Specific flow injection sandwich binding assay for IgG using protein A and a fusion protein.
A sandwich-type flow-injection binding assay for quantitation of various IgG's was developed. The assay is based on the pseudoimmunological reaction between protein A from Staphylococcus aureus and immunoglobulin G from different species. Protein A immobilized on a solid support and a fusion protein of protein A and beta-galactosidase from Escherichia coli are used for detection. The fusion protein is produced with a temperature-inducible recombinant E. coli strain. A sandwich is formed by subsequent injection of IgG and fusion protein into the buffer stream flowing through the immobilized protein A column. The amount of enzyme activity bound is proportional to the amount of IgG bound and is measured by pumping a lactose solution as substrate for beta-galactosidase through the protein A column. Lactose is converted to glucose and galactose. The detector is an enzyme thermistor that measures the heat evolved in the enzymatic conversion of glucose by coimmobilized glucose oxidase and catalase. The assay takes 16 min at a flow rate of 0.6 mL min-1 with a lower detection limit of 33 pmol per injection of rabbit IgG. The precision of replicate measurements has a standard deviation of 4-5%, and the column can be used for more than 50 cycles.
Brandes
W
W
Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Germany.
Maschke
H E
HE
Scheper
T
T
D000818:Animals; D002138:Calibration; D004926:Escherichia coli / Q000201:enzymology; D017022:Flow Injection Analysis / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A; D001616:beta-Galactosidase
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D001616:beta-Galactosidase