The level ELISA kit is a "sandwich-type" of enzyme immunoassay. In the microtiter plates, TNF is captured by monoclonal antibodies coated to polystyrene microtiter wells. The drug, present in the patient sample, the calibrator or the controls, binds to the TNF on the microtiter plate. Non-bound material is then removed by washing. Subsequently, a horseradish peroxidase-labeled monoclonal anti-drug antibody is added. This antibody binds to the drug/TNF/anti-TNF complex present in the microtiter well. After removal of non-bound HRP conjugate by washing, substrate solution is added to the wells. A coloured product is formed in proportion to the amount of adalimumab present in the sample, calibrator and controls. After the reaction has been terminated by the addition of a stop solution, absorbance is measured in a microtiter plate reader. From the absorbance of samples and those of the calibrator curve, the concentration of drug can be determined by interpolation with the calibrator curve.
Clinical optimal ranges, e.g. therapeutic ranges, were determined at Sanquin Diagnostic Services with the adalimumab ELISA for rheumatoid arthritis: 5–8 µg/mL (Pouw et al. 2015) and in dermatology (Psoriasis): 3.5–7 µg/mL (Menting et al. 2015).
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