A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

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A new enzyme immunoanalytical concept that can be used for point-of-care testing has been investigated. Enzyme as a tracer requires a separate reaction step for signal generation, which follows the completion of immune complex formation with analyte (e.g., Hepatitis B surface antigen) in a sample. This has been a major factor limiting its utilization within the laboratory. We carried out such sequential processes employing chromatographic analysis, using two crosswise-arranged membrane pads in vertical and horizontal directions. The vertically arranged pads were the same as those in the usual format for pregnancy testing, for instance, with the exception of the use of horseradish peroxidase (HRP) as tracer. By placing the horizontally arranged pads on each lateral side of the signal generation pad in the vertical arrangement, they were employed to supply substrate to the enzyme present in the immune complexes. The substrate flow was initiated after the antigen-antibody bindings to produce a signal, which was typically a color change in proportion to the analyte concentration. Under optimal conditions, the use of HRP labeling increased the detection capability of the assay approximately 30 times compared to that of gold colloids. Potential advantages of using the concept investigated are (1) provision of a rapid and simple immunoassay, (2) satisfaction of a clinical need for highly sensitive determination of analyte, and (3) utilization of relatively inexpensive, portable quantitation means.

A number of studies have been done on the orientation of surface-immobilized antibodies since it plays a significant role in the performance of immunoassays. Here, we present a new study by which the orientation of differently immobilized antibodies was directly probed by using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). For greater control over the orientations of intact IgG and F(ab ) 2 antibody fragment, they were either site-directly biotinylated at the hinge region or randomly biotinylated at the amino groups, and were then immobilized onto a streptavidin-terminated surface. According to the PCA results from ToF-SIMS spectra, site-directly and randomly biotinylated IgG became 'end-on' oriented (Fc is closer to the surface) at a gradual rate as immobilization concentration increased, while site-directly biotinylated IgG became oriented at a slightly faster rate. Furthermore, site-directly biotinylated F(ab ) 2 quickly became 'end-on' oriented even at a low concentration of 1 µg/ml as immobilization concentration increased, whereas randomly biotinylated F(ab ) 2 was not oriented at all over any concentration. Our results show that a ToF-SIMS partnership with multivariate analysis is useful for interpreting protein orientations in terms of surface amino acid profiles.

The molecular orientation of antibody layers formed on separate solid matrices (e.g., gold-coated glass substrate) was characterized by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS) in static mode. For comparison, three different antibody species, IgG, F(ab')(2), and Fab, were prepared, biotinylated in random and site-directed fashions, and immobilized on distinct streptavidin-coated surfaces. ToF-SIMS analyses of each antibody layer revealed that the secondary ion intensity peaks measured at the mass-to-charge (m/z) ratio 253, 325, and 647 were unique to the site-directly immobilized antibodies. The ions in the three peaks were detected neither from the streptavidin layer nor from the randomly prepared antibody, indicating that the insolubilized antibody layers constructed in the two different manners had distinct molecular arrangements. The antibody preparations were further tested for their binding characteristics in sandwich-type immunoassays, which showed that the site-directed antibodies consistently enhanced the detection capability comparing to those randomly prepared. Based on the analytical results of both the ToF-SIMS analysis and sandwich-type immunoassays, the site-directed antibody species were immobilized on the surfaces in a more orientated manner, with their antigen binding sites exposed to the bulk solution, than when random immobilization was used.