Preparation and application of a specific single-chain variable fragment against artemether

Area of Science:

• Biotechnology
• Immunology
• Analytical Chemistry

Background:

• Artemether is a key component in artemisinin-based combination therapies, crucial for malaria treatment.
• Monoclonal antibodies offer high specificity for drug detection, but their application can be limited.
• Developing recombinant antibody fragments like single-chain variable fragments (scFv) can enhance utility and production.

Purpose of the Study:

• To clone and express an artemether-specific single-chain variable fragment (scFv).
• To develop a sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemether detection.
• To validate the developed icELISA by comparing its results with ultra-performance liquid chromatography (UPLC).

Main Methods:

• Cloning of VH and VL genes from an artemether-specific monoclonal antibody (mAb 2G12E1).
• Construction of scFv into various prokaryotic expression vectors, with successful soluble expression achieved using pMAL-p5x.
• Development of an indirect competitive ELISA using the anti-artemether scFv.

Main Results:

• Soluble expression of anti-artemether scFv was achieved using the pMAL-p5x vector, maintaining sensitivity and specificity.
• The developed icELISA demonstrated a low IC50 value of 4.33 ng/mL and a working range of 1.05-22.65 ng/mL.
• Artemether quantification using icELISA showed high consistency with results obtained via UPLC analysis.

Conclusions:

• The anti-artemether scFv is a valuable tool for artemether detection and monitoring.
• The developed icELISA provides a reliable method for quantifying artemether content in pharmaceutical preparations.
• This genetically engineered antibody fragment has potential applications in drug quality control and mechanistic studies.