1. Immunoassays
Immunoassays have found widespread application in the field of clinical diagnostics for the detection and measurement of drugs, vitamins, hormones, proteins, metabolites, microogranisms, and other substances of interest (analytes) in biological and non-biological fluids. Typically, these analytes occur in micromolar (10.sup.-6 M) or less concentration.
Immunoassays generally incorporate antibodies and antigens as reactants, at least one of which is labeled with a signal producing compound (e.g. radioisotope, fluorophore, etc.). Following mixture with the sample and incubation, specific antibody/antigen reactions occur (specific binding). The reaction mixture is subsequently interrogated to detect free and specifically-bound labeled reactant, enabling a measurement of the analyte in the sample.
Immunoassays can be divided into two general categories, homogeneous and heterogeneous. In a homogeneous immunoassay, the signal emitted by the specifically-bound labeled reactant is different from the signal emitted by the free labeled reactant. Hence, bound and free can be distinguished without physical separation.
The archetypal homogeneous immunoassay is the enzyme-multiplied immunoassay technique (EMIT), which is disclosed in U.S. Pat. No. 3,817,837. In this technology, analyte present in patient sample and analyte/enzyme conjugate compete for a limited amount of anti-analyte antibody. Specific binding of antibody to the conjugate modulates its enzymatic activity. Hence, the amount of enzyme activity is proportional to the amount of analyte in the sample. Homogeneous immunoassays have the advantage of being rapid, easy to perform, and readily amenable to automation. Their principal disadvantages are that they are relatively prone to interferences, are generally limited to low molecular weight analytes and are generally limited in sensitivity to approximately 10.sup.-9 M.
In a heterogeneous immunoassay, the signal emitted by the bound labeled reactant is indistinguishable from the signal emitted by the free labeled reactant. Therefore, a separation step is required to distinguish between the two.
Typical heterogeneous immunoassays include the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA). In the RIA, radiolabeled analyte and analyte present in patient sample compete for a limited amount of immobilized (solid phase) anti-analyte antibody. The solid phase is washed to remove unbound labeled analyte and either the bound or the free fraction is analyzed for the presence of labeled reactant. ELISA assays are performed analogously. In this case, though, the signal is an enzyme instead of a radioisotope. Heterogeneous immunoassays typically employ at least one reagent immobilized on a solid phase. Since the kinetics of reaction between an immobilized antibody (or antigen) and its binding site tend to be slower than the kinetics of the same reaction occurring in solution, long incubation times are frequently required. When the multiple wash steps often needed are considered, it can be appreciated that heterogeneous assays tend to be time-consuming and labor-intensive. However, they are in general more sensitive than homogeneous assays and less prone to interferences, since interfering substances can be removed in the wash steps.
Solids used to immobilize reactants in immunoassays have included controlled pore glass and preformed polymers such as polyvinyls, polyacrylamides, polydextrans and polystyrene.
Numerous separation methods are known in the art and have been used in heterogeneous immunoassays. These include centrifugation, filtration, affinity chromatography, gel permeation chromatography, etc.
The homogeneous immunoassay methods of the prior art are generally prone to interferences, of limited sensitivity and have a limited range of antigen sizes. The heterogeneous immunoassays of the prior art, while increasing the sensitivity and minimizing interferences, tend to be time consuming and labor intensive. These difficulties generally arise from the added step of physical separation and the need for numerous washes to decrease background interference.
There is a need in the art for an immunoassay method which is sensitive to sub-micromolar concentrations of analyte; which has fast reaction kinetics; and which minimizes the number of manipulations necessary to achieve a result.
2. Polymer Chemistry
A reaction fundamental to polymer chemistry is the initiation of end-to-end covalent linkages between soluble organic monomers leading to the formation of larger polymeric molecular structures (polymers). Synthetic polymers can be formed from a single monomeric species (homopolymer) or from a mixture of different monomers (co-polymer). Linear, branched, or cross-linked structures are possible. By varying the chemical composition or ratios of the monomers, it is possible to form either soluble or insoluble polymers which comprise a broad range of chemical and physical structures. For example, water-soluble monomers (such as acrylamide) can be copolymerized to form water-soluble homopolymers. They can also be copolymerized with less water-soluble monomers (such as N-alkyl or N, N-dialkyl acrylamides) or with cross-linking monomers (such as N, N'-methylenebisacrylamide) to form water-insoluble copolymer structures. Some water-soluble monomers (such as hydroxyethyl methacrylate or acrylonitrile) can be homopolymerized to form water-insoluble homopolymers.
In the fields of biochemistry and immunology, water-insoluble polymers (such as polysaccharides or polyacrylics, sometimes cross-linked) have been commonly used as solid phase supports with passively absorbed, physically entrapped, or covalently-linked proteins in affinity chromatography, enzyme immobilization, and immunoassay. See, for example, U.S. Pat. Nos. 3,957,741; 4,257,884; 4,195,129; 4,225,784; 4,181,636; 4,401,765; and 4,166,105.
To date, the documented coupling of a polypeptide to a polymer has occurred under circumstances in which the polypeptide was provided in soluble form and the polymer was provided as a preformed soluble or preformed insoluble material. While these polymers are of utility in providing a surface upon which selective biochemical or immunological reactions can occur, the polymers are of limited value in that the spacing, steric accessibility, and number of polypeptides bound per unit length of polymer cannot be precisely or reproducibly controlled. Lot-to-lot variation is commonly encountered during the manufacture of such solid phase polymer/reactant matrices. In certain end-use applications where reproducibility and standardization are essential (e.g. immunoassays), this variation in composition of the solid-phase polymer/reactant matrices presents a critical problem. Consequently, there is a need in the art for a method to specifically tailor or molecularly engineer polymer compounds incorporating controlled quantities of reactants.