Methods for Improving Assays of Biological Samples

Abstract

Methods for improving assays of biological samples are provided, including assays of small volume biological samples, such as blood samples obtained from a fingerstick. The methods include steps of selecting a baseline assay; providing a first revised assay by altering an aspect, a reagent, or a step of the baseline assay; comparing the results of the baseline assay and first revised assay; and identifying the first revised assay as an improved assay if the results of the first revised assay are similar to, are substantially the same as, or are better than, the results of the baseline assay. The methods may be iterated. Alterations include reducing sample volume; setting temperature; reducing step number and duration; altering reagent composition and number; and altering detection. Improved assays may reduce the duration, cost, or complexity of the assay, may improve assay sensitivity, accuracy, or reliability, and may provide synergistic improvements.

1 . A method for improving an assay for use in an automatic assay device, said assay comprising detecting the presence of, detecting the absence of, or quantifying the amount of, an analyte in a biological sample, said method comprising:
 Selecting a baseline assay performed at a baseline assay temperature on a baseline volume of biological sample, and comprising one or more steps selected from pre-treating, diluting, aliquotting, separating, transporting, mixing, incubating, reacting, labeling, and detecting, the duration of said steps comprising baseline step durations, the time period required to perform said baseline assay in its entirety comprising a total baseline assay duration, and the performance of said baseline assay providing baseline results;   Providing a first revised assay based on said baseline assay by:   Providing a first reduced volume of said biological sample, or a portion thereof, for use in the performance of said first revised assay, said first reduced volume comprising a smaller volume than said baseline volume; then   Reducing the duration of, or eliminating, a pretreatment step applied to said biological sample or portion thereof, if any; then   Diluting said first reduced volume of biological sample by at least 10-fold to provide diluted biological sample; then   Altering the steps of said baseline assay by one or more of:   i) Altering a reagent composition or reducing the number of reagents used in the performance of the assay;   ii) Providing an altered assay temperature different than said baseline assay temperature; and   iii) Shortening the duration of a step, eliminating a step, combining two or more steps, altering the sequence of performance of steps, or combinations thereof; then   Performing said first revised assay at said altered assay temperature on an aliquot of said diluted biological sample to provide first revised results; then   Determining a difference, or lack thereof, between said baseline results and said first revised results; and   Identifying the first revised assay as an improved assay if the first revised results are as good as, or better than, the baseline results,   Whereby the baseline assay is improved if the first revised assay is identified as an improved assay.   
     
     
         2 . The method of  claim 1 , further comprising the following steps:
 Providing a second revised assay based on said first revised assay by:   Providing a second reduced volume of said biological sample, or a portion thereof, for use in the performance of said second revised assay, said second reduced volume comprising a smaller volume than said first reduced volume if said first reduced volume comprises a volume of more than 250 μL, and wherein said second reduced volume comprises said first reduced volume if said first reduced volume comprises a volume of no more than 250 μL; then   Diluting said second reduced volume of biological sample, or portion thereof, by at least 10-fold to provide diluted biological sample; then   Altering the steps of said first revised assay by one or more of:   i) Altering a reagent composition or reducing the number of reagents used in the performance of the assay;   ii) Altering the assay temperature; and   iii) Shortening the duration of a step, eliminating a step, combining two or more steps, altering the sequence of performance of steps, or combinations thereof; then   Performing said second revised assay on an aliquot of diluted biological sample to provide second revised results; then   Determining a difference, or lack thereof, between said baseline results and said second revised results; and   Identifying the second revised assay as an improved assay if the second revised results are as good as, or better than, the baseline results,   Whereby the baseline assay is improved if the second revised assay is identified as an improved assay.   
     
     
         3 . The method of  claim 1 , comprising altering at least one step selected from the steps of treating, mixing, and incubating. 
     
     
         4 . The method of  claim 1 , wherein said altering at least one step comprises altering a reagent used in a step. 
     
     
         5 . The method of  claim 4 , wherein altering a reagent used in a step comprises one or more of: eliminating a reagent; combining two or more reagents to provide a single combined reagent; eliminating an ingredient from a reagent; replacing an ingredient in a reagent;
 adding an ingredient to a reagent; increasing the amount of an ingredient in a reagent; decreasing the amount of an ingredient in a reagent; altering the pH or osmolarity of a reagent; and   combinations thereof.   
     
     
         6 . The method of  claim 1 , wherein said altering assay temperature comprises setting assay temperature to between about 32° C. and about 37° C. 
     
     
         7 . The method of  claim 1 , wherein said aliquot has a volume comprising about 10 μL or less. 
     
     
         8 . The method of  claim 1 , wherein said altering comprises altering the duration of a step. 
     
     
         9 . The method of  claim 1 , further comprising transporting said biological sample, a reagent, or both within a housing comprising a detector. 
     
     
         10 . The method of  claim 1 , wherein said biological sample comprises a biological sample selected from blood, serum, plasma, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, fluid obtained from a spinal tap, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluid, ocular fluids, a throat swab, tissue, breath, hair, finger nails, skin, biopsy samples, placental fluid, amniotic fluid, cord blood, lymphatic fluid, fluid from a body cavity, sputum, pus, microbiota, meconium, breast milk and other secretions and excretions. 
     
     
         11 . A method for improving a baseline assay to provide an improved assay for use in an automatic assay device, said baseline assay comprising detecting the presence of, detecting the absence of, or quantifying the amount of, an analyte in a biological sample having a sample volume, said method comprising:
 Providing a revised assay by altering said baseline assay by:   Setting said sample volume to about 250 μL or less;   Diluting said volume of biological sample by at least 10-fold to provide diluted biological sample;   Setting the assay temperature of the revised assay to between about 32° C. and about 37° C.;   Altering a step, altering the sequence of performance of steps, or both;   Eliminating a reagent or combining two or more of said reagents to provide no more than 2 reagents for use in said revised assay;   Reducing the duration of a step, eliminating a step, or combining two or more steps, effective that the time required to perform the revised assay is reduced as compared to the time required to perform the baseline assay;   Requiring that said detection step comprises an optical detection step;   Performing said revised assay on an aliquot of diluted biological sample to provide results of said revised assay; and   Comparing the results of said revised assay with the results of said baseline assay,   Wherein the baseline assay is improved if the results of the first revised assay are as good as, or better than, the baseline results.   
     
     
         12 . The method of  claim 11 , wherein said optical detection step comprises optical detection selected from optical detection using a photomultiplier tube (PMT), a photodiode, a charge-coupled device (CCD), a photon counting detector, a camera, a microscope, and arrays and combinations thereof. 
     
     
         13 . The method of  claim 11 , wherein said optical detection comprises colorimetric detection, fluorimetric detection, luminometric detection, turbidometric detection, absorbance detection, light scattering detection, or combinations thereof. 
     
     
         14 . The method of  claim 11 , wherein the time required to perform said improved assay is less than about 30 minutes. 
     
     
         15 . The method of  claim 11 , wherein the time required to perform said improved assay is less than about 20 minutes. 
     
     
         16 . The method of  claim 11 , wherein the time required to perform said improved assay is less than about 10 minutes. 
     
     
         17 . The method of  claim 11 , wherein the volume of said aliquot of diluted biological sample comprises about 10 μL or less. 
     
     
         18 . The method of  claim 11 , wherein the volume of said aliquot of diluted biological sample comprises about 5 μL or less. 
     
     
         19 . The method of  claim 11 , wherein performing the improved assay comprises three or fewer assay steps. 
     
     
         20 . The method of  claim 11 , wherein the cost of the reagents required for performing the improved assay is less than the cost of reagents required for performing the baseline assay. 
     
     
         21 - 60 . (canceled)