Analysis of several fluorescent detector molecules for protein microarray use.
The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE-antibody complex. The two array architectures tested consisted of an array of murine Fc-biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc-biotin array, detector signal intensity (background adjusted) was as follows: RPE-antibody complex > fluospheres > RPE > liposomes > Alexa 546: for the IgG array: RPE/antibody complex > RPE > fluospheres > Alexa546 > liposomes. The RPE-antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc-biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE-antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE-antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays.
Wiese
Rick
R
Genometrix Inc, 2700 Research Forest Drive, The Woodlands, TX 77381, USA. rick.wise@perbio.com
D000818:Animals; D001710:Biotin / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D010799:Phycoerythrin / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011817:Rabbits; D012680:Sensitivity and Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008081:Liposomes; C089187:cyanine dye 3; D010799:Phycoerythrin; D001710:Biotin