Patent Application
20080220980
The invention relates to assay devices and methods for constructing assay devices for detecting the presence of an analyte in a biological sample and the quantity of same.
Quality standards for immunoassays have traditionally been driven by external calibration reference standards. Current methods of analysis for typical immunodiagnostic assays provide diagnostic test results based on generally accepted external standard reference measurements. A number of known discrepancies have become apparent to be quantitation errors induced when assays are carried out, leading to variations in test results. For example, the concept of assay sensitivity attempts to characterize sensitivity by classic statistical analysis based on repeated measurement of low concentration samples to confirm that the sample result is not statistically different from zero. As the standard error incurred is inversely proportional to the square root of the number of actual measurements, this method does not actually measure the inherent assay sensitivity. Further refinement has led to some improvements. Known in the art as analytical sensitivity, the zero standard is measured several times and the limit of sensitivity becomes a concentration equating to 2-3 standard deviations (SD) from the mean (M). However, the precision for this theoretical determination may be incorrect by an order of magnitude. The concomitant fitting of any derived external calibration curve(s) does not create a true value dynamic dose response curve that can lead to considerable error in the actual sensitivity.
To further measure the accuracy of such analytical measurement, accuracy is used to define how close the average measured value is to the true value. The difference in measurement is known as the bias or degree of accuracy. Bias may vary over the range of the assay. It is known in the art that methods for measuring this true value need to be developed.
The repeatability of an assay or the estimated error in an analytical assay is known in the art as the percentage coefficient of variation (% CV). Automated assay analysis machines can be affected by variations in sample concentration, temperature, heat and edge effects, incomplete suspension of particles and solid phase precipitation. Precision effects also result from fraction separation and counting errors. In optical systems error is due to effects of turbidity, presence of fluorophores, deterioration of lamps and detectors and the deterioration, over time, of reagents. These factors generally lead to significant decreases in signal to noise ratio. Mechanical manipulation errors can result from poor pipetting and instrument stand-by periods.
As a direct result, the assessment for precision of any analytical method requires the measurement of resulting variability at known and relevant concentrations by using defined or standard control solutions to create baseline calibration standards. Accurate determination of such calibrators is based on measurement of known concentrations in dilution series at predetermined intervals, which are then interpolated. Commercially available, as well as in-house prepared reference solutions or reference standards are available, but are often calibrated with standard or pooled matrices, which may vary considerably from actual patient test samples. Part of the solution to overcome these errors is to plot the precision against a wide range of concentrations to obtain a precision profile, or calibration, of the assay.
Cross reactivity, assay specificity, bias causing interference, alterations in antigen, antibody, binding sites, low dose (competitive assay) and high dose (sandwich assay) hook effects, heterophilic antibody interference, endogenous interfering auto-antibodies, complement, rheumatoid factor, interference in solid phase antibody binding, endogenous signal generating substances, enzyme inhibitors, catalysts and co-factors have also been shown to express confounding activity in assays, including cross reactivity, matrix effects and carry over of sample in automated immunoassay instruments and samplers.
For diagnostic applications, the quality control samples may not reflect actual clinical concentrations in the patient, may not reflect the spectrum of present analytes and interfere with the sample matrix to no longer reflect the content of the patient samples. The quality control samples may measure performance at discrepant intervals of concentration which may not reflect clinical decision points.
There is therefore a need for an immunoassay that can be reliably calibrated.
The invention is directed to a method for internal dynamic calibration of an assay device for determining the concentration of an analyte in a sample where the assay device has an assay surface. A plurality of calibration dots containing pre-determined quantities of the analyte are printed on the assay surface. A test dot containing a reagent for binding said analyte is also printed on the assay surface. The analyte is labeled with a detectable marker complex prior to introduction onto the assay device. The analyte—detectable marker complex binds to the reagent in the test dot. The amount of antigen in the sample is proportional to the intensity of detectable marker in the test dot. The calibration dots contain differing pre-determined quantities of the analyte. Any unlabeled detectable marker will bind to the calibration dots. An internally calibrated calibration curve is thus prepared. The intensity of detectable marker in the test dot can be compared to the calibration curve to obtain an absolute value.
The method provides a quantitative analysis that is carried out rapidly using a single assay device with the ability to contain a known minimum volume of test fluid and also to have the ability for flowing fluid through the device in order to meet a known concentration of analyte as a function of analyte concentration per tested volume. Both the calibration dots and test dots are printed within a single assay device which then needs only the application of a single, premixed solution containing the analyte and an excess of detecting reagent which is preferably an antibody.
The invention further includes a method for obtaining dynamic true dose response curves by printing both calibrator and test samples onto a common test platform device. The test platform has a minimum of one test dot. Each test dot has multiple corresponding calibration dots. The signal obtained from the total number of comparative concentration dynamic calibration dots, at indexed X/Y co-ordinates is integrated to form the dynamic internal calibration true dose response curve. In a similar process, the unknown test dot label response reading is also integrated over all obtained readings. The use of multiple test arrays or matrices in platform format predicates a confidence limit approaching one hundred percent in having obtained the correct test result. The common test platform is exposed to the same test fluid and because the calibrator and test samples are exposed simultaneously to the same test fluid, accurate measurement of the concentration of analyte present in the test sample is determined by the resulting true dose response calibration curve. The invention provides the surprising result that the various errors, incurred using known state of the art methods, are not reflected when a test sample is processed with the disclosed method and device.
According to one aspect of the invention, there is provided a method of determining an amount of analyte in a sample solution comprising the following steps:
The present method is for calibrating an assay device. A preferred assay device is shown in
The bound antibodies are preferably spaced apart to make each bound antibody available for binding to the test antigen free of stearic hindrance from adjacent antigen complexes. Preferably, a non-reactive protein separates the bound antibodies in the test dots.
The reading area 16 has calibration dots 22 printed thereon. The calibration dots include a pre-determined amount of said analyte for reacting with un-reacted reagent in a vessel, conjugated with a detectable marker. The calibration dots allow the intensity of the label to be correlated to the amount of the antigen present. The intensity of label in the test dots can then be used to derive the quantity of antigen present.
The calibration dots have a concentration of the analyte that corresponds to a dynamic range of the analyte. Dynamic range is the concentration of analyte normally found in the patient. The quantitation needs to be within this lowest and highest concentration and relate to the clinically relevant concentrations.
Many of the problems associated with current methods typical for immunoassays derive from the assay calibration being determined by introducing external standard reference samples for calibration. The present method provides more accurate results by not using these standard external calibration samples.
In developing a platform device for measuring the quantity of a respective analyte, instead of using external standards to generate a calibration or base line, both calibration dots as well as test dots at unknown concentration are printed onto the same test platform. The calibration test dots are printed at known concentrations of analyte. The test dots are printed, also at predetermined X-Y locations, containing only capture reagent which is preferably an antibody specific for the analyte under investigation. The test sample is then conjugated with an excess of marker reagent, which is preferably an antibody, and analyte. The marker antibody has previously been conjugated with a respective fluorescent label, emitting at a suitable wavelength (e.g. 650 nanometers). The marker antibody/antigen complex as well as the free, remaining marker antibody is then flowed over the test platform using laminar flow. A person skilled in the art will appreciate however that other assay devices that do not rely on laminar flow may also be employed. For example, an assay device in the form of a vessel where the antibody/antigen complex as well as the remaining free marker antibody move through the device by diffusion limited kinetics may also be employed for the purposes of the present invention.
In this fashion, marker antibody/unknown concentration of antigen complexes are bound by the capture antibody test dots, whereas free marker antibodies bind to the pre-printed antigen dots at known concentrations. Both the test dots of unknown concentration to be measured and the calibration dots of known concentrations encompassing the dynamic range of the analyte are exposed to the same test fluid sample at the same time. The laminar flow effectively places the analyte components within proximity of the respective binding sites to promote optimal adhesion kinetics for the respective association constants.
The test platform is examined in a reader for determination of the respective concentrations of fluorescent label attached to the dots on the platform when activated by suitable wavelength irradiation. The calibration dots, preferably originally printed at up to ten different concentrations of analyte, result in producing a dynamic internal true dose response curve providing very accurate calibration reference for the assay. The intensity reading obtained from the test dots of unknown concentration is compared to this calibration line. The unknown test concentrations accurately and efficiently interpolate into the dynamic internal true dose response calibration obtained from the known calibration spots.
Each assay device tested provides similarly accurate and reproducible results confirming that the present method for on-platform dynamic calibration provides an accurate, enhanced and sensitive determination of analyte in both quantitative as well as qualitative assays for diagnostic use and detection of analytes in clinical use for humans and animals. This immediate and significant benefit demonstrates that these assays, when processed using the described method (Dynamic Internal Calibration™), do not reflect the errors described in association with current other methods for running these assays while using externally derived calibration standards.
The ability to calibrate and test simultaneously also enhances the confidence level in assuring that the obtained measurements are true. This methodology of the present invention, allows for several different analyte tests to be run on the same assay device at the same time. Several sets of test dots or test arrays for testing for different analytes as well as multiple sets of corresponding calibration dots or calibration arrays for the different analytes can be run on the same assay device at the same time. In order to reach a better than 99% confidence level for a test, the test needs to be run a minimum of three times. Multiple panels of different tests may also be used and run, all at the same time, on a single test platform. The surprising results of the present invention prove that errors in the tests are eliminated, reproducibility is enhanced, the dynamic range for any test is easily extended to cover a required analyte concentration range, sensitivity and coefficient of variance is dramatically improved. The combination of these advantages over prior state of the art also results in considerable saving in time to test results. It is an important advantage that the format of the present invention is device independent and may be applied by those skilled in the art.
The assay device and method as described, effectively represent a novel, quantitative and fast method for the accurate determination of analyte and or marker concentrations, typically for diagnostic clinical markers associated with disease processes. The immediate benefit of rapid, accurate measurement of marker concentration allows for rapid dynamic detection of marker concentration as an indicator of a disease process such as increasing, steady or decreasing concentration, as well as rapid quantitative monitoring of drug efficacy in modifying gene expression for the production of specific marker proteins to indicate drug efficacy.
The sandwich immunoassay matrix incorporates a capture antibody that is specific for the antigen of interest and a fluorescence conjugated secondary antibody for detection of analytes and immune complexes. Human chorionic gonadotropin (HCG), a marker of pregnancy in humans, was used as antigen for testing of this platform assay. Currently, the dynamic analytical range for this test is between 1 to 150 fmol/uL (280-37,600 mIU/mL) with an assay volume of 5 μL. Comparison between the calculated HCG concentration using the device compared with known HCG concentrations has excellent agreement between values (
The assay principle is based on quantitative, non-competitive, heterogeneous immunoassays. First generation devices were printed with a series of antigen dots at decreasing concentrations for standard curve auto-calibration; followed by capture antibody dots. Test sample containing the antigen was processed with lyophilized detecting antibody already conjugated to the respective indicator or dye. The test sample reacted with label for 2 minutes and was then dispensed into the chip assay device to be inserted into the reader. The fluorescent intensity of each dot was measured. The reader software compares the fluorescence of the capture antibody dots having unknown antigen concentration with those of the true dose response standard curve having known concentrations and calculates the concentration of the test antigen.
Each of four 10×10 matrices containing 100 test dots as shown in
As shown in
As shown in
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments of the invention described above. Such equivalents are intended to be encompassed in the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2,475,240 | Jul 2004 | CA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA05/01147 | 7/20/2005 | WO | 00 | 3/26/2008 |
