| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland.
a Author for correspondence. Fax 358-2-333 8050; e-mail susann.eriksson{at}utu.fi
Background: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies.
Methods: The capture Fab fragment, which binds free
prostate-specific antigen (PSA) and PSA in complex with
1-antichymotrypsin on an equimolar basis, is
site-specifically biotinylated and attached to the solid phase in
streptavidin-coated microtitration wells. The Fab fragment that detects
only free PSA is site-specifically labeled with a fluorescent europium
chelate, and the Fab fragment that detects both free and
serpin-complexed PSA in an equimolar fashion is labeled with a
fluorescent terbium chelate. Time-resolved fluorescence is used to
measure both europium and terbium signals in one well.
Results: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 µg/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2–11% and 4–10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0.99) were not significantly different from zero, and the slopes were 0.95–1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay.
Conclusions: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Y. Xu and Q. Li Multiple Fluorescent Labeling of Silica Nanoparticles with Lanthanide Chelates for Highly Sensitive Time-Resolved Immunofluorometric Assays Clin. Chem., August 1, 2007; 53(8): 1503 - 1510. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zhang, C. Zhang, Z. Xing, and X. Zhang Simultaneous Determination of {alpha}-Fetoprotein and Free {beta}-Human Chorionic Gonadotropin by Element-Tagged Immunoassay with Detection by Inductively Coupled Plasma Mass Spectrometry Clin. Chem., July 1, 2004; 50(7): 1214 - 1221. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Tarkkinen, T. Palenius, and T. Lovgren Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution Clin. Chem., February 1, 2002; 48(2): 269 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Harma, P. Tarkkinen, T. Soukka, and T. Lovgren Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments Clin. Chem., November 1, 2000; 46(11): 1755 - 1761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Scorilas, A. Bjartell, H. Lilja, C. Moller, and E. P. Diamandis Streptavidin-Polyvinylamine Conjugates Labeled with a Europium Chelate: Applications in Immunoassay, Immunohistochemistry, and Microarrays Clin. Chem., September 1, 2000; 46(9): 1450 - 1455. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
