The present invention is concerned with assay procedures which utilize immunochemical agents such as antigens and antibodies as reactants, as is particularly directed to agglutination immunoassays involving latex carrier particles.
Of the many heterogeneous and homogeneous immunological assay methods now available, agglutination immunoassays continue to be widely used in biology and medicine for the detection of small quantities of an antibody or antigen of interest in a fluid test sample. The agglutination reaction involves the in vitro aggregation of microscopic carrier particles. This aggregation is mediated by the specific reaction between antibodies and antigens, one of which is immobilized on the surface of the carrier particles. In one format, a fluid containing the ligand of interest is introduced into a suspension of the sensitized carrier particles and the appearance of aggregation is noted as indicative of the ligand.
One especially valuable use of the agglutination assays is in the detection of a ligand or analyte of interest in human body fluids such as serum. The agglutination reaction may then be used in several different modes to detect an antigen or antibody (the ligand of interest) as follows:
with an antigen immobilized carrier particle for the detection of a specific antibody as the ligand of interest; PA1 with an antibody immobilized carrier particle for the detection of specific antigen or hapten as the ligand of interest; PA1 in an agglutination inhibition mode using antigen immobilized particles: a fixed quantity of antibody is mixed with a dilution of the test sample containing the ligand of interest. This reaction mixture is then combined with the antigen immobilized carrier particles. The degree to which the ligand of interest (the antigen) in the test sample inhibits the aggregation of the carrier particles that would otherwise have occurred, indicates the concentration of ligand present in the sample; PA1 in an agglutination inhibition mode with antibody immobilized particles: a fixed quantity of antigen is mixed with a dilution of the test sample containing the ligand of interest--a specific antibody--which inactivates a portion of the antigen. This reaction mixture is then combined with the antibody immobilized carrier particles. The degree to which the ligand (the antibody) present in the test sample inhibits the aggregation of carrier particles, in comparison to that which would otherwise have occurred, indicates the concentration of antibody present.
It has been found, however, that many body fluids such as serum often contain other undefined substances, in addition to the particular ligand or analyte of interest, which cause or inhibit agglutination and thus cause interferences and errors in the assay. Such interferences are nominally called non-specific in that the nature of the interferring agent(s) and the mechanism by which they interfere are poorly understood and no particular causative agent or set of conditions is attributable for these effects. Moreover, interferences of these types cannot be corrected by comparison of the assay results with a similar assay using a sample not containing the ligand or analyte in question as a blank sample because the blank may not be truly representative of the particular serum under test and often the interference is so great that no specific reaction takes place. As a result, much time and effort has been expended in the search for means of eliminating non-specific interferences.
Currently known methods of reducing non-specific interferences in agglutination assays include the following: massive dilution of the test sample up to at least 20-fold; addition of detergents such as are taught in U.S. Pat. No. 4,060,597; rigorous pre-treatment of the test sample including heat treatment for 30 minutes at 56.degree. C. as described by Merz et al, J. Clin. Micro., vol. 5, pg 596, 1977: enzymatic treatment with proteases reaction as described by Collet-Cassart et al, Clin. Chem., vol. 27, 0g 1205, 1981; treatment with reducing/oxidative reagents as described by Cambiaso et al, J. Immuno. Meth., vol. 28, pg 13, 1979 and separation of components using ion exchange chromatography as described in U.S. Pat. No. 4,270,923. While these procedures individually and as a whole are effective, they are time consuming and they can carry with them the undesirable effect of drastically reducing the potential sensitivity and accuracy of the immunoassay as a result of the required manipulations. Another approach has been the addition of specific chemical agents to reduce non-specific interferences in agglutination reactions such as is described within U.S. Pat. No. 4,362,531. The described agents, however, include a wide range of dissimilar and unrelated compounds which are effective to varying degrees in relation to one another. For these reasons, the search for additives to agglutination reaction mixtures which reduce or eliminate the effects of non-specific serum interferences in such immunoassays continues.
USPTO Backgrounds
USPTO Backgrounds