. Κατά το πρώτο μέρος πραγματοποιείται αυτοματοποιημένα, με τη βοήθεια της Τεχνικής των Διαδοχικών Εγχύσεων, εκχύλισηκαι προσυγκέντρωση του αναλύτη με χρήση μικροστηλών εκχύλισης στερεής φάσηςOASIS HLB. Στη συνέχεια, διαμέσου της βαλβίδας πολλαπλής επιλογής, η θειόληπροωθείται στο βρόγχο της βαλβίδας έγχυσης της χρωματογραφίας, εγχύεται σεαναλυτική στήλη αντίστροφης φάση και διαχωρίζεται από τα λοιπά συστατικά τουυποστρώματος υπό ισοκρατικές συνθήκες έκλουσης. Τέλος, μετά την έξοδό της απότη στήλη, η καπτοπρίλη συναντά τα αντιδραστήρια παραγωγοποίησης μεθυλικόςεστέρας του προπιολικού οξέος ή αιθυλικός εστέρας του προπιολικού οξέος σε ρεύμασυνεχούς ροής, όπου και πραγματοποιείται εν ροή η αντίδραση παραγωγοποίησης. Τοτελικά σχηματιζόμενο προϊόν οδηγείται σε φασματοφωτομετρικό ανιχνευτή, όπου καιανιχνεύεται. Όμοια με την προηγούμενη μέθοδο οι δύο διατάξεις λειτουργούνανεξάρτητα η μία από την άλλη και ελέγχονται από ηλεκτρονικό υπολογιστή. Ηπροτεινόμενη μέθοδος δίνει τη δυνατότητα προσδιορισμού όχι μόνο της ανηγμένηςμορφής της καπτοπρίλης, αλλά και της συνολικής της ποσότητας, καθώς η παρουσίααναγωγικών αντιδραστηρίων στο μίγμα που εγχύεται στη στήλη δεν επηρεάζει τηναντίδραση παραγωγοποίησης. Σε σύγκριση με παλαιότερες μεθόδους προσδιορισμούτης καπτοπρίλης μετά από παραγωγοποίηση η προτεινόμενη μέθοδος πλεονεκτεί ωςπρος την ευαισθησία για την εφαρμογή της σε δείγματα ούρων και την απλότητα πουτης χαρίζει ο αυτοματοποιημένος χαρακτήρας της. Η προτεινόμενη μέθοδοςεπικυρώθηκε ως προς τη γραμμικότητα (150 μg L-1- 2500 μg L-1), τα όριαανίχνευσης και ποσοτικού προσδιορισμού (10 μg L-1και 35 μg L-1αντίστοιχα), τηνακρίβεια και την εκλεκτικότητα και εφαρμόστηκε με επιτυχία στον προσδιορισμόκαπτοπρίλης σε δείγματα ούρων.3) Στην τρίτη πειραματική εργασία προτείνεται μια νέα φασματοφωτομετρικήμέθοδος για τον προσδιορισμό γλουταθειόνης και κυστεΐνης σε δείγματα μαγιάς.Αρχικά πραγματοποιήθηκε ο χρωματογραφικός διαχωρισμός των δύο θειολών μεχρήση χρωματογραφικής στήλης υδρόφιλων αλληλεπιδράσεων και στη συνέχεις οιθειόλες παραγωγοποιήθηκαν εν-ροή με τον μεθυλικό εστέρα του προπιολικού οξέος.Τα τελικά σχηματιζόμενα παράγωγα ανιχνεύθηκαν στα 285 nm. Η μέθοδος είναισύμφωνα με τη βιβλιογραφία μια από τις πρώτες μεθόδους που πραγματοποιείαντίδραση παραγωγοποίησης μετά από χρωματογραφικό διαχωρισμό σε σύστημαυδρόφιλων αλληλεπιδράσεων. Από τη μια η ικανότητα της Υγρής ΧρωματογραφίαςΥδρόφιλων Αλληλεπιδράσεων να συγκρατεί ικανοποιητικά και να διαχωρίζει πολικέςενώσεις και από την άλλη η αύξηση της ευαισθησίας ως αποτέλεσμα τωναντιδράσεων παραγωγοποίησης μετά τη χρωματογραφική στήλη προσέφεραν τηδυνατότητα ανάλυσης των δειγμάτων χωρίς επιδράσεις λόγω του υποστρώματος. Ηπροτεινόμενη μέθοδος επικυρώθηκε ως προς τη γραμμικότητα (έως και 200 μg L-1), αόρια ανίχνευσης και ποσοτικού προσδιορισμού 0,6 × 10-6 mol L-1και 2,0 × 10-6 mol L-1αντίστοιχα), την ακρίβεια και την εκλεκτικότητα και εφαρμόστηκε με επιτυχίαστον προσδιορισμό γλουταθειόνης και κυστεΐνης σε δείγματα μαγιάς

Micelles Mediated Zone Fluidics Method for Hydrazine Determination in Environmental Samples

An automated flow method for the determination of hydrazine based on the concept of zone-fluidics has been developed. The analyte reacts under flow conditions with p-dimethylamino benzaldehyde (25 mmol L−1) in micellar medium (100 mmol L−1 SDS) to form a stable derivative (460 nm). Micelles mediated catalysis excludes the use of highly acidic environment typical for this kind of reaction. Following careful examination of chemical and instrumental variables, the method allows the determination of hydrazine at the low micromolar level (0.3–10 μmol L−1) in water samples. Real sample analyses (drinking and boiler feed water) resulted in satisfactory results in terms of accuracy with the percent recoveries being in the range of 82–114%

MOESM9 of A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

Additional file 9: Figure S5. KLK9 free monomer detection by the newly developed KLK9 ELISA upon the formation of serpinA3–KLK9 heterocomplexes. Mat-KLK9 (0.5 μg) was incubated either alone (control) or with different amounts of the human recombinant serpinA3 inhibitor (R&D systems) [at molar ratios (KLK9/SerpinA3): 1/0.2, 1/0.5, 1/1 and 1/2] in 50 mM Tris–HCl (pH 8.0) for 1 h at 37 °C. The samples were further diluted with 6% BSA and the assay was performed by following the described protocol (see “Methods” section). The values of the sample containing no inhibitor (control) was arbitrarily defined as 100 % recovery. The samples containing serpinA3–KLK9 complexes were expressed as % recovery of KLK9 compared to the control

Thyroid dysfunction and autoantibodies in early pregnancy are associated with increased risk of gestational diabetes and adverse birth outcomes

Context: Maternal thyroid dysfunction, especially in early pregnancy, may lead to pregnancy complications and adverse birth outcomes. Few population-based prospective studies have evaluated these effects and results are discrepant. Objective: We examined the association of thyroid function and autoimmunity in early pregnancy with adverse pregnancy and birth outcomes. Setting and Participants: The study used data from the prospective mother-child cohort >Rhea> study in Crete, Greece. A total of 1170 women with singleton pregnancies participated in this analysis. Maternal serum samples in the first trimester of pregnancy were tested for thyroidhormones (TSH, free T4, and free T3) and thyroid antibodies (thyroid peroxidase antibody and thyroglobulin antibody). Multivariable log-Poisson regression models were used adjusting for confounders. Main Outcome Measures: Outcomes included gestational diabetes, gestational hypertension/preeclampsia, cesarean section, preterm delivery, low birth weight, and small-for-gestational-age neonates. Results: The combination of high TSH and thyroid autoimmunity in early pregnancy was associated with a 4-fold increased risk for gestational diabetes [relative risk (RR) 4.3, 95% confidence interval (CI) 2.1- 8.9)] and a 3-fold increased risk for low birth weight neonates (RR 3.1,95%CI 1.2- 8.0) after adjustment for several confounders. Women positive for thyroid antibodies without elevated TSH levels in early pregnancy were at high risk for spontaneous preterm delivery (RR 1.7, 95% CI 1.1-2.8), whereas the combined effect of high TSH and positive thyroid antibodies did not show an association with preterm birth. Conclusions: High TSH levels and thyroid autoimmunity in early pregnancy may detrimentally affect pregnancy and birth outcomes. Copyright © 2012 by The Endocrine Society.This work was supported by the European Uniuon Integrated Projects NewGeneris, Sixth Framework Programme (Contract FOOD-CT-2005-016320), and Chicos, Seventh Framework Programme (Contract Health-F2-2009-241604).Peer Reviewe

Maternal Weight Status, Cord Blood Leptin and Fetal Growth: a Prospective Mother-Child Cohort Study (Rhea Study)

{Background: Leptin is an adipocyte-secreted hormone that regulates energy homeostasis, while its role in fetal programming remains poorly understood. We aimed to evaluate the effect of maternal weight status on cord blood leptin levels and their combined effect on fetal growth. Methods: We included 638 mother-child pairs from the prospective mother-child cohort `Rhea' study in Crete, Greece with singleton pregnancies, providing cord blood serum samples for leptin analysis and complete data on birth outcomes. Multivariable logistic and linear regression models were used adjusting for confounders. Generalised additive models were used to explore the form of the relationship between cord leptin and continuous birth outcomes. Results: Log cord leptin was positively associated with birthweight \{beta-coef: 176.5 {[}95\% confidence interval (CI): 133.0, 220.0]\}, ponderal index (beta-coef: 1.0 {[}95\% CI: 0.6, 1.4]) and gestational age (beta-coef: 0.7 {[}95\% CI: 0.5, 0.8]). Excessive weight gain during pregnancy was associated with a threefold increased risk for cord hyperleptinaemia \{relative risk (RR): 3.0, {[}95\% CI: 1.5, 6.3]\}. Maternal pre-pregnancy overweight/obesity {[}body mass index (BMI) >= 25 kg/m(2)] increased the risk of giving birth to a hyperleptinaemic neonate (RR: 2.1 {[}95\% CI: 1.4, 3.2] and the effect of log leptin on birthweight (beta-coef: 219.1 {[}95\% CI: 152.3, 285.9] compared with women with a BMI <25 kg/m(2) (beta-coef: 150.5 {[}95\% CI: 93.1, 207.9]. Conclusions: Higher cord blood leptin levels are associated with increased size at birth and gestational age, while maternal pre-pregnancy BMI and weight gain during pregnancy represent significant indicators of cord blood leptin.

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

Abstract Background TEX101 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. Methods Mass 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. Results We 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. Conclusions We 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