Construction of an antibody microarray based on agarose-coated slides.
The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.
Lv
Lin-Li
LL
Institute of Nephrology, Zhongda Hospital, Southeast University, Nanjing, Jiangsu, China.
Liu
Bi-Cheng
BC
Zhang
Chun-Xiu
CX
Tang
Zu-Ming
ZM
Zhang
Lu
L
Lu
Zu-Hong
ZH
D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D018932:Chemokine CCL2 / Q000032:analysis* / Q000276:immunology; D007118:Immunoassay; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012685:Sepharose / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry
D000906:Antibodies; D002232:Carbocyanines; D018932:Chemokine CCL2; C089187:cyanine dye 3; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin