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Updated: Apr 25, 2026
Generation of Monoclonal Antibodies Against Natural Products
Published on: April 6, 2019
Preparation and identification of monoclonal antibodies against ω-conotoxin MVIIA
Yanling Yang1, Yanling Ma, Heng Li
1Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, School of Life Sciences, Fujian Agriculture and Forestry University , Fuzhou, China .
Abstract:
ω-Conotoxins MVIIA (ω-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against ω-conotoxin MVIIA. In this study, an MAb against ω-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a (+), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against ω-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against ω-CTX MVIIA was 7.33×10(9) L/mol, and the cross-reaction test showed that the MAb specifically bound ω-CTX MVIIA. The MAb could be used as a specific antagonist for ω-CTX MVIIA in the physiological study on the CaV channels in the nervous system.
Insights
Researchers developed a novel monoclonal antibody (MAb) for immunoassay against ω-conotoxin MVIIA, a peptide toxin. This specific MAb offers a new tool for studying ω-conotoxin MVIIA
Area of Science:
- Neuroscience
- Immunology
- Biochemistry
Background:
- ω-Conotoxins MVIIA (ω-CTX MVIIA) are peptide blockers of N-type voltage-gated calcium channels.
- These toxins show potential as analgesics for pain management.
- Currently, no specific monoclonal antibodies (MAbs) exist for ω-CTX MVIIA immunoassay.
Purpose of the Study:
- To develop and characterize a monoclonal antibody (MAb) for specific detection of ω-conotoxin MVIIA.
- To establish a tool for physiological studies involving ω-CTX MVIIA and CaV channels.
Main Methods:
- Chemical synthesis of conotoxin-coding DNA and cloning into expression vectors (pGEX-6p-1, pET32a(+)).
- Expression and purification of GST-CTX fusion protein for immunization of BALB/c mice.
- Hybridoma technology: spleen cell-myeloma cell fusion (SP2/0), screening (indirect ELISA), and cloning (limiting dilution) to obtain MAb-producing hybridoma (4A12).
Main Results:
- A hybridoma cell line (4A12) producing a specific MAb against ω-CTX MVIIA was successfully established.
- The MAb is of the IgM subclass and exhibits high affinity (7.33×10^9 L/mol) for ω-CTX MVIIA.
- Cross-reactivity tests confirmed the MAb's specific binding to ω-CTX MVIIA, with no significant cross-reaction.
Conclusions:
- A novel, specific monoclonal antibody (MAb) against ω-conotoxin MVIIA has been developed.
- This MAb serves as a specific antagonist for ω-CTX MVIIA.
- The MAb is a valuable tool for physiological studies of CaV channels in the nervous system.
