High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay.
An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.
Hage
D S
DS
Department of Laboratory Medicine, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Kao
P C
PC
D000906:Antibodies / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007118:Immunoassay; D008163:Luminescent Measurements; D010281:Parathyroid Hormone / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D010281:Parathyroid Hormone