Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-01-13
1999-11-09
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 5, 435 6, 435 71, 435 79, 422 8201, 422 8202, 427 211, 436518, 436525, 436528, 436807, G01N 3353, G01N 2700, G01N 33543, A61L 5103
Patent
active
059812036
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to certain sandwich-type immunoassay devices and their use. In particular, the invention relates to an enzyme immunoassay using an electrochemical means of detection that allows for a non-separation assay format.
BACKGROUND ART
Enzyme immunoassays (EIA) have become important analytical methods in clinical chemistry laboratories for the selective detection of drugs, hormones, and proteins at trace levels. As specific examples, such methods are now used routinely to detect diagnostically important blood proteins, including creatine kinase-MB to detect the occurrence of myocardial infarctions, prostate-specific antigen (PSA) to screen for prostate cancer, and human chorionic gonadotropin (hCG) to confirm pregnancy. Further, such methods can be used to detect the protein coat of a specific virus. Thus, the invention also has utility for assays in agriculture and the food processing industry, for example, in-line monitoring of a food processing plant.
One of the must useful of the immunoassays is the two-antibody sandwich technique, which is used primarily to determine the presence and concentration of an antigenic analyte in an unknown sample. Two-antibody assays can be relatively quick, particularly if a source of pure antigen is available. The assay requires two antibodies that bind to non-overlapping epitopes on the antigenic analyte. Either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies can be used.
To use the conventional two-antibody assay, a "capture" antibody is purified and bound to a solid phase, and the antigenic analyte in a standard or test sample solution is allowed to bind to the capture antibody. Unbound analyte proteins are then typically removed by washing. A second antibody, which is labeled with an enzyme, is allowed to bind to the immuno-bound analyte. After another wash step to remove the excess enzyme-labeled antibody, the amount of enzyme label bound to the matrix is determined, usually by adding a substrate on which the enzyme acts and measuring the rate of product generated. The rate that product is generated is directly proportional to the amount of the antigenic analyte present in the sample.
This sandwich-type assay is superior to other types of solid phase immunoassays with respect to sensitivity, specificity and kinetics. These advantages arise from the fact that excess amounts of the capture antibody and the enzyme-antibody conjugate are used relative to the low levels of the analyte commonly present, thus driving the equilibrium of the binding reaction toward the formation of the sandwich structure.
An EIA method can be heterogeneous or homogeneous, depending on whether or not washing steps are required to separate free and bound enzyme label. EIA's can also be competitive or non-competitive, depending on the availability of antibody binding sites. Of these different EIA methods, non-competitive heterogeneous sandwich assays have the advantage of using two co-existing determinant sites on the same antigenic analyte to be detected. As a result, these sandwich-type assays generally exhibit good specificity when identifying the presence and/or amount of protein analytes. Further, due to the innate amplification properties of enzymes, the use of EIA offers excellent sensitivity.
Even though sandwich-type EIA's are used widely in clinical laboratories, such methods usually require multiple wash steps to separate the excess amounts of reagents used from the sandwich being formed. This has created a need for complex instrumentation to do a high volume of immunoassay tests. Thus, the need to limit the wash steps has made it very difficult to adapt these assays into portable test systems that would be desirable for detecting diagnostically important proteins in field locations, such as doctors' offices, emergency vehicles, and hospital "crash" carts.
Another problem has been the length of time required for incubation to accomplish sufficient binding between wash step
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Duan Chuanming
Meyerhoff Mark E.
Housel James C.
Swartz Rodney P.
The Regents of the University of Michigan
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