para-Nitrophenylphosphate (pNPP) is a non-proteinaceous chromogenic substrate for alkaline and acid phosphatases used in ELISA and conventional spectrophotometric assays.

[1] Phosphatases catalyze the hydrolysis of pNPP liberating inorganic phosphate and the conjugate base of para-nitrophenol (pNP).

[5] A PNPP assay involves mixing the sample with a PNPP-containing mixture and permitting the reaction to run its course for a predetermined period of time.

After that point, the process is halted through the addition of a stop solution, often made of a potent alkali like sodium hydroxide.

[7] To get precise and credible findings, the assay parameters must be carefully regulated because the reaction is sensitive to factors such as pH, temperature, and the varying degree of inhibitors or stimulants.