Quantitative measurement of mouse IgG subclasses with the use of heteroantisera: the importance of allotype considerations
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Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.
Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.
