For IgA HLC assays, polyclonal IgA was purified from pooled normal human sera by ammonium-sulfate precipitation, anion-exchange chromatography, and immunoaffinity chromatography. Low-level contamination with alternate classes of immunoglobulins was removed by immunoaffinity adsorption and/or protein G affinity chromatography. Fractionation of κ from λ light chain forms was achieved using both protein L and anti–light chain immunoaffinity matrices.
Purified IgAκ and IgAλ were >99% pure by silver-stained SDS-PAGE, (Figure 9.4
) and light chain specific as indicated by enzyme immunoassays and Western blot analysis (Figure 9.5
). Contamination with other immunoglobulin and light chain types was <0.5% by enzyme immunoassay. Finally, the total protein concentration of the primary standards (determined using the bicinchoninic acid method) and CRM 470 plus DA470k were used to assign IgAκ and IgAλ values to the IgA HLC internal reference standard (Figure 9.7
). Similar methods were used for the calibration of the IgG and IgM HLC assays.