Immunochromatographic analysis of proteins
Abstract
The technique of immunochromatographic analysis is developed for the quantitative determination of specific antibodies and for characterization of proteins. Either a single column high performance affinity chromatography (HPAC) or HPAC and reversed-phase chromatography (HPAC-RPC) tandem approaches can be used for specific antibody determination. These techniques are demonstrated for the quantification of antibodies to human growth hormone (hGH) in serum from patients. When the HPAC approach is employed, a protein G affinity column is used to capture the antibodies. Texas Red$\sp{\rm TM}$-labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves were established 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%. The precision of 15% RSD can be achieved at the MDC and is better above the detection limit. The linear dynamic range of the method is approximately two orders of magnitude. Total fluorescence recovery from the affinity column is $\ge$96%. In the HPAC-RPC tandem column approach, a protein G affinity column is used to separate IgG-bound hGH-TR from unbound antigen. Antigen-antibody complexes were then desorbed onto a RPC column where they were separated and quantified. Both the HPAC and HPAC-RPC techniques give results in absolute units of concentration. The assay results of these two approaches are consistent and correlate well 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 an improved MDC for anti-hGH in serum of 50 ng/ml. Size-exclusion column was also used in conjunction with an affinity column to examine antigen variants. A high performance protein G column on which either mono- or polyclonal antibodies were immobilized was used to analyze hGH, hGH-derivatives and aggregates.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.