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Affinity columns containing anti-DNA Id+ human myeloma proteins adsorb human epibodies from intravenous gamma
R C Williams1, C C Malone, G Fry
1University of Florida School of Medicine, Gainesville, USA.
Objective:
To study eluates of intravenous gamma globulin (IVGG) prepared from affinity columns of human cationic IgG myeloma proteins bearing anti-DNA idiotype (Id) markers 16/6, F4, 3I, and 8.12 for possible anti-Id (combining site) blocking activity.
Methods:
Anti-DNA idiotypic antibody activity was studied in 3 preparations of IVGG containing high, medium, and low levels of IgG anti-F(ab')2, and in 4 other commercial IVGG preparations. Affinity-purified IgG anti-DNA (APAD) from systemic lupus erythematosus (SLE) patients was biotinylated, and binding to DNA coated on enzyme-linked immunosorbent assay plates was used to measure anti-DNA antibody activity. IVGG was adsorbed to Sepharose 4B affinity columns linked to a panel of cationic human IgG myeloma proteins positive for anti-DNA Id markers 16/6, F4, 3I, and 8.12. Material adsorbing to such columns was eluted at low pH (2.5) and after neutralization, tested for its ability to inhibit biotinylated APAD reacting with DNA.
Results:
Only 0.05-0.9% of IVGGs bound firmly to Id affinity columns. These IVGGs were then eluted, using pH 2.5 glycine-saline and eluates neutralized to pH 7.4. Column flowthrough and eluate fractions were compared for their ability to block SLE APAD reacting with DNA. Significant inhibition of SLE APAD combining sites was observed with eluates from anti-DNA Id affinity columns; however, no correlation between IVGG anti-F(ab')2 activity and true anti-Id blocking of APAD was apparent. No residual anti-Id activity remained in column flowthrough fractions. No anti-Id blocking activity was recorded for IVGG eluates from human cationic myeloma columns devoid of the 4 anti-DNA Id markers. DNase treatment of IVGG or Id column eluates did not affect anti-Id blocking activity. Thus, all detectable anti-DNA idiotypic antibody capable of blocking SLE anti-DNA combining sites bound to Id+ affinity columns. Column eluates also showed some relative concentration of IgG anti-DNA activity, which was of lower affinity for DNA than antibodies also present in eluates which blocked anti-DNA combining sites.
Conclusion:
The presence of both anti-DNA and antiidiotypic (anti-combining site) activity in human anti-DNA Id column eluates indicates that epibodies from IVGG are relatively concentrated when this strategy is used. This approach may lead to a new strategy for treatment of SLE nephritis.