Reducing non-specific binding in immunoassays performed on polymeric solid phases

#1. Field of the Invention

#This invention resides in the field of immunoassays performed in a heterogeneous format with a polymeric solid phase. In particular, this invention addresses the problem of non-specific binding to the solid phase in serum assays and the false positive readings that are caused by such binding.

#2. Description of the Prior Art

#This example illustrates the use of water-soluble polymeric blocking agents in an assay for anti-thyroglobulin (anti-Tg) in human serum. The assay was a flow cytometry immunoassay in which the solid phase consisted of carboxylate surface-modified polymeric magnetic particles, 8 μm in diameter, with a surface layer prepared from vinyl benzene carboxylic acids and vinyl alkyl carboxylic acids. The particles were coated with thyroglobulin (Tg) at a coating density of 1 μmg/cm2. Detection of binding of anti-Tg was achieved by use of a labeled antibody consisting of phycoerythrin covalently bound to the F(Ab′)2 fragment of polyclonal antibody specific for human IgG. Other assay components included a particle and conjugate diluent for suspending the particles and diluting the conjugate, consisting of an aqueous solution of proteinaceous buffered saline containing detergents and preservatives, a sample of human serum that had been stripped in charcoal and was therefore negative for both anti-Tg and anti-thyroid peroxidase (anti-TPO), and a wash buffer consisting of buffered saline and detergent.

#The general assay protocol consisted of combining 5 μL of the sample with 100 μL of a suspension of the particles in the particle diluent which also included a candidate blocking agent at a concentration of 0.1% by weight. The combined sample and particles were incubated for 15 minutes at 37° C., then separated by magnetic separation and washed four times with 300 μL each of wash buffer, each time followed by a three-minute magnetic separation. Labeled antibody (50 μL) was then added, and the suspension incubated for 15 minutes at 37° C., followed by washing twice with 300 μL each of wash buffer alternating with three-minute magnetic separations. The particles were then resuspended in 35 μL wash buffer and read by flow cytometry fluorescence detection.

#A series of candidate blocking agents were used as well as prior art blocking agents and a blank in which no blocking agents had been added beyond those present in the sample diluent and particle diluent. For each agent and the blank, the protocol was performed twice, once with the charcoal-stripped serum as the sample and once with the wash buffer as the sample, and a relative fluorescence intensity (RFI) value was obtained for each repetition. From these values, a percent change (δ), i.e., the false positive effect of the proteins in the charcoal-stripped serum as measured by the difference between the charcoal-stripped serum value and that of the wash buffer divided by that of the wash buffer, was calculated.