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Updated: Jun 3, 2026
Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium
Published on: May 18, 2021
Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine
Xing Yao1, Fu-Chu Qian, Li-Cheng Dai
1Department of Surgery, Huzhou Central Hospital, Hongqi, Huzhou, Zhejiang Province, China.
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.
Insights
Researchers developed rabbit monoclonal antibodies targeting human midkine (MK). These antibodies were validated for specificity and affinity, showing potential for research and clinical applications in cancerous tissues.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Human midkine (MK) is a protein implicated in various biological processes, including cancer.
- Development of specific antibodies against MK is crucial for its research and clinical applications.
Purpose of the Study:
- To generate and characterize rabbit monoclonal antibodies (MAbs) targeting human midkine (MK).
- To evaluate the specificity, affinity, and binding capability of the developed MAbs in cancerous tissues.
Main Methods:
- Amplification and cloning of the MK gene into a GST-fusion protein expression vector.
- Expression and purification of the N-terminally GST-tagged MK fusion protein in E. coli.
- Immunization of rabbits and generation of hybridomas using the rabbit hybridoma technique.
- Screening and characterization of MAbs using ELISA, Western blotting, SDS-PAGE, and immunohistochemistry.
Main Results:
- Successfully expressed and purified GST-MK fusion protein.
- Generated hybridoma cell lines secreting anti-MK MAbs.
- Confirmed MAb identity and specificity via Western blotting.
- Achieved high MAb titer (1:64,000) and affinity constant (3.0 × 10^9 M⁻¹).
- Demonstrated MAb binding to MK protein in cancerous tissues using Western blotting and immunohistochemistry.
Conclusions:
- Rabbit monoclonal antibodies against human midkine (MK) were successfully prepared and validated.
- The developed MAbs exhibit high specificity and affinity, suitable for research and clinical applications.
- These antibodies show promise for detecting MK in cancerous tissues, aiding further research and diagnostics.
