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Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.

We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane. Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation. To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin. After incubation of the reagent with the DNA sample for 1 h at 37 degrees C to form a complex of streptavidin--biotin--SSB--DNA--anti-DNA--urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex. The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a light-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex. This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.

Culture medium from fibroblasts of cystic fibrosis patients and controls was examined for the ability to inhibit (calcium plus magnesium)-activated ATPase and (sodium plus potassium)-activated ATPase. The ATPase systems used were both a solubilised preparation from dog-fish and a membrane associated preparation from human erythrocyes. Contrary to other reports the medium from cystic fibrosis fibroblasts did not inhibit ATPase activity.

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbate's action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.