Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.
Fatemeh AfsharnooriMehdi Forouzandeh MoghadamPublished in: Medical oncology (Northwood, London, England) (2023)
Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified V H /V κ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.
Keyphrases
- pseudomonas aeruginosa
- peripheral blood
- immune response
- atrial fibrillation
- binding protein
- chronic kidney disease
- wastewater treatment
- type diabetes
- south africa
- genome wide
- multiple sclerosis
- transcription factor
- papillary thyroid
- bone marrow
- skeletal muscle
- dendritic cells
- copy number
- end stage renal disease
- smoking cessation
- molecular docking
- drug induced
- long non coding rna
- amino acid
- recombinant human