Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography

Immuno-chromatographic Analysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus

“…17 The four membrane pads used in assembling the rapid test kit were as follows (from the bottom): a glass fiber membrane (4 × 20 mm) (Ahlstrom, Whatman, Kent, UK) for sample application, a glass fiber membrane (4 × 7 mm) (Ahlstrom, Whatman, Kent, UK) for the release of the detection antibody labeled with colloidal gold, a NC membrane (4 × 25 mm) (Millipore, Billerica, MA) for signal generation, and a cellulose membrane (4 × 50 mm) (Ahlstrom, Whatman, Kent, UK) for continuous sample absorption. The sample application pad was prepared by immersing in 10 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100 and drying at 55 o C for 4 h. The signal generation pad was prepared by spotting a monoclonal antibody (1 µL) selected as the capture binder on the NC membrane and then allowing it to incubate at 37 o C for 20 min.…”

“…The detection antibodies, clone #35 and #1 specific to HPV-16 E7 and HPV-18 E7 proteins, respectively, were separately coupled to HRP via a chemical reaction as previously described. 17 Briefly, the antibodies were first reduced using DTT (10 mM, final concentration) and the excess reagent was removed on a Sephadex G-15 gel filtration column (GE Healthcare, Waukesha, WI). HRP was activated with a 50-fold molar excess of succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (SMCC; Pierce, Rockford, IL), and the excess reagent was immediately removed in the same manner.…”

“…Antibody labeling with colloidal gold: A monoclonal antibody selected as the detection binder was labeled with colloidal gold (the mean diameter: 30 nm) as previously reported. 17 Briefly, the antibodies dialyzed in 10 mM phosphate buffer were conjugated with colloidal gold that was synthesized using the sodium citrate method. Unreacted antibodies were removed by centrifugation and the antibody-gold conjugates were then resuspended in Casein-PBS such that the gold density was concentrated by 20-fold relative to the initial gold density.…”

“…The major factors, including the concentration of the capture and detection antibodies, and the lateral flow rate along the immuno-strip, were optimized as described in our previous reports. 17 Standard samples of the target proteins were prepared and used to analyze the immunochromatographic assay according to the pre-determined protocol. The results of this experiment revealed that the produced signals were proportional to the analyte concentration and detectable by the naked eyes at an analyte concentration as low as 2 ng/mL for the both of HPV-16 and 18 E7 proteins (Table 1).…”

Development of optical biosensor technologies for cardiac troponin recognition

“…To achieve the increasing demand of quick troponin diagnosis and consequently the clinical therapeutics, a plethora of methods have been used for detection of the troponin family, including enzyme-linked immunosorbent assay (ELISA), 25 chemiluminescent immunoassay, 26 fluoroimmunoassay, 27 electrical detection, 28 surface plasmon resonance (SPR) detection, 29 and colorimetric protein array. 30 These optical biosensors for cardiac troponin detection, in comparison with electrochemical biosensors for troponin detection, are more bulky and expensive and also require difficult labeling procedures.…”