Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective.
Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 microg/mL). PSA at 50 nM (1.5 microg/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R2 > 0.99) and reproducibility (coefficient of variation < or =15%), and the detection limit could be improved to 0.14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.
Järås
K
K
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, Malmö, Sweden.
Ressine
A
A
Nilsson
E
E
Malm
J
J
Marko-Varga
G
G
Lilja
H
H
Laurell
T
T
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002457:Cell Extracts / Q000737:chemistry; D005260:Female; D005453:Fluorescence; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008297:Male; D016062:Porosity; D017430:Prostate-Specific Antigen / Q000097:blood*; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D017430:Prostate-Specific Antigen; D012825:Silicon