Production and confirmation of polyclonal antibody against tamm horsfall protein and its application in kidney stone disease diagnosis

Abstract

Increasing numbers of the kidney stone cases, all over the world, urgently demands simple and reliable method of detection, in which measuring the expression of biomarker level has already gained considerable success. Tamm Horsfall Protein (THP), biomarker for kidney stone, acts as the major inhibitors of stone formation (crystal growth) in normal urine but due to its abnormality its inhibitory effect is lost and growth of stone is promoted. Herein, we have isolated THP by four different methods namely Sodium chloride, Ammonium sulphate, Acetone, Polyethylene glycol and purified from human urine samples by column chromatography which were confirmed by SDS PAGE band at 97 kDa. PAGE analysis revealed that four protein bands were seen in Ammonium sulphate followed by sodium chloride method. Also decreased excretion of THP was found in case of stone patients and also aggregation of THP was more in stone patients than compared to normal which was established by SEM. Polyclonal antibodies against purified THP protein (antigen) was produced and confirmed. Our results indicate that this is the easy way to screen and differentiate the normal subjects from the renal stone patients excreting abnormal THP levels. Thus, the present method holds huge promises to design a diagnostic kit for detection of kidney stone in future.