An enzyme immunoassay for buffalo serum ferritin

Buffalo ferritin has been isolated and purified from liver using conventional biochemical techniques such as thermal denaturation, ammonium sulphate fractionation, Sephacryl S‐300 gel filtration and DEAE‐blue gel affinity chromatography. Native gel‐electrophoresis of affinity‐purified ferritin followed by iron staining showed a single band corresponding to rat liver ferritin. The yield and the iron content of purified ferritin were 10.7 mg per 500 g of liver and 7% respectively. As a glycoprotein, buffalo ferritin has 12.2% neutral carbohydrate. Polyclonal antibodies raised against purified buffalo liver ferritin in rabbits were used successfully in the development of competitive indirect ELISA using alkaline phosphatase as an enzyme label. The sensitivity range of the assay was 5–30 ng with intraand inter‐assay coefficients of variation (CVs) of 3.1% and 9.2% respectively. The assay was reproducible and could be applied under field conditions for the evaluation of ferritin levels in buffalo. Using this method, the range of circulating ferritin levels in 10 normal buffalo was 1.12–3.68 μg ml^‐1. Cross‐reactivity studies by ELISA also suggest the applicability of the method to the evaluation of ferritin levels in other domestic species such as sheep and cattle.