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Objective 1: Produce pure histamine dehydrogenase (HDH) enzyme in-house for the new higher sensitivity MaxSignal histamine kit. Milestone 1a: Produce 400 units of HDH with identical activity and stability of enzyme obtained externally. Milestone 1b: Produce at least 4000 units (about 1 gram) of commercial grade HDH enzyme in-house for the new higher sensitivity MaxSignal histamine kit. <P>Objective 2: Produce high quality recombinant melamine degrading enzyme for use as a detection reagent in the MaxSignal rapid melamine detection kit. Milestone: Produce 500 mg of melamine degrading enzyme (enough for about 1000 kits) of equivalent activity to the enzyme used in Phase I to develop the rapid test assay. <P>Objective 3: Develop a rapid sample preparation method to extract melamine from milk or other food samples with high recovery for our high-throughput enzyme-based test kit. Milestone: Develop a new high-throughput compatible high recovery sample prep method for melamine extraction compatible using standard colorimetric detection. <P>Objective 4: Develop a higher sensitivity detection method for use in our plate-based melamine assay. Milestone: Develop a microplate compatible ammonia detection method (compatible with our enzymatic detection assay) capable of detecting <0.5 ppm ammonia. <P>Our main goal is to commercialize the two enzyme-based toxin detection tests we invented during our Phase 1 work. Although our new toxin detection methods work very well, we are currently unable to produce the required enzyme components at a large enough scale for mass production. Our first two objectives address this need. To commercialize our new kits, we will scale up our production methods used to make the two critical enzyme reagents required for our new test kit products. <P>The last two technical objectives (3 and 4) focus on the developing new sample preparation and detection methods for our new melamine test kit. We will focus on the development of sample prep and procedures which are compatible with the activity and optimal reaction conditions for the melamine degrading enzyme. <P>Although our new melamine test currently works well, accomplishing these goals will enable us to will allow or customers to use our kit to identify melamine in more sample types with higher sensitivity.
Non-Technical Summary: The safety of the public food supply is an emerging and important public health issue in the United States. Recent, widely-reported food and feed contaminations by pathogens and banned substances such as melamine have fueled an increased demand for improved assays to screen and protect the food supply. The antibody-based ELISA assay is a powerful, cost-effective method to detect trace analytes, such as banned substances, in food samples. While ELISA assays can detect many different types of banned substances, a number of low molecular weight analytes, such as melamine or histamine (a marker for seafood samples contaminated the bacteria which cause scombroid poisoning) are too small for use as antigens to develop high-quality ELISA assays. Consequently there are currently no rapid, reliable screening methods to detect many low molecular weight banned substances in food and feed supplies. In contrast to antibodies, protein enzymes can bind small molecules (MW < 100) very specifically. Surprisingly however, the potential advantages of using enzymes to detect banned substances in food and feed supplies has never been thoroughly investigated. In our Phase I research, we invented two new rapid tests to detect melamine and histamine. In Phase II, we will develop robust detection kits using theses enzyme-based assays to detect banned substances, such as melamine, in food and feed. We will also enhance our enzyme manufacturing capability to internally produce larger quantities of low cost high activity enzymes. Successful commercial development of these kits during Phase II will result in much needed improved rapid testing products for melamine and histamine and provide a solid foundation for future development of other assays using enzymes from bacteria which metabolize banned substances in the environment. These assays will be an effective new tool in developing improved procedures for comprehensive yet cost-effective screening of food samples to better ensure the safety of our food and feed supplies. <P> Approach: Our first goals will be accomplished by developing internal capabilities and competencies to express recombinant enzymes using high density fermentation procedures. We will first clone both the histamine dehydrogenase and melamine degrading enzyme genes in bacterial expression plasmids. Next, we will systematically optimize enzyme production in shake flask cultures by varying the culture growth conditions as well as the cell density at induction. After we have maximized expression of the proteins in shake flasks, we will transfer the optimized protocol to a fermentor bioreactor system which we establish in-house to permit high density bacterial fermentations. For each of the two target enzymes, we will optimize the growth and induction conditions to obtain the highest levels of active enzyme. We will compare the stability and function of our enzymes produced in-house to those of enzymes proeviously obtained in smaller scale from outside sources. After validation of our production, we will commercialize lower cost, high performance Histamine and Melamine kits using our enzymes manufactured in-house. In parallel, we will develop optimized sample preparation and product detection methods for our enzymatic melamine detection assay. For sample prep, we will explore various extraction, precipitation and enzymatic degradation steps while for detection, we will optimize the reaction conditons for product detection as well as examine enhanced sensitivity caused by improved reaction catalysts as well as enzymatic detection schemes.