Abstract
Antigen Used for Immunization
Human klotho was PCR amplified using a pcDNA3.1 plasmid containing the cDNA across the sequence corresponding to amino acids 55-261. The resulting fragment was cloned into pGEX-6P2 (GE Healthcare, Piscataway, NJ) in frame with glutathione-S-transferase (GST) at the BamHI/XhoI restriction sites. Plasmid was transformed into Escherichia coli strain BL21 (Agilent, San Jose, CA) and GST-klotho was induced by the addition of 0.4 mM IPTG for 16 h at room temperature. Bacteria were pelleted, lysed in STE (100 mM NaCl, 10 mM Tris [pH 7.6], 1 mM EDTA). Lysates were treated with lysozyme (10 mg/mL) for 15 min followed by the addition of 5 mM DTT, and 1.5% Sarcosyl. Following sonication, cleared supernatants were incubated with GST-agarose beads (Pierce, Rockford, IL) overnight. The GST beads were washed in NENT (0.5% NP-40, 1 mM EDTA, 20 mM Tris [pH7.6], and 100 mM NaCl) and GST-KL eluted with 10 mM glutathione. The concentration of fusion protein was estimated by comparison to BSA on Coomassie stained gel.
Method of Immunization
Rats received a primary immunization totaling 200 μg of GST-KL emulsified in complete Freund's adjuvant administered subcutaneously (100 μL per site) in the thigh area of each rear leg. Fourteen days after the primary immunization, rats were boosted with 200 μg of klotho in incomplete Freund's adjuvant administered subcutaneously near the initial immunization sites. The final boost, given on day 20, consisted of 100 μg of GST-KL in saline injected subcutaneously at the base of the tail.
Parental Cell Line Used for Fusion
Mouse myeloma P3U1.
Selection and Cloning Procedure
Initial selection was performed by ELISA to detect GST (GST protein) and to detect klotho (utilizing the immunizing peptide KL-GST). Hybridomas reacting to KL-GST only were further purified and tested.
Heavy and Light Chains of Immunoglobulin
IgG2a.
Specificity
Mass spectrometry was conducted after IP to confirm specific binding to klotho.
Specific Antigen Identified
Klotho expression was confirmed by Western blot, immunocytochemistry, immunohistochemistry, and finally immunoprecipitation followed by mass spectrometry.