A comparison of three strategies for biopanning of phage‐scFv library against diphtheria toxin

Abstract

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low‐yield positive results. In this study, three different strategies including soluble antibody‐capturing, pH‐stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme‐linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single‐chain fragment variable (scFv) fragments were purified by nickel‐nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody‐capturing method yielded 15 positive phage‐scFv clones against diphtheria toxoid. Conventional panning and pH‐stepwise elution model resulted from nine and five positive phage‐scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody‐capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two‐fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody‐capturing method is an efficient method for isolation of specific scFv fragments.

Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection

“…Selection of antibodies against human PCSK9. The recombinant human scFv phage library was screened against PcSK9 as previously reported (13,14). Immunotubes were coated with detection antigen (purified PCSK9) diluted to 100 µg/ml (2 ml/immunotube) and incubated at 4˚C overnight.…”

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

“…In a recent example, four different biopanning strategies, including the immobilization of recombinant protein to polystyrene beads and different cell-based methods, evaluated the selection output when incorporating various depletion and blocking steps [ 38 ]. Lakzaei and colleagues compared the effects of an antigen in solution to the same antigen immobilized on a carrier surface and found that the soluble approach was more efficient for isolating functional antibody fragments against the target [ 39 ]. However, comparative studies reported in the literature mainly focus on the optimization of strategies, including pH-elution parameters [ 39 , 40 ], the impact of depletion and negative selection [ 41 ], and the use of different antigen capture reagents [ 42 , 43 , 44 ].…”

“…Lakzaei and colleagues compared the effects of an antigen in solution to the same antigen immobilized on a carrier surface and found that the soluble approach was more efficient for isolating functional antibody fragments against the target [ 39 ]. However, comparative studies reported in the literature mainly focus on the optimization of strategies, including pH-elution parameters [ 39 , 40 ], the impact of depletion and negative selection [ 41 ], and the use of different antigen capture reagents [ 42 , 43 , 44 ]. Here, we show that biopanning strategies employing the surface-tethered recombinant ECD of an integral type I membrane protein were more effective in isolating a functional binder compared to biopanning with the full-length protein in the cellular membrane environment.…”

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

“…Here, we compared three different panning procedures: panning in MTP with directly immobilized antigen, panning in solution with biotinylated antigen and capture panning with human Fc-tagged antigen. Compared to the panning in MTP the panning in solution is slightly more complex but allows the target to be present in its native form which can lead to improved epitope accessibility [26,32]. The protocol with captured antigen is comparable to the protocol with directly immobilized antigen but potentially allows better target accessibility.…”