Affinity Chromatography with Immunochemical Detection Applied to the Analysis of Human Methionyl Granulocyte Colony Stimulating Factor in Serum

An on-line, automated, HPLC method was developed for the separation and detection of recombinant human methionyl granulocyte colony stimulating factor (GCSF) and GCSF modified with poly(ethylene glycol) (PEG-GCSF) in rat serum. The automated method consists of an initial immunoaffinity chromatograph...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1996-09, Vol.68 (18), p.3077-3082
Main Authors: Miller, Karen J, Herman, Alan C
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Biochemistry
Biological and medical sciences
Calibration
Chromatography, Affinity
Chromatography, High Pressure Liquid
Filgrastim
Fundamental and applied biological sciences. Psychology
Granulocyte Colony-Stimulating Factor - blood
Humans
Immunity (Disease)
Immunochemistry
Pharmacology
Protein hormones. Growth factors. Cytokines
Proteins
Rats
Recombinant Proteins
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Summary:An on-line, automated, HPLC method was developed for the separation and detection of recombinant human methionyl granulocyte colony stimulating factor (GCSF) and GCSF modified with poly(ethylene glycol) (PEG-GCSF) in rat serum. The automated method consists of an initial immunoaffinity chromatography step, a microbore reverse phase separation, and postcolumn immunochemical detection. Calibration curves were constructed with a lower limit of 1.5 ng of GCSF (80 fmol) and 7.5 ng of PEG-GCSF in 120 μL of rat serum. In vivo serum samples from rats dosed with PEG-GCSF were also analyzed. The development of the method is discussed as well as its general application to the detection of metabolites of recombinant proteins in body fluids.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac960201e