Determination of antibodies to human growth hormone in serum by protein G affinity—reversed-phase tandem column chromatography with fluorescence detection

Abstract

High-performance protein G affinity (HPAC) and reversed-phase chromatographic (RPC) columns were used in tandem for the determination of antibodies to human growth hormone (hGH) in human serum. Serum samples were incubated with excess Texas Red-labeled hGH (hGH-TR) in solution. Protein G affinity was used to separate IgG-bound hGH-TR from unbound antigen. Antigen-antibody complexes were then dissociated and desorbed onto an RPC column, where they were separated and determined. The HPAC-RPC technique gives results in absolute units of concentration which are consistent with binding capacity values determined by radioimmunoassay. However, in this tandem column chromatographic approach, the serum background fluorescence is separated from the fluorescence of the labeled hGH. This results in a minimum detectable concentration for anti-hGH in serum of 50 ng ml −1. The analytical recovery was 98 ± 4% for three replicates with 200 ng ml −1 of GHC101. The relative standard deviations for the analysis of serum samples ranged from 1 to 8%. Sample analysis times are of the order of 50 min.