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).
Department of Electrical Measurements, Lund Institute of Technology, Lund University, P.O. Box 118, S-221 00 Lund, Sweden.
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Finnskog
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Ressine
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Laurell
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Marko-Varga
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