A new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry.
A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.
Robers
M
M
Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.
Rensink
I J
IJ
Hack
C E
CE
Aarden
L A
LA
Reutelingsperger
C P
CP
Glatz
J F
JF
Hermens
W T
WT
D017304:Annexin A5 / Q000032:analysis; D015415:Biomarkers / Q000097:blood; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000032:analysis / Q000097:blood; D011232:Chemical Precipitation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis / Q000097:blood; D008051:Lipid Bilayers; D018996:Myelin P2 Protein / Q000032:analysis / Q000097:blood; D009203:Myocardial Infarction / Q000097:blood / Q000175:diagnosis; D009363:Neoplasm Proteins; D010743:Phospholipids; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012825:Silicon; D025521:Tumor Suppressor Proteins
D017304:Annexin A5; D015415:Biomarkers; D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015850:Interleukin-6; D008051:Lipid Bilayers; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D010743:Phospholipids; D011506:Proteins; D025521:Tumor Suppressor Proteins; D012825:Silicon