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[Expression of antibody Fab against platelet IIb/IIIa in E.coli and characterization of its biological activity]
Yong-chang Yang1, Chun-ning Lai, Yan Wang
1Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Aim:
To develop a Fab antibody against platelet GPIIb/IIIa.
Methods:
A mouse anti-human GPIIb/IIIa monoclonal antibody (mAb) P140 was selected by the indirect immunofluorescence assay (IFA) and platelet aggregation inhibition test. The Fd and light chain genes were cloned from the hybridoma cells secreting the mAb P140. The genes of P140 Fab were inserted into plasmid p3MH to construct recombinant expression plasmid p3MH/P140kappa-Fd. After digestion with the restriction enzyme, the recombinant plasmid was transformed into E.coli XLI-Blue. The expressed product was purified by TALON metal affinity resin. Purified P140 Fab was characterized by SDS-PAGE, ELISA, Western blot and platelet aggregation inhibition test.
Results:
SDS-PAGE analysis showed that relative molecular mass (M(r)) of P140 Fab was 47x10(3). The results of ELISA, Western blot and platelet aggragation inhibition test indicated that P140 Fab could specifically bind to platelet and inhibit platelet aggragation in dose-dependent manner. The mean value of IC(50) was 16.85 mg/L.
Conclusion:
A soluble anti-platelet GPIIb/IIIa antibody P140 Fab was prepared successfully, which lays the foundation for further clinical application.
Insights
Researchers successfully developed a Fab antibody targeting platelet GPIIb/IIIa. This soluble antibody effectively inhibits platelet aggregation, paving the way for potential clinical applications.
Area of Science:
- Immunology
- Biotechnology
Background:
- Platelet GPIIb/IIIa is a critical receptor involved in platelet aggregation.
- Developing targeted antibodies can offer therapeutic strategies for platelet-related disorders.
Purpose of the Study:
- To develop a functional Fab antibody fragment against human platelet GPIIb/IIIa.
- To characterize the binding and inhibitory activity of the developed antibody.
Main Methods:
- Cloning and expression of Fd and light chain genes from a hybridoma producing anti-GPIIb/IIIa mAb P140.
- Construction of recombinant plasmid p3MH/P140kappa-Fd and transformation into E. coli.
- Purification of P140 Fab using TALON metal affinity resin and characterization via SDS-PAGE, ELISA, Western blot, and platelet aggregation inhibition assays.
Main Results:
- Successfully prepared and purified soluble P140 Fab antibody with a molecular weight of 47x10(3).
- Demonstrated specific binding to platelets and dose-dependent inhibition of platelet aggregation.
- Determined the IC(50) value for platelet aggregation inhibition to be 16.85 mg/L.
Conclusions:
- A soluble anti-platelet GPIIb/IIIa antibody fragment (P140 Fab) was successfully prepared.
- The P140 Fab exhibits specific binding and inhibitory activity against platelet aggregation.
- This successful preparation provides a foundation for future clinical investigations and applications.
