Antibody-bound cell microarray for immunophenotyping: surface modification and lymphocyte subpopulations.
Antibody-based cell microarrays may serve as a high-throughput diagnostic tool that requires precise surface design providing high biological specificity, high reliability, and high validity. A fundamental study on the quantitative evaluation of immunophenotyping using a cell microarray on which antibodies specific to cluster of differentiation (CD) antigens of blood cells are fixed was performed. The microarray, which was prepared by photomicropatterning self-assembled monolayers of alkanethiols, consisted of carboxyl group (3-mercaptopropionic acid)-packed domains regularly distributed in the methyl group (1-dodecanethiol)-packed matrix phase. This was verified by X-ray photoelectron spectroscopy and water wettability measurements. The patterned carboxylated domains of 1.0 mm or 0.2 mm diameter were covalently fixed with the anti-CD4 or anti-CD8 antibody by coupling reaction using a water-soluble carbodiimide and hydroxysuccinimide. Precision antibody fixation at almost complete conversion was verified by confocal laser scanning microscopy coupled with a specific dye staining technique. Peripheral blood mononuclear cells expressing CD4 or CD8 antigen, respectively, adhered on the anti-CD4 or anti-CD8 antibody-fixed domains of the microarray at a very high specificity, which was verified with flow cytometric analysis and an antibody-coated magnetic-bead-based cell isolation technique. The CD4/CD8 subset ratios determined using the antibody-fixed microarrays were very close to those obtained by flow cytometric analysis. These results indicate that microarrays, on which antibodies specific to cell surface antigens are covalently fixed, provide a solid basis of the high-throughput quantitative evaluation of immunophenotyping in medical diagnosis.
Fujii
Yasuo
Y
Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, Higashiku, Fukuoka 812-8582, Japan.
Anderson
James M
JM
Matsuda
Takehisa
T
D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002448:Cell Adhesion; D002478:Cells, Cultured; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D016131:Lymphocyte Subsets / Q000276:immunology*; D046228:Microarray Analysis / Q000379:methods*; D013053:Spectrophotometry; D013499:Surface Properties
D000906:Antibodies; D000941:Antigens