Comparison of antibody array substrates and the use of glycerol to normalize spot morphology.
Antibody microarrays are a high-throughput proteomic technology used to examine the expression of multiple proteins in complex solutions. Antibody microarrays can be manufactured on a variety of commercially available activated glass or coated slides. The goal of this study was to compare Hydrogeltrade mark, nitrocellulose, aldehyde-silane and epoxy-silane slides to determine the amount of antibody bound. The optimal substrate was defined as one that bound the greatest amount of antibody with minimal background. Our studies found that epoxy-silane enhanced surface (ES) slides gave the greatest degree of binding along with a minimal background. However, larger antibody microarrays showed variability in spot size, high intra-spot coefficient of variation and drying artifacts. Increasing the amount of glycerol in the spotting buffer caused a dose-dependent improvement in overall spot morphology. Glycerol was tested on 128 different antibodies and showed decreased: mean spot diameter, intra-spot coefficient of variation and drying artifacts. These studies revealed that the optimal slide substrate was epoxy-silane ES microarray slides. Furthermore, glycerol could normalize spot size, decrease intra-spot coefficient of variability, decrease drying artifacts and increase antibody-spotting density.
Olle
Eric W
EW
Pfizer Global Research and Development, Safety Sciences, Ann Arbor, MI 48105, USA.
Messamore
James
J
Deogracias
Michael P
MP
McClintock
Shannon D
SD
Anderson
Timothy D
TD
Johnson
Kent J
KJ
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D003101:Collodion / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry*; D006041:Goats; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D007118:Immunoassay; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007375:Interleukin-1 / Q000276:immunology; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry*
D000906:Antibodies; D007074:Immunoglobulin G; D007375:Interleukin-1; D012821:Silanes; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D003101:Collodion; D005990:Glycerol