Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54388—Immunochromatographic test strips based on lateral flow
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

• This invention relates to chemical analysis tests, and in particular, to a chromatographic assay device and methods for detecting the presence and/or concentration of various analytes in bodily fluids, such as saliva.
• the assay device and the methods described in this disclosure allow increased sensitivity of results by preventing analyte loss through analyte adsorption to solid materials used in the assay.
• Chromatographic immunoassay with visual read-out has often been used in detecting many analytes. See U. S. Patent Nos. 5,559041; 4,943,522; 5,602,040; 5,5591,645, 5,037,736 and 4,098,876, and Wheeler, Prof. Nurse 14:571 (1999) , which are incorporated herein by reference, as if fully set forth.
• a sample containing the analyte to be tested is added to a solid support.
• the solid support contains a movable labeled specific antibody to the analyte, as well as an immobilized binding reagent.
• the sample containing the analyte migrates to the zone containing the movable labeled specific antibody. There, the analyte binds to the antibody forming a labeled antibody-analyte complex. Next, the labeled antibody- analyte complex migrates to the zone containing the immobilized binding reagent.
• the immobilized binding reagent is an antibody to the analyte and is capable of capturing the labeled antibody-analyte complex to form a labeled antibody-analyte-immobilized antibody sandwich.
• the immobilized binding reagent is a binding pair of the labeled antibody.
• This binding pair binds to only labeled antibodies that are not bound by the analyte.
• the binding pair of the labeled antibody binds to unbound free labeled antibodies.
• any unbound labeled antibody or labeled antibody- analyte complex migrates further down the chromatographic unit past the immobilized binding reagent zone. Detection of the label within the immobilized binding reagent zone shows the presence and or/quantity of the particular analyte in the sample solution. See Dresser, D. W.
• Chromatographic immunoassays with visual read-out have the advantage of being instrument free and rapid in terms of obtaining assay results. It is because of this very recognized advantage that a number of chromatographic assay devices have become available for use over-the-counter (OTC) . For instance, the HCG test for pregnancy is commonly available to consumers over the counter. See Ekins, R.P. (1976) , Hormone Assays and their Clinical Application. 4th edition (eds. Galfre, G. and Milstein, C , and Meth. Enzymol., 73:3-48 (1981). Furthermore, many chromatographic immunoassay devices are being used for point-of-care (POC) testing. Such chromatographic immunoassay POC tests include the test for streptococcus and the one for tetrahydrocannabinol (THC) , the active component of marijuana.
• THC tetrahydrocannabinol
• chromatographic immunoassay devices use porous materials at the sample addition area. These materials cause some of the analyte to become adsorbed to the surface of these porous materials before the analyte contacts the assay reagents of the device. This adsorption often results in poor assay sensitivity.
• a number of POC tests for detecting • THC constitute chromatographic immunoassay devices that use porous materials at the sample addition area.
• the THC- analyte is adsorbed onto the porous surface before it contacts the assay reagent.
• most of the available POC tests for THC have poor assay sensitivity and precision.
• This invention involves a method and device for prevention of loss of analytes through adsorption to solid materials used in the analysis by adding an antibody to a solution containing an analyte which will bind with the analyte, and then applying the analyte-antibody complex in the solution to a solid support.
• the solid support of the assay consists of a separate chromatography unit having a sample addition zone, a test zone, and an absorption zone.
• a sample mixed with a first antibody is added to the sample addition zone, from where it migrates via capillary flow onto the test zone.
• the test zone contains either an immobilized analyte or analog of the analyte or an immobilized second antibody.
• the immobilized analyte or analog of the analyte binds to unbound labeled antibody, whereas the immobilized second antibody binds to the first- antibody-analyte complex in the solution. Any labeled antibody that is not bound at the immobilized antibody or immobilized antigen zone flows through onto the absorption zone. Adding the antibody capable of binding to the hydrophobic regions of the analyte in the sample containing the analyte prior to applying the sample solution to the chromatography unit decreases adsorption of the analyte to the solid materials of the chromatography unit. This increases sensitivity and enhances precision of the assay measurement .
• Figure 1 shows a chromatographic assay strip 10 having a sample addition zone 11, a test zone 12, and a receiving zone 13.
• Figure 2 shows a chromatography assay strip 20 having a sample addition zone 21, a test zone 22, a receiving zone 23, and an antibody labeling zone 24.
• An antibody labeling reagent is supported at zone 24.
• Figure 3 depicts a container for the solubilizing reagent.
• the assay materials of the invention comprise a chromatographic assay device and a solubilizing reagent containing an antibody specific for the analyte.
• the chromatographic assay device of the invention comprises a porous chromatographic strip with an upstream end and a downstream end. At the upstream end of the porous strip is a sample addition zone for receiving a sample solution. A test zone having an immobilized binding reagent is disposed downstream of the sample addition zone. In addition to the test zone toward the downstream end of the assay strip there is an absorption zone. All zones of the porous strip are physically in flow communication so that a sample solution applied to the sample addition zone will flow through the test zone until it reaches the absorption zone.
• the materials of the zones of the assay strip are chosen from a group of materials that includes cellulose, fiberglass, nylon, polyester, etc. The material for the sample addition zone must be capable of receiving a sufficient amount of sample solution.
• Fiberglass, polyester, and cellulose paper are among the preferred materials for the sample addition zone.
• the material for the test zone must have the capability of immobilizing a minimum of 10 ⁇ g/cm 2 of protein.
• Nitrocellulose membrane is one of the preferred materials for the test zone.
• the material for the absorption zone is capable of receiving the sample solution with all unbound reagents at the test zone.
• the preferred materials for the absorption zone and the sample addition zone include cellulose paper.
• the antibody of the solubilizing reagent is capable of binding to the analyte molecule and blocking the hydrophobic regions of the analyte, thus preventing the analyte from being adsorbed to the surface of the sample addition zone of the chromatographic assay strip.
• the assay device and the solubilizing reagent are for a competitive chromatographic immunoassay.
• the solubilizing reagent consists of a labeled antibody.
• the binding reagent at the test zone consists of the analyte or analogues of the analyte, which are capable of binding antibodies to the analyte.
• a preferred binding reagent for a THC test is THC-bovine serum albumin (BSA) conjugate.
• BSA THC-bovine serum albumin
• the binding reagent is immobilized at the test zone by adsorption or covalent binding. Nitrocellulose membrane is capable of binding large concentration of proteins by passive adsorption.
• the solublizing reagent consists of an unlabled antibody.
• the binding reagent at the test zone consists of the analyte or an analogue of the analyte, capable of binding antibodies to the analyte.
• the solid support or chromatograhic unit of this assay device further comprises an antibody-labeling zone having an antibody- labeling reagent.
• the antibody-labeling zone is positioned downstream from the sample addition zone and upstream from the test zone.
• a labeling reagent capable of binding the antibody of the analyte is movably supported at the antibody-labeling zone.
• the labeling reagent is a binding protein of the antibody conjugated with a reporter substance
• the binding protein of the antibody is chosen from a group of proteins including antibodies, protein A, and protein G.
• the label of the labeling reagent is chosen from a group of fluorescent, chemiluminescent, bioluminescent compounds, radioactive isotopes, enzymes, and particular color particles . Such particular particles include colloidal gold, colloidal silver, carbon black, latex beads, etc., which can be seen by the naked eye.
• the label is coupled to a binding protein via various means including covalent binding and physical adsorption, which have been described in previous publications. See U.S. Patent Nos.
• the solubilizing reagent to be used with a device having an antibody-labeling zone consists of a specific antibody of the analyte, which antibody can be labeled by the labeling reagent at the antibody-labeling zone .
• the assay device and reagent are for a sandwich chromatographic immunoassay.
• the binding reagent at the test zone is an antibody of the analtye.
• the solubilizing assay reagent consists of a labeled antibody that recognizes a different epitope of the analtye than the antibody of the binding reagent.
• both antibodies can bind the same analyte to form a labeled antibody-analyte-binding antibody "sandwich".
• a sample solution containing the analyte is first exposed to the solubilizing reagent. If the analyte is present in the sample solution, the analyte binds to the antibody in the solubilizing reagent to form an antibody-analyte complex. The resulting solution is then applied to the sample addition zone of the assay device. The labeled antibody- analyte complex will migrate to the test zone. If the antibody in the solubilizing reagent is unlabeled, the assay device consists of an antibody-labeling zone. The antibody- analyte complex is labeled by the antibody-labeling reagent at the antibody labeling zone before the solution migrates to the test zone. In either case, the antibody at the test zone is a labeled antibody and the antibody-analyte complex is a labeled antibody-analyte complex.
• the labeled antibody binds to the binding reagent at the test zone, and the labeled antibody-analyte complex flows through the test zone.
• the tracer signal at the test zone is inversely proportional to the concentration of analyte in the sample solution.
• the presence or quantity of the analyte in the sample solution is determined by measuring the tracer signal at the test zone.
• the labeled antibody flows through the test zone and the labeled antibody-analyte complex binds to the binding reagent of the test zone.
• the tracer signal at the test zone is proportional to the concentration of the analyte in the assay sample.
• the presence or quantity of the analyte in the sample solution is determined by measuring the tracer signal at the test zone.
• Example 1 Moving of Fluorescent dye labeled THC on Fiberglass Membrane: ⁇ 9-Tetrahydrocannabinol was labeled with Cy5 dye.
• the THC moiety of the labeled compound is hydrophobic.
• Fiberglass or porous polyester membrane materials have large surface areas, which can absorb even more THC-Cy5.
• Control sample solution is lOmM PBS containing lOnM of THC-Cy5, 0.2% BSA, and 0.5 ⁇ g/ml mouse IgG.
• a test sample solution is lOmM PBS containing lOnM of THC-Cy5, 0.2% BSA, and 0.5 ⁇ g/ml of mouse anti-THC monoclonal antibody.
• THC-Cy5 Migration of THC-Cy5 on fiberglass and polyester membrane materials: lO ⁇ l of THC-Cy5 control/test sample solution was spiked in the center of each membrane strip at the point that is 15mm to one end (end A) of the strip. 5 drops of 0.2% BSA- PBS solution was gradually applied onto each strip from end A. When the solution migrates from end A to the other end, end B, the strips were scanned with a fluorescence scanner.
• Example 2 Chromatographic immunoassay of L- ⁇ 9-carboxy- tetrahydrocannabinol (THCA) in urine.
• a line of approximately 0.2 ⁇ g of THC-BSA conjugate was placed on the nitrocellulose membrane at the point approximately 10 mm from the overlap area,
• the antibody-gold sol conjugate was re-suspended in 1 ml of 0.01 mole/L sodium phosphate buffered saline containing 0.1% BSA, 1% trehalose, and 0.05% Tween 20. 0.01 ml aliquots of the antibody-gold sol conjugate solution were dispensed into 2 ml glass vials and freeze-dried.
• 0.2 ml of urine sample was added into a glass vial having freeze-dried antibody-gold sol conjugate.
• the antibody-gold sol conjugate was reconstituted by vortex mixing.
• the nitrocellulose end of a chromatographic immunoassay strip was dipped into the glass vial containing urine sample mixed with antibody-gold sol conjugate.
• the assay result was read after 10 minutes.
• the presence of a red line at the test area of the assay strip was interpreted as a negative test result.
• the absence of red line at the test area of the assay strip was interpreted as a positive test result.
• Example 3 Chromatographic immunoassay of ⁇ 9- tetrahydrocannabinol (THC)
• Solutions of varied THC concentrations were made by diluting THC into 0.2% BSA-PBS solution. The same device and protocol for testing THCA in urine was used in testing THC solutions.
• a 5 x 30 mm plastic backed nitrocellulose strip was positioned on the middle area of a 5 x 76 mm one-sided glued vinyl strip.
• a 5 x 25 Whatman filter paper strip and a 25 mm #33 fiberglass strip were separately positioned on the ends of the vinyl strip. Both the filter paper and the fiberglass have 2 mm overlapping with the nitrocellulose strip.
• 10 ul of goat anti-mouse IgG antibody-gold sol conjugate solution was applied at one spot of the fiberglass 5 mm from the nitrocellulose-fiberglass overlap area.
• a line of approximately 0.2 ug of THC-BSA conjugate was placed on the nitrocellulose membrane at the point approximately 10 mm from the nitrocellulose-filter paper overlap area.
• the assay result was read after 10 minutes.
• the presence of a red line at the test area of the assay strip was interpreted as a negative test result.
• the absence of a red line at the test area of the assay strip was interpreted as a positive test result.

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  • 2001-08-09 NZ NZ524107A patent/NZ524107A/en unknown
  • 2001-08-09 CA CA002419551A patent/CA2419551A1/en not_active Abandoned
  • 2001-08-09 AU AU2001286451A patent/AU2001286451A1/en not_active Abandoned
  • 2001-08-09 WO PCT/US2001/025262 patent/WO2002014869A2/en not_active Application Discontinuation
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