T. Beumer et al., CONVECTION DURING INCUBATION OF MICROPLATE SOLID-PHASE IMMUNOASSAY - EFFECTS ON ASSAY RESPONSE AND VARIATION, Analytical chemistry, 68(8), 1996, pp. 1375-1380
In solid phase immunoassays, the incubation period is used to specific
ally bind free analyte molecules of low concentration to immobilized c
ountermolecules. Many of these assays use elevated temperatures during
incubation, based on the assumption that the reaction thus is stimula
ted by enhancing the association constant. In this paper we demonstrat
e that elevated incubation temperatures are not likely to have any sig
nificant effects on the association reaction. Furthermore we show that
in many situations, uncontrolled thermally induced free convection pl
ays an important role in mass transport of analyte toward the solid ph
ase. Convection is a function of assay geometry and the properties of
the materials used. Our experiments show that it is a major source of
interassay variation which never before has been described in the cont
ext of immunoassay incubation. Finally we define the conditions under
which forced convection will improve assay response and reduce interas
say variation. Applying forced convection during sample incubation of
a two-step HBsAg assay, we obtained an increase in response rate per t
ime unit by a factor of similar to 5. Interassay variation was reduced
from 5-10% to 1-2%. Assay sensitivity improved by a factor of 2-6 wit
hout the slightest modification in assay chemistry.