The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice (1)
In mice, antibodies of the IgG2a, subclass are the predominant isotype produced to viral infections and in cytokine-induced Th1-type responses. The murine Igh1-b allele is the most divergent of the IgG2a heavy chains. The protein of this allele is distinctly different from other IgG2a allotypes by its elution profile from protein A. The Igh1-b allele in mouse strains such as C57BL/6, C57BL/10, SJL and NOD is not an allele but is derived from a separate gene whose product is designated as the novel isotype IgG2c. In these mouse strains the IgG2a gene is deleted, whereas in inbred strains with the Igh1-a allele, like BALB/c, the IgG2c gene is deleted. Commercial sera against the IgG2a isotype are raised exclusively against BALBrc IgG2a myeloma proteins. Because there is a 16% difference in the amino acid sequences between IgG2a and IgG2c, the question arises how reliably do anti-IgG2a sera react with IgG2c.
Commercial anti-IgG2a serum recognized IgG2a antibodies in BALB/c serum as well as purified IgG2a protein from a BALBrc myeloma, but it showed little crossreactivity with NOD and C57BLr10 sera. Conversely, anti-serum raised to recombinant protein detected IgG2c in NOD and C57BLr10 sera, but did not react with BALB/c serum or purified IgG2a.
Anti-IgG2a serum of the Mouse Typer sub-iso- typing panel detected IgG2a in BALB/c serum, but showed poor cross-reactivity to IgG2c in NOD serum.
Commercial antiserum detected the heavy chain of the IgG2a isotype in sera of BALB/c, CBA/H and 129/J mice, but not NOD and C57BL/10 mice. IgG2a antibodies from mice of the Igh-1b allotype eluted from Protein A at pH 4.5, while other allotypes eluted at pH 5.0 consequently using differential pH (i.e., binding at pH 7.2 and elution at pH 4.0) both IgG2c or IgG2a would be eluted.
It is essential to use IgG2c- specific antiserum in order to quantify accurately isotypic responses in C57BL/6, C57BL/10, NOD, and presumably other mouse strains with the Igh1-b allele. Binding of IgG to protein A is mediated by the junctional region of the CH2 and CH3 domains of the heavy chain, with three sites being critical in this binding. Murine IgG2a these sites would be the MIS 140–142 , QHQ 197–199, and NHH 322–324 positions. There is only one residue that differs between IgG2a and IgG2c in these sites (H at position 324 is replaced by L). The presence or absence of high IgG2a levels has been proposed as a prototypic example of the Th1/Th2 paradigm in both examples, which C57BL/6 mice are resistant to leishmaniasis and NOD mice develop spontaneous insulin dependent diabetes.