The purpose of this study is 1) to examine the presence of the antigen in serum by employing a newly developed ELISA immunoassay that quantifies the total antigen and bound antigen (antigen-antibody complex) using polyclonal chicken antibodies directed against an IgM-binding epitope of the new antigen, and 2) to determine the location of the antigen in carcinoma and normal cells. Sera from healthy volunteers (n = 147) and cancer patients (n = 26) were compared for both bound and total antigen concentrations using the new ELISA. Healthy volunteers were subdivided into three groups: those with a personal history of cancer (n = 13), those with no personal or family history of cancer (n = 36) and those with a family history of cancer (n = 97). Ovarian, breast, colon carcinoma tissues and their normal counterparts and cultured ovarian and prostatic carcinoma cells were subjected to immunofluorescence using IgY antibodies and goat anti-chicken fluorescent secondary antibodies. Basic imaging was performed on tissue sections while confocal microscopy was performed on cultured cells. Furthermore, immunohisto-chemical staining using an anti-chicken HRP-conjugated secondary antibody was performed on 16 normal ovarian tissues, 53 ovarian adenocarcinomas, and 3 borderline ovarian tumors. Statistical analysis revealed significant differences in cancer patients' bound and total antigen levels compared to that of healthy volunteers (p < 0.005). Bound and total antigen levels of cancer patients were also significantly higher than those of the healthy volunteers with no personal or family history of cancer and those with a family history of cancer (p < 0.01). However, no significant difference existed between the bound (p > 0.120) and total antigen levels (p > 0.076) of cancer patients and patients with a personal cancer history. Immunohistochemical staining of ovarian tissues revealed a significant difference in the lumenal staining of the carcinomas compared to that of the normal ovarian tissues. Furthermore, fluorescence imaging revealed that the antigen is localized to the cell membranes of the carcinoma cells but is absent from the normal tissues. Confocal microscopy further emphasized the antigen's association with the membrane and also revealed some filamentous cortical staining.

Thymidine kinase 1 (TK1) is a cancer biomarker which has diagnostic and prognostic potential in a variety of malignancies. TK1 is significantly elevated in the serum and tumor tissue of most malignancies. This increase in TK1 can be detected in the very early stages of malignancy, including in pre-malignant disease with an increased risk for progression. Several studies have demonstrated that elevated TK1 is found in serum months before any clinical symptoms of malignancy. It has also been demonstrated that TK1 is elevated months before clinical recurrence of malignancy. This work first sought to demonstrate the early nature of TK1 expression in breast tumor tissue and pre-malignant tissue. We found that TK1 is elevated in breast hyperplasia tissue and breast carcinoma tissue. In this study we also identified some cases of ‘normal’ tumor margins (considered normal by current pathological standards) which also had elevated TK1 expression. Conversely, true normal breast tissue from noncancerous individuals had no reported elevation in TK1 expression. This study illustrated that TK1 is elevated in pre-malignant breast hyperplasia tissue, as well as some 'normal' tumor margins. TK1 expression was significantly elevated in lung, prostate, colon, esophagus, stomach, liver, and kidney tissues. This work further investigated TK1 expression in a variety of malignant tissue including the two leading causes of cancer mortality in men: lung and prostate cancer. In our study, TK1 was significantly elevated in lung and prostate cancer but not significantly elevated in prostate hyperplasia tissue. TK1 expression also increased with increasing grade in prostate carcinoma tissue. Overall, this work demonstrated that TK1 is a good universal marker of malignancy and is elevated in early cancer development. Despite the potential for TK1 as both a screening and monitoring treatment tool, there have been significant challenges associated with developing a clinically relevant method of TK1 detection. This work proposes one clinically relevant method of detection, namely a TK1 ELISA. Using preoperable lung cancer patients and normal controls, we developed a sensitive and specific ELISA which shows highly statistically significant differences in serum TK1 levels between stage 1 and stage 2 lung cancer compared with normal controls. In fact, this TK1 ELISA is more sensitive and accurate than the traditional TK radioassay, which was unable to detect differences in TK1 between early stage lung cancer and normal patients. Although elevated TK1 is not lung cancer specific, we reported significantly elevated TK1 levels in lung cancer sputum. Screening of sputum and serum for TK1 may be one method for the early detection of lung cancer. Overall, we report TK1 has promising diagnostic potential in a variety of malignancies. We also propose one sensitive and specific method to detect TK1 levels which may easily be adapted to meet current clinical applications. We hope this work will help propel TK1 forward into clinical view in the coming years.

Current screening methods for drug analysis with urine samples includes examination of the sample with an immunoassay. These methods are used to determine the concentration of drug metabolites contained within the sample prior to further confirmatory testing. Drug testing plays a crucial role in maintaining safe workplace environments and safety of individuals. However, a positive result can lead to heavy consequences for the employee including suspension or removal from the workplace. Therefore, a majority of individuals add commonly known products into the sample to evade detection by developing a false negative result. Although specimen integrity examinations are performed to identify tampering of the sample, these results are typically biased on the experience of the examiner. The purpose of this study was to develop an analytical screening technique that will detect the drug of interest as well as the presence of any additional products that may be added into the sample via Direct Analysis in Real Time – High Resolution/Mass Spectrometry (DART-HR/MS) which is an ambient ionization source that produces fast mass spectrum results that can provide semi-quantitative information of the target metabolite concentration. Although there are various studies that indicate the ability of the DART to detect drug compounds, there are no known studies that have examined how real-world urine samples are analyzed. Additionally, there are no current studies that take into consideration adulteration of the urine sample using the DART method. The results obtained in the study showed the ability for DART to identify molecular protonated peaks indicative of dextroamphetamine and/or the presence of masking agents. While the other target drugs could not be identified using this method, the identification of dextroamphetamine, adulterant products and the deuterated internal standard show promise in using this as a screening technique prior to confirmatory tests. Future work is currently being conducted to optimize the protocol for the evaluation of THC, cocaine and benzodiazepines.

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Coagulation factor XI plays an important role in the consolidation phase of blood coagulation. Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD~110 nM) affinity via residues (K252, K253 and K255) located in the apple 3 (A3) domain of the molecule. In contrast, the enzyme, factor XIa (FXIa), was shown to bind to heparin with significantly higher affinity (~1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). The interaction between heparin and FXIa potentiates the inhibition of FXIa by protease nexin-2 by 10-fold. In addition, related polyanions heparin and dextran sulfate inhibit the catalytic activity of FXIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI in heparin binding. During this project, wtFXI, FXIR504A, FXIK505A, FXIR507A, FXIR529A, FXIR530A, FXIR532A, and FXIR586A have been expressed and purified. All mutants were homogenous and identical to wtFXI on SDS-PAGE, clotting assays and 1G5 monoclonal antibody binding studied by SPR. In addition, monomeric FXI C321S/K331A was expressed and purified. Utilizing an ELISA assay, no difference in the affinity for heparin between FXIa and FXI was found. Surface plasmon resonance (SPR) data collected for FXI clearly indicate a complex interaction which does not conform to a simple 1:1 Langmuir binding model making it difficult to obtain quantitative information. The complexity of FXI interactions with heparin is likely to arise from the multivalent nature of the binding, in which both protein and heparin have multiple binding sites. Two positively charged residues in the FXI catalytic domain, FXIR507A and FXIR532A, were found to be particularly important for interaction with heparin. The FXIR507A and FXIR532A mutants demonstrated ~ 65% and ~50% decreases respectively in total number of heparin binding sites based on ELISA. Also, the apparent dissociation constants for FXIR507A (KDapp ~13 nM) and FXIR532A (KDapp ~21 nM ) were 6 and 10-fold increased respectively compared with 2.1 nM for the wtFXI. Mutant FXIR586A also demonstrated a defect in affinity (KDapp ~ 13 nM) without an effect on the Bmax. The monomeric FXIC321S/R331A was also characterized for its ability to bind heparin compared with wtFXI. Surprisingly, the monomeric FXI displayed defective binding to heparin according to ELISA (KDapp ~ 30 nM) and SPR methods. Thus, the unique homodimeric structure of FXI in addition to the residues both in its catalytic and A3 domain chains are necessary for high-affinity heparin binding. / Biochemistry

Prey-mediated effects of imidacloprid were evaluated for Laricobius nigrinus Fender and Sasajiscymnus tsugae Sasaji and McClure after feeding on hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). Two methods were evaluated for detecting imidacloprid in hemlock tissues: a commercially available enzyme linked immunoassay (ELISA) kit and a high performance thin-layer chromatography technique for detecting and quantifying imidacloprid residues in hemlock wood and needle tissues. ELISA is advantageous because of its cost, sensitivity, and ease of use. However, matrix effects in the form of false positives and overestimated imidacloprid concentrations were evident in hemlock wood and needle tissue extracts. Matrix effects could be reduced by dilution with water, effectively raising the lower detection range of the kit from 0.2 to 200 ppb. High performance thin-layer chromatography was accurate, quick, easy to use, and matrix effects were not evident. However, the technique is sensitive in the lower ppm range and tissue samples from field-treated hemlocks are often in the ppb range, making this technique less desirable than more sensitive analytical methods. Lethal and sublethal effects on both predators were evident after eastern hemlock branches infested with HWA were spiked with imidacloprid in the laboratory. HWA mortality increased with dosage and time, and its 30 d LC50 was determined to be 242 ppb. Both predator species exhibited reduced survivorship and fitness parameters after feeding on HWA from the treated branches. In a topical application bio-assay, 6 d imidacloprid LD50 values for L. nigrinus and S. tsugae were 2.43 and 1.82 µg/g, respectively. Imidacloprid and its major metabolites in hemlock tissues were analyzed by liquid chromatography-tandem mass spectrometry. Imidacloprid recovery from beetle cadavers was correlated with beetle mortality from feeding on treated hemlock branches. Olefin was the primary imidacloprid metabolite recovered from hemlock wood tissues. When predators fed on HWA from field-treated trees, impacts on survivorship and fitness were variable. In 2005, significantly higher proportions of both species of beetles were affected by feeding on control branches compared with treated branches. In 2006, beetles feeding on HWA from some of the trees treated in the field exhibited longer fliptimes compared with beetles feeding on controls, although beetle mortality was not significant among treatements. In the field, imidacloprid controlled HWA populations 1-3 years post-treatment. Hemlock health improved in the highest dosage group, with significantly greater lengths of new shoots compared with shoots from control trees. Eastern hemlock trees primarily metabolized imidacloprid into the olefin metabolite, which can have increased insecticidal toxicity compared with imidacloprid. Imidacloprid was detected in beetle cadavers after feeding on HWA from treated branches, suggesting that prey-mediated impacts of systemic imidacloprid are possible on nontarget predators. However, because of HWA's sensitivity to imidacloprid, in field situations predators are more likely to be affected by reduced adelgid density and quality. / Ph. D.