Abstract
Antigen Used for Immunization
Recombinant human His6-tagged PEX5L (long isoform) produced in Escherichia coli.
Method of Immunization
Female BALB/c mice were subcutaneously injected with 0.1 mg of PEX5 antigen in PBS and Freund's complete adjuvant and boosted four times with 0.1 mg antigen each.
Parental Cell Line Used for Fusion
P3-X63-Ag8.653.
Selection and Cloning Procedure
Hybridomas were selected in HAT media. Cell culture supernatants were screened by ELISA using purified recombinant HsPEX5L as antigen.
Heavy and Light Chains of Immunoglobulin
Not determined.
Specificity
The specificities of clones 9D12 and 3F6 were determined under denaturing conditions by Western blot analyses using whole cell lysates of human fibroblasts. Both MAbs detect both isoforms of the cycling peroxisome import receptor, PEX5L and PEX5S. As shown by indirect immunofluorescence microscopy, MAb 3F6 recognizes the predominant cytosolic form of PEX5 whereas MAb 9D12 favorably detects the peroxisome-associated state of the receptor.
Specific Antigen Identified
Peptide scan analyses using synthetic 15-mer peptides covering the full-length sequence of HsPEX5L revealed the following epitopic regions:
MAb 3F6: AHPWLSDY (aa 305 to 312)
MAb 9D12: QDYNET (aa 133 to 138)