Utilization of recombinant Fab fragments in a cTnI immunoassay conducted in spot wells.
OBJECTIVE
To evaluate the performance of a new cTnI immunoassay utilizing site-specifically biotinylated recombinant Fab fragments on recently established spot wells.


METHODS
Two different cTnI-specific recombinant site-specifically biotinylated Fab fragments were produced. The performance of the new sandwich-type cTnI immunoassay in spot wells was evaluated in terms of binding capacity, assay kinetics and assay sensitivity and compared with a cTnI immunoassay carried out in conventional microtitration wells. Furthermore, the functionality of the recombinant Fab fragments was compared to the corresponding monoclonal antibodies in assay with one, two or three capture antibodies.


RESULTS
The signal-to-background level was improved, providing an analytical detection limit of 0.002 microg/l with a surface of two capture Fab fragments. The spot wells increased the signal levels 2-fold and a further 4-fold improvement was detected with the Fab fragments already after 5 min assay time.


CONCLUSIONS
The spot-concept in combination with site-oriented capture Fab fragments carries great promise as a very useful approach to improve the immunoassay performance of future point-of-care cTnI assays.
Ylikotila
Johanna
J
University of Turku, Department of Biotechnology, Tykistökatu 6A, Biocity 6th floor, FI-20520 Turku, Finland. Johanna.ylikotila@utu.fi
Hellström
Johanna L
JL
Eriksson
Susann
S
Vehniäinen
Markus
M
Välimaa
Lasse
L
Takalo
Harri
H
Bereznikova
Anastasia
A
Pettersson
Kim
K
D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007700:Kinetics; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D019210:Troponin I / Q000032:analysis*
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D019210:Troponin I