Table 2.

Representation of the different selection strategies and elution methods used in antibody Phage Display.

Library	Method	Reference
Synthetic scFv library	Selection in immunotubes coated with antigens. The phage preparation was added to tubes and after extensive washing, the bound phage was eluted with triethylamine.	[45]
Combinatorial naive scFv	Selection against an immobilized antigen on a roller bench. Bound phages were eluted with triethylamine.	[46]
Synthetic Fab library	Selection with seven rounds of panning, using trypsin elution.	[47]
Combinatorial scFv library	Anti-Golgi selections were carried out on streptavidin-coated magnetic beads. Biotinylated Golgi membranes were bound to streptavidin magnetic beads. After incubation, phages were eluted twice by incubating beads with triethylamine.	[48]
Combinatorial scFv library	Selection with immobilized antigens, reducing the antigen concentration over the rounds.	[49]
Combinatorial scFv library	Selection of specific antibodies against human platelets. Phages bound to the platelets were eluted with two distinct strategies: (1) Standard elution via incubation with 0.1 M glycine (pH = 2.2), followed by neutralization with Tris HCl (pH = 8). (2) Competitive elution via incubation with highly saturating concentrations (2 mg/mL) of abciximab, an anti-platelet competitor antibody.	[50]
Immune scFv library	Selection of antibodies, using sulfatide liposomes as antigen.	[51]
Combinatorial scFv library	Selection using inactivated measles virus as antigen	[52]
Combinatorial Fab library	Selection on streptavidin magnetic beads and biotin-conjugated antigen. Separation of the bound phages via pull down of the beads. Elution via competition through use of saturated antigen solution.	[53]
Human scFv library	Panning after a first negative selection with control antigen. Unbound phages were subjected to further incubation with VZV cell lysate antigen. The bound phages were eluted with 500 mL triethylamine followed by neutralization with 500 mL Tris/HCl pH 5.5, and they were amplified for a new round of selection.	[54]
Human scFv library	Panning decreasing concentrations of the antigen. The bound phages were detached via the direct addition of 1 mL of XL1-Blue cells.	[55]
Naïve scFv library	Selection against adherent IGROV-I cells. The cells were incubated with pre-warmed medium at 37 °C for 15 min to allow endocytosis of surface-bound phage. To remove phage bound to the extracellular matrix or to the culture plate, adherent cells were trypsinized. Subsequently, to remove phage bound to the cell surface, the cells were stripped three times with low-pH glycine buffer (150 mM NaCl, 100 mM glycine pH 2.5). Internalized phages were recovered from within the cells by lysing with 100 mM triethylamine for 4 min at 4 °C and neutralizing with 0.5 M TrisHCl.	[56]
Three scFv libraries	The phage library was incubated with 108 PBMC. Cells were then centrifuged, washed three times, and labeled with anti-BDCA3 antibody (dendritic cells). Approximately 105 cells were purified with magnetic-activated and subsequently fluorescence-activated cell sorting. Sorted cells were washed once with PBS, and cell-bound phages were eluted with HCl solution and then neutralized.	[57]
Immune mice scFv-Fc library	Phage library was used against rat liver endothelial cell plasma membranes.	[58]
Synthetic scFv library	Antibody selection, using biotinylated antigen captured by streptavidin–agarose resin. Selection can take place through centrifugation or on a column, separating the non-specific phages and the binding phages.	[59]
Commercial scFv library	Selection using cells containing the antigen of interest and control cells that are arranged on a flat surface (slide). Cells containing the antigen were previously and partially covered with a gold disc. Phages were incubated with both cells. Subsequently, non-target-cell binding phages were inactivated with UV irradiation. During irradiation, the phages bound to the antigen-bearing target cell were protected by the gold disc (radiation shielding).	[60]
Combinatorial immune Fab library	BRASIL selection. The phage library was incubated with normal cells and this system was applied in the top of an organic matrix, which was centrifuged. After, the supernatant with unbound phages was incubated with cancer cells, in the same method. Then, the cell pellet with bound phages was used directly to infect bacterial cells.	[37]
Immune llama VHH library	Masked selection: This selection consisted of incubating the phage library with antigens containing non-relevant (unwanted) epitopes, and then the non-relevant binding phages were eluted, and their antibody fragments were produced in soluble form. A new round of selection occurred, incubating antigens containing the relevant (desired) epitope with the non-relevant soluble antibody population to block (mask) the non-relevant epitopes; then, the original phage library was added to the selection.	[61]
Commercial combinatorial Fab library	Two libraries, one with Kappa chain and the other with Lambda chain, were mixed and used to select antibodies against protein F pre-fusion and post-fusion of RSV, performing a negative selection with pre-fusion protein and a positive post-fusion, and, in parallel, selection in reverse (negative for post-fusion and positive for pre-fusion).	[62]
scFv naive library	Selection of binding phages to a monolayer of corneal epithelial cells and elution with trypsin, with the support of a peristaltic pump.	[63]
Synthetic human VHH library	Selection of specific phages using a streptavidin-based Mass Spectrometry Immunoassay (MSIA Streptavidin D.A.R.T.) selection method. Metal pins containing streptavidin were attached to biotinylated antigen. The mixture was incubated with the phage and then washed to remove non-binding phage. Each wash cycle consisted of direct injection into an LC-MS in pulses with increasing speed. Afterward, the metal pins were incubated with a glycine-HCl solution, and the eluted phages were subjected to 300 cycles of low-speed injection and dispensing. The eluted fractions were immediately neutralized.	[64]
Synthetic scFv library	Selection with astringency with increasing Tween concentration, instead of the number of washes.	[65]
Immune rabbit scFv library	Selection with whole immobilized cells of the bacteria Campylobacter jejuni.	[66]
Naive scFv library	Selection with brain epithelial cells in solution.	[67]
Synthetic scFv library	Affinity column selection. Live Listeria monocytogenes cells were immobilized on a matrix in an immobilization column. The phage library was applied in the column. Elution was conducted with acid solution and centrifugation.	[68]
Synthetic scFv library	Selection of internalized phages in breast cancer cells. The phage library was incubated with cells and after removing unbound phages via washes, the specific phages were internalized. The internalized phages were eluted using cell lysis.	[69]
Synthetic scFv library	In vivo selection of atherosclerotic aorta antigen-specific scFv. The phage library was injected into ill rabbits and the phages circulated for one hour. The animals were sacrificed, and PBS was applied to ensure removal of free phages in the blood. The aorta was removed, and the phages were eluted.	[70]
Naive scFv library	In vivo selection in mice, with 3 h of phage circulation. Phages binding to tumor organs (lung and breast) and control organs (brain, muscles, pancreas) were analyzed.	[71]
Naive scFv library	Selection against immobilized antigen with astringency by gradually increasing intense washes in each round.	[72]
Combinatorial naive scFv library	Selection of specific antibodies against a biotinylated antigen, immobilized on a streptavidin surface, using two elution strategies: (1) Standard elution via incubation with 0.1 M glycine (pH = 2.2), followed by neutralization with Tris HCl (pH = 9). (2) Competitive elution via incubation with highly saturating concentrations (2 mg/mL) of non-biotinylated antigen (free antigen).	[39]
Immune murine scFv library	Selection of antibodies against a mixture of leukocyte cells from leukemia patients, with a previous depletion step with leukocyte cells from healthy donors. In the selection, the non-specific phages were removed with centrifugation and the cell-surface-bound phages were eluted with trypsin.	[73]
Naïve Fab library	Selection using a Phage Display library, in which the Fabs are attached to the phage by a covalent bond with a capture protein (SpyCatcher) expressed on the surface of the phage, instead of the classic form of gene fusion of the antibody gene and the gene of a virus protein, avoiding the subcloning step of the library construction (smaller phagemids). The selection was performed with different times of incubation of the library,, with an immobilized antigen in each round.	[74]
Synthetic camelid nanobody library	Panning using a single selection round, with a strong astringency method of twenty washes with PBS and four additional washes with acid buffer. The bound phages were eluted via competitive elution with the free antigen.	[75]