Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection.
The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves are established by using a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The precision is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is greater than or equal to 96%. Sample analysis times are on the order of 20 min. The HPAC technique gives results in absolute units of concentration that correlate well with binding capacity values determined by radioimmunoassay.
Riggin
A
A
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Regnier
F E
FE
Sportsman
J R
JR
D000906:Antibodies / Q000097:blood*; D002846:Chromatography, Affinity; D006801:Humans; D009419:Nerve Tissue Proteins; D011863:Radioimmunoassay; D013002:Somatomedins / Q000276:immunology*; D013050:Spectrometry, Fluorescence
D000906:Antibodies; C029152:G-substrate; D009419:Nerve Tissue Proteins; D013002:Somatomedins