Abstract:
A method for performing time resolved homogeneous assays using a long-lifetime luminescent dye as a donor. A reaction well containing a sample portion, donor reagent, and acceptor reagent and a matrix well containing a sample portion and donor reagent are excited and the resulting emission from each is measured at a single wavelength associated with the acceptor. The measurement obtained from the matrix well is used to provide a correction for the measurement obtained from the reaction well. The sample may be a biological fluid such as an oral fluid.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority of U.S. Provisional Application No. 61/271,263, filed Jul. 17, 2009. The aforementioned application is herein expressly incorporated by reference in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to biological homogeneous assays. In particular, the invention relates to a corrected method for biological homogeneous assays. 
         [0004]    2. Description of Related Art 
         [0005]    Ullman et al. first applied luminescence resonance energy transfer (LRET) technology to produce a homogeneous immunoassay using fluorescein and rhodamine as donor and acceptor, respectively. The use of conventional organic fluorophore pairs for homogeneous LRET applications proved not to be practical because the lifetimes of the donor and acceptor were too short, translating to background levels that were too high to make the technology practical. Ideally, a donor luminophore should combine high brightness, high stability and a long luminescence lifetime to be practical for high sensitivity diagnostic applications. The longer luminescent lifetimes of lanthanide donors make them appealing for LRET applications because time-gating techniques can remove the background levels that hamper conventional LRET technologies. 
         [0006]    Variations in sample properties including color, viscosity, pH, presence/absence of solids, etc. have the potential to impact assay results. With a heterogeneous assay, this is not usually a concern when the sample result is read by absorbance or luminescence because the sample is washed from the reaction site before the sample is read. With a homogeneous assay, the differences in sample properties can affect the luminescence values. Samples with the same amount of analyte can and do yield different raw luminescent data depending on the properties of the sample. Interfering constituents in samples can absorb the excitation light, donor emission, absorbance emission more or less than a control sample, thereby affecting sample results. 
         [0007]    Luminescent lanthanide compounds are known to have long lifetimes (on the order of milliseconds), while conventional organic dyes have lifetimes in the range of nanoseconds. This unique property enables the time gated removal of short-lived background interference that hampers conventional luminescence techniques. As shown in Prior Art  FIG. 1A , an excitation flash is delivered to a complex biological matrix followed by a short delay (for example 100 μs) to allow unwanted interfering background noise to dissipate. Then data is collected during an integration window that is much longer than the undesirable background (for example 400 μsec) yielding a signal almost free of interference. 
         [0008]    Lanthanide dyes can act as donors with conventional organic dye acceptors. When the donor and acceptor are brought into close proximity, excitation energy absorbed by the donor is transferred to the acceptor by a Förster mediated dipole-dipole coupling. The emitted light from the acceptor can be measured in a time-resolved mode, yielding low-noise LRET data compared to data obtained from conventional LRET pairs. 
         [0009]    The improved data quality is readily apparent in Prior Art  FIG. 2  when comparing the emission of fluorescein (conventional settings) with the emission of an exemplary lanthanide dye, LUMI4-TB (a terbium chelate trademarked by Lumiphore, Inc.) using time-resolved settings. LUMI4-TB is a member of a new class of luminescent hydroxyisophthalamide chelates that combines a high quantum yield (60%) and absorption coefficient and exhibits high stability in aqueous environments. The properties and structure of luminescent hydroxyisophthalamide chelates of lanthanides are discussed in U.S. patent application Ser. No. 12/521,919, “Multi-Color Time Resolved Fluorophores Based on Macrocyclic Lanthanide Complexes,” Butlin, Corneillie, and Xu; filed Jan. 25, 2008. 
         [0010]    Prior Art  FIG. 2  demonstrates the reduced background and improved linear range achieved with time-resolved luminescence. In this experiment, fluorescein exhibits a linear range down to ˜1.0 nM (conventional settings). Time-resolved measurements of LUMI4-TB exhibit a much larger linear range (down to below 10 pM), which is achieved by gating out short-lived interference attributable to the lamp source, optics, matrix components, etc. 
         [0011]    Prior Art  FIG. 3  shows the absorption and emission spectra for an exemplary lanthanide dye, LUMI4-TB. Broad absorption of the sensitizing 2-hydroxyisophthalamide chelating unit is centered at 340 nm. The emission spectrum is characteristic of luminescent terbium complexes with the dominant peaks centered at 545 nm and 490 nm. The large separation between absorption and emission peaks (Stokes Shift) eliminates the reabsorption of emitted luminescence that hampers the performance of conventional organic dyes. 
         [0012]    A schematic representation of an exemplary competitive, homogeneous assay technology is shown in Prior Art  FIGS. 4A ,  4 B,  4 C, and  4 D. As shown in  FIG. 4A , an antibody  405  is coupled to an acceptor luminophore  410  (e.g., fluorescein) to serve as an acceptor reagent with binding sites  415  that are specific for analyte  420  and the competitor conjugate donor  425  that is derived from the analyte component and a luminophore (e.g., LUMI4-TB). As shown in  FIG. 4A , the concentration of the analyte  420  is low relative to the concentration of the competitor conjugate donor  425 . 
         [0013]    As shown in  FIG. 4B , excitation energy is first absorbed by the competitor conjugate donor  425 . When the donor and acceptor are brought into close proximity (e.g., through binding to the antibody  405 ), energy is transferred from the competitor conjugate donor  425  to the acceptor  410  by a Förster mediated dipole-dipole coupling. The acceptor  410  subsequently produces a LRET signal at the wavelength of the acceptor luminophore  410 . In the presence of a low amount of analyte the LRET signal is near its maximum, as the competitor conjugate donor  425  binds unimpeded to the analyte-specific antibody  405 . 
         [0014]    As shown in  FIG. 4C  and  FIG. 4D , the concentration of the analyte  420  is high relative to the concentration of the competitor conjugate donor  425 , and the LRET signal is low, since the analyte  420  out competes the competitor conjugate donor  425  that carries the lanthanide donor. 
         [0015]    For laboratory tests, homogeneous assay methods are preferable to heterogeneous methods because test procedures are simple and fast, requiring no washing steps. LRET is a homogeneous method that does not require enzyme inactivation or reformation to generate a signal and avoids some of the limitations of other homogeneous assay technologies. This assay platform relies on LRET between complementary molecules labeled with either a lanthanide donor or an acceptor (e.g., fluorescein). The key component to a practical LRET assay is the donor luminophore, which ideally should combine high brightness, high stability and low background to attain the sensitivity required for detecting drugs of abuse in oral fluid. The longer lifetimes of lanthanide donors make them appealing for LRET applications because time-gating techniques remove the background levels that hamper conventional LRET technologies. LUMI4-TB incorporates four isophthalamide chelating units and achieves an unparalleled level of brightness when coordinated to terbium. The luminescent lanthanide technology has been combined with high affinity and high specificity antibodies for drugs to develop this oral fluid drug assay technology. 
         [0016]    Oral fluid has become an important matrix for testing because samples are relatively easy to collect, are more reflective of recent use and more closely tracking blood levels, the reference standard. Testing for drugs in oral fluid presents a challenge for current homogeneous laboratory testing technologies and point-of-care testing (POCT) products, which were developed for testing in urine. While detection levels for laboratory and lateral flow POCT assays for urine are in the 50-2000 ng/mL range for drugs, the levels in oral fluid are sometimes an order of magnitude or more lower. More sensitive technologies are needed. 
         [0017]    In order to correct for the variability between samples that can occur in a homogeneous assay, techniques involving additional measurements may be used. For example, U.S. Pat. No. 6,861,264, “Method of Measuring the Luminescence Emitted in a Luminescent Assay,” Mabile et al, issued Mar. 1, 2005, teaches the use of measurements taken at two difference wavelengths emitted by a single sample volume. 
         [0018]    Thus there is a need for a rapid, homogeneous assay method for measuring parent drug compounds in oral fluid. There is also a need for a method of measurement correction that is applicable at a single wavelength. 
       BRIEF SUMMARY OF THE INVENTION 
       [0019]    The present invention provides a method for performing time resolved homogeneous assays using a long-lifetime luminescent dye as a donor. A reaction well containing a sample portion, donor reagent, and acceptor reagent and a matrix well containing a sample portion and reference luminophore (e.g., donor reagent) are excited and the resulting emission from each is measured at a single wavelength associated with the acceptor. The measurement obtained from the matrix well is used to provide a correction for the measurement obtained from the reaction well. The sample may be a biological fluid such as an oral fluid. 
         [0020]    In one embodiment of the invention, the donor reagent includes a lanthanide dye. The dye may be a hydroxisophthalamide-based luminescent or a macrocyclic hydroxisophthalamide-based luminescent (e.g., LUMI4-TB). 
         [0021]    In another embodiment, the correction is obtained by a calculation involving the measured intensities of the reaction well and relevant matrix well. In a further embodiment the corrected intensity is obtained by taking the ratio of the reaction well intensity to the matrix well intensity. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0022]    Prior Art  FIG. 1  shows the temporal evolution of a luminescent process involving a long-lifetime dye. 
           [0023]    Prior Art  FIG. 2  shows a plot of luminescent intensity versus concentration for fluorescein and LUMI4-TB. 
           [0024]    Prior Art  FIG. 3  shows the absorption and emission spectra for LUMI4-TB. 
           [0025]    Prior Art  FIG. 4A  shows a schematic representation of a system with an antibody-luminophore and competitor conjugate donor in the presence of a relatively low concentration of analyte. 
           [0026]    Prior Art  FIG. 4B  shows a schematic of the luminescent behavior of the system shown in  FIG. 4A . 
           [0027]    Prior Art  FIG. 4C  shows a schematic representation of a system with an antibody-luminophore and competitor conjugate donor in the presence of a relatively high concentration of analyte. 
           [0028]    Prior Art  FIG. 4D  shows a schematic of the luminescent behavior of the system shown in  FIG. 4C . 
           [0029]      FIG. 5  shows a flow diagram for a multi-volume homogeneous assay in accordance with an embodiment of the present invention. 
           [0030]      FIG. 6  shows a flow diagram for a multi-volume homogeneous assay incorporating a reference in accordance with an embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0031]    A single assay in accordance with the present invention involves the optical excitation of at least two discrete volumes (e.g., reaction well and matrix well) and the measurement of the luminescent response at a single wavelength. The preparation of a reaction well solution involves mixing an antibody-luminophore reagent and sample, followed by the addition of a competitor-luminophore conjugate. The preparation of a matrix well involves the mixing of a sample and a reference luminophore (e.g., competitor-luminophore conjugate). 
         [0032]    In the examples that follow, multiple assays are performed for seven drugs using a single microtiter plate. The details provided in the examples should not be construed as limiting the subject of invention. Each drug assay requires a unique antibody-luminophore reagent that is specific for the drug being evaluated. To simplify the testing procedure and improve sample throughput, the various competitor conjugates are formulated into a single competitor solution. The assay technology yields a rapid result with only two reagents (antibody-luminophore and competitor luminophore conjugate) and is amenable to automation in a laboratory setting. 
         [0033]    In a homogeneous LRET experiment, there are two dyes: donor and acceptor. When donor and acceptor are brought into close proximity, LRET occurs. The close proximity may be obtained through antibody-antigen binding events, receptor-ligand interactions, etc. The reaction should be composed of a donor luminophore, acceptor luminophore and sample. The matrix should be composed of the sample and a luminophore that absorbs and emits at the same or similar wavelengths as those being used in the reaction well. 
         [0034]    A simple ratio of the value for the reaction well divided by the value for the matrix well value yields a value corrected for sample differences, although more complicated mathematics can be used. For example, correct raw data for sample interference using the equation Cx=Sx/Mx where Cx is the corrected result, Sx is the reaction signal and Mx is the matrix signal for the same sample. 
         [0035]    Exemplary Instrument System 
         [0036]    A time resolved luminescence reader capable of accurate reading of 96 well microplates (PerkinElmer ENVISION microplate reader) was used for measurements in the following examples. The instrument provided an excitation capability at 340 nm with a 30-60 nm band pass, and 520 nm centered emission detection with 5-30 nm bandpass filters. The instrument should have timing control, the capability for delay (e.g., 100 usec), integration window (e.g., 100 usec) and cycle settings (e.g., 10-100 cycles). A computer interfaced to the reader captures and reports results. 
       Example 1 
       [0037]    Seven different assays (AMP, COC, MET, MOR, PCP, THC, XTC) were performed with a coffee colored oral fluid sample that was successively diluted with a colorless, clear buffer yielding sample dilutions of 2×,  4 ×,  8 ×,  16 ×,  32 × and 64×. All of these samples were negative for the drug analyzed. 
       Materials 
       [0038]    Donor Reagent: LUMI4-TB conjugated to analyte competitor (analyte-Tb conjugates combined into single multi-analyte competitor solution) 
         [0039]    Acceptor Reagent: Fluorescein acceptor conjugated to analyte-specific antibodies 
         [0040]    oral fluid sample 
         [0041]    dilution buffer 
       Assay Procedure 
       [0042]    1. Dispense 25 μL of each sample into all 9 wells of a single row in the microtiter plate. 
         [0043]    2. Dispense 25 μL of each calibrator into all 9 wells of rows 7 (Negative control) and 8 (positive control). 
         [0044]    3. Add 75 μL of Acceptor Reagents AMP, COC, MET, MOR, PCP, THC, MDMA and the Blank Matrix Solution to columns 1-8, respectively. 
         [0045]    4. Gently mix plate on shaker at room temperature for 5 minutes. 
         [0046]    5. Add 75 μL of Multi-Donor Reagent to all wells in each column. 
         [0047]    6. Mix plate gently for at least 30 seconds and read plate at least 3 minutes after addition of Multi-Donor Reagent. 
         [0048]    The reaction well for each drug included: sample (25 uL), acceptor reagent (75 uL), donor reagent (75 uL). The first matrix well included: sample (25 uL), donor reagent (75 uL), solution added (75 uL) to yield same volume as reaction wells. The second matrix well (less volume) included: sample (25 uL) and donor reagent (75 uL). No “makeup” solution was added to the second matrix well. 
         [0049]    The time-resolved luminescence plate reader instrument was set for a 100 μs delay, a 100 μs integration window and 100 cycles. A computer interfaced to the reader captured and reported the results shown in Table 1 below. 
       Data Treatment 
       [0050]    The correction was made using luminescence measurements at a single wavelength (single emission bandpass filter is used). Reaction and matrix wells are read at a wavelength where acceptor emission can be detected (520 nm in this example). Note that donor and acceptor emission are visible in the acceptor wavelength window in this example. Any changes in the luminescence value for the sample is also reflected in the value for the matrix. Dividing these values effectively corrects the luminescence reading for sample differences. A control or calibrator sample is measured the same way (reaction well and matrix well). 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                   
                   
                 Raw data 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 Dilution of 
                 AMP 
                 COC 
                 MET 
                 MOR 
                 PCP 
                 THC 
                 XTC 
                 MATRIX 
                 MATRIX 
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 same volume 
                 less volume 
               
               
                   
               
               
                 1 
                  2x 
                 11815 
                 9192 
                 15967 
                 21295 
                 12709 
                 5275 
                 12984 
                 3046 
                 3321 
               
               
                 2 
                  4x 
                 15904 
                 12393 
                 21359 
                 27098 
                 16825 
                 6802 
                 17703 
                 3630 
                 4038 
               
               
                 3 
                  8x 
                 17957 
                 14666 
                 24639 
                 31305 
                 19392 
                 8155 
                 20624 
                 4301 
                 4535 
               
               
                 4 
                 16x 
                 20154 
                 16008 
                 27135 
                 34108 
                 21489 
                 8993 
                 23330 
                 4835 
                 5171 
               
               
                 5 
                 32x 
                 21220 
                 16465 
                 28161 
                 35250 
                 21829 
                 9355 
                 23702 
                 4935 
                 5285 
               
               
                 6 
                 64x 
                 21880 
                 16949 
                 28771 
                 36310 
                 22510 
                 9980 
                 24484 
                 5143 
                 5478 
               
               
                 Negative control 
                   
                 22202 
                 17153 
                 29528 
                 35911 
                 23158 
                 10537 
                 25246 
                 5162 
                 6288 
               
               
                 Positive Control 
                   
                 11587 
                 12696 
                 15224 
                 22217 
                 14012 
                   
                 15535 
                 5237 
                 6080 
               
               
                   
               
             
          
           
               
                 Corrected data - Reaction well value/Matrix (same volume) value 
               
             
          
           
               
                   
                 Dilution of 
                 AMP 
                 COC 
                 MET 
                 MOR 
                 PCP 
                 THC 
                 XTC 
                   
                   
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
               
               
                   
               
               
                 1 
                  2x 
                 3.88 
                 3.02 
                 5.24 
                 6.99 
                 4.17 
                 1.73 
                 4.26 
                   
                   
               
               
                 2 
                  4x 
                 4.38 
                 3.41 
                 5.88 
                 7.47 
                 4.63 
                 1.87 
                 4.88 
                   
                   
               
               
                 3 
                  8x 
                 4.18 
                 3.41 
                 5.73 
                 7.28 
                 4.51 
                 1.9 
                 4.8 
                   
                   
               
               
                 4 
                 16x 
                 4.17 
                 3.31 
                 5.61 
                 7.05 
                 4.44 
                 1.86 
                 4.83 
                   
                   
               
               
                 5 
                 32x 
                 4.3 
                 3.34 
                 5.71 
                 7.14 
                 4.42 
                 1.9 
                 4.8 
                   
                   
               
               
                 6 
                 64x 
                 4.25 
                 3.3 
                 5.59 
                 7.06 
                 4.38 
                 1.94 
                 4.76 
                   
                   
               
               
                 Negative control 
                   
                 4.3 
                 3.32 
                 5.72 
                 6.96 
                 4.49 
                 2.04 
                 4.89 
                   
                   
               
               
                 Positive Control 
                   
                 2.21 
                 2.42 
                 2.91 
                 4.24 
                 2.68 
                   
                 2.97 
                   
                   
               
               
                 AVERAGE  
                 (1-6) 
                 4.19 
                 3.3 
                 5.63 
                 7.17 
                 4.43 
                 1.87 
                 4.72 
                   
                   
               
               
                   
                 SD 
                 0.17 
                 0.15 
                 0.22 
                 0.18 
                 0.15 
                 0.07 
                 0.23 
                   
                   
               
               
                   
                 CV 
                 0.04 
                 0.04 
                 0.04 
                 0.02 
                 0.03 
                 0.04 
                 0.05 
                   
                   
               
               
                 Negative Control  
                 REL ERR 
                 −0.03 
                 −0.01 
                 −0.02 
                 0.03 
                 −0.01 
                 −0.09 
                 −0.03 
               
               
                   
               
             
          
           
               
                 Corrected data - Reaction well value/Matrix (less volume) value 
               
             
          
           
               
                   
                 Dilution of 
                 AMP 
                 COC 
                 MET 
                 MOR 
                 PCP 
                 THC 
                 XTC 
                   
                   
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
                 0 ng/mL 
               
               
                   
               
               
                 1 
                  2x 
                 3.56 
                 2.77 
                 4.81 
                 6.41 
                 3.83 
                 1.59 
                 3.91 
                   
                   
               
               
                 2 
                  4x 
                 3.94 
                 3.07 
                 5.29 
                 6.71 
                 4.17 
                 1.68 
                 4.38 
                   
                   
               
               
                 3 
                  8x 
                 3.96 
                 3.23 
                 5.43 
                 6.9 
                 4.28 
                 1.8 
                 4.55 
                   
                   
               
               
                 4 
                 16x 
                 3.9 
                 3.1 
                 5.25 
                 6.6 
                 4.16 
                 1.74 
                 4.51 
                   
                   
               
               
                 5 
                 32x 
                 4.02 
                 3.12 
                 5.33 
                 6.67 
                 4.13 
                 1.77 
                 4.48 
                   
                   
               
               
                 6 
                 64x 
                 3.99 
                 3.09 
                 5.25 
                 6.63 
                 4.11 
                 1.82 
                 4.47 
                   
                   
               
               
                 Negative control 
                   
                 3.53 
                 2.73 
                 4.7 
                 5.71 
                 3.68 
                 1.68 
                 4.01 
                   
                   
               
               
                 Positive Control 
                   
                 1.91 
                 2.09 
                 2.5 
                 3.65 
                 2.3 
                   
                 2.56 
                   
                   
               
               
                 AVERAGE  
                 (1-6) 
                 3.89 
                 3.06 
                 5.23 
                 6.65 
                 4.11 
                 1.73 
                 4.38 
                   
                   
               
               
                   
                 SD 
                 0.17 
                 0.16 
                 0.22 
                 0.16 
                 0.15 
                 0.09 
                 0.24 
                   
                   
               
               
                   
                 CV 
                 0.04 
                 0.05 
                 0.04 
                 0.02 
                 0.04 
                 0.05 
                 0.05 
                   
                   
               
               
                 Negative Control  
                 REL ERR 
                 0.1 
                 0.12 
                 0.11 
                 0.16 
                 0.12 
                 0.03 
                 0.09 
               
               
                   
               
             
          
         
       
     
       Discussion of Results 
       [0051]    In this assay, the luminescence is inversely proportional to the concentration of analyte. In general, higher values indicate less analyte is present with a negative sample having the highest value. This sample was known to be free of all of the drug analytes tested. Yet, it is clear that the raw luminescent values in all of the assays including the matrix wells increase as the sample is diluted and the color of the sample becomes lighter. The color of the sample is affecting the result. The raw data falsely indicate that Sample 1 is more positive than the positive control for COC, MOR, PCP and XTC. Sample 2 even appears to be positive for COC when looking at the raw data. However, the corrected data (using either the same volume or less volume matrix) yields the expected negative results for all samples, and the average value for all samples for a given analyte are very close to the Negative Control as expected. 
       Example 2 
       [0052]    Seven different assays (AMP, COC, MET, MOR, PCP, THC, XTC) were performed with a coffee colored oral fluid sample that was successively diluted with a colorless, clear buffer yielding sample dilutions of 2×, 4×, 8×, 16×, 32× and 64×. The samples were initially negative for the drug analyzed. Once the samples were diluted with buffer, the samples were then split into aliquots. Some aliquots were spiked at one of the following drug concentrations: COC: 10 ng/mL, 20 ng/mL, MOR: 40 ng/mL 
         [0053]    Materials 
         [0054]    Donor Reagent: LUMI4-TB conjugated to analyte competitor (analyte-Tb conjugates combined into single multi-analyte competitor solution) 
         [0055]    Acceptor Reagent: Fluorescein acceptor conjugated to analyte-specific antibodies 
         [0056]    oral fluid sample 
         [0057]    dilution buffer 
         [0058]    analyte solutions for spiking 
       Assay Procedure 
       [0059]    1. Dispense 25 μL of each sample into all 7 wells of a single row in the microtiter plate. 
         [0060]    2. Dispense 25 μL of each calibrator into all 7 wells of rows 7 (Negative control) and 8 (positive control). 
         [0061]    3. Add 75 μL of Acceptor Reagents COC (columns 1-3), MOR (columns 4-5) and the Blank Matrix Solution (column  6 ) to the indicated columns. 
         [0062]    4. Gently mix plate on shaker at room temperature for 5 minutes. 
         [0063]    5. Add 75 μL of Multi-Donor Reagent to all wells in each column. 
         [0064]    6. Mix plate gently for at least 30 seconds and read plate at least 3 minutes after addition of Multi-Donor Reagent. 
         [0065]    The Time-resolved luminescence plate reader instrument was set for a 100 μs delay, a 100 μs integration window and 100 cycles. A computer interfaced to the reader captured and reported results shown in Table 2. 
         [0066]    The reaction well for each drug included: sample (25 uL), acceptor reagent (75 uL), donor reagent (75 uL). The first matrix well included: sample (25 uL), donor reagent (75 uL), solution added (75 uL) to yield same volume as reaction well. The second matrix well (less volume) included: sample (25 uL) and donor reagent (75 uL). No “makeup” solution was added to the second matrix well. 
       Data Treatment 
       [0067]    The correction was made using luminescence measurements at a single wavelength (single emission bandpass filter is used). Reaction and matrix wells are read at a wavelength where acceptor emission can be detected (520 nm in this example). Note that donor and acceptor emission are visible in the acceptor wavelength window in this example. Any changes in the luminescence value for the sample is also reflected in the value for the matrix. Dividing these values effectively corrects the luminescence reading for sample differences. A control or calibrator sample is measured the same way (reaction well and matrix well). 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
             
             
               
                   
                   
                 Raw data 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Dilution of 
                 COC 
                 COC 
                 COC 
                 MOR 
                 MOR 
                 Matrix 
                 Matrix 
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 20 ng/mL 
                 10 ng/mL 
                 0 ng/mL 
                 40 ng/mL 
                 same vol 
                 less volume 
               
               
                   
               
               
                 1 
                  2x 
                 9658 
                 7099 
                 8086 
                 21904 
                 11076 
                 3039 
                 2831 
               
               
                 2 
                  4x 
                 12140 
                 9002 
                 9597 
                 27188 
                 12982 
                 3756 
                 3383 
               
               
                 3 
                  8x 
                 14776 
                 10182 
                 11302 
                 31252 
                 15695 
                 4199 
                 3785 
               
               
                 4 
                 16x 
                 15391 
                 10913 
                 12647 
                 33129 
                 16291 
                 4641 
                 4102 
               
               
                 5 
                 32x 
                 16846 
                 11539 
                 13153 
                 34851 
                 17122 
                 4763 
                 4569 
               
               
                 6 
                 64x 
                 18084 
                 12382 
                 13862 
                 35906 
                 17935 
                 5227 
                 4843 
               
               
                   
                 Negative control 
                 17999 
                 17242 
                 17162 
                 35296 
                 34420 
                 5294 
                 4770 
               
               
                   
                 Positive Control  
                 12508 
                 12121 
                 12013 
                 19345 
                 19081 
                 5243 
                 4965 
               
               
                   
               
             
          
           
               
                 Corrected data - Reaction well value/Matrix (same volume) value 
               
             
          
           
               
                   
                 Dilution of 
                 COC 
                 COC 
                 COC 
                 MOR 
                 MOR 
                   
                   
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 20 ng/mL 
                 10 ng/mL 
                 0 ng/mL 
                 40 ng/mL 
               
               
                   
               
               
                 1 
                  2x 
                 3.18 
                 2.34 
                 2.66 
                 7.21 
                 3.64 
                   
                   
               
               
                 2 
                  4x 
                 3.23 
                 2.4 
                 2.56 
                 7.24 
                 3.46 
                   
                   
               
               
                 3 
                  8x 
                 3.52 
                 2.42 
                 2.69 
                 7.44 
                 3.74 
                   
                   
               
               
                 4 
                 16x 
                 3.32 
                 2.35 
                 2.73 
                 7.14 
                 3.51 
                   
                   
               
               
                 5 
                 32x 
                 3.54 
                 2.42 
                 2.76 
                 7.32 
                 3.59 
                   
                   
               
               
                 6 
                 64x 
                 3.46 
                 2.37 
                 2.65 
                 6.87 
                 3.43 
                   
                   
               
               
                   
                 Negative control 
                 3.4 
                 3.26 
                 3.24 
                 6.67 
                 6.5 
                   
                   
               
               
                   
                 Positive Control 
                 2.39 
                 2.31 
                 2.29 
                 3.69 
                 3.64 
                   
                   
               
               
                 AVERAGE  
                 (1-6) 
                 3.37 
                 2.38 
                 2.67 
                 7.2 
                 3.56 
                   
                   
               
               
                   
                 SD 
                 0.15 
                 0.04 
                 0.07 
                 0.19 
                 0.12 
                   
                   
               
               
                   
                 CV 
                 0.05 
                 0.02 
                 0.03 
                 0.03 
                 0.03 
                   
                   
               
               
                 Negative Control  
                 REL ERR 
                 −0.01 
                   
                   
                 0.08 
                   
                   
                   
               
               
                   
               
             
          
           
               
                 Corrected data - Reaction well value/Matrix (less volume) value 
               
             
          
           
               
                   
                 Dilution of 
                 COC 
                 COC 
                 COC 
                 MOR 
                 MOR 
                   
                   
               
               
                 Sample 
                 oral fluid 
                 0 ng/mL 
                 20 ng/mL 
                 10 ng/mL 
                 0 ng/mL 
                 40 ng/mL 
               
               
                   
               
               
                 1 
                  2x 
                 3.41 
                 2.51 
                 2.86 
                 7.74 
                 3.91 
                   
                   
               
               
                 2 
                  4x 
                 3.59 
                 2.66 
                 2.84 
                 8.04 
                 3.84 
                   
                   
               
               
                 3 
                  8x 
                 3.9 
                 2.69 
                 2.99 
                 8.26 
                 4.15 
                   
                   
               
               
                 4 
                 16x 
                 3.75 
                 2.66 
                 3.08 
                 8.08 
                 3.97 
                   
                   
               
               
                 5 
                 32x 
                 3.69 
                 2.53 
                 2.88 
                 7.63 
                 3.75 
                   
                   
               
               
                 6 
                 64x 
                 3.73 
                 2.56 
                 2.86 
                 7.41 
                 3.7 
                   
                   
               
               
                   
                 Negative control  
                 3.77 
                 3.61 
                 3.6 
                 7.4 
                 7.22 
                   
                   
               
               
                   
                 Positive Control 
                 2.52 
                 2.44 
                 2.42 
                 3.9 
                 3.84 
                   
                   
               
               
                 AVERAGE  
                 (1-6) 
                 3.68 
                 2.6 
                 2.92 
                 7.86 
                 3.89 
                   
                   
               
               
                   
                 SD 
                 0.17 
                 0.08 
                 0.1 
                 0.32 
                 0.16 
                   
                   
               
               
                   
                 CV 
                 0.05 
                 0.03 
                 0.03 
                 0.04 
                 0.04 
                   
                   
               
               
                 Negative Control  
                 REL ERR 
                 −0.02 
                   
                   
                 0.06 
               
               
                   
               
             
          
         
       
     
       Discussion of Results 
       [0068]    In this assay, the luminescence is inversely proportional to the concentration of analyte. In general, higher values indicate less analyte is present with a negative sample having the highest value. Samples were spiked with analytes at the concentrations indicated in the top row of the results tables above. It is clear that the raw luminescent values for a given spike level group in all of the assays including the matrix wells increase as the sample is diluted and the color of the sample becomes lighter. The color of the sample is affecting the result. For the COC 0 ng/mL group, the raw data falsely indicate that Samples 1 &amp; 2 were more positive than the positive control for COC. Note that the corrected values using either matrix yield the expected result (all samples are negative in this group). The corrected data (using either matrix to correct) are consistent for a given group as the oral fluid color is diluted. 
         [0069]    Cocaine was spiked at 20 ng/mL in the positive control for the cocaine tests. As can be seen in the 20 ng/mL COC test column, the raw data yield differing results for samples spiked at the same level, and the values correlate with the intensity of the color of the sample. The correction factor removes the color bias, and all of the samples test near the level of the positive control. The raw data for samples spiked with 10 ng/mL cocaine show the same trend. The corrected values indicate that there is less than 20 ng/mL in the samples, while the raw data for these same samples indicate that the 2×, 4× and 8× samples have greater than 20 ng/mL cocaine and the 16×, 32× and 64× samples have less than 20 ng/mL drug. 
         [0070]    For the MOR 0 ng/mL group, raw luminescent values increase as the sample is diluted and the color of the sample becomes lighter. Note that the corrected values using either matrix yield the expected result (all samples are negative in this group). 
         [0071]    Morphine was spiked at 40 ng/mL in the positive control for the morphine tests. As can be seen in the 40 ng/mL MOR test column, the raw data yield differing results for samples spiked at the same level, and the values correlate with the intensity of the color of the sample. The correction factor removes the color bias, and all of the samples test near the level of the positive control. 
       Example 3 
       [0072]    Seven different assays (AMP, COC, MET, MOR, PCP, THC, XTC) were performed with oral fluid samples, which contained drug analytes or interfering species. The samples results were compared against cutoff calibrators. The cutoff levels were used for determining whether a sample was Positive (POS) or Negative (−) for a specific analyte. The cutoff levels used were as follows: AMP −50 ng/mL, COC −20 ng/mL, MET −50 ng/mL, MOR −40 ng/mL, PCP −10 ng/mL, THC −4 ng/mL, XTC −50 ng/mL. The assay results were compared with the results of quantitative analysis by GC/MS. 
       Materials 
       [0073]    Specific Acceptor Reagents: Amphetamine (AMP), Cocaine (COC), Methamphetamine (MET), Opiates (OPI), Phencyclidine (PCP), Δ9-THC (THC) and MDMA. Antibodies conjugated to an acceptor fluorophore in buffer with preservative. 
         [0074]    Multi-Donor Reagent: Donor luminophore conjugated to amphetamine, cocaine, methamphetamine, opiate, phencyclidine, Δ9-THC and MDMA drug derivatives in buffer with preservative. 
         [0075]    Matrix Blank Reagent 
         [0076]    Neat oral fluid samples 
       Assay Procedure 
       [0077]    1. Allow reagents and assay components to warm room temperature (20-25° C., 68-77° F.). 
         [0078]    2. Mix oral fluid samples thoroughly before use. 
         [0079]    3. Dispense 25 μL of each sample into all 8 wells of a single column in the microtiter plate. Up to 10 samples may be assayed in each plate in columns 1-10. 
         [0080]    4. Dispense 25 μL of each calibrator into all 8 wells of columns 11 (Negative) and 12 (Cutoff). 
         [0081]    5. Add 75 μL of Acceptor Reagents AMP, COC, MET, OPI, PCP, THC, MDMA and the Blank Matrix Solution to rows 1-8, respectively. 
         [0082]    6. Gently mix plate on shaker at room temperature for at least 5 minutes. 
         [0083]    7. Add 75 μL of Multi-Donor Reagent to all wells in each row. 
         [0084]    8. Mix plate gently for at least 30 seconds and start reading plate 3-5 minutes following addition of the Multi-Donor Reagent. 
         [0085]    The Time-resolved luminescence plate reader instrument was set for a 100 μs delay, a 100 μs integration window and 100 cycles. A computer interfaced to the reader captured and reported results shown in Table 3. 
       Calibration and Quality Control 
       [0086]    A cutoff calibrator is required to determine whether the concentration of the target analyte is above or below the cutoff level in an oral fluid sample. Positive and negative controls should be within +50% and −50% of the individual analyte cutoffs and users should follow the appropriate federal, state and local guidelines concerning the running of external quality controls. 
       Data Treatment 
       [0087]    Correct raw data for sample interference using the equation Cx=Sx/Mx where Cx is the corrected result, Sx is the sample, calibrator or control signal and Mx is the matrix signal for the same sample, calibrator or control. Compare matrix-corrected sample, control and negative calibrator values to those of the matrix-corrected cutoff calibrator signal. Corrected samples and controls with a value less than the corrected cutoff calibrator are considered presumptive positive. Corrected samples and controls with values equal to or greater than the corrected cutoff calibrator are considered negative. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
             
             
               
                 520 nm Raw Data 
               
             
          
           
               
                 AMP 
                 17574 
                 13582 
                 15593 
                 32021 
                 16523 
                 17348 
                 18967 
                 35263 
                 43899 
                 22352 
               
               
                 COC 
                 38040 
                 37218 
                 38615 
                 30636 
                 38005 
                 36209 
                 29775 
                 36430 
                 41798 
                 32676 
               
               
                 MET 
                 15166 
                 15399 
                 16186 
                 33965 
                 16266 
                 14638 
                 20835 
                 37051 
                 54050 
                 24676 
               
               
                 MOR 
                 36432 
                 36367 
                 37451 
                 30562 
                 37695 
                 34815 
                 17573 
                 28693 
                 40909 
                 20282 
               
               
                 PCP 
                 34770 
                 34842 
                 35413 
                 28960 
                 35753 
                 33437 
                 14050 
                 27805 
                 38396 
                 16510 
               
               
                 THC 
                 35966 
                 36591 
                 36809 
                 29924 
                 37452 
                 34817 
                 30446 
                 36456 
                 40326 
                 32159 
               
               
                 XTC 
                 28212 
                 16204 
                 28446 
                 27219 
                 20748 
                 22127 
                 18062 
                 28133 
                 38420 
                 21103 
               
               
                 MATRIX 
                 8442 
                 8272 
                 8667 
                 6641 
                 8647 
                 8133 
                 9829 
                 9697 
                 9689 
                 9915 
               
               
                   
               
             
          
           
               
                 520 Corrected Data [(520 sample /520 Matrix ] 
               
             
          
           
               
                   
                 917 
                 920 
                 926 
                 936 
                 937 
                 990 
                 PLUS 50 
                 MINUS 50 
                 NEG 
                 CUTOFF 
               
               
                   
               
               
                 AMP 
                 2.082 
                 1.642 
                 1.799 
                 4.822 
                 1.911 
                 2.133 
                 1.930 
                 3.636 
                 4.531 
                 2.254 
               
               
                 COC 
                 4.506 
                 4.499 
                 4.455 
                 4.613 
                 4.395 
                 4.452 
                 3.029 
                 3.757 
                 4.314 
                 3.296 
               
               
                 MET 
                 1.796 
                 1.862 
                 1.868 
                 5.114 
                 1.881 
                 1.800 
                 2.120 
                 3.821 
                 5.578 
                 2.489 
               
               
                 MOR 
                 4.316 
                 4.396 
                 4.321 
                 4.602 
                 4.359 
                 4.281 
                 1.788 
                 2.959 
                 4.222 
                 2.046 
               
               
                 PCP 
                 4.119 
                 4.212 
                 4.086 
                 4.361 
                 4.135 
                 4.111 
                 1.429 
                 2.867 
                 3.963 
                 1.665 
               
               
                 THC 
                 4.260 
                 4.423 
                 4.247 
                 4.506 
                 4.331 
                 4.281 
                 3.098 
                 3.760 
                 4.162 
                 3.243 
               
               
                 XTC 
                 3.342 
                 1.959 
                 3.282 
                 4.099 
                 2.399 
                 2.721 
                 1.838 
                 2.901 
                 3.965 
                 2.128 
               
               
                   
               
             
          
           
               
                 Interpretation of Results 
               
             
          
           
               
                   
                 917 
                 920 
                 926 
                 936 
                 937 
                 990 
                 PLUS 50 
                 MINUS 50 
                 NEG 
                 CUTOFF 
               
               
                   
               
               
                 AMP 
                 POS 
                 POS 
                 POS 
                 — 
                 POS 
                 POS 
                 POS 
                 — 
                 — 
                 — 
               
               
                 COC 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 POS 
                 — 
                 — 
                 — 
               
               
                 MET 
                 POS 
                 POS 
                 POS 
                 — 
                 POS 
                 POS 
                 POS 
                 — 
                 — 
                 — 
               
               
                 MOR 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 POS 
                 — 
                 — 
                 — 
               
               
                 PCP 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 POS 
                 — 
                 — 
                 — 
               
               
                 THC 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 POS 
                 — 
                 — 
                 — 
               
               
                 XTC 
                 — 
                 POS 
                 — 
                 — 
                 — 
                 — 
                 POS 
                 — 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
       Correlation of Results by GC/MS 
       [0088]    Sample 917—Homogeneous Assay POS for AMP, MET
       GC/MS results—AMP 57 ng/mL, MET 600 ng/mL       
 
         [0090]    Sample 920—Homogeneous Assay POS for AMP, MET, XTC
       GC/MS results—AMP 336 ng/mL, MET 4435 ng/mL   Homogeneous Assay POS for XTC is due to cross-reactivity of that antibody with MET at high concentrations.       
 
         [0093]    Sample 926—Homogeneous Assay POS for AMP, MET
       GC/MS results—AMP 110 ng/mL, MET 449 ng/mL       
 
         [0095]    Sample 936—Homogeneous Assay NEG for all drugs tested 
         [0096]    Sample 937—Homogeneous Assay POS for AMP, MET
       GC/MS results—AMP 77 ng/mL, MET 2127 ng/mL       
 
         [0098]    Sample 990—Homogeneous Assay POS for AMP, MET
       GC/MS results—AMP 58 ng/mL, 993 ng/mL       
 
         [0100]    PLUS 50—Controls containing 150% cutoff concentrations tested positive by homogeneous assay for all drugs as expected. 
         [0101]    MINUS 50—Controls containing 50% cutoff concentrations tested negative by homogeneous assay for all drugs as expected. 
         [0102]    NEG—Negative calibrator tested negative by homogeneous assay for all drugs as expected. 
         [0103]    CUTOFF—Cutoff calibrator was used to determine cutoff level for determining positive and negative test results. 
       Discussion of Results 
       [0104]    In this example the corrected data correctly identified the presence of AMP and MET at concentrations above 50 ng/mL when compared to GC/MS quantitation. For sample 936, the raw data indicated that COC and THC were positive relative to the cutoff calibrator. The corrected data indicates that this sample was negative for COC and THC. 
         [0105]      FIG. 5  shows a flow diagram  500  for an embodiment of a corrected homogeneous assay using at least two wells and a single wavelength for characterization. The assay uses one matrix well and may use one or more reaction wells. Although the examples provided above referred specifically to wells in a microtiter plate, a “well” in the general sense refers to a discrete volume of liquid. 
         [0106]    At steps  505   a  and  505   b , a sample is added to the respective reaction and matrix wells. The sample is a biological fluid (e.g., oral fluid) for which the presence of a specific analyte is to be determined. 
         [0107]    At step  510   a  an acceptor reagent including an analyte-specific antibody-luminophore is added to the reaction well. The luminophore may be an organic dye (e.g., fluorescein). At optional step  510   b  a buffer may be added to the matrix well in an amount similar to the amount of acceptor reagent added to the reaction well in step  510   a.    
         [0108]    At steps  515   a  and  515   b  the respective reaction and matrix wells may be mixed and incubated. Incubation typically involves time-at-temperature, and mixing may be assisted by gentle shaking. When the reaction and matrix wells reside on a single microtiter plate, steps  515   a  and  515   b  will typically be identical; however, since the reaction well involves a binding reaction with the acceptor reagent that is not present in the matrix well, the degree of mixing (if used) and the time-at-temperature may not necessarily be identical. 
         [0109]    At steps  520   a  and  520   b  a donor reagent is added to the respective reaction and matrix wells. The donor reagent includes at least one competitor conjugate donor. The competitor conjugate donor may include a lanthanide dye. 
         [0110]    At steps  525   a  and  525   b  the respective reaction and matrix wells may be mixed and incubated. Incubation typically involves time-at-temperature, and mixing may be assisted by gentle shaking. When the reaction and matrix wells reside on a single microtiter plate, steps  515   a  and  515   b  will typically be identical; however, since the reaction well involves a competitive binding reaction between the acceptor reagent, and the sample and donor reagent that is not present in the matrix well, the degree of mixing (if used) and the time-at-temperature may not necessarily be identical. 
         [0111]    At steps  530   a  and  530   b  the respective signal intensity of the reaction well and matrix well are measured at a characteristic wavelength associated with the acceptor reagent luminophore. As can be seen from the above examples and Prior Art  FIG. 3 , the emission of the donor luminophore (e.g., LUMI4-TB) at the acceptor wavelength (e.g., 520 nm) may be more than an order of magnitude lower than the emission at a peak wavelength associated with the donor luminophore (e.g., ˜550 nm for LUMI4-TB). 
         [0112]    At step  535  a correction is applied to the intensity measurement obtained from the reaction well using the intensity measurement obtained from the matrix well. For example, the reaction well intensity value may be divided by the matrix well intensity value to produce the corrected value. 
         [0113]      FIG. 6  shows a flow diagram  600  for an embodiment of a corrected homogeneous test assay that utilizes the corrected value obtained from the homogeneous assay shown in  FIG. 5 . The corrected homogeneous test assay includes a series of steps similar to the steps shown in  FIG. 5 , except that the sample, which is typically of an unknown composition, is replaced by a reference with a known composition (e.g., predetermined analyte content). 
         [0114]    At steps  605   a  and  605   b , a reference is added to the respective reaction and matrix wells. At step  610   a  an acceptor reagent including an analyte-specific antibody-luminophore is added to the reaction well. The luminophore may be an organic dye (e.g., fluorescein). At optional step  610   b  a buffer may be added to the matrix well in an amount similar to the amount of acceptor reagent added to the reaction well in step  610   a.    
         [0115]    At steps  615   a  and  615   b  the respective reaction and matrix wells may be mixed and incubated. Incubation typically involves time-at-temperature, and mixing may be assisted by gentle shaking. When the reaction and matrix wells reside on a single microtiter plate, steps  615   a  and  615   b  will typically be identical; however, since the reaction well involves a binding reaction with the acceptor reagent that is not present in the matrix well, the degree of mixing (if used) and the time-at-temperature may not necessarily be identical. 
         [0116]    At steps  620   a  and  620   b  a donor reagent is added to the respective reaction and matrix wells. The donor reagent includes at least one competitor conjugate donor. The competitor conjugate donor may include a lanthanide dye. 
         [0117]    At steps  625   a  and  625   b  the respective reaction and matrix wells may be mixed and incubated. Incubation typically involves time-at-temperature, and mixing may be assisted by gentle shaking. When the reaction and matrix wells reside on a single microtiter plate, steps  615   a  and  615   b  will typically be identical; however, since the reaction well involves a competitive binding reaction between the acceptor reagent, and the sample and donor reagent that is not present in the matrix well, the degree of mixing (if used) and the time-at-temperature may not necessarily be identical. 
         [0118]    At steps  630   a  and  630   b  the respective signal intensity of the reaction well and matrix well are measured at a characteristic wavelength associated with the acceptor reagent luminophore. As can be seen from the above examples and Prior Art  FIG. 3 , the emission of the donor luminophore (e.g., LUMI4-TB) at the acceptor wavelength (e.g., 520 nm) may be more than an order of magnitude lower than the emission at a peak wavelength associated with the donor luminophore (e.g., ˜550 nm for LUMI4-TB). 
         [0119]    At step  635  a correction is applied to the intensity measurement obtained from the reaction well using the intensity measurement obtained from the matrix well. For example, the reaction well intensity value may be divided by the matrix well intensity value to produce the corrected value. At step  640  the corrected reference value obtained in step  635  is compared to the corrected sample value obtained in step  535  of  FIG. 5 . 
         [0120]    While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention.