Estimation of polyclonal Ig G 4 hybrids in normal human serum

Summary The in vivo or in vitro formation of I g G 4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal I g G 4 hybrids in normal human serum and comment on the existence of I g G 4 molecules with different immunoglobulin light chains. Polyclonal I g G 4 was purified from pooled or individual donor human sera and sequentially fractionated using light‐chain affinity and size exclusion chromatography. Fractions were analysed by SDS – PAGE , immunoblotting, ELISA , immunodiffusion and matrix‐assisted laser‐desorption mass spectrometry. Polyclonal I g G 4 purified from normal serum contained I g G 4 κ , I g G 4 λ and I g G 4 κ / λ molecules. Size exclusion chromatography showed that I g G 4 was principally present in monomeric form (150 000 MW). SDS – PAGE , immunoblotting and ELISA showed the purity of the three I g G 4 samples. Immunodiffusion, light‐chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the I g G 4 κ / λ molecules. The amounts of I g G 4 κ / λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two I g G 4 heavy chains plus one κ and one λ light chain. Polyclonal I g G ( I g G 4‐depleted) was similarly fractionated according to light‐chain specificity. No evidence of hybrid I g G κ / λ antibodies was observed. These results indicate that hybrid I g G 4 κ / λ antibodies compose a substantial portion of I g G 4 from normal human serum.