I'm assaying saliva samples using a competitive immunoassay with HRP-labeled steroid hormone on microplates and TMB as the chromogenic substrate with H2O2. However, I'm getting oxidation of TMB by some samples (and it is sample specific) even without the presence of HRP-labeled steroid (or anti-hormone antibody, etc). I can add saliva to the wells with Trizma buffer (no antibody or HRP-lablel), then wash the plate and add TMB-H2O2 in sodium acetate buffer (pH5) and still get strong colour development consistently with some samples (but blank wells with Trizma buffer only and no saliva are always colourless). I'm trying different wash buffer recipes to remove whatever is in the saliva sample that is oxidizing the TMB. Any ideas what might be in the saliva causing the oxidation of the TMB that I'm having difficulty washing away and what I might add to my wash buffer recipe to solve the problem?
Laura H. Graham In peroxidase reactions, the enzyme catalyzes the two-electron oxidation of TMB by H2O2, whereas in the Fenton reaction, Fenton reagents (such as Fe2+ or Cu1+) react with H2O2 to produce highly oxidative hydroxyl radicals (•OH), which subsequently oxidize TMB to oxTMB.
Hi Laura H. Graham
Without getting too deep into the chemistry weeds, it's worth paying attention to the mouth hygiene habits of the saliva provider.
Many toothpastes - specifically those designed for enamel protection - contain high amounts of nitrate, usually as several % of sodium nitrate. Bacteria in the mouth may be able to reduce some residual nitrate to nitrite, which is a weaker oxidant but a much more active one.
Furthermore, some mouth wash solutions contain fairly strong oxidants, e.g. sodium chlorite (NaClO2). Finally, the occasional germophobe will actually use a dilute H2O2 mouth rinse.
Rinsing after toothbrushing is often imperfect, especially since some people (and, I think, dentists) believe that the curative properties of the toothpaste are enhanced by not rinsing or rinsing very superficially.
Ah, another clever theory killed by a cruel fact...
Not human ruminants by any chance?
Kidding aside, I trust you already pay attention to any correlation of the results (and their variance) with the type of feed, feeding time and time between feeding and sampling.
That is going to be looked at. In the meantime i need to develop a wash buffer capable of washing the guilty parties out of the microplate wells before adding the TMB substrate buffer.
Hi Laura H. Graham
I read a bit about your problem ans it seems to me that the most likely oxidizing contaminant - in your non-toothbrushing ruminants' mouths - is nitrite, which can be formed by conversion from nitrate by bacteria in the rumen. (Cud of course then travels from the rumen into the mouth.) See link below to the relevant pages in the Merck Veterinary Manual.
https://www.bing.com/ck/a?!&&p=95d00d831b61e6aeabaf8e3f982e78e81c6a2abf21d5fc5631cd2b118dd1ae5eJmltdHM9MTc0MTY1MTIwMA&ptn=3&ver=2&hsh=4&fclid=1654d470-0b00-6c72-0d35-c1190a726d77&u=a1aHR0cHM6Ly93d3cubWVyY2t2ZXRtYW51YWwuY29tL3RveGljb2xvZ3kvbml0cmF0ZS1hbmQtbml0cml0ZS1wb2lzb25pbmcvbml0cmF0ZS1hbmQtbml0cml0ZS1wb2lzb25pbmctaW4tYW5pbWFscw&ntb=1
Nitrite can give a color reaction with TMB, and TMB is even used for nitrite detection - see for example
Article A Derivative of 3,3',5,5'-tetramethylbenzidine for Highly Ro...
You can purposely add a few ppm sodium nitrite to your saliva samples and see if you get a consistent false positive. Then you can start looking for ways to remove it. Urea in a moderately acidic solution (pH ~2) does it, but I don't have the reference readily available.
I hope this helps.
Emanuel Cooper
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