Many non-isotopic assay systems depend on photometric assessment to attain results. The wavelength of these assays would be according to the design of the assay. For instance, Emit® monitors the activity of the enzyme G6PD by the conversion of NAD enzyme to NADH. The reaction is absorbed at the wavelength 340nm. However, other compounds that get absorbed at similar wavelengths can interfere with the result accuracy. This mechanism was conducted by Wagner and Linder who proposed the interference of Emit® by using salicyclates. During the reaction at 340nm, other compounds such as mefenamic acid and metronidazole also produce absorbance, thereby causing invalid results. Similarly, in FPIA assays, compounds having fluoresce such as radiologic dyes can also cause interferences.
Activity Of Enzymes
In the immunoassays that utilize enzymatic activity to detect compounds, the physiochemical aspects can affect the enzymatic activity related to the assay reaction. Many enzymes have their pH according to the optimal activity, but variation of pH beyond the normal range can affect the performance of the assay. Even though urine samples have buffering potential to adapt to physiological changes, the addition of adulterants to the sample affect the pH of the urine significantly, hence affecting the usefulness of the assay test. Moreover, the presence of chemicals that can inhibit or corrupt the enzymes can critically influence the assay and produce interferences.
Antigen-Antibody Binding Behavior
There is an utmost importance of antibodies whenever any interaction in an immunoassay is concerned. This is so because immunoassays depend on the reaction between antigens and antibodies which leads to quantifiable results. The binding of antigen and antibody takes place due to the following basic forces between the molecules: hydrophobic interactions, van der Waal-London’s dipole bonds and ionic coulombic bonds. The binding activity can be disturbed by major changes in the pH level, viscosity of the specimen, ionic strength and other factors that affect the affinity of the site of binding. To check this phenomenon, high concentrations of chemicals such as sodium chloride, sodium hypochlorite, sodium bicarbonate and hydrogen peroxide were added to urine sample to observe their effect on the performance of the immunoassay reagent.