Isocratic reporter-exclusion immunoassay using restricted-access adsorbents

Abstract

We introduce analyte-dependent exclusion of reporter reagents from restricted-access adsorbents as the basis of an isocratic reporter-exclusion immunoassay for viruses, proteins, and other analytes. Capto™ Core 700 and related resins possess a noninteracting size-selective outer layer surrounding a high-capacity nonspecific mixed-mode capture adsorbent core. In the absence of analyte, antibody-enzyme reporter conjugates can enter the adsorbent and be captured, and their signal is lost. In the presence of large or artificially-expanded analytes, reporter reagents bind to analyte species to form complexes large enough to be excluded from the adsorbent core, allowing their signal to be observed. This assay principle is demonstrated using M13 bacteriophage virus and human chorionic gonadotropin as model analytes. The simple isocratic detection approach described here allows a rapid implementation of immunoassay for detection of a wide range of analytes and uses inexpensive, generally-applicable, and stable column materials instead of costly analyte-specific immunoaffinity adsorbents.

Figure 1:

Illustration of reporter-exclusion assay for large analytes such as viruses. (a) In the absence of large viral analyte, small protein reporters such as antibody-enzyme conjugates can penetrate the core of the resin particles and bind to the internal mixed-mode ligands. (b) In the presence of virus particles, antibody-enzyme conjugates bind to the viruses, are excluded by the size-selective shell, and can give detectable signal.

Figure 2:

Flow-rate dependence of reporter capture in a reporter-exclusion assay for M13 virus particles. (a) In absence of M13 phage the HRP/Anti-M13 conjugate is almost entirely captured by the Capto Core 700 adsorbent at a high linear flow velocity of 122 cm/h, the uncaptured conjugate is detected as background HRP activity (gray bars). (b) At the same flow rate, for the sample containing M13 phage, the phage particles bind the conjugate and the HRP activity of excluded complex (gray bars) is detected and is much higher in later fractions than in the absence of phage. The blue trace indicates the absorbance of each fraction at 280 nm. (c) Loading HRP/Anti-M13 conjugate at a lower linear flow velocity of 31 cm/h (residence time 7 min) results in very low background HRP activity.

Figure 3:

Variation of reporter capture efficiency with phage concentration. Various concentrations of M13 phage incubated with HRP/Anti-M13 conjugate were separated at linear flow velocity 31 cm/h on the Capto Core column.

Figure 4:

Schematic of small-analyte reporter-exclusion immunoassay using restricted-access adsorbent. (a) In the absence of analyte, the free reporter is small enough to be captured by the adsorbent. (b) If present, analyte bridges the reporter onto phage, forming a large, excluded complex of anti-analyte M13 phage, sandwiched analyte, and anti-analyte reporter.

Figure 5:

Detection of hCG using M13 phage anti-hCG conjugate as an excluded capture carrier with HRP/Anti-β-hCG as reporter. Flow-through assays were performed with HRP conjugate alone, and with phage and conjugate without or with 10 ng/mL hCG. The HRP activity was analyzed using 1-Step^™ Ultra TMB-ELISA substrate.