METHODS AND DEVICES TO ENHANCE SENSITIVITY AND EVALUATE SAMPLE ADEQUACY AND REAGENT REACTIVITY IN RAPID LATERAL FLOW IMMUNOASSAYS

• US 20120094276A1
• Filed: 12/23/2011
• Published: 04/19/2012
• Est. Priority Date: 11/04/2009
• Status: Active Grant

• Current Assignee: Thomas Matthew Buchanan
• Original Assignee: Thomas Matthew Buchanan
• Inventors: Buchanan, Thomas M.
• Granted Patent: US 8,309,366 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0825 Test strips
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/5029 using swabs
• CPC Class Codes: G01N 33/54389 with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

Methods and devices for rapid lateral flow immunoassays to detect specific antibodies within a liquid sample while also validating the adequacy of the liquid sample for the presence of immunoglobulin and the integrity and immunoreactivity of the test reagents that detect the antibodies of interest, without requiring instrumentation. The methods and devices provide for delivery of a diluted liquid sample to a single location that simultaneously directs the liquid flow along two or more separate flow paths, one that serves as a positive control to confirm that all critical reagents of the test are immunoreactive, and that the sample being tested is adequate, and the other to detect specific antibodies if present.

Claims

1. A method for detecting an analyte in a sample and checking the reactivity of the method reagents comprising:

(a) collecting the diluted sample into a porous diluted-sample membrane having a point of collection, wherein the diluted sample flows toward points distant from the point of collection;

(b) conducting the diluted sample from a first portion of the diluted-sample membrane into a first flow path that provides flow from the diluted-sample membrane upstream to an absorbent pad downstream, wherein the first flow path comprises porous membranes and reagents to evaluate reagent reactivity comprising in flow order;

(i) one or more mobilizable markers for the class of analytes being detected, (ii) one or more mobilizable binding partners specific to downstream immobilized ligands being used to detect the analytes of interest in the diluted-sample, the mobilized binding partners capable of being recognized by the mobilized marker, (iii) one or more immobilized ligands for the analytes of interest, and (iv) an absorbent pad in direct fluid communication with the membrane containing immobilized ligands or connected thereto via an outflow wick membrane; and

(c) conducting the diluted sample from a second portion of the diluted-sample membrane into a second flow path that provides flow from the diluted-sample membrane upstream to an absorbent pad downstream, wherein the second flow path comprises porous membranes and reagents comprising in flow order;

(i) the same mobilizable markers as in the first flow path, (ii) no mobilizable binding partners specific to downstream immobilized ligands, (iii) the same immobilized ligands for the analytes of interest as in the first flow path, and (iv) an absorbent pad in direct fluid communication with the membrane for immobilized ligands or connected thereto via an outflow wick membrane.

2. The method of claim 1, wherein recombinant protein A is immobilized on the first, second, or both flow paths, downstream from the immobilized antigen ligand or ligands that bind specific antibodies from the diluted sample, and is used to evaluate whether immunoglobulin is present in the diluted sample.

3. The method of claim 1, wherein the mobilizable marker or markers are selected from the group consisting of protein G, protein L, and protein A, each bound to one or more observable components selected from the group consisting of colloidal gold, magnetic spheres, and fluorescent markers.

4. The method of claim 1, wherein the absorbent pad is capable of receiving and holding the entire volume of diluted sample added to the diluted sample membrane.

5. The method of claim 1, wherein diluted sample is collected into the diluted sample membrane such that a range of different sample dilutions become localized in different regions of the diluted sample membrane prior to migrating down each flow path.

6. The method of claim 1, wherein the porous membrane containing mobilizable marker or markers is positioned so that only a portion of this membrane contacts the diluted sample membrane in the direct flow line consisting of the shortest distance between the diluted sample and immobilized ligand or ligands for analytes of interest in the second flow path, and the remaining mobilized marker or markers contact diluted sample and flow downstream later via an indirect flow line to reach immobilized ligands later than flow down the direct flow line.

7. The method of claim 6 wherein the diluted sample membrane comprises different dilutions of sample in different regions, resulting in a greater variation in the concentration of diluted sample analytes that contact a given concentration of mobilized markers than occurs with a diluted sample membrane containing a uniform concentration of diluted sample.

8. The method of claim 1, wherein evaporation of the diluted liquid sample preferentially occurs in the region of the absorbent pad.

9. A method for detecting immunoglobulin in a lateral flow immunoassays using markers comprised of a combination of protein G and protein L bound to observable components, wherein the immunoglobulin tagged by these protein G and protein L molecules is subsequently bound to immobilized recombinant protein A.

10. The method of claim 9, wherein the observable components are selected from the group comprised colloidal gold, fluorescent markers, and magnetic spheres.

11. A method for providing mobilizable markers to lateral flow immunoassays during a longer time period than occurs with direct flow of sample past mobilizable markers along the shortest route flow path between sample and immobilized ligands for analytes of interest, comprising locating some mobilizable markers adjacent the direct flow shortest route, this adjacent location requiring a longer indirect route for sample to flow past mobilizable markers en route to downstream immobilized ligands for analytes of interest.

12. The method of claim 11, wherein different concentrations of sample are delivered to different portions of the indirect longer flow path, comprising a range of different concentrations of sample contacting a given concentration of mobilizable markers and this mixture of mobilized markers and sample reaching the downstream immobilized ligands after the initial mixture of direct route sample and markers has reached the immobilized ligands.

13. A method for preventing backflow of liquid in a lateral flow immunoassay device, comprising positioning an absorbent pad at the end of a liquid flow path, wherein the absorbent pad has a capacity to receive and hold the total liquid volume added to the device.