Table 1.

Other typical methods used for studies characterizing the genetics of human (auto-)antibody responses

Method	Production of mAb	Sequencing	Selection bias1	Advantages	Disadvantages
Antibody phage display	scFv, Fab (Ig)	Directly from phage display vector	+	Relative ease of screening, sequencing, and production of soluble protein; depth of coverage; ease of adapting isotype-specific applications	Random pairing of VH and VL, tedious Ig production (subcloning required)
Heterohybridoma	Ig	RT-PCR	+	In vivo VH/VL pairing, production of full-length Ig	EBV/unselected fusions often yield IgM, instability phenomena and inefficient isolation, expensive culturing
Single B-cell PCR	(Ig)	RT-PCR	+	In vivo VH/VL pairing	Difficult mAb production (subcloning), difficult for rare clones
Deep sequencing	None	From any nucleotide source	–	No selection bias, calculation of frequencies	Necessary trade-off of sequencing length and read number; no pairing of VH and VL for analysis

Advancements and combinations of the methods detailed above may pave the way for future studies of unparalleled depth and accuracy (see DeKosky et al., 2013, and others).

EBV, Epstein–Barr virus; scFv, single-chain variable fragment; VH, variable heavy; VL, variable light.

¹Selection bias may result from differences in phage growth rates, instability of cell fusions, or limited numbers of B cells analyzed by single B-cell PCR. These effects may lead to nonrepresentative (biased) distributions of the parameters analyzed.