Clinical laboratory chemical diagnostic tests are an important component of health care delivery. The utilization of these tests by physicians to monitor drug levels where only a narrow therapeutic range exists, to guide decisions on treatment and surgical options, and to screen patients for the early detection of disease has rapidly increased the number of tests performed annually. With almost 6 billion tests performed in 1976 and 12.2 billion estimated to be performed in 1986 [Luning Prak Associates Survey, 1980], speed, accuracy, and cost control are important objectives. The desire to measure such analytes as drugs, hormones, and metabolites at micromolar (.mu.M) to picomolar (pM) levels in complex body fluid matrices has led to the development of sophisticated test methodology which can be implemented by automated techniques at reasonable cost.
Broadly applicable, accurate screening assays are therefore needed to monitor the presence and quantity of biological materials. Various methods have been utilized in the past including liquid and gas chromatography, mass spectrometry, and numerous bioassay techniques. These methods are time consuming and not easily applied in large-scale, automated screening programs.
In recent years, a number of immunoassay techniques have been developed to take advantage of the specificity of antibody reactions while avoiding the complicating features of radiochemical labelling. Agglutination reactions involving bivalent antibodies and antigens or haptens of clinical interest have been utilized in both visual and quantitative assays with a wide variety of bacteria, red blood cells, or polymer particles. Agglutination results from the growth of antibody-antigen bridged particle aggregates to produce an extensive network which can be detected. Agglutination can result by adding the specific binding partner, either antibody or antigen, to the suspension of particles with immobilized antigen or antibody. At low concentrations of the specific binding partner, small aggregates consisting only of a few particles are produced. The presence of both free and particle-immobilized reagent results in an inhibition of aggregation by the specific binding partner.
Several systems of clinical analysis have been developed using particle immobilized reagents which generally provide specificity through their method of preparation, and quantitative information from the aggregation kinetics obtained in the presence of the analyte of interest. In these systems, the analyte of interest constitutes the antigen or hapten against which specific binding antibody is prepared. Particle-based reagent systems can provide a sensitive, flexible measurement system for quantifying such medically important materials as cardiac glycosides, antibiotics, therapeutic drugs, hormones, and vitamins. In addition, methods of analysis for toxins, food and packaging additives and environmental pollutants at low concentrations are required.
U.S. Pat. No. 4,174,952, issued to Cannell et al., discloses a particle-based immunoassay measurement method in which the ratio of intensity of light scattered at two different angles, the so-called anisotropy ratio, is determined as a relative measure of the distribution of particle sizes. This method requires the use of initially monodisperse particle reagents in order to detect a change in the anisotropy ratio at low levels of aggregation. A broad distribution of particle sizes, as generally found in a polymer latex, would have low sensitivity for detection of analyte or antibody induced aggregates because only a small change in the anisotropy ratio will be observed. Similarly, the method is primarily useful during the early stages of aggregation where the rate of change of the anisotropy ratio is great.
U.S. Pat. No. 4,184,849, issued to Cambiaso et al. discloses a method of particle-based immunoassay utilizing selective counting techniques with two different particulate reagents. The particles in this case are required to be at least a factor of two different in size, and each particle suspension must be uniform in size. The technique involves a measurement of the fraction of unaggregated (monomer) particles of one size in the system. The measurement of the disappearance of unaggregated particles requires long incubation times, however.
U.S. Pat. No. 4,191,739, issued to Uzgiris et al. discloses the use of a mixture of two antibody-coated latex particle suspensions to measure analyte concentration. The choice of particle sizes is, however, critical requiring a particle volume ratio of about 1.5:1 for the two particles. The assay is based upon counting particles of a size which would not exist but for an antigen-antibody interaction. The system must therefore be carefully designed with respect to particle size and requires relatively sophisticated electronic equipment to carry out the measurements.
U.S. Pat. No. 4,080,264, issued to Cohen et al. discloses a particle-based immunoassay technique utilizing quasi-elastic light scattering. Direct measurement of the mean diffusion coefficient of the particles can be related to particle size through the Einstein and Stokes equations. The presence of target analyte in the medium leads to aggregation and an increase in particle size which is related to analyte concentration. In general, however, the technique is utilized only with uniform-sized particle latex suspensions and reserved for measurements in the early stages of interaction where there is a large change in the mean diffusion coefficient. Analysis of the data also requires an assumption about the distribution of particle aggregate sizes present.
U.S. Pat. No. 4,279,617 issued to Masson et al. discloses a two-particle immunoassay and selective particle counting to analyze human sera for small quantities of antibodies indicating allergy, infection, or autoimmune diseases. The method only monitors the concentration of unaggregated first or second particulate reagent, and requires that one of the two particle reagents be of particle size at least 0.6 micrometer or greater to obtain reliable particle counting.