Toward optimized antibody microarrays: a comparison of current microarray support materials.
With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.
Angenendt
Philipp
P
Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.
Glökler
Jörn
J
Murphy
Derek
D
Lehrach
Hans
H
Cahill
Dolores J
DJ
D000180:Acrylic Resins / Q000737:chemistry; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D004355:Drug Stability; D004356:Drug Storage; D005340:Fibrinogen / Q000276:immunology; D005898:Glass / Q000737:chemistry; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000906:Antibodies; D002232:Carbocyanines; D005340:Fibrinogen; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011107:Polylysine; D011108:Polymers; D000099166:Polyacrylamides; C089187:cyanine dye 3; C085321:cyanine dye 5