Establishment of the anti-klotho monoclonal antibodies and detection of klotho protein in kidneys

A novel gene, klotho (kl), which is involved in the development of a syndro me resembling human aging in mice, was recently identified. The kl gene enc odes a single-pass membrane protein whose extracellular domain carries homo logy to beta-glucosidases, There also exists a splice variant of hi mRNA wh ich encodes a putative secreted protein in both human and mouse. In this st udy, to characterize the physiological roles of Klotho protein, we establis hed three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG(2)a), KM2119 (rat IgG(2)b), an d KM2365 (mouse IgG(1)), In Western blots, KM2076 and KM2119 specifically r ecognized a 130 kDa Klotho protein in the mouse and human kidney membrane f ractions, To detect the human Klotho protein, the sandwich-type ELISA syste m with KM2076 and KM2365 was established, Using the ELISA system, we detect ed the human Klotho protein as low as 20 ng/ml in the supernatant of Chines e hamster ovary cells (CHO cells), introduced the human klotho gene, KM2076 and KM2119 specifically gave a positive staining by immunohistochemical st aining in paraffin or frozen sections of the kidneys from wild-type mice bu t not in those from hi mice. Strong staining was observed especially in cor tical renal tubules of the mouse kidney, where expression of klotho transcr ipts overlaps, KM2076 also showed a similar reaction pattern in the paraffi n sections of rat and human kidneys. The mAbs established in this paper wil l serve as useful analytical, pathological, and diagnostic tools to disclos e the role of Klotho protein in the suppression of a syndrome resembling hu man aging, (C) 2000 Academic Press.