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United States Department of Agriculture

Agricultural Research Service

Title: Apparent Thixotropic Properties of Saline/Glycerol Drops with Biotinylated Antibodies on Streptavidin-Coated Glass Slides: Implications for Bacterial Capture on Antibody Microarrays

Authors
item Albin, David
item Gehring, Andrew
item Reed, Sue
item Tu, Shu-I

Submitted to: Sensors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 11, 2009
Publication Date: February 16, 2009
Citation: Albin, D.M., Gehring, A.G., Reed, S.A., Tu, S. 2009. Apparent Thixotropic Properties of Saline/Glycerol Drops with Biotinylated Antibodies on Streptavidin-Coated Glass Slides: Implications for Bacterial Capture on Antibody Microarrays. Sensors. 9:995-1011.

Interpretive Summary: Harmful bacterial may be present in foods. In addition, bacteria and many other pathogens may be intentionally introduced into the food supply. Therefore, we have developed a screening test that is potentially capable of detecting hundreds or thousands of harmful bacteria in a single sample. The test uses microarrays, glass microscope slides containing spots with molecules called antibodies that specifically capture harmful bacteria from a sample. Then, a second antibody, containing a label that can be easily detected and measured, is used to attach to any captured, harmful bacteria. In addition to this method development, and in an effort to optimize the method, we examined several aspects of this test, including various ways to attach capture antibodies to the microscope slide, and timing of different parts of the test. The findings of this research improve testing methods, which will be used by food producers, distributors, and retailers, for harmful bacteria in foods.

Technical Abstract: The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (~108 cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (~106 cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay.

Last Modified: 10/1/2014
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