This is the assay, so this is an example of what it would look like if melamine was present. This would be like there’s no melamine; you can drink the milk and then these would be like definitely there’s melamine there. We have to take care of that. Some time ago we had noticed the news reports coming out of China about melamine adulteration of milk. And we became aware that there was industrial interest in developing a rapid test kit means to detect melamine present in the milk. And we had described previously an enzyme that deaminates melamine so that specifically acts on melamine and doesn’t touch other chemicals, even ones that are similar in structure and we realized that this could be used to develop a test kit for melamine. The protein content per unit volume of milk is generally pretty constant and so as a result of this, people have relied on a very simple test where they just detect total nitrogen in the milk as an indicator of the amount of protein. So that means that any nitrogenous compound will show up as positive and elevate the apparent protein content in this test. So people took advantage of this and what they did was to take the milk, dilute it one-to-one with water so they would now double the amount of milk they had so they could double their profits and they added the chemical melamine into the milk to mimic protein, so then the test would make it look like there’s plenty of protein there, but half of it was actually melamine. Naturally there were companies that jumped in and wanted to devise methods in testing for melamine and so the approach that was selected – because it was really an accepted and well-worked method – was to use things like gas chromatography, mass spectrometry, or LC, liquid chromatography mass spectrometry. These are methods that can detect melamine. However, they require very expensive equipment and a dedicated laboratory. So this isn’t something you’d set up at a dairy collection point or somebody could carry on a truck with them, I mean, you really need large, expensive equipment in order to accomplish this, and trained technicians to run the equipment. The melamine deaminase in our lab was initially discovered by Jennifer Seffernick, who was at the time a graduate student here in the department of Biochemistry, Molecular Biology and Biophysics and Jennifer really did this because we were studying triazine herbicide degradation and she had reasoned that an enzyme that would deaminate melamine would resemble these other enzymes. So she did this for kind of fundamental basic research understanding of where these enzymes come from. Naomi Kreamer, a graduate of College of Biological Sciences, a recent graduate in biochemistry, she took over the project and has done a very nice job in terms of purifying the enzyme, studying its properties, finding out how to make it maximally active because if you have the maximum activity out of the enzyme, you can use less of the enzyme. So from a practical standpoint that’s good because you keep the costs down of raw materials that go into the kit. So melamine deaminase is the enzyme that Bioo Scientific is using for their kit. So I’m looking at it from more of a basic research standpoint. So I’m looking at it… so for them what I did is I prepared the enzyme, I brought it up through several different stages of purification and sent it to them and they kind of decided which one would be best for the kits. What I’ve done since then is sort of fine tune the purification more and then also I’m doing more basic research for the enzyme. So, what I’m doing right now is I’m making up solutions at various melamine concentrations so I can do kinetics. And then also I’m making standards for the HPLC. This particular approach of using enzymes is a little bit different. You can use specific enzymes that will recognize only very certain select set of chemicals, maybe only one. And will do some sort of reaction with it that will produce some product that can be detected usually with some kind of color test. You do a chemical reaction that leads to something that you can see with the eye – some red, blue, yellow color – very easy to spot. This is something where then you don’t even need any to tell actually yes or no if the chemical is present. You don’t even need a piece of equipment. You just look at it and say, oh it’s blue, therefore, there’s melamine in it.

Detecting melamine in the field
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