Preparation and Preliminary Characterization of Rabbit Monoclonal Antibodies Against Human Midkine

Abstract

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0 × 10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.

Effect of Listeria monocytogenes inoculation, sodium acetate and nisin on microbiological and chemical quality of grass carp Ctenopharyngodon idella during refrigeration storage

“…As described previously, prokaryotic expressed MK protein were immunize into rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique, then hybridoma cell line secreting anti-MK mAb was obtained and the monoclonal antibodies were purified (Yao et al, 2011).…”

Detection of serum midkine levels in cancer patients using rabbit anti-human midkine monoclonal antibodies

“…The rabbit anti-human midkine monoclonal antibodies were preserved in our laboratory. As described previously, prokaryotic expressed MK protein were immunize into rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique, then hybridoma cell line secreting anti-MK mAb was obtained and the monoclonal antibodies were purified (Yao et al, 2011).…”