title: Reduction of non-specific binding in immunoassays requiring long incubations.
abstract: Despite the studies so far about the non-specific binding of antibody molecule to the plastic of solid phase in enzyme-linked immunoassays, background binding in microwell Elisa continues to be a troublesome problem.Non-specific immunoglobulin from an undiluted serum sample can adhere to the surface of a 'blocked' plate to result in a maximal signal in an antigen capture assay for specific antibody to render analysis virtually impossible in undiluted serum when using labelled anti-species antibodies. Yet it is desirable in many circumstances that the maximum sensitivity achievable by the simple expedient of using a concentrated sample (undiluted serum) be exploited, for example in the analysis of antibodies to HIV in the interest of earlier diagnosis. To circumvent this problem we have developed an alternative strategy in which a biotinylated capture reagent is preincubated with the serum sample for the necessary time after which the biotinylated ligand/antibody complex is itself rapidly captured in streptavidin-coated wells at 4°C, with subsequent detection with labelled anti-species immunoglobulin. This manoeuvre enables the capture ligand to be incubated with undiluted serum sample for long time periods resulting in improved specificity of detection. By this means we describe a general method to improve the specificity of serum antibody immunoassays which will be expected to produce the benefit of more rapid diagnosis by signalling antibody production earlier in the abnormal state. Furthermore, our new method could be used to reduce non-specific binding in other immunological assays such as antibody arrays to which much attention has recently been paid.
authors.affiliation: Department of Medical Biotechnology, and Cellular and Molecular Research Centre, Tehran University of Medical Sciences, Hemmat Campus, Hemmat Express Way, Tehran, Iran. mfarajol@tums.ac.ir
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Farajollahi
authors.forename: David B
authors.initials: DB
authors.lastname: Cook
authors.forename: Sepideh
authors.initials: S
authors.lastname: Hamzehlou
authors.forename: Colin H
authors.initials: CH
authors.lastname: Self
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D011485:Protein Binding