The establishment of hybridoma cell lines producing monoclonal antibodies against human and zebrafish steroid and xenobiotic receptor (SXR, NR1I2)

The steroid and xenobiotic receptor (SXR, NR1I2) also known as pregnane x receptor (PXR), is a member of the nuclear receptor (NR) superfamily. The proteins ability to induce and up regulate expression of a large network of genes that regulate enzymes involved in oxidation (phase I), conjugation (phase II) and transport (phase III) make the study of SXR of great importance because of the role played in xenobiotic protection. A major challenge in the study of SXR is the lack of quality commercial antibodies that specifically recognize the protein. Due to the lack of well-functioning commercial antibodies we sought to develop highly specific monoclonal antibodies (mAbs) against human (Homo sapiens) and zebrafish (Danio rerio) SXR through hybridoma technology. Towards this goal two Balb/c mice were immunized five times with partially purified recombinant full-length human SXR (hSXR) protein and two Balb/c mice were immunized with partially purified recombinant full-length zebrafish SXR (zfSXR) protein. The fusion of splenocytes from mice with serum expressing anti-SXR activities and myelomas was conducted through the use of polyethylene glycol (PEG). Resulting hybrids were selected for by hypoxanthine, aminopterin and thymidine (HAT) selection system and hybrids secreting anti-SXR antibodies were identified through indirect antibody capture enzyme-linked immunoassay. Eight positive hybridoma cultures with five secreting anti-human SXR antibodies and three secreting anti-zebrafish SXR antibodies were sub-cloned by limiting dilution. Antibodies from the hybridoma clones were assessed through western blot analysis. Seven clones were shown to detect linear SXR in a prokaryotic expression system, while four clones also detected linear SXR in a eukaryotic expression system. Antibodies from the final hybridoma clones were shown to be cross-reactive with SXR from other species.