Integrated protein microchip assay with dual fluorescent- and MALDI read-out.
A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).
Finnskog
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Department of Electrical Measurements, Lund Institute of Technology, Lund University, P.O. Box 118, S-221 00 Lund, Sweden.
Ressine
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A
Laurell
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T
Marko-Varga
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G
D000803:Angiotensin I / Q000097:blood / Q000737:chemistry / Q000276:immunology; D000804:Angiotensin II / Q000097:blood / Q000737:chemistry / Q000276:immunology; D000805:Angiotensin III / Q000031:analogs & derivatives* / Q000097:blood / Q000276:immunology; D000808:Angiotensinogen / Q000031:analogs & derivatives* / Q000097:blood / Q000276:immunology; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012825:Silicon / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D007074:Immunoglobulin G; D011506:Proteins; C079544:angiotensinogen (1-14); D000808:Angiotensinogen; D000804:Angiotensin II; D000805:Angiotensin III; C056764:angiotensin III, Ile(7)-; D000803:Angiotensin I; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; D012825:Silicon