I am using a strategy in which the primary Ab1 is immobilized on magnetic particles. Ab1 binds the target Ag. While the secondary Ab2 is conjugated with "peroxide" enzyme. After binding the the Ag, the conjugated Ab2 is supposed to catalyze the substrate (TMB:H2O2) oxidation/reduction process. I am using amperiometric technique using 0.15 V, screen printed gold W.E. I don't see any reasonable change in 0% and 40% Ag concentration. To be honest, it shouldn't make any difference because I can see the TMB color change visually of similar intensity for control and assay, while it gives clear difference when I do it using luminol substrate in ELISA. This is the issue, can anyone help me with it?
Well, I believe that the HRP subsrate TMB does not release an electroactive product after enzymatic hydrolysis. It releases an optically active product that is detectable at Amax = 370nm and 652nm and in the presence of a quencher like HCl or H2SO4 that changes to yellow that is detectable at Amax = 450nm.
HRP label can be used with luminol which releases luminescence that can be detected at 428 nm but this is low intensity. It can be enhanced by perfroming the process in the presence of phenols such as p-iodophenol.
If you want an immunoassay with electrochemical detection, you must use alkaline phosphatase label with para-aminophenol as the enzyme substrate. Check these papers:
a) Z. P. Aguilar and M. Sirisena, “Development of Automated Amperometric Detection of Antibodies against Bacillus anthracis Protective Antigen,” Anal.Bioanal.Chem. 2007,389,507-515.
b) Z. P. Aguilar, “Small Volume Detection of Plasmodium falciparum Using a 50-Micrometer Diameter Cavity with Self-contained Electrochemistry”, ECS Trans, 2007, 3(40), 1-7.
Let me know if you need more help.
HRP followed by ammperometric detection
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