Kari Kukanskis

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed "immunoRCA. " In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

limit (15 and 22 below the limits for the Stratus and Immulite assays, respectively) and 42 (12%) had both results below the respective detection limits. Regression analysis was performed on the 268 remaining samples: Immulite cTnI 1.84 (Stratus cTnI) 1.1 g/L; r 0.977; S yx 4.7 g/L. The high slope might result from a lack of standardization between cTnI assays (2 ) and/or a difference in the reactivities of the antibodies used to the various circulating forms of the protein (3-6 ). We routinely used an upper reference limit (URL) of 0.6 g/L for the Stratus cTnI assay. To estimate the corresponding value for the Immulite cTnI assay, we established the relationship between the two assays in 137 samples with Stratus cTnI values 5 g/L: Immulite cTnI 1.51 (Stratus cTnI) 0.27; r 0.924. The estimated Immulite URL (based on the regression) corresponding to a Stratus value of 0.6 g/L was 1.18 g/L (Fig. Using these cutoffs, we studied 80 patients admitted to the intensive care unit (37 with acute myocardial infarction, 22 with unstable angina, and 21 with chest pain). Heparinized samples were collected on admission. The Stratus (0.6 g/L) and estimated Immulite (1.18 g/L) URLs gave specificities of 93% [95% confidence interval (CI), 69.6 -98.8%] and 95% (95% CI, 76.2-99.9%), respectively, for acute coronary syndrome (vs 21 chest pain patients) and sensitivities of 95% (95% CI, 85.9 -98.9%) and 92% (95% CI, 81.3-97.2%), respectively.

s at 95 C followed by 30 s at 70 C. The sequencing gel was made of 8% Long Ranger gel solution (BMA, Rockland, ME, USA) and containing 7 M urea (Research Organics, Cleveland, OH, USA) and 1 TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA . Na 2 [pH 8.4]). Three microliters of formamide loading dye (from the Amersham Pharmacia Biotech kit) were added to the samples after the cycle sequencing reaction. The loading membranes (sample loader) were from Genetic BioSystems (San Diego, CA, USA). The samples were heated for 2.5 min at 95 C and either immediately spotted onto the tabs of a preheated (95 C) membrane loader (hot sample application) or cooled down in ice-cold water bath before spotting (regular sample application).