Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of analytes that may be present in a test sample. For instance, immunoassays utilize mechanisms of the immune systems, wherein antibodies are produced in response to the presence of antigens that are pathogenic or foreign to the organisms. These antibodies and antigens, i.e., immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism that may be used to determine the presence or concentration of that particular antigen in a biological sample.
There are several well-known immunoassay methods that use immunoreactants labeled with a detectable component so that the analyte may be detected analytically. For example, “sandwich-type” assay formats typically involve mixing the test sample with detection probes conjugated with a specific binding member (e.g., antibody) for the analyte to form complexes between the analyte and the conjugated probes. These complexes are then allowed to contact a receptive material (e.g., antibodies) immobilized within the detection zone. Binding occurs between the analyte/probe conjugate complexes and the immobilized receptive material, thereby localizing “sandwich” complexes that are detectable to indicate the presence of the analyte. This technique may be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich-type assays are described in. by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the “competitive-type” assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.
Many of these assays rely upon calibration to provide valid and meaningful results, particularly for semi-quantitative and quantitative detections. In external calibration systems, a standard curve is usually obtained from standard samples containing a series of a known amount of analyte, and the results obtained from the samples are then compared with the standard curve to extract the presence and/or amount of the analyte in the sample. The external calibration method is relatively easy to design and simple to implement. However, it is often subject to interference from environmental and batch-to-batch variations, and is thus unreliable.
Some internal calibration systems have thus been developed to overcome these problems. For example, U.S. Pat. No. 5,387,503 to Selmer, et al. describes an internal calibration technique that involves mixing a sample of a known volume with a predetermined amount of a calibrator analyte and contacting the mixture with a solid support. The solid support contains a reagent capable of selectively binding the test analyte in a first discrete area and a reagent capable of selectively binding the calibrator analyte in a second discrete area. A mixture of a labeled reagent for the test analyte and a similarly labeled reagent for the calibrator analyte are also applied to the solid support. The amount of test analyte in the sample is determined by comparing the levels of labeled reagent bound to the test and calibrator analytes, respectively. Unfortunately, such internal calibration techniques are not readily incorporated into lateral flow devices, which involve heterogeneous separation of the analyte using chromatographic methods. In addition, the requirement for pre-mixing the assay reagents is burdensome and overly complicated, particularly for point-of-care applications in which the ultimate user is not a trained medical professional or technician.
As such, a need currently exists for an accurate internal calibration system for lateral flow assays that is accurate, yet relatively inexpensive, simple, and easy to use.