Objectives: We systematically investigated normally or subclinically increased thyroid stimulating hormone (TSH) values associated with unexpectedly increased thyroxine (FT4) and free triiodothyronine (FT3) in findings of patients without any thyroid disease. Moreover, we looked for alternatives to overcome such states with an improved diagnostic procedure and to investigate the pathogenetic background of the respective patients. Methods: Samples with TSH concentrations within the range of 0.4–10 mU/L combined with increased concentrations of FT4 (n=120; Cobas, Roche) were collected over a period of around six years. Cobas FT4 results were compared with measurements from Liaison (DiaSorin) and Architect (Abbott) FT4 assays. For further validation all samples were measured for total thyroxine (TT4) (Cobas, Roche). Finally, FT3 and TT3 as complementary parameters were measured in samples with leftover material. To overcome potential analytical disturbances from stimulating heterophilic antibodies, we used heterophilic blocking tubes (HBTs). Results: From the 120 samples with increased FT4 concentrations by Cobas, 51/120 were also increased by Liaison, and 26/120 by Architect. However, the measurement of TT4 indicated only n=10/120 increased values. The number of increased FT3 (n=71) measurements was higher in Architect>Cobas>Liaison (28>27>9). TT3 levels of 70/71 samples were within the reference interval. HBTs were inappropriate to reduce unspecific immunoreactivity in our samples. No clear pathogenetic background could be elucidated in the anamnesis of individual patients. Conclusions: To overcome dubious constellations of TSH, FT4, and FT3, it is helpful to measure TT4 and TT3 for control or to use an immunoassay with an alternative assay design for the respective parameters.:I. Abkürzungsverzeichnis 4 I. Einleitung 5 1.1 Hintergrund 5 1.2 Regelkreis 7 1.3. Methodik zur Diagnostik von Schilddrüsenhormonen 8 1.5. Störfaktoren 9 1.6 Fragestellungen/Ziele 11 2 Publikationsmanuskript 13 3 Zusammenfassung der Arbeit 23 4 Literaturverzeichnis 27 II Anhang 37 III Darstellung des eigenen Beitrags 40 IV Erklärung über die eigenständige Abfassung der Arbeit 41 V Curriculum vitae 42 VI Publikationen 44 VII Danksagung 45

The antiseizure medication levetiracetam is used to treat epilepsy with significant success, the medication concentration in serum may be affected by other co-administered medication. Levetiracetam is excreted renally, and the halftime is depending on the renal function which is often correlated to age. The clinical chemistry laboratory at Sundsvall hospital did previously send samples for levetiracetam analysis to Karolinska University Hospital in Stockholm. There was a wish to start analysing the medication locally to reduce analysis time and thereby increase patient safety. ARK Diagnostic markets a kit for quantitative analysis of levetiracetam on automated chemistry analysers. The sample may be taken in a few different test tubes with slightly different characteristics. The aim of this study was to verify the ARK kit on Cobas Pro c503 at Sundsvall hospital and investigate the effects of different test tubes. To assess the accuracy and precision of the method, serum was spiked with levetiracetam from a liquid solution to construct dilution series for testing linearity and sample materials, and by running internal controls to assess the repeatability and reproducibility of the method. The results show that the method does in fact give accurate measurements of the levetiracetam concentration and the results does not vary more than acceptable between measurements nor over time and that different sample type does not show a clinically important difference in result.

Immunoassays basieren auf der Anwendung von Antikörpern, welche selektiv den zu messenden Analyten binden. Die Richtigkeit der erhaltenen Ergebnisse hängt maßgeblich von der Selektivität der Antikörper ab und kann durch Interferenzen gestört werden. In dieser Arbeit wurde eine Methode entwickelt, bei der die Probe mittels Hochleistungsflüssigkeitschromatographie (LC) in Fraktionen aufgetrennt wird und diese Fraktionen anschließend mittels Enzyme-linked Immunosorbent Assay (ELISA) vermessen werden. Dieses Verfahren wurde als LC-ELISA bezeichnet. Das erhaltene Profil aus im ELISA gemessener Analytkonzentration in Abhängigkeit von der Elutionszeit wurde als LC-ELISAgramm bezeichnet und bietet die Möglichkeit, Interferenzen zu erkennen, welche beim ELISA unentdeckt bleiben. Als Modellanalyten für die zu untersuchenden ELISAs dienten Sulfamethoxazol (SMX), Carbamazepin (CBZ) und Estron (E1). Dabei wurden verschiedene Umweltmatrices wie Oberflächenwasser und Abwässer mit dem jeweiligen ELISA vermessen. Es wurde ein Ansatz zur Unterscheidung von spezifischen und unspezifischen Interferenzen in Umweltproben aufgezeigt. Durch diesen Ansatz und Anwendung der sauren Hydrolyse der Probe war es möglich, einen bisher unbekannten SMX-Metaboliten zu detektieren und dessen wahrscheinliche Kreuzreaktivität mit 460 ± 150 % abzuschätzen. Es wurde zudem ein neuer Tracer in einer linearen 13-Stufen-Synthese entwickelt, wobei neuartig die Konjugation der Peroxidase an der N1-Position des SMX erfolgte. / Immunoassays are based on the use of antibodies that selectively bind the analyte. The trueness of the results obtained depends to a great extent on the selectivity of the antibodies and can be affected by interferences. In this study, a method was developed in which the sample is separated into fractions by using high-performance liquid chromatography (LC) and these fractions are measured using an enzyme-linked immunosorbent assay (ELISA). This method was referred to as LC-ELISA. The profile obtained from the measured analyte concentration by ELISA as a function of the elution time was referred to as LC-ELISAgram and offers the possibility to detect interferences which otherwise remain undetected during the ELISA. Sulfamethoxazole (SMX), carbamazepine (CBZ) and estrone (E1) were used as model analytes for the ELISA and LC-ELISA measurements. Various environmental matrices such as surface water and wastewater were examined for their interference in the respective ELISA. The good quantification properties of the validated LC-ELISA have been used to demonstrate an approach to distinguish between specific and non-specific interferences from environmental samples. By this approach and application of acidic hydrolysis of the sample, it was possible to detect a previously unknown metabolite of SMX and estimate its cross-reactivity to probably 460 ± 150%. Furthermore, a new tracer was developed in a linear 13-step synthesis, which resulted in the novel conjugation of the peroxidase at the N1-position of SMX. The new hapten was also used for the synthesis of a novel immunogen.

We demonstrate the improvement of fluorescence immunoassay (FIA) diagnostics in deploying a newly developed compact diode-pumped solid state (DPSS) laser with emission at 315 nm. The laser is based on the quasi-three-level transition in Nd:YAG at 946 nm. The pulsed operation is either realized by an active Q-switch using an electro-optical device or by introduction of a Cr<SUP>4+</SUP>:YAG saturable absorber as passive Q-switch element. By extra-cavity second harmonic generation in different nonlinear crystal media we obtained blue light at 473 nm. Subsequent mixing of the fundamental and the second harmonic in a β-barium-borate crystal provided pulsed emission at 315 nm with up to 20 μJ maximum pulse energy and 17 ns pulse duration. Substitution of a nitrogen laser in a FIA diagnostics system by the DPSS laser succeeded in considerable improvement of the detection limit. Despite significantly lower pulse energies (7 μJ DPSS laser versus 150 μJ nitrogen laser), in preliminary investigations the limit of detection was reduced by a factor of three for a typical FIA.

Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar.<br /> Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. <br /> Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet.<br /> Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid Fructosyl-Valin-Histidin-Leucin-Threonin-Prolin (glkPP) als Kompetitor und Affinitätsligand immobilisiert und so eine regenerierfähige Oberfläche geschaffen. Beim Sandwich-Immunoassay wurde im ersten Schritt Gesamt-Hämoglobin an die mit Haptoglobin (Hp) modifizierte Festphase angereichert und im zweiten Schritt der gebundene HbA1c-Anteil nachgewiesen. <br /> Für die Konstruktion des HbA1c-Immunosensors wurden Affinitätsmatrizen durch Modifizierung von Cellulose-Dialysemembranen mit glkPP bzw. Hp hergestellt. Grundlegend studiert wurde die Aktivierung der Cellulose-Membranen mit 1,1'-Carbonyldiimidazol (CDI) und 1-Cyano-4-dimethylaminopyridintetrafluoroborat (CDAP) als Aktivierungsagenzien. Eine gerichtete Immobilisierung der Liganden wurde realisiert, indem glkPP über dessen C-Terminus (einzige Carboxylatgruppe) und Hp über dessen periodat-oxidiertem Kohlenhydratrest an die amino- oder hydrazidfunktionalisierte Membranen kovalent gekoppelt wurden. <br /> Mit dem Einsatz der glkPP- und Hp-modifizierten Membranen in der elektrochemischen Messzelle war erstmalig der biosensorische Nachweis von HbA1c möglich. Als Transduktor diente eine Pt-Elektrode, an der das von der GOD generierte H2O2 umgesetzt und ein mit der HbA1c-Konzentration korrelierendes Stromsignal erzeugt wurde. Die Immunosensoren zeigten Ansprechzeiten von 3 s. Mit dem Immunosensor auf Basis des indirekt-kompetitiven Testprinzips wurde eine Kalibrationskurve für HbA1c im Bereich von 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %) mit Assayzeiten von 60 min und mit dem Immunosensor im Sandwich-Format eine Kalibrationskurve im Bereich von 0,5-5 &#181;g/ml (7,8-78 nM; 5-50 % HbA1c vom Gesamt-Hb, CV 6-10 %, 3 h) aufgenommen. / Hemoglobin-A1c (HbA1c) is a hemoglobin subtype formed by non-enzymatic reaction of glucose with the N-terminus of the beta-polypeptide chains. As it reflects the glycemic status of diabetics over the preceding 8-12 weeks, the determination of HbA1c has become an established procedure in the management of diabetes mellitus. It is measured as the percentage of total hemoglobin. Up to 5 % HbA1c are considered as normal whereas in diabetic subjects it could be elevated from 5-20 %. In addition to amperometric biosensors for glucose self monitoring which have been successfully applied in diabetes management, biosensors for HbA1c would be an useful supplement for a comprehensive diabetes control. <br /> <br /> Objective of this work was to develop and compare amperometric biosensors for determination of HbA1c based on enzymatic and immunochemical methods. <br /> <br /> For the enzyme based HbA1c assay a novel fructosamine oxidase (FAO) derived from marine yeast Pichia pastoris, strain N1-1 was utilized. It recognizes and oxidatively degrades fructosyl-valine (FV) which corresponds to the glycated N-terminus of the beta-chain of HbA1c and therefore is regarded as a model compound for HbA1c. Hydrogen peroxide which is liberated by the FAO during FV conversion was indicated optically in a horseradish peroxidase (POD) coupled reaction and electrochemically. For the biosensor the FAO was embedded in polyvinyl alcohol-stylbazole (PVA-SbQ) and fixed it in front of a Pt-electrode. So far, the measuring range of FV did not cover the clinically relevant range of HbA1c. Low specificity was assumed since enzyme activity also was obtained with glycated peptides, e.g. fructosyl-valine-glycine, not corresponding to the glycated N-terminus of the hemoglobin-beta-chain.<br /> <br /> For the immunosensor two immunoassays formats - heterogeneous sandwich and heterogeneous competitive - were tested. The assays were designed as follows: The competitive immunoassay was based on the immobilized synthetic glycated pentapeptide fructosyl-valine-histidine-leucine-threonine-proline (glkPP) utilized as HbA1c analogue. The peptide has an amino acid sequence corresponding to the N-terminus of the hemoglobin beta-chains and is capable for competition together with the HbA1c of the sample for the amount of a glucose oxidase (GOD)-labelled anti-HbA1c antibody. In the sandwich-type assay haptoglobin (Hp), a natural hemoglobin binding molecule with antibody characteristic properties, was used as bioreceptor for enrichment of total hemoglobin onto the surface. In a subsequent step the HbA1c fraction was quantified by a GOD-labelled HbA1c specific antibody. <br /> <br /> Cellulose dialysis membrane was used as the solid support for immobilization of Hp and glkPP near the sensor surface. For activation of the membrane two reagents, 1,1&prime;-carbonyldiimidazole (CDI) and 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), were compared with respect to the degree of activation and coupling efficiency. Site-directed immobilization of Hp and glkPP was achieved by coupling Hp via its carbohydrate residue and glkPP via its C-terminus to the activated membrane using a bis-amine or bis-hydrazide spacer. <br /> <br /> The affinity membranes were placed in front of a modified Clark-type hydrogen peroxide electrode in an electrochemical measuring cell and HbA1c analysis was carried out within the stirred cell. Detection of the bound GOD-label was achieved by measurement of the electrocatalytic oxidation of hydrogen peroxide at +600 mV vs. Ag/AgCl. The indication was done in only 3 s. For the competitive principle a typical inhibition curve with a linear range between 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %, 60 min per sample) HbA1c was obtained. Due to the high functional stability of the peptide multiple regeneration of the affinity surface was possible without loss of binding capacity. With the sandwich assay configuration the clinically relevant range could easily be covered (calibration curve: 5-50 % HbA1c corresponding to 7,8-78 nM, CV 6-10 %, 3 h per sample).

Ziele: Die Arbeit hatte die Etablierung und Validierung einer robusten, spezifischen und sensitiven GC-NCI-MS Methode zum Ziel, um Testosteron und 17α-OH-Progesteron im Serum zuverlässig quantifizieren zu können. Anschließend wurde die Methode mit dem Siemens ADVIA Centaur Immunoassay zur Testosteronanalyse verglichen. Hintergrund der Arbeit ist die mangelnde Richtigkeit und Sensitivität der Steroidhormonanalyse mit Immunoassays, inbesondere bei Hormonanylsen in geringen Konzentrationen, wie z.B. Testosteron bei Frauen und Kindern. Methode: Die Methode umfasste die Zugabe von deuterierten Internen Standard zu 1 ml Serum, gefolgt von Flüssig-Flüssig-Extraktion mit Ethylacetat und einem Clean-up mittels Festphasenextraktion. Die angereicherten und aufgereinigten Proben können mit Pentafluor¬benzyl¬hydroxylamin-hydrochlorid und MSTFA/TMCS (99:1) erfolgreich derivatisiert und im Anschluss in die GC-MS injiziert werden. Die Methode zeigt eine exzellente chromatographische Trennung. Testosteron und 17-OH-Progesteron wurden im Selected Ion Monitoring detektiert, die Quantifizierung erfolgte durch den Vergleich der Verhältnisse der Peakflächen zwischen Internen Standard und Analyten. Für den Methodenvergleich, zwischen der GC-MS Methode und dem Siemens ADVIA Centaur Immunoassay, wurde Testosteron in 10 Proben von Männern und 20 Proben von Kindern und Frauen analysiert. Ergebnisse: In dem für Männer physiologischen Konzentrationsbereich (2,62 -9,29 ng/ml) konnte eine sehr gute Übereinstimmung der Analysenergebnisse beider Methoden gezeigt werden (r= 0,97). Im Gegensatz dazu fiel im niedrigen Konzentrationsbereich aus Seren von Frauen und Kindern (0,05 - 0,51 ng/ml) eine geringe Übereinstimmung (r= 0,77) der Messergebnisse auf, wobei hier mit dem untersuchten Immunassay im Mittel um 158,9 % höhere Konzentrationen erzielt wurden. Schlussfolgerung: Der Methodenvergleich spiegelt die aktuelle Problematik in der Testosteronanalytik wider. So werden Messdifferenzen zu massenspektrometrischen Referenzmethoden auf eine mangelnde analytische Spezifität und Sensitivität, als auch ungenügende Validierung der Immunoassays zurückgeführt. Wenn niedrige Konzentrationen erwartet werden, wie bei der Testosteronanalyse in Serum von Frauen und Kindern, oder wenn zweifelhafte Analysenergebnisse vorliegen, sollte auf massenspektrometrische Referenzmethoden zurückgegriffen werden.

Biotin är ett vitamin som finns naturligt i livsmedel och det dagliga intaget nås via födan. Höga doser biotintillskott samt höga doser biotin i läkemedel, kan leda till biotininterferens i kliniska immunokemiska analyser. Roche Diagnostics® vill införa nya reagens med högre tröskel för biotininterferens för att minska risken för biotininterferens vid analys av patientplasma. Därför var syftet med studien att metodverifiera fyra nya reagens från Roche Diagnostics® som används vid diagnostisering och behandling av hjärtsvikt, sepsis, och prostatacancer. De fyra reagensen, Elecsys® proBNP II, Elecsys® BRAHMS PCT, Elecsys® total PSA samt Elecsys® free PSA metodverifierades för att användas på Cobas® e801. Studiematerialet bestod av 20 patientprover av litiumheparinplasma per reagens (totalt 80 patientprover). Resultatet av verifieringen av Elecsys® proBNP II visade en korrelation till det befintliga reagenset på r = 0,9998 och Bland-Altman analys visade en spridning av resultaten på &lt; 10 %; inomserieprecisionsstudien gav CV 1,56 %. Elecsys® BRAHMS PCT hade en korrelation på r = 0,9997 och Bland-Altman analysen visade en spridning på &gt; 10 %; inomserieprecisionsstudien gav CV 1,70 %. För Elecsys® total PSA och free PSA fanns korrelationen till det befintliga reagenset på r = 1 respektive 0,9997 och Bland- Altman analysen visade en spridning på &lt; 10 % hos båda reagensen. Inomserieprecisionsstudien gav CV 0,44 % respektive CV 2,67 %. Resultaten för samtliga reagens uppvisar god korrelation till det befintliga reagenset och en hög mätnoggrannhet vilket talar för att de fyra nya reagensen kan tas i bruk. / Biotin is naturally found in foods, and we obtain this vitamin through our daily diet. Biotin supplements as well as high doses of biotin in drugs can lead to biotin interference in clinical immunochemical analyzes. Therefore, the purpose of this study was to methodically verify four new reagents from Roche Diagnostics® with a higher threshold for biotin interference, used in the diagnosis and treatment of heart faliure, sepsis and prostate cancer. The four reagents, Elecsys® proBNP II, Elecsys® BRAHMS PCT, Elecsys® total PSA and Elecsys® free PSA were method-verified for use on Cobas® e801. The study material consisted of 20 patient samples of lithium heparin plasma per reagent. In total 80 samples were analyzed.The result of the verification of Elecsys® proBNP II showed a correlation to the existing reagent of r = 0.9998 and Bland-Altman analysis showed a distribution of the results of &lt;10 %. The withinseries precision study yielded CV 1.56 %. Elecsys® BRAHMS PCT had a correlation of r = 0.9997 and the Bland-Altman analysis showed a distribution of &gt; 10 %. The withinseries precision study gave CV 1.70 %. For Elecsys® total PSA and free PSA, the correlation to the existing reagent was r = 1 and 0.9997, respectively, and the Bland-Altman analysis showed a distribution of &lt;10 % in both reagents. The withinseries precision study yielded CV 0.44 % and CV 2.67 % respectively.The results for all reagents show a good correlation to the existing reagent and a high accuracy of measurement, which indicates that the four new reagents can be used.

Tillväxthormon och insulinliknande tillväxtfaktor-1 är blodprover som tas vid misstanke om akromegali. Immulite 2000 XPi är ett instrument som använder chemiluminescent microparticle immunoassay metodik för att kvantitativt mäta koncentrationen av GH och IGF-1. Analys av biokemiska markörer påverkas av flera olika faktorer. En viktig sådan faktor är hemolys. Hemolys innebär att de röda blodkropparna går sönder, vilket leder till frisättning av dess innehåll såsom hemoglobin i serum/plasma. Syftet med detta projekt var att undersöka hur hemolys påverkar resultatet av GH- och IGF-1-prover på immulite 2000 XPi. Utifrån erhållna resultat kommer klinisk kemi vid Universitetssjukhuset Örebros laboratorie rutiner vid hemolytiska prover på GH eller IGF-1 att uppdateras . Hemolys tillverkades och tillsattes till olika serumprover med låg och hög nivå av GH respektive IGF-1 för att erhålla prover med varierande grad av hemolys. Resultatet visade att ökande hemolysindex korrelerar med GH- respektive IGF-1-koncentrationer, med undantag för den låga GH koncentrationen som inte uppvisade någon korrelation till hemolysindex. En 10%:ig skillnad av GH och IGF-1- koncentrationer uppnåddes vid en ökning av hemolysindex med 555,40 mg/dL för GH respektive 333,3 mg/dL för IGF-1.Utifrån resultaten var hemolysgränsen likvärdig med tillverkarens gränser. Därför kan klinisk kemi på Universitetssjukhuset Örebro fortsätta med nuvarande hemolysgränser. / Growth hormone and insulin-like growth factor-1 are blood samples taken upon suspicion of acromegaly and for follow-up of acromegaly treatment. Immulite 2000 XPi is an instrument that uses chemiluminescent microparticle immunoassay method to quantitatively measure the concentration of GH and IGF-1. Analysis of biochemical markers is affected by several different factors. An important such factor is hemolysis. Hemolysis means that the red blood cells break, whose contents leak into the serum/plasma. The study aimed to investigate how hemolysis affects the results of GH and IGF-1 samples on the Immulite 2000 XPi. The results will determine the routines for hemolytic samples for GH or IGF-1 at Örebro University Hospital. Hemolysis was produced and added to various serum samples with low and high levels of GH and IGF-1 to obtain samples with varying degrees of hemolysis. The results showed that increasing hemolysis index correlates with GH and IGF-1 concentrations, with the exception of low GH concentration, which did not show any correlation to hemolysis index. A 10% difference in GH and IGF-1 concentrations was achieved with an increase in hemolysis index of 555.40 mg/dL for GH and 333.3 mg/dL for IGF-1. Based on the results, the hemolysis limit was equivalent to the manufacturer's limits. Therefore, clinical chemistry at Örebro University Hospital can continue with current hemolysis limits.

Progesterone in the mare declines to almost undetectable concentrations in late gestation. It’s metabolized into several pregnanes, some circulating at very high concentrations. Although the function of many pregnanes remains unclear, 5α-dihydroprogesterone and allopregnanolone are bioactive. Measurements of pregnanes in late gestation are typically by immunoassay, although results are confounded by cross-reactivity with related pregnanes. Conversely, liquid chromatography tandem mass spectrometry (LC-MS/MS) allows differentiation of individual pregnanes. The purposes of these studies were: 1) to evaluate the ability of a 5α-reductase inhibitor, dutasteride, to alter pregnane metabolism and pregnancy outcome, 2) to evaluate changes in target pregnanes in late gestation by LC-MS/MS in mares with ascending placentitis, and 3) compare immunoassay and LC-MS/MS detection of pregnanes in late gestation. Our findings suggest that dutasteride significantly altered pregnane metabolism without effects on pregnancy outcome. Pregnane measurement by LC-MS/MS resulted in a significant (p<0.05) differences in assay results, while correlation was observed between immunoassay measurements and actual progesterone concentrations by LC-MS/MS. These studies demonstrate the complexity of pregnane metabolism in late gestation in the mare and the necessity of LC-MS/MS to detect specific changes that immunoassays cannot differentiate.

Luminescent quantum dot (QD) based probes have gained significance in the last decade for optical imaging of cells, tissues and in bioassays as alternatives to conventional organic fluorophores. The main objective of my PhD dissertation was to develop luminescent quantum dot based bioassays for real time monitoring of enzyme activity and simultaneous detection of several biomarkers. The quantum dot based bioassays developed will be potential tools in identification and diagnosis of several ailments that interfere with normal living conditions of human beings. In Chapter 2 new liposome encapsulated quantum dot based fluorescence resonance energy transfer (FRET) probes have been fabricated and characterized for monitoring the enzymatic activity of phospholipase A 2. The probes were able to detect the enzyme activity as low as 0.0075 U/mL (PLA2 = 1500 U/mg) in 30 min. Further these FRET probes were also used to screen the inhibition efficiencies of phospholipase A2 inhibitors. Chapter 3 focuses on the first time synthesis and characterization of liposome encapsulated InP/ZnS quantum dots while preserving the integrity of the liposomes. Results from the experiments to assess photostability and effect of pH on the optical properties of InP/ZnS QD-liposomes showed greater advantages over InP/ZnS quantum dots demonstrating their utility as a potential tool in several biological applications such as bio imaging, bioassays and in immunoassays. Chapter 4 discusses the development of fluorescence based immunoassay for simultaneous detection of the cardiac biomarkers troponin T and troponin I using CdSe/ZnS quantum dots. The assay achieved a detection limit was 0.1 pg/mL for both biomarkers troponin xi T and I. The method was highly specific for the both the biomarkers with no observed cross reactivity. The multiplex assay was able to detect two biomarkers simultaneously that will yield a high throughput diagnostic tool for heart attack. A similar method discussed as above was used in chapter 5 for the simultaneous detection of atherosclerosis biomarkers. The detection limits achieved in this study are comparable to the detection limits of the biomarkers reported so far. Incorporation of QDs in silica beads before conjugation to antibodies might improve detection limits that will also improve risk assessment.