Published research in the Journal of Analytical Toxicology and the American Society for Clinical Pathology has confirmed that the presence of Zinc Sulfate in adulterated urine samples can influence the testing results using EMIT and ELISA immunoassay testing when testing for Cannabinoids (THC), Cocaine (Benzoylecgonine), Methamphetamines, Opiates (Morphine, Methadone, and Propoxyphene), Phencyclidine (PCP), and Ethanol (Alcohol Dehydrogenase). This research included adding Zinc Sulfate directly to urine samples. In 2006, the Substance Abuse and Mental Health Service Administration (SAMHSA) released an advisory that the use of Ethyl glucuronide (EtG) as a new biomarker as an indicator for the past-use of alcohol was promising and warranted more research. Ethyl glucuronide is a direct metabolite of the biotransformation of ethanol in the human body. This compound is excreted in urine and can be used as a specific biomarker for the ingestion of alcohol. Because EtG is only produced when ethanol is metabolized, there are no false positives due to fermentation and a much longer detection window exists for its detection. Scientific literature states that EtG can be present in urine long after ethanol has been eliminated. Testing for EtG is commonly referred to as the “80 hour test” for the ability of EtG to be measured up to 80 hours after consuming alcohol. It was hypothesized that if the presence of Zinc Sulfate added to urine falsely reduced urine alcohol level when measuring for Alcohol Dehydrogenase enzyme, will the presence of Zinc Sulfate added to SurineTM falsely reduce the urine alcohol level when measuring for EtG? Since it is very likely that EtG would still be present in the body after ethanol has been eliminated, samples contained either no ethanol or 5% (5g/dL) of ethanol. Samples were spiked at 10mg/mL, 15mg/mL or contained 0mg/mL of Zinc Sulfate. Additionally, duration testing was conducted to see if there was any observed differences between testing the samples fresh and then after a one week duration in a refrigerator and brought to room temperature prior to testing. Two different immunoassay EtG tests were used to perform the analysis. It was concluded that Zinc Sulfate directly added to the sample affected one of the immunoassay test regardless of whether EtG or ethanol were present, by fading the Test and Control regions. Additionally, it is concluded that SurineTM samples containing Zinc Sulfate could easily be distinguished from samples free of Zinc Sulfate because of the presence of a white cloudy precipitate.

Recently, point-of-care diagnostics has gained great attention because it can improve patient’s quality of life. Electrochemical diagnostic systems are promising because of their miniaturizability and low-cost. However, fabrication of such devices requires special skills as well as expensive equipment and supplies. This thesis is based on a research project aimed at fabricating electrochemical sensors combing wax printing and inkjet printing or wax printing and hand painting. The electrochemical sensors can be used for measuring different kinds of electrochemical analytes like dopamine, uric acid by electrochemical methods like amperometry, which can show great calibration curve. The LOD of dopamine, uric acid, ascorbic acid, Nile Blue, hydrogen peroxide and ferrocene is 0.015 µM, 7.3 µM, 30 µM, 1.3 µM, 8 nM and 30 µM, respectively. Further, we can modify the electrochemical sensor by using multiwall carbon nanotube in order to improve the sensitivity of the electrochemical sensors. This modified electrochemical sensor can also be used as immunoassay by sandwich format ELISA for detecting carcinoembryonic antigen (CEA), which has been designated as a reliable biomarker for several types of cancers. We found that the CNT modified hand-painting device can detect CEA down to 0.6 ng/mL, which is three times lower than the cut-off value of diagnosis, i.e. 5 ng/mL in blood.

The aim of the research was to use the principles of immunoassays and fluorescence spectroscopy to detect the presence of 4-amino-N-(4,5- dimethyl-2-pyrimidinyl)benzenesulfonamide, commonly known as sulfamethazine, in food.

Thesis: M.S., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1975 / Vita. / Includes bibliographical references. / M.S. / M.S. Massachusetts Institute of Technology, Department of Nutrition and Food Science

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The strategy required for the development of the immunochemical reagents needed for a non-competitive assay for the detection of haptens was investigated. For this work, these reagents were termed "anti-complex" antibodies. To preserve the integrity of such an assay, these "anti-complex" antibodies should not react with either the free anti-hapten antibody or the free hapten and they should not compete with the hapten for binding to the anti-hapten antibody. Anti-hapten hybridomas were developed using standard hybridoma technology. The development of anti-complex hybridomas was investigated using standard hybridoma technology and the novel approach of intrasplenic immunization. Both immunization protocols addressed the three questions concerning the strategy for developing such hybridomas, namely the type of immunization (snygeneic or allogeneic), the physico-chemical nature of the hapten in the complex (free or conjugated) and the physico-chemical nature of the anti-hapten antibody in the complex (intact or fragmented). (Abstract shortened with permission of author.)

The components involved in an immunoassay were investigated in order to improve the detection limits of the ELISA and to make the assay adaptable to a flow injection analysis (FIA) configuration. The goal being the total automation of the ELISA procedure which is long, tedious and has high standard deviation. The antibody purification and cleavage methods were studied with special emphasis on obtaining products with highest immunological activity. The antibody-enzyme coupling reactions using homobifunctional reagents and heterobifunctional reagents were studied in order to attempt the preparation of highly characterized reagents. The fragments of IgG were coupled to polymeric supports via the hinge thiol groups to retain the maximum immunological activity. This method was found to be superior to those methods involving coupling via amino group. These reagents were used in the development of a sandwich ELISA for bovine IgG. The range of assay was in the 20-1000 femtomole range with a linear dynamic range of 2 orders of magnitude and an accuracy of 2-5%. A competitive ELISA based on the use of immobilized anti-human IgG Fab' fragments was developed. The linear dynamic range for this assay was found to be less than one order of magnitude. The detection limit was in the low picomole range with an accuracy of 2-5%. Based on the principle used in the two assays an enzyme immobilization scheme was developed for the reversible immobilization of these enzymes. Which was subsequently utilized in the determination of substrate in the picomole range in a reagent less FIA technique. The goals of this research project were realized in that the FIA system utilized in this work was capable of carrying out totally automated ELISA assays with an accuracy far surpassing the conventional plate ELISA assays.

This dissertation develops technology for microfluidic point-of-care (POC) immunoassay devices, divided into three papers, and explores the use of a quartz crystal microbalance for real time monitoring of blood coagulation in a fourth paper. The concept of POC testing has been well established around the world. With testing conveniently brought to the vicinity of the patient or testing site, results can be obtained in a much shorter time. There has been a global push in recent years to develop POC molecular diagnostics devices for resource-limited regions where well equipped centralized laboratories are not readily accessible. POC testing has applications in medical/veterinary diagnostics, environmental monitoring, as well as defense related testing. In the first paper, we demonstrated the use of latex immunoagglutination assays within a microfluidic chip to be an effective and sensitive method for detecting the bovine viral diarrhea virus. In the second paper the feasibility and general ease of integrating liquid core optical components onto a microfluidic lab-on-a-chip type device, for point-of-care AI diagnosis is demonstrated. In the third paper particle agglutination assays, utilizing light scattering measurements at a fixed angle from incident light delivery, for pathogen detection are explored in both Rayleigh and Mie scatter regimes through scatter intensity simulations and compared to experimental results. In the fourth paper a quartz crystal microbalance was used for real-time monitoring of fibrinogen cross-linking on three model biomaterial surfaces.