There is a desire in the field of clinical chemistry to determine the concentrations of proteins, drugs, organisms and other analytes in biological fluids for the purpose of diagnosing and monitoring disease. For example, during myocardial infarction proteins are released from the heart. Detecting the presence, concentration and time course of release of such proteins can aid in the diagnosis of a heart attack.
Most clinically relevant proteins that are currently detected in biological fluids are present at concentrations greater than 1 picogram/ml. Prostate specific antigen (PSA), for example, is a serum protein useful in the detection of prostate disease that is normally present in males at concentrations of about 0-4 ng/ml. PSA levels above 4 ng/ml are suspicious for prostate disease, particularly prostate cancer. This concentration range is readily detected by conventional immunoassay technology. Following prostate removal, however, the concentration of PSA drops to levels that are undetectable by conventional technology. Increasing PSA levels in cancer patients that have undergone prostate removal is indicative of relapse. An assay with femtogram/ml sensitivity is required to monitor these patients.
It is estimated that there are approximately 35,000 genes and as many as 500,000 proteins in the human species. The increased diversity of proteins versus genes can be accounted for post transcriptional (e.g., splicing) and posttranslational (e.g., phosphorylation, glycosylation) modifications. Such modifications can significantly alter protein function. Thus, even subtle differences may be clinically relevant. Only 290 proteins have been identified in human plasma even though there are thousands of spots seen in 2D gels. The human plasma proteome may contain hundreds of thousands of proteins that are present at concentrations too low to detect by current technology. Methods to detect the majority of these proteins are not currently available.
The difficulty of detecting low concentrations of certain analytes is compounded by the relatively small sample sizes that can be utilized in a clinical assay. Therefore, most immunoassays for protein analytes rely on heterogeneous methodology such as ELISA (enzyme linked immunosorbent assay), in which antibody-bound analyte is physically separated from unbound analyte. Heterogeneous detection methods are complicated and require multiple steps (e.g., binding to a solid phase and repeated washing steps) to separate the bound analyte from the unbound. These steps lead to non-specific binding and lowered sensitivity; they can be costly and time consuming. Thus, there is a need for a high sensitivity homogenous assay which avoid non-specific binding.
Homogeneous immunoassays (those which do not require a physical separation of the bound-species and the free-species) have been described for small molecules, such as drugs. These assays include SYVA's FRAT® assay, EMIT® assay, enzyme channeling immunoassay, and fluorescence energy transfer immunoassay (FETI) (Dade Behring, Deerfield, Ill.); enzyme inhibitor immunoassays (Hoffman LaRoche and Abbott Laboratories): fluorescence polarization immunoassay (Dandlicker), among others. All of these methods have limited sensitivity, and only a few, including FETI and enzyme channeling, are suitable for large multiepitopic analytes. Thus, there exists a need for a sensitive, homogeneous method for the detection of large and/or complex analytes present in biological and clinical samples.