Table 1.

Overview of immunization and biopanning strategies for the identification of cell plasma membrane-protein targeting nanobodies.

Immunization and Biopanning Strategies	Advantages	Disadvantages	References
Transfected cells	-Presents membrane protein in its native form -Applicable for both immunization and panning procedures	-Generation of nanobodies directed towards undesired membrane proteins by the host animal -Requires the construction of transfected cells expressing high levels of the protein of interest	[17,71,84,85,86,87,88]
Membrane extracts	-Presents membrane protein in its native form -Enables to identify intracellular and extracellular binders -Applicable for both immunization and panning procedures	-Generation of nanobodies directed towards undesired membrane proteins by the host animal -Requires the construction of transfected cells expressing high levels of the protein of interest	[71,89,90]
Nanodiscs	-Presents membrane protein in its native form -Enables to identify intracellular and extracellular binders -Does not imply the risk of retrieving unspecific nanobodies that target other components present on cells	-Might require the construction of transfected cells expressing high levels of the protein of interest -Complex membrane model (antigen-dependent)	[91,92,93,94,95,96]
Virus-like particles	-Presents membrane protein in its native form -Applicable for both immunization and panning procedures -Does not imply the risk of retrieving unspecific nanobodies that target other components present on cells	-Complex membrane model (antigen-dependent)	[71,97,98,99]
cDNA immunization	-Presents membrane protein in its native form (in vivo) -No need to construct specific membrane models -No generation of nanobodies directed towards undesired membrane proteins by the host animal	-Difficult retrieval of desired nanobodies due to a weak immune response of the host animal	[68,84,100,101]
Detergent solubilized proteins	-Applicable for both immunization and panning procedures -No need to construct specific membrane models	-Detergents may cause an altered structure and function of the cell plasma membrane proteins	[92]
Endogenous proteins at the cell surface	-Presents membrane protein in its native form -Applicable for both immunization and panning procedures -No need to construct specific membrane models	-Difficult retrieval of desired nanobodies due to a weak immune response of the host animal -Generation of nanobodies directed towards undesired membrane proteins by the host animal -Low expression levels of the antigen of interest	[71,100]