NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE

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Tony Rook
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NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE

Please find the link for the following protocol:

NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE

Link Reference:
http://aactg.s-3.com/pub/download/imm/imneopte.pdf

PRINCIPLE AND PURPOSE OF THE TEST:

Neopterin [D-erythro-neopterin] belongs to the group of pteridines. Neopterin as well as other pteridines are derived in vivo from guanosinetriphophate (GTP). The enzyme GTP-cyclohydrolase-I catalyses this reaction in monocytes and macrophages (fig.1). Neopterin is exclusively
excreted by activated macrophages (fig.2), and can be etermined in serum, plasma and other body fluids by immunoassay.

The principal of the ELItest Neopterin test procedure is that of an Enzyme immunoassay (EIA) based on the competition of unlabelled neopterin of the standards, controls samples, patients samples and enzyme conjugated (Neopterin/alkaline phosphatase conjugate) for the binding sites of the anti-neopterin-antibodies (polyclonal, sheep). An antigen-antibody complex is formed and bound to the solid phase (coated well). The following intensive washing ensures the complete removal of all unbound components. The addition of 4-nitrophenyl-phosphate substrate solution starts the enzyme reaction, where the alkaline phosphatase being part of the neopterin/enzyme conjugate catalyses the separation of the phosphate of the 4-nitrophenyl-phosphate, thus setting free the yellow 4- nitrophenol. Pipetting of sodium hydroxide stops the enzymatic reaction.

The intensity of the color (measured in optical densities or OD) depends on the amount of enzyme bound in the wells at a constant reaction time and consequently is inversely proportional to the neopterin concentration in the patient sample. Thus high neopterin values correspond to low amounts
of optical density. The optical density is measured by a microtiter plate reader with a wavelength of 405 nm. For calculation of results a standard curve is created (optical Density versus concentrations of neopterin standards), allowing the determination of the neopterin concentrations within the patient samples.

The following clinical indications for determination of neopterin can be emphasized:

Monitoring of HIV infected individuals.

Surrogate marker for the effects of antiviral therapy in HIV
infected Individuals.

Screening of donated blood and bone marrow for avoidance
of HIV infection.

Testing of infants born to HIV+ mothers.

Follow up of transplant recipients for rejection.