Presentation Title

Thread/Paper-Based Enzyme-Linked Immunosorbent Assay (ELISA)

Faculty Mentor

Frank A. Gomez

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 10:15 AM

Location

9-263

Session

Physical Sciences 3

Type of Presentation

Oral Talk

Subject Area

physical_mathematical_sciences

Abstract

We demonstrate the use of a novel microfluidic thread/paper-based analytical device (µTPAD) for the detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis. Fabrication of the µTPAD consist of six pieces of nylon thread, three pieces of flannel, and three pieces of circular paper chromatography analysis sites. For system one, nitrocellulose (NC) was spotted on the detection sites. Biotinylated goat anti-mouse IgG was spotted and dried on the analysis sites, prior to subjecting it to a series of Tris-Tween washes, increasing streptavidin-alkaline phosphatase (Strep-ALP) concentrations, p-nitrophenyl phosphate (p-NPP), and stop p-NPP solution, realizing a yellow color at the detection sites. The detection sites were then scanned and reported as inverse yellow intensity versus concentration of Strep-ALP. The yellow color intensity increases as Strep-ALP concentration increases. For system two, primary rabbit IgG antibody was spotted and dried on the analysis sites, followed by a solution of Tween-BSA, ALP-conjugated secondary antibody, a series of PBS washes, and a solution of the colorimetric substrate for ALP, realizing a purple color at the detection sites. The platforms were scanned and analyzed yielding a correlation between the purple color produced, which was analyzed as inverse blue intensity, and the concentration of rabbit IgG. The purple color increased as the concentration of the primary antibody increased. The design of the µTPAD enables triplicate data collection. The device is low-cost, easy-to-use, requires a minimal amount of material, and facilitates an ELISA.

Summary of research results to be presented

To demonstrate the efficacy of the microfluidic thread/paper-based analytical devices (µTPADs), we measured antibody-antigen recognitions in an ELISA. Biotinylated goat anti-mouse IgG and rabbit IgG antibodies were used as the two model systems. For both systems data was gathered in triplicates. For the detection of biotinylated goat anti-mouse IgG (system one), a yellow color at the detection sites indicated the completion of the ELISA. The intensity of the yellow color corresponds to how much Strep-ALP bound to the biotinylated goat anti-mouse IgG. Average inverse yellow intensity was plotted as a function of Strep-ALP concentrations. A linear range of detection was observed for concentrations of Strep-ALP between 3.75 X 10-4 to 1.75 X 10-3 mg/mL (R2 = 0.9824). These lower Strep-ALP concentrations produce a product in a linear fashion as the enzyme has not yet reached a Vmax. At higher concentrations of Strep-ALP, saturation is achieved and the inverse yellow intensity stops changing significantly. For the detection of rabbit IgG antibodies (system two), a purple color at the detection sites indicated the completion of the ELISA. The intensity of the purple color (quantified as blue) was proportional to how much secondary antibody bound to the primary rabbit IgG. The corrected average blue inverse intensity was plotted as a function of the log of the rabbit IgG concentration. An IC50 value of 1034 fmol/zone was obtained. The limit of detection (LOD) was determined to be 3.75 X 10-4 mg/mL for the first system and 0.7 fmol/zone for the second system.

This document is currently not available here.

Share

COinS
 
Nov 18th, 10:00 AM Nov 18th, 10:15 AM

Thread/Paper-Based Enzyme-Linked Immunosorbent Assay (ELISA)

9-263

We demonstrate the use of a novel microfluidic thread/paper-based analytical device (µTPAD) for the detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis. Fabrication of the µTPAD consist of six pieces of nylon thread, three pieces of flannel, and three pieces of circular paper chromatography analysis sites. For system one, nitrocellulose (NC) was spotted on the detection sites. Biotinylated goat anti-mouse IgG was spotted and dried on the analysis sites, prior to subjecting it to a series of Tris-Tween washes, increasing streptavidin-alkaline phosphatase (Strep-ALP) concentrations, p-nitrophenyl phosphate (p-NPP), and stop p-NPP solution, realizing a yellow color at the detection sites. The detection sites were then scanned and reported as inverse yellow intensity versus concentration of Strep-ALP. The yellow color intensity increases as Strep-ALP concentration increases. For system two, primary rabbit IgG antibody was spotted and dried on the analysis sites, followed by a solution of Tween-BSA, ALP-conjugated secondary antibody, a series of PBS washes, and a solution of the colorimetric substrate for ALP, realizing a purple color at the detection sites. The platforms were scanned and analyzed yielding a correlation between the purple color produced, which was analyzed as inverse blue intensity, and the concentration of rabbit IgG. The purple color increased as the concentration of the primary antibody increased. The design of the µTPAD enables triplicate data collection. The device is low-cost, easy-to-use, requires a minimal amount of material, and facilitates an ELISA.