ABSTRACT
A novel washing scheme following antigen-antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation <10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were >0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing.
Subject(s)
Biomarkers/blood , Chromatography, Paper/methods , Fluorescent Dyes/analysis , Immunoassay/methods , Myocardial Infarction/blood , Fluorescent Dyes/chemistry , Humans , Linear Models , Reproducibility of Results , Signal-To-Noise RatioABSTRACT
To test the feasibility of semiquantitative immunohistochemical staining (IHC) with large sized gold conjugated secondary antibody (gold-2 degrees Ab), we used beads covered with a known amount of primary antibody and a secondary antibody conjugated with gold colloid particles (20 and 40 nm in diameter), and we compared the results to those obtained by enzyme IHC. Beads coated with 6 graded amounts of mouse IgG molecules showed 6 levels of color intensity. The graded color intensities could readily be distinguished. The color developed as soon as we added gold-2 degrees Ab, and the intensities were stable for 1 wk. Enzyme IHC using identical beads showed dregs of pigment after incubation in DAB for 5 min. The large sized gold-2 degrees Ab showed strong signals on cell surfaces; application of the large sized gold-2 degrees Ab to paraffin-embedded tissue sections was also feasible. The color was bright red and was easier to differentiate from hemosiderin pigment than the color developed by enzyme IHC. In conclusion, gold IHC with large sized gold-2 degrees Ab is superior to enzyme IHC for quantification of antigens via IHC. Gold IHC is especially recommended for tissues with many macrophages, such as bone marrow and spleen.
Subject(s)
Fluorescent Antibody Technique, Indirect/methods , Gold Colloid/chemistry , Animals , CD5 Antigens/analysis , Feasibility Studies , Humans , Immunoconjugates , Immunoenzyme Techniques , Leukocytes, Mononuclear/chemistry , Mice , Particle Size , Rats , Staining and LabelingABSTRACT
OBJECTIVES: The exquisite performance characteristics of an immunochemical fecal occult blood test (IFOBT) are well understood. We evaluated the diagnostic validity of a new IFOBT for colorectal neoplasia in patients undergoing colonoscopy and compared its results with two other commercially available IFOBTs. DESIGN AND METHODS: Eighty-five consecutive patients referred for colonoscopy were studied. We performed three different IFOBTs, namely, HM-Jack, Instant-View, and a newly developed OcculTech on each fecal specimen. RESULTS: OcculTech was easy to perform and had a sensitivity, specificity, and positive predictive value of 58.3%, 76.3%, and 27.9%, respectively, for the detection of colorectal cancers and >or=1 cm adenomas. OcculTech showed higher sensitivity than the automatic analyzer, HM-Jack. No improvement was obtained by combining tests. CONCLUSIONS: The OcculTech test had performance characteristics comparable to the two other IFOBTs. This study confirms the usefulness of the OcculTech test for colorectal neoplasia screening.
