Antibody microarrays: an evaluation of production parameters.
Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.
Kusnezow
Wlad
W
Functional Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany. w.kusnezow@dkfz.de
Jacob
Anette
A
Walijew
Alexandra
A
Diehl
Frank
F
Hoheisel
Jörg D
JD
D000042:Absorption; D000906:Antibodies / Q000737:chemistry*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry; D004306:Dose-Response Relationship, Immunologic; D005898:Glass; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D008956:Models, Chemical; D020411:Oligonucleotide Array Sequence Analysis; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D011485:Protein Binding; D012984:Software; D013048:Specimen Handling; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors
D000906:Antibodies; D019941:Cyclin-Dependent Kinase Inhibitor p16; D008164:Luminescent Proteins; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma