LIQUID-CHROMATOGRAPHY BASED ENZYME-AMPLIFIED IMMUNOLOGICAL ASSAYS IN FUSED-SILICA CAPILLARIES AT THE ZEPTOMOLE LEVEL

Through the use of fused-silica capillaries it was shown that reducing the liquid volume of an enzyme-amplified immunological assay increases the rate of amplification and sensitivity of the assay by several orders of magnitude. Human immunoglobulin G (hIgG) captured on protein G-coated 100-mum-i.d. columns was saturated with F(ab) anti-hIgG conjugated to alkaline phosphatase (ALP). Conjugated enzyme captured by the antigen was subsequently assayed in a stop-flow incubation with p-nitrophenyl phosphate. p-Nitrophenol produced in the stop-flow incubation was then swept to the detector and quantitated at 405 nm. The detection limit in the stop-flow mode was approximately 3 fmol. Three problems were identified in this flow-through, capillary assay format. The first was that the rate of immunological complex formation within the capillary was too slow. Preforming the immunological complex before application to the column increased the sensitivity by 2 orders of magnitude. Another problem was that the rate of mass transfer within the capillary limited capture of the preformed immunological complex. This problem was solved by stop-flow incubation of the complex in the column. The combination of preformation of the immunological complex and stop-flow binding within the column reduced the detection limit to approximately 3 amol. Finally, reducing the amount of F(ab)-ALP used in the assay minimized nonspecific binding of the conjugated enzyme and reduced the detection limit further to 333 zmol.