Estimation of serum xanthine oxidase (XO)

Xanthine Oxidase (XO) levels in serum were measured by ELISA using a commercially available ELISA kit, which was purchased from MyBioSource company, USA (Human XO ELISA Kit, Cat No: MBS774009). This kit was used to measure the levels of XO in serum, based on the principle of ELISA. Briefly, standards and serum samples were added to the wells that were pre-coated with specific antibodies and then HRP-Conjugate solution was added to the wells to form an immune complex. After incubation, the unbound enzyme was removed through washing. The substrates were added, then the solution colour turned blue, and finally changed into yellow. The colour intensity was positively correlated with XO levels present in the serum sample. The absorbance (OD) of each micro-well was measured at 450 nm within 15 min by an ELISA reader (Apollo 11 LB 913, Berthold, Germany). Finally, XO concentrations in the samples were calculated according to the standard graph prepared in the excel sheet.