Table 1. Primer for the isotype-specific amplification of the complete variable regions.

Forward primer	Reverse primer	Primersequence 5′-3′	Approximated product size in bp
BoLH_BACK		ACCCACTGTGGACCCTCCTC
	BoIgMCH2_FOR	TGCCGTCACCAGAGAGGCTGT	795
	BoIgDCH2_FOR	TGCGTGCTGACCGCCTTGTT	805
	BoIgG1-3CH1_FOR	GGCACCCGAGTTCCAGGTCA	536
	BoIgECH1_FOR	GCCCAGCCTTACACGGGCTT	467
	BoIgACH1_FOR	GCCAGCACGGCAGGGAAGTT	574
GAPDH_for		TGGTCACCAGGGCTGCT
	GAPDH_rev	GGAGGGGCCATCCACAGTCT	527

One universal forward primer was used for annealing within the leader region. For each isotype, a reverse primer was generated for specific amplification. The annealing sites were selected in the first constant region (IGCH1), with the exception of IgM and IgD. Both isotypes share high homologies in the IGCH1 and therefore, specific reverse primers were generated for binding in the second constant region. The IgG subtypes were not distinguished further. Primers for bovine GAPDH served as cDNA quality control.