ES2264580T3

Device for assay of antibodies in oral fluids

Abstract

An apparatus for collecting and lateral flow chromatography of an oral fluid, said apparatus comprising: a capillary matrix having a surface exposed to receive oral fluid; a lateral flow chromatography strip, said lateral flow chromatography strip being in combination with said capillary matrix such that when said capillary matrix receives oral fluid, said capillary matrix absorbs said oral fluid and administers said oral fluid in a receiving area of said lateral flow chromatography strip; and a substantially planar blocking strip positioned between the capillary matrix and the lateral flow chromatographic strip.

ES2264580T3, drawing sheet 1 — Sheet 1 of 2

Term

Term ended

Projected expiry passed 29 March 2019, 7.5 years ago.

83 claims: 4 independent, 79 dependent

1
ES 2 264 580 T3 REIVINDICACIONES 1. Un aparato para la recolección y la cromatografía de flujo lateral de un fluido oral, comprendiendo dicho aparato:una matriz capilar que tiene expuesta una superficie para recibir fluido oral;una tira de cromatografía de flujo lateral, estando dicha tira de cromatografía de flujo lateral en combinación con dicha matriz capilar tal que cuando dicha matriz capilar recibe fluido oral, dicha matriz capilar absorbe dicho fluido oral y administra dicho fluido oral en una zona de recepción de dicha tira de cromatografía de flujo lateral;y una tira de bloqueo sustancialmente planar colocada entre la matriz capilar y la tira cromatográfica de flujo lateral.
2
El aparato de la reivindicación 1, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente la morfología de dicha matriz capilar.
3
El aparato de la reivindicación 2, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente el tamaño medio de poro de dicha matriz capilar.
4
El aparato de la reivindicación 2, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente el volumen hueco de dicha matriz capilar.
5
El aparato de la reivindicación 2, en el que dicha matriz capilar tiene un tamaño medio de poro que varía de aproximadamente 40 pm a aproximadamente 250 pm.
6
El aparato de la reivindicación 2, en el que dicha matriz capilar tiene un volumen hueco de menos del aproximadamente 60%/cm 3 .
7
El aparato de la reivindicación 1, en el que dicha matriz capilar comprende un plástico.
8
El aparato de la reivindicación 7, en el que dicha matriz capilar comprende un plástico seleccionado del grupo constituido por un polietileno de alta densidad (HDPE), un polietileno de peso molecular ultra elevado (UHMW), un polipropileno (PP), un fluoruro de polivinilideno (PVDF), un politetrafluoroetileno (PTFE), un nailon 6 (N6) y una polietersulfona (PES).
9
El aparato de la reivindicación 7, en el que dicho plástico es hidrófilo o está tratado para que sea hidrófilo.
10
El aparato de la reivindicación 1, en el que dicha matriz capilar, cuando entra en contacto con una mucosa oral absorbe fluido oral de dicha cavidad oral y libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral en menos de aproximadamente 1 minutos.
11
El aparato de la reivindicación 10, en el que dicha matriz capilar, cuando entra en contacto con una mucosa oral absorbe fluido oral de dicha cavidad oral y libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral en menos de aproximadamente 30 segundos.
12
El aparato de la reivindicación 10, en el que dicha matriz capilar es saturada con fluido oral en menos de aproximadamente 1 minuto.
13
El aparato de la reivindicación 1, en el que dicha matriz capilar es saturada por menos de aproximadamente 500 jul.
14
El aparato de la reivindicación 1, en el que dicha matriz capilar libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral sin la compresión de dicha matriz capilar.
15
El aparato de la reivindicación 14, en el que se libera suficiente fluido oral como para saturar dicha zona de recepción.
16
El aparato de la reivindicación 1, en el que dicha tira de bloqueo comprende un tampón.
17
El aparato de la reivindicación 1, en el que dicha tira de bloqueo evita el reflujo de los reactivos desde dicha tira de cromatografía de flujo lateral hacia dicha matriz capilar.
18
El aparato de la reivindicación 1, que comprende además:una carcasa que tiene una cavidad, en la que dicha tira de cromatografía de flujo lateral se extiende por el interior de la cavidad a lo largo de la carcasa hasta una zona de inspección sobre la carcasa;y al menos una zona de inspección desde un exterior de la carcasa hasta la tira de cromatografía lateral que permite la inspección visual de los reactivos en los puntos seleccionados sobre la tira cromatográfica lateral. ES 2 264 580 T3
19
El aparato de la reivindicación 18, en el que dicha carcasa actúa como un mango para introducir dicha matriz capilar en dicha cavidad oral.
20
Un procedimiento para la detección o la cuantificación de uno o más analitos en un fluido oral, comprendiendo dicho procedimiento las etapas de:i) introducir el aparato de la reivindicación 1 en la cavidad oral de un mamífero, tal que dicha matriz capilar entre en contacto con una superficie de mucosa oral, por medio de lo cual dicha matriz capilar absorbe fluido oral y administra dicho fluido oral en una zona de recepción de dicha tira de cromatografía de flujo lateral;ii) leer una señal sobre dicha tira de cromatografía de flujo lateral que indica la presencia, la ausencia o la cantidad de dicho uno o más analitos.
21
El procedimiento de la reivindicación 20, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente la morfología de dicha matriz capilar.
22
El procedimiento de la reivindicación 21, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente el tamaño medio de poro de dicha matriz capilar.
23
El procedimiento de la reivindicación 21, en el que la saturación de dicha matriz capilar con un fluido oral no altera sustancialmente el volumen hueco de dicha matriz capilar.
24
El procedimiento de la reivindicación 21, en el que en el que dicha matriz capilar tiene un tamaño medio de poro que varía de aproximadamente 40 μm a aproximadamente 250 pm.
25
El procedimiento de la reivindicación 21, en el que dicha matriz capilar tiene un volumen hueco de menos de aproximadamente 60%/cm 3 .
26
El procedimiento de la reivindicación 20, en el que dicha matriz capilar comprende un plástico.
27
El procedimiento de la reivindicación 26, en el que dicha matriz capilar comprende un plástico seleccionado del grupo constituido por un polietileno de alta densidad (HDPE), un polietileno de peso molecular ultra elevado (UHMW), un polipropileno (PP), un fluoruro de polivinilideno (PVDF), un politetrafluoroetileno (PTFE), un nailon 6 (N6) y una polietersulfona (PES).
28
El procedimiento de la reivindicación 26, en el que dicho plástico es hidrófilo o está tratado para que sea hidrófilo.
29
El procedimiento de la reivindicación 20, en el que dicha matriz capilar, cuando entra en contacto con una mucosa oral absorbe fluido oral de dicha cavidad oral y libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral en menos de aproximadamente 1 minuto.
30
El procedimiento de la reivindicación 29, en el que dicha matriz capilar, cuando entra en contacto con una mucosa oral absorbe fluido oral de dicha cavidad oral y libera de aproximadamente 100 pl a aproximadamente 200 pl de dicho fluido oral en dicha tira de cromatografía de flujo lateral en menos de aproximadamente 1 minuto.
31
El procedimiento de la reivindicación 29, en el que dicha matriz capilar, cuando entra en contacto con una mucosa oral absorbe fluido oral de dicha cavidad oral y libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral en menos de aproximadamente 30 segundos.
32
El procedimiento de la reivindicación 29, en el que dicha matriz capilar es saturada con fluido oral en menos de aproximadamente 1 minuto.
33
El procedimiento de la reivindicación 20, en el que dicha matriz capilar es saturada por menos de aproximadamente 500 pl.
34
El procedimiento de la reivindicación 20, en el que dicha matriz capilar libera dicho fluido oral en dicha zona de recepción de dicha tira de cromatografía de flujo lateral sin la compresión de dicha matriz capilar.
35
El procedimiento de la reivindicación 34, en el que se libera suficiente fluido oral como para saturar dicha zona de recepción.
36
El procedimiento de la reivindicación 20, en el que dicha tira de bloqueo comprende un tampón.
37
El procedimiento de la reivindicación 20, en el que dicha tira de bloqueo evita el reflujo de los reactivos desde dicha tira de cromatografía de flujo lateral hasta dicha matriz capilar.
38
El procedimiento de la reivindicación 20, en el que dicho aparato comprende además:ES 2 264 580 T3 una carcasa que tiene una cavidad, en la que dicha tira de cromatografía de flujo lateral se extiende por el interior de la cavidad a lo largo de la carcasa hasta una zona de inspección sobre la carcasa;y al menos una zona de inspección desde un exterior de la carcasa hasta la tira de cromatografía lateral que permita la inspección visual de los reactivos en los puntos seleccionados situados sobre la tira cromatográfica lateral.
39
El procedimiento de la reivindicación 38, en el que dicha carcasa actúa como un mango para introducir dicha matriz capilar en dicha cavidad oral.
40
Un equipo para la detección de un analito en un fluido oral, comprendiendo dicho equipo:el aparato de la reivindicación 1 para la recolección y la cromatografía de flujo lateral de un fluido oral;y materiales informativos para dicho aparato.
41
El aparato de la reivindicación 1, que comprende además:una carcasa;una cavidad en la carcasa, en la que la tira de cromatografía lateral se extiende en la cavidad desde la cavidad a lo largo de la carcasa hasta una zona de inspección sobre la carcasa, teniendo la tira de cromatografía lateral reactivos para unirse a analitos de prueba;al menos una zona de inspección desde un exterior de la carcasa hasta la tira cromatográfica lateral para permitir la inspección visual de los reactivos en los puntos seleccionados sobre la tira cromatográfica lateral;en el que la matriz capilar se comunica desde la carcasa hasta la cavidad oral por un extremo y tiene comunicación con la tira cromatográfica lateral por el otro extremo, y en el que la matriz capilar es hidrófila.
42
El aparato de la reivindicación 41 que comprende además:una matriz capilar hidrófila que define una matriz de canales de un material que tiene partículas esféricas.
43
El aparato de la reivindicación 41 que comprende además:una matriz capilar hidrófila que define una matriz de canales de un material seleccionado del grupo que incluye polímeros plásticos y poliestireno.
44
El aparato de la reivindicación 41, en el que la matriz capilar hidrófila no aumenta su volumen durante el transporte del fluido oral.
45
Un procedimiento de transportar analitos de prueba en fluido oral desde una cavidad oral hasta una tira cromatográfica lateral que comprende las etapas de:proporcionar una tira cromatográfica lateral;proporcionar una carcasa con una cavidad en la carcasa;proporcionar una tira cromatográfica lateral que se extiende por el interior de la cavidad a lo largo de la carcasa hasta una zona de inspección sobre la carcasa;proporcionar una matriz capilar hidrófila que se comunica desde la carcasa con la cavidad oral por un extremo y que tiene comunicación con la tira cromatográfica lateral por el otro extremo;proporcionar una tira de bloqueo sustancialmente planar entre la matriz capilar y la tira cromatográfica de flujo lateral;comunicar la matriz capilar hidrófila por un extremo con la boca de la persona por se analizada;y observar la tira cromatográfica lateral para la inspección de los reactivos en los puntos seleccionados sobre la tira cromatográfica lateral.
46
Un procedimiento de transporte de fluido acuoso desde la cavidad oral hasta una tira cromatográfica lateral según la reivindicación 45 y que comprende la etapa adicional de:proporcionar al menos una zona de control desde el exterior de la carcasa hasta la tira cromatográfica lateral para indicar la presencia de un mínimo de fluido por ser muestreado recibido desde el lecho corto absorbente a la tira cromatográfica lateral. ES 2 264 580 T3
47
Un procedimiento de transporte de fluido acuoso desde la cavidad oral hasta una tira cromatográfica lateral según la reivindicación 45 y que comprende la etapa adicional de:matriz capilar hidrófila que define una matriz de canales de un material que tiene partículas esféricas.
48
Un procedimiento de transporte de fluido acuoso desde la cavidad oral hasta una tira cromatográfica lateral según la reivindicación 45 y que comprende la etapa adicional de:matriz capilar hidrófila que define una matriz de canales de un material seleccionado del grupo que incluye polímero plástico y poliestireno.
49
Un procedimiento de transporte de fluido acuoso desde la cavidad oral hasta una tira cromatográfica lateral según la reivindicación 45 y que comprende la etapa adicional de:la matriz capilar hidrófila proporcionada no aumenta su volumen durante el transporte del fluido acuoso.
50
El aparato de la reivindicación 1, que comprende además una tira de conjugado colocada entre dicha matriz capilar y dicha tira cromatográfica de flujo lateral.
51
El aparato de la reivindicación 50, en el que los reactivos cromatográficos están dispuestos en dicha tira de conjugado.
52
El aparato de la reivindicación 1, en el que dicha tira de bloqueo comprende un detergente.
53
El aparato de la reivindicación 1, en el que dicha tira de bloqueo comprende un reactivo de bloqueo.
54
El aparato de la reivindicación 53, en el que dicha tira de bloqueo se selecciona del grupo constituido por albúmina de suero bovino (ASB), desoxicolato y n-lauroilsarcosina.
55
El procedimiento de la reivindicación 20, en el que dicha tira de bloqueo comprende un detergente.
56
El procedimiento de la reivindicación 20, en el que dicha tira de bloqueo comprende un reactivo de bloqueo.
57
El procedimiento de la reivindicación 56, en el que dichos reactivos de bloqueo se seleccionan del grupo constituido por albúmina de suero bovino (ASB), desoxicolato y n-lauroilsarcosina.
58
El procedimiento de la reivindicación 45, en el que dicha tira de bloqueo comprende un detergente.
59
El procedimiento de la reivindicación 45, en el que dicha tira de bloqueo comprende un reactivo de bloqueo.
60
El procedimiento de la reivindicación 59, en el que dicho reactivo de bloqueo se seleccionan del grupo constituido por albúmina de suero bovino (ASB), desoxicolato y n-lauroilsarcosina.
61
El aparato de la reivindicación 1, en el que dicha tira cromatográfica de flujo lateral contiene al menos un reactivo.
62
El aparato de la reivindicación 61, en el que dicho reactivo es una etiqueta detectable.
63
El aparato de la reivindicación 62, en el que dicha etiqueta detectable se selecciona del grupo constituido por:perlas magnéticas, tintes fluorescentes, enzimas y etiquetas colorimétricas.
64
El aparato de la reivindicación 63, en el que dichos tintes fluorescentes son seleccionados del grupo constituido por:isotiocianato de fluoresceína, rojo texas, rodamina, proteína verde fluorescente.
65
El aparato de la reivindicación 63, en el que una de dichas etiquetas colorimétricas es oro coloidal.
66
El aparato de la reivindicación 20, en el que dichos analitos son anticuerpos seleccionados del grupo constituido por:anticuerpos del VIH, anticuerpos del VLTH, anticuerpos de Helicobacter pylori, anticuerpos de la hepatitis, anticuerpos de las paperas, anticuerpos de la rubéola, cotinina, cocaína, benzoilecgonina, benzodizazpina, tetrahidrocanabinol, nicotina, teofilina de etanol, fenitoína, acetaminofén, litio, diazepam, nortriptilina, secobarbital, fenobarbitol, teofillina, testosterona, estradiol, 17-hidroxiprogesterona, progesterona, tirosina, hormona estimulante del tiroides, hormona estimulante de los folículos, hormona luteinizante, factor de crecimiento transformante de tipo alfa, factor de crecimiento epidérmico, factor de crecimiento de tipo insulina I y II, factor de inhibición de la liberación de hormonas de crecimiento, IgA, glucosa, colesterol, cafeína y globulina de unión a corticosteroides.
67
El aparato de la reivindicación 41, en el que al menos uno de dichos reactivos es una etiqueta detectable. ES 2 264 580 T3
68
El aparato de la reivindicación 67, en el que dicha etiqueta detectable se selecciona del grupo constituido por:perlas magnéticas, tintes fluorescentes, enzimas y etiquetas colorimétricas.
69
El aparato de la reivindicación 68, en el que dichos tintes fluorescentes son seleccionados del grupo constituido por:isotiocianato de fluoresceína, rojo texas, rodamina, proteína verde fluorescente.
70
El aparato de la reivindicación 68, en el que una de dichas etiquetas colorimétricas es oro coloidal.
71
El aparato de la reivindicación 41, en el que dichos analitos son anticuerpos seleccionados del grupo constituido por:anticuerpos del VIH, anticuerpos del VLTH, anticuerpos de Helicobacter pylori, anticuerpos de la hepatitis, anticuerpos de las paperas, anticuerpos de la rubéola, cotinina, cocaína, benzoilecgonina, benzodizazpina, tetrahidrocanabinol, nicotina, teofilina de etanol, fenitoína, acetaminofén, litio, diazepam, nortriptilina, secobarbital, fenobarbitol, teofillina, testosterona, estradiol, 17-hidroxiprogesterona, progesterona, tirosina, hormona estimulante del tiroides, hormona estimulante de los folículos, hormona luteinizante, factor de crecimiento transformante de tipo alfa, factor de crecimiento epidérmico, factor de crecimiento de tipo insulina I y II, factor de inhibición de la liberación de hormonas de crecimiento, IgA, glucosa, colesterol, cafeína y globulina de unión a corticosteroides.
72
Un dispositivo para recolectar y analizar fluidos, que comprende:una carcasa que tiene un extremo de recepción de fluidos y una parte de análisis;una tira de análisis por flujo lateral sustancialmente contenida en la parte de análisis, conteniendo la tira de análisis al menos un reactivo que es usado para detectar una entre la presencia y la ausencia de al menos un analito en un fluido;y una tira de recolección para transportar el fluido desde una fuente de fluido hasta la tira de análisis, incluyendo la tira de recolección: un primer extremo estrecho contenido en la carcasa y en comunicación fluida con la tira de análisis;un segundo extremo agrandado que sobresale del extremo receptor de fluidos;y una tira de bloqueo acoplada entre y en comunicación fluida con la tira de análisis de flujo lateral y la tira de recolección.
73
El dispositivo de la reivindicación 72, en el que la tira de recolección comprende una matriz capilar adaptada para una absorción rápida de fluido procedente de una fuente de fluido a la tira de análisis.
74
El dispositivo de la reivindicación 72, en el que la fuente de fluido es una cavidad oral.
75
El dispositivo de la reivindicación 72, en el que el segundo extremo es uno en forma de paleta y tiene una forma sustancialmente bulbosa.
76
Un dispositivo para el análisis de fluido oral, que comprende:una parte de análisis que alberga una tira de análisis de flujo lateral, conteniendo la tira de análisis al menos un reactivo que es usado para detectar una entre la presencia y la ausencia de al menos un analito en un fluido;y una parte de cuello que se extiende desde la parte de análisis, formando la parte de cuello un canal para la administración de fluido en la tira de análisis, estando el canal definido por una primera parte estrecha proximal a la parte de análisis y una segunda parte que incluye una abertura para recibir el fluido oral, en la que la segunda parte incluye una anchura del canal que es sustancialmente mayor que la anchura del canal en el extremo estrecho;una tira de recolección en comunicación fluida con la tira de análisis lateral, teniendo la tira de recolección una primera parte dispuesta en el canal y una segunda parte que sobresale hacia fuera desde la abertura de la parte de cuello;y una tira de bloqueo acoplada entre y en comunicación fluida con la tira de análisis de flujo lateral y la tira de recolección.
77
El dispositivo de la reivindicación 76, en el que la segunda parte del miembro absorbente tiene forma de paleta.
78
El dispositivo de la reivindicación 76, en el que la anchura de la parte de cuello se afila desde la anchura de la abertura hasta la anchura del extremo estrecho.
79
El dispositivo de la reivindicación 72, en el que la tira de análisis es una tira de inmunocromatografía.
80
El dispositivo de la reivindicación 72, que comprende además un indicador de la idoneidad de la muestra. ES 2 264 580 T3
81
El dispositivo de la reivindicación 72, en el que el reactivo es una pareja de unión inmunoespecífica que lleva una etiqueta detectable.
82
El dispositivo de la reivindicación 76, en el que el reactivo es una pareja de unión marcada enzimáticamente.
83
El dispositivo de la reivindicación 72, en el que el reactivo comprende uno entre un antígeno y un anticuerpo.

Independent claims83

131 paragraphs in 10 sections, as filed

ES 2 264 580 T3

DESCRIPTION

Collection device for single-stage analysis of oral fluids.

This invention relates to commonly used side strip chromatography analysis for oral fluids. A single-stage, continuous-line, rapid analysis format is disclosed and a single unit suitable for oral specimen collection and testing. More specifically, a hydrophilic capillary matrix is disclosed as a transport for oral fluids to a lateral chromatographic strip. This allows rapid analysis of oral fluids while keeping a disposable testing device in the patient's mouth.

Numerous analytical procedures have been developed to determine the presence or absence and / or quantify the amount of various analytes in tissues and fluids of organisms. Currently, most diagnostic tests are performed with either blood, urine, stool, or tissue biopsy. However, tests based on these materials pose a substantial invasion of privacy and pose a significant security risk (particularly with blood tests). In contrast, the collection of oral fluids including saliva and / or mucosal transudate for testing involves a relatively small invasion of privacy, is relatively safe, and can be performed quickly with relative ease.

The idea of using oral fluid in a screening procedure has been around for some time in scientific and clinical research. A multitude of investigators have investigated the use of oral fluid as a possible clinical specimen for the diagnosis of specific disease states or altered metabolic activity (see, eg, Annl. New York Acad. Sci., Vol. 694: " Saliva as a Diagnostic Fluid ”, Malamud and Tabak, eds., NY Acad. Sci. Pub. (1993)). There is a predominance of tests suggesting that oral fluids could be extremely useful samples for the detection of certain analytes. The basic technological premise is that the analytes present in the blood will pass through the oral mucosa and / or the salivary glands into the oral cavity where they can be detected. Furthermore, it is assumed that the analyte concentration in the oral fluid will be indicative of the blood concentration. There is, therefore, considerable interest in the development of devices for the collection, transport, and handling of oral fluid samples, as well as in the development of fluid-based assays; specifically, assays for various antibodies and metabolites.

Commonly, in testing samples such as blood, urine, or fecal material, there is an abundant supply of test material, with high volumes of analytes available for analysis. Furthermore, as the analyzes of such materials are carried out outside the body , there is no case of contamination of the body with the test reagents.

This is illustrated, for example, in the analysis device described by Zoeten et al. in US Patent 5,611,995 issued March 18, 1997. In this device, an absorbent body with a handle is provided and held in the stream of urine being expelled from the body. Once the absorbent body is saturated, it is inserted into a holding device with a test strip. The saturated short bed comes into contact with a test strip and is compressed, depositing the urine on the test strip for analysis. A space on the side of the clamping device that holds the test strip ensures evaporation of excess fluid to prevent backflow along the test strip.

Previously described analyzes of biological samples, in particular, analyzes of analytes in oral fluid, have generally required at least two different actions. First, the sample is collected, eg, blood or urine. The collection sample is then well stored, eg, for later analysis in a laboratory, or it is analyzed in or by an analysis device which is commonly a device other than the collection device. Such tests, which require multiple components, are usually expensive to manufacture and cumbersome for home use.

In addition, particularly with regard to the analysis of oral fluid samples, oral fluid is usually in short supply, particularly in circumstances where the subject of the analysis is under stress (e.g., when analyzing drugs or diseases that pose a threat for life) which can make it difficult to use such multi-component tests. Additionally, attempts to stimulate oral fluid production (eg, through the use of citric acid or other salivating agents) result in increased saliva production, which can actually dilute the analyte concentration.

When common absorbent pads are used to retrieve oral fluid, they generally must be compressed to release the retained oral fluid. The manipulations associated with the compression step can result in sample contamination. Furthermore, such "traditional" short beds have a significant void volume, requiring that the sample usually be collected in a significantly larger volume than actually required for the analyte analysis itself.

The present invention is specified in the claims.

This invention provides improved devices and methods for one-step oral fluid collection, and detection and / or quantification of analytes in oral fluid. The devices and procedures require extremely low volumes of oral fluid, and do not require subsequent handling of samples after

ES 2 264 580 T3 collection. The adequate collection of the sample is immediately verified and the risk of contamination of the sample is minimized. The analyzes are direct, fast and do not require complicated steps. The devices and procedures are therefore ideally suited for use at home, at work or in the office, and generally do not require the presence of qualified medical personnel.

Unlike prior art oral fluid collection devices, which typically use an absorbent pad of paper, cellulose, cotton, or sponge, and require compression of the collection pad to release the fluid sample Orally, the devices of this invention utilize a relatively rigid capillary matrix, also referred to as a capillary matrix. The capillary matrix, when introduced into the oral cavity of a mammal (eg, a human) absorbs oral fluid (eg, by capillary action) and delivers it to the receiving area of a strip of lateral flow chromatography. Oral fluid is rapidly released from the capillary matrix to the lateral flow chromatography strip without any manipulation (eg, compression) of the matrix.

In one embodiment, this invention provides an apparatus for lateral flow chromatography of an oral fluid. The apparatus comprises a capillary matrix having an exposed surface for introduction into an oral cavity; and a lateral flow chromatography strip, wherein the lateral flow chromatography strip is attached to the capillary matrix such that when the capillary matrix comes into contact with an oral mucosa of an oral cavity, the capillary matrix absorbs the oral fluid and delivers it into a receiving area of a lateral flow chromatography strip. In another embodiment, the apparatus comprises a capillary matrix having an exposed surface for receiving oral fluid; and a lateral flow chromatography strip, wherein the lateral flow chromatography strip is in communication with the capillary matrix such that when the capillary matrix receives the oral fluid, the capillary matrix absorbs the oral fluid and delivers it to an area reception of said lateral flow chromatography strip.

In a preferred embodiment, the capillary matrix is composed of a different material from the material that comprises the lateral flow chromatography strip or the receiving zone or the short sample bed of such strip. The capillary matrix is composed of a material such that saturation of the capillary matrix with an oral fluid does not substantially alter the morphology of the capillary matrix. Thus, neither the average pore size nor the hollow volume of the capillary matrix is substantially altered. Furthermore, the volume of the capillary matrix is substantially constant. The saturation of the capillary matrix generally exerts a volumetric change of less than 30%, preferably less than 25%, more preferably less than 20%, most preferably less than about 15%, 10%, 5% or even less than about 1%. The capillary matrix preferably has an average pore size ranging from about 40 µm to about 250 µm, more preferably from about 60 µm to about 200 µm, and most preferably, from about 80 µm to about 120 µm, and a volume gap less than approximately 60 Jul / cm3. Particularly preferred porous matrix materials have pore sizes ranging from about 45 pm to about 90 pm, from about 90 pm to about 130 pm, or from about 80 pm to 120 pm. Preferred capillary matrix materials are plastics (e.g., porous matrices of a high density polyethylene (HDPE), an ultra-high molecular weight polyethylene (UHMW), a polypropylene (PP), a polyvinylidene fluoride (PVDF) ), a polytetrafluoroethylene (PTFE), a nylon 6 (N6) or a polyethersulfone (PES)). Plastics can be hydrophilic or treated (eg, with a surfactant such as sodium N-methyl cocoyl taurate) to be hydrophilic.

In a preferred embodiment, the capillary matrix, when it comes into contact with an oral mucosa, absorbs the oral fluid from said oral cavity and immediately releases that oral fluid in said receiving zone of said lateral flow chromatography strip in less than approximately 1 minute. no compression, alteration of fluid or air pressure or other manipulation of the matrix material. Administration comprises about 100 µl to about 200 µl of oral fluid on said lateral flow chromatography strip in less than about 1 minute. The capillary matrix, when in contact with an oral mucosa, absorbs the oral fluid from the oral cavity and releases said oral fluid in said receiving zone of said lateral flow chromatography strip, preferably, in less than about 30 seconds. Under these conditions, the capillary matrix is preferably saturated with an oral fluid in less than about 1 minute, and saturation commonly uses less than about 500 µl of oral fluid. Generally speaking, the capillary matrix will release enough oral fluid to saturate the receiving area of the chromatographic strip.

The apparatus may optionally further include a blocking strip positioned between the capillary matrix and the lateral flow chromatographic strip. The blocking strip can contain a blocking reagent (eg, BSA, deoxycholate, n-lauroylsarcosine sodium, etc.) and / or one or more buffers. The blocking strip can also prevent the reagents from refluxing from the lateral flow chromatography strip into the capillary matrix.

The apparatus may optionally further include a conjugate strip containing one or more chromatographic reagents (eg, labeled microparticles). In addition, a single strip can serve as both a blocking strip and a conjugate strip.

The apparatus may further comprise a housing having a cavity, wherein said lateral flow chromatographic strip extends in the cavity along the housing to an inspection zone on the housing; and at least one inspection zone from an exterior of the housing to the lateral chromatographic strip to allow visual inspection of the reagents at selected points on the lateral chromatographic strip. The housing can act as a handle to introduce the capillary matrix into the oral cavity.

ES 2 264 580 T3

In a particularly preferred embodiment, a testing device is disclosed comprising a single unit, continuous line, one-stage rapid testing format suitable for oral specimen collection and testing. More specifically, a hydrophilic capillary matrix is provided as a transport for oral fluids to a lateral chromatographic strip. The lateral chromatographic strip is inserted into a cavity defined in a housing and is disposed along the housing to an inspection zone. A hydrophilic capillary matrix protrudes from the shell to an outer oral collection zone of the shell at one end and communicates with the lateral chromatographic strip at the other end. This hydrophilic capillary matrix defines a matrix of defined conduits between non-absorbent materials, such as either plastic spheres or foams. The outer surfaces of the matrix are hydrophilic either by being naturally hydrophilic or by being treated to be hydrophilic. The dimension of the interstitial matrix is such that the forces of capillary action cause the materials to fall immediately into the hydrophilic capillary matrix. The hydrophilic capillary matrix immediately releases the oral fluid onto the lateral chromatographic strip. Prevention of backflow into the oral cavity from the lateral chromatographic strip occurs naturally due to the tortuous ducts of the porous absorption material. By observing the side chromatographic strip when the entire test device is in the mouth, immediate test results are obtained.

In another embodiment, this invention provides a method for the detection or quantification of one or more analytes in an oral fluid. The procedure involves the steps of: i) introducing into the oral cavity of a mammal any of the oral fluid analysis devices described herein, such that the capillary matrix comes into contact with a surface of the oral mucosa, by means of which which the capillary matrix absorbs the oral fluid and delivers it into a receiving zone of a lateral flow chromatography strip; and ii) reading a signal on the lateral flow chromatography strip indicating the presence, absence or quantity of one or more analytes.

This invention also provides kits for the detection of an analyte in an oral fluid. The kits include an apparatus for the collection and lateral flow chromatography of an oral fluid as described herein, as well as information materials that describe the use of the apparatus.

Definitions

As used herein, the term "analyte" is used to refer to a residue that is to be detected in a given assay. Analytes can be atoms (elements), molecules, or groups of molecules. Analytes commonly detected in the assays of this invention include, but are not limited to, antibodies, antigens, growth factors, enzymes, therapeutic drugs, drugs, and the like. Particularly preferred analytes include antibodies and antigens relevant to infectious and non-infectious diseases.

As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Recognized immunoglobulin genes include the genes for the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as the thousands of genes for the immunoglobulin variable regions. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

The basic (antibody) structural unit of immunoglobulin is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The N terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain (VL) ”And“ variable heavy chain (VP) ”Refer to these light and heavy chains respectively.

Antibodies can exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds of the hinge region to produce F (ab) '2, a Fab dimer that is itself a V-linked light chainP-CP1 via a bisulfide bond. The F (ab) '2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thus converting the F (ab) 'dimer2 in a Fab 'monomer. The Fab 'monomer is essentially a Fab with part of the hinge region (see, "Fundamental Immunology", WE Paul, ed., Raven Press, NY (1993) for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digest of an intact antibody, it will be appreciated by one of ordinary skill that such Fab 'fragments can be synthesized de novo either chemically or by using recombinant DNA methodology. Therefore, the term "antibody" as used herein also includes antibody fragments either produced by modifying whole antibodies or synthesized de novo using recombinant DNA methodologies.

The term "oral fluid", as used herein, refers to one or more fluids found in the oral cavity individually or in combination. These include, but are not limited to, saliva and mucosal transudate. It is recognized that oral fluid (eg, saliva) may comprise a combination of fluids from a number of sources (eg, parotid gland, submandibular gland, sublingual gland, accessory glands, gingival mucosa and buccal mucosa), and the term "oral fluid" includes fluids from each of these sources individually or in combination. The term "saliva" refers to a combination of oral fluids

ES 2 264 580 T3 such as that normally found in the mouth, in particular after chewing. The term "mucosal transudate", as used herein, refers to fluid produced by passive diffusion of serum components from the interstices of the oral mucosa into the oral cavity. Mucosal transudate usually forms a component of saliva.

The terms "capillary matrix" and "porous matrix" are used herein to refer to a highly porous material characterized by a pore size small enough that the material rapidly absorbs an aqueous solution (eg, fluid. oral) predominantly through capillary action or 'absorption'.

The term "absorption" is used to refer to the absorption of a fluid predominantly by adsorption and capillary action.

The term "lateral flow chromatography strip" refers to a test strip used for lateral flow chromatography. Commonly, lateral flow (chromatographic) analyzes involve the application of a liquid sample suspected of containing an analyte by being detected in a lateral flow (immunochromatographic) test strip application site. The strip is composed of a matrix material (eg, paper, nitrocellulose, etc.), see, eg, US Patent No. 5569608) through which the fluid sample and the suspended or dissolved analyte therein it can flow by capillary action from the application zone to a detection zone in which a visible signal, or the absence of such signal, reveals the presence or absence of the analyte. If the detection of the analyte uses an antibody or an antibody fragment, the analysis may be called a lateral flow immunochromatography assay, and the strip, a lateral flow immunochromatography strip.

A "receiving zone or the short bed for samples of said lateral flow chromatography strip" refers to the zone of the lateral flow chromatography strip where a sample is applied first.

A "signal on said lateral flow chromatography strip" refers to an indication, normally, in a certain predefined region of the chromatography strip that indicates the presence, absence, or an amount of analyte, or a sufficient amount of sample. on the chromatography strip. The signal can be colorimetric, fluorescent, electroluminescent, radioactive, etc.

As used herein, "housing" refers to any member that coats or supports, but does not react with, the lateral flow chromatographic strip.

As used herein, "cavity" refers to any receiving volume on or within the housing to hold the lateral chromatographic strip.

The "lateral flow chromatographic strip" is any absorbent member capable of transporting analyte and reagents to the visual inspection zone. The strip can be nitrocellulose, cellulose acetate, paper, nylon, cellulose, or any other highly absorbent material.

As used herein, "an immunoassay" is an assay that uses an antibody or antigen to specifically bind to the analyte. Immunoassay is characterized by the use of specific binding to a certain antibody, as opposed to other physical or chemical properties, to isolate, target, and quantify the analyte.

The phrase "specifically binding to an analyte" or "specifically immunoreactive with", when referring to an antibody, refers to a binding reaction that determines the presence of the analyte in the presence of a heterogeneous population of molecules such as proteins. and other biological compounds (ie, such as those that can be found in oral fluid). Therefore, under designated immunoassay conditions, specified antibodies bind to a certain analyte and do not bind in a significant amount to other analytes present in the sample. A variety of immunoassay formats can be used to screen for antibodies specifically immunoreactive with a particular analyte. For example, solid phase ELISA immunoassays are automatically used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) "Antibodies, A Laboratory Manual", Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

A "tag" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Tags useful in the present invention include magnetic beads (eg, Dynabeads®), fluorescent dyes (eg, fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like), radio tags (eg. ,3H, 1251, 35Yes, 14C or 32P), enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric labels such as colloidal gold, colored glass, or plastic beads (eg, polystyrene, polypropylene, latex, etc). Patents teaching the use of such labels include US Patent Nos: 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. The means of detecting such tags are known to those skilled in the art. Thus, for example, it is possible to detect radio tags using photographic film or scintillation counters; fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzyme tags are normally detected by providing the enzyme with a substrate and detecting the reac5 product.

ES 2 264 580 T3 tion produced by the action of the enzyme on the substrate, and the colorimetric labels are detected simply by visualizing the colored label.

Fig. 1 is a schematic embodiment of an oral collection device of this disclosure illustrating generally from left to right an oral fluid transport wick, a blocking short bed, a gold conjugate short bed, a lateral chromatographic strip with an observation window and, finally, a short final absorption bed, all contained in a simple housing;

fig. 2 is a schematic embodiment of an alternate oral sample collection device similar to that of FIG. 1, which omits the blocking short bed and has the gold conjugate applied to the end of the wick material; and fig. 3 is a three-dimensional rendering of the test device with the wick cover removed for use in the oral cavity of a human.

This invention provides a device for the rapid one-stage collection and detection of analytes in oral fluid. In a preferred embodiment, the device is introduced into the oral cavity (eg, preferably juxtaposed with the oral mucosa) where it absorbs oral fluid. After a period of time, the device is detached from the oral cavity, and one or more indicators contained in the device are read (eg, by visual inspection or by detection on a "reader") to provide an indication. of the presence or absence and / or the amount of one or more analytes of interest. The device thus provides a non-invasive, single-step rapid analysis for the detection of one or more analytes of interest.

The test devices and methods of this invention can be used for the detection (positive or negative, and / or quantification) of almost any analyte present in oral fluid. Furthermore, devices and procedures can be used to detect one or more analytes simultaneously. Such analytes may include, but are not limited to, HIV antibodies, HTLV antibodies, Helicobacter pylori antibodies, hepatitis antibodies, measles antibodies, mumps antibodies, rubella antibodies, cotinine, cocaine, benzoylecgonine, benzodizazpine, tetrahydrocannabinol, nicotine, ethanol theophylline, phenytoin, acetaminophen, lithium, diazepam, nortriptyline, secobarbital, phenobarbitol, theophylline, testosterone, estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, transforming growth factor-type alpha, epidermal growth factor, insulin type I and II growth factor, growth hormone release inhibiting factor, IgA and sex hormone binding globulin; and other analytes including glucose, cholesterol, caffeine, cholesterol, corticosteroid-binding globulin, EPA, or DHEA-binding glycoprotein, the methods being particularly suitable for the detection of HIV antibodies.

The analysis device of this invention is based on the exclusive cooperation between a capillary matrix (W in Figure 1) and a lateral flow chromatographic strip (C in Figure 1). The device is constructed such that the capillary matrix can be introduced into the oral cavity and, in a preferred embodiment, juxtaposed with the oral mucosa. The capillary matrix acts as a receptor body or short bed that rapidly absorbs oral fluid, eg, through capillary action, and delivers the oral fluid on a lateral flow immunochromatography strip (eg, C in figure 1). The immunochromatography strip then provides an indication of the presence, absence, or amount of one or more analytes in the oral fluid.

The device may be conveniently assembled such that the capillary matrix comprises a receptor short bed for introduction into the oral cavity, while the lateral flow immunochromatography strip comprises the handle of the device. One or more areas of the handle may comprise indicators for reading (eg, a colorimetric signal) of the results of the analysis. Of course, there are other formats of the device that are suitable and will be readily recognized by those skilled in the art.

Capillary matrix

The material of the capillary matrix (the porous matrix) is preferably selected to provide a number of unique properties to the analysis device. Such properties include, but are not limited to, relatively low void volume, sufficient pore size to provide rapid and efficient delivery of the oral fluid on the test strip, non-reactivity or low reactivity with the oral fluid or analytes. , easy release of oral fluid onto the immunochromatographic test strip, and a short, non-deformable (when wetted) collection bed.

Because oral fluid can be in short supply (patients often have a “dry mouth” during the test), it is desirable to maximize the amount of oral fluid that is transported from the oral cavity (eg, the oral mucosa) to the lateral flow chromatography strip. This is achieved through the use of a capillary matrix that has a minimal void volume. The capillary matrix should have a hollow volume of less than about 65% / cm3, preferably less than about 57% / cm3, more preferably less than about 48% / cm3with less than about 40% / cm being most preferable3, 35% / cm3 or even 25% / cm3. Capillary matrices that have such low void volumes typically deliver a significant amount of oral fluid absorbed onto the lateral flow chromatography strip.

ES 2 264 580 T3

The matrix itself must be relatively small in dimension. Specifically, it is preferable that the interstices are of a dimension in which the capillary forces cause the fluid to fall into the capillary matrix. Thus, the capillary matrix is also selected to have an average pore size small enough to provide rapid absorption of the oral fluid with which it is in contact (eg, by capillary action). The small pore size also works to exclude particulate matter present in the fluid sample. However, the pore size is also selected to be large enough that the viscous oral fluid does not clog the capillary matrix and instead is transported rapidly through the matrix to the lateral flow chromatography short bed. . Preferred materials have an average pore size ranging from about 40 µm to about 250 µm, more preferably, from about 60 µm to about 200 µm, and most preferably, from about 80 µm to about 120 µm.

In addition to having a pore size (channel size) that results in rapid absorption of oral fluid, the surfaces of the capillary matrix should be chemically compatible with rapid absorption of oral fluid. Therefore, the capillary matrix materials themselves are hydrophilic or are treated to be hydrophilic (eg, by the addition of a surfactant also referred to as a detergent or wetting agent). That is, the water must flow through and be attracted to the surfaces of these materials.

While there are a number of suitable materials that are naturally hydrophilic (eg, clean sintered glass or molten glass beads), other suitable materials (eg, plastics) are commonly hydrophobic (eg, not moisten easily). However, such hydrophobic materials can be routinely treated with a wetting agent (ie, surfactant / detergent) and thereby rendered hydrophilic (wetting). However, since the capillary matrix is used in the oral cavity, the treatment detergent is required to be known to be non-harmful to the mammalian subject (eg, to the human body) and to preferably be approved for such use. by the relevant regulatory authority (e.g., the US Food and Drug Administration). In another preferred embodiment, it is possible to render a porous plastic material (eg, polyethylene or polypropylene foam) hydrophilic by taking the raw matrix material and placing it in a dilute aqueous solution of an approved detergent such as N -methyl-cocoyl-sodium taurate. Thereafter, the treated material is dried, leaving the surfaces of the matrix apparently coated with a thin layer of detergent.

Although sodium N-methyl cocoyl taurate is preferred, it will be understood that other detergents can also be used. It is only required that the detergent, for the safety of oral mammalian exposure, does not interfere with the test on the lateral chromatographic strip C, and produces the required hydrophilic properties on the outer surfaces of the matrix.

In addition, to rapidly absorb and transport the oral fluid onto the lateral flow chromatography strip, the capillary matrix material is selected to preferably immediately release the fluid onto the chromatography strip. This should be accomplished quickly without compression of the matrix material itself. Therefore, in a preferred embodiment, the capillary matrix delivers and releases the oral fluid onto the lateral flow chromatography strip without any manipulation (eg, without squeezing or compressing the capillary matrix).

From the foregoing, it should be clear that preferred capillary matrix materials have interstitial spacing that facilitates absorption of oral fluid through capillary attraction in combination with adsorption on the material. This causes the oral fluid collected from the mouth to be transported to the lateral chromatographic strip instead of staying in the mouth. At the same time, when the oral fluid reaches the lateral chromatographic strip, it is absorbed by the strip instead of remaining in the capillary matrix.

In a preferred embodiment, the hydrophilic capillary matrix is an essentially non-absorbent matrix that absorbs liquid by capillary action. In such absorption, the volume of the material is not appreciably affected. Furthermore, the capillary matrix material is relatively rigid such that its morphology remains essentially unchanged during analysis (eg, when saturated with oral fluid). Therefore, saturation of the matrix with an oral fluid does not substantially alter the mean pore size and void volume of the porous matrix. Furthermore, saturation of the capillary matrix with an oral fluid results in a volume change of less than 30%, preferably less than 25%, more preferably less than 20%, most preferably less than about 15%, 10%, 5%, or even less than about 1%.

In a particularly preferred embodiment, the capillary matrix can act as a barrier against the reflux of reagents from the lateral flow chromatography strip into the capillary matrix. This can be achieved, for example, when the chromatographic strip has a larger volume for fluid storage than the capillary matrix. Additionally, or alternatively, when the lateral flow chromatography strip is more hydrophilic than the capillary matrix, the capillary matrix can also act as a reflux barrier.

The capillary matrix materials are selected such that they are not chemically reactive with either the oral fluid or the analytes contained therein. Matrix materials compatible with oral fluid are known to those of skill in the art, and include, but are not limited to, glass, resins, and various plastics.

In a preferred embodiment, the properties described above are achieved through the use of porous plastic materials for the capillary matrix. Suitable porous plastic materials include, but are not limited to, porous matrices of high density polyethylene (HDPE), ultra high molecular weight (UHMW) polyethylene,

ES 2 264 580 T3 polypropylene (PP), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), nylon 6 (N6) and polyethersulfone (PES). In a preferred embodiment, the porous matrix materials are either hydrophilic on their own (to easily absorb oral fluid) or are treated (eg, with a surfactant / detergent) to be hydrophilic.

Such porous plastics are commercially available (see, eg, Porex Technologies, Fairburn, GA). Particularly preferred porous plastics are detergent (surfactant) treated polyethylene and / or polypropylene. Treatment typically involves soaking the hair matrix in a surfactant / detergent and then allowing it to dry naturally or by force drying the material.

Particularly preferred porous matrix materials are Porex X-4588, 80-120 µm pore size at 0.061 cm (0.024 inch) thickness of polypropylene. Also suitable are Porex X-4903 at a thickness of 0.159 cm (0.0625 inches), pore size of 45-90 μιη, and Porex X-4913 at a thickness of 0.159 cm (0.0625 inches) and a pore size of 90-130 μm. In a preferred embodiment, these materials are treated with sodium N-methylcocoyl taurate. The materials of the capillary matrix are soaked in the detergent which is then dried on the surface that comprises the porous matrix.

It will be understood that the Porex7 materials that are used do not retain large volumes of oral fluid. For example, the following data is considered:

TABLE 1

Porex® X-4903 Hollow Volume Middle Pore

Porex X-4903 1 cm x 1 cm x 0.1588 cm

<td> #</td><td>Dry weight (g)</td><td>Weight + H2 O (g)</td><td>H weight2O (g)</td><td>% volume / cm3</td>

TABLE 2

Porex® X-4913 Hollow Volume Coarse Pore

Porex X-4913 1 cm x 1 cm x 0.1588 cm

<td> #</td><td>Dry weight (g)</td><td>Weight + H2 O (g)</td><td>H weight2O (g)</td><td>% volume / cm3</td>

In the above, we assume that 1 cm3 = 1 ml = 1 g of H2OR

ES 2 264 580 T3

At the same time, the hydrophilic capillary matrix W does not have a high relative oral fluid retention. For example, it immediately delivers its fluid to the lateral chromatographic strip C and the absorbent short bed A. It will be understood that the hydrophilic capillary matrix W acts more like a conduit than an absorbent; the material is immediately discharged from the wick.

Identification of suitable materials for the porous matrix

It will be understood that the rate of absorption, transport to the lateral flow chromatography shortbed, and release into the oral fluid shortbed is a function of both the composition of the capillary matrix and its shape (eg, the surface exposed to the oral mucosa, the transverse surface and the surface in contact with the lateral flow chromatography strip). The rates are also affected by the mean pore size, the hydrophilicity of the capillary matrix material, and the relative absorbance characteristics of the capillary matrix and the lateral flow chromatography short bed.

These parameters can be optimized according to standard procedures known to those skilled in the art. In one embodiment, this is accomplished by assembling a test device having the desired capillary matrix material and a lateral flow immunochromatography strip. The test device can then be contacted with an oral fluid sample solution (eg, natural oral fluid or synthetic oral fluid, see, US Patent 5,695,929 and co-pending). USSN 08 / 608,431).

Contacting can be accomplished by actual introduction of the receiving part (eg, short bed / receptor face) of the capillary matrix into the oral cavity and contacting an oral mucosa (eg, a human or animal under test). Alternatively, contacting can be accomplished by touching the capillary matrix with the test fluid arranged on a surface or a bowl or other container, immersing part or all of the capillary matrix in the test fluid, or contacting the capillary matrix with a test body (eg, a sponge, cloth, smear, etc.) impregnated with the test fluid.

Typically, the oral fluid sample will be in contact with the capillary matrix for the desired period of time to run the analysis (e.g., less than 5 minutes), and then the lateral flow chromatography strip can be read in in terms of the presence, absence or amount of analyte and / or in terms of the amount of oral fluid absorbed. This will determine if the analysis offers adequate sensitivity and specificity.

In addition, it is possible to determine the time period for the absorption of the oral fluid in the capillary matrix and the lateral flow chromatography strip. Similarly, the total sample volume required to adequately saturate the lateral flow chromatography strip and the amount of fluid retained by the capillary matrix can also be established (e.g., weighing the various components before and after analysis). .

In a preferred embodiment, the capillary matrix, when inserted and held in the mouth is saturated with oral fluid in less than about 5 minutes, preferably, in less than about 3 minutes, more preferably, in less than about 1 minute, being most preferably in less than about 30 seconds. Similarly, the capillary matrix will release enough fluid on the chromatography strip to properly run the analysis (a run of the analysis according to the specifications of the chromatographic strip) in less than about 10 minutes, preferably, in less than about 5 minutes, plus preferably in less than about 3 minutes and most preferably, in less than about 2 minutes or even in less than about 1 minute without compressing the porous matrix.

A porous matrix will be made that is saturated to less than about 500 µl, more preferably less than about 300 µl, and most preferably less than about 100 µl.

It will be recognized that when only the absorption and release properties of the oral fluid are to be analyzed, there is no need to use a full chromatographic analysis. The base material of the capillary matrix, the lateral flow chromatography strip and, if desired, the blocking short bed can be assembled. The rate of absorption and delivery of fluid in the short bed can be quantified by immersing or contacting the short bed of the capillary matrix with a fluid sample (e.g., natural or synthetic oral fluid) and quantifying the rate of absorption. and administration of fluid to the short bed and / or the amount retained in the capillary matrix (eg, weighing the various elements after certain preselected times of exposure to the test fluid). A combination of materials and shaped elements is preferred that provides the maximum delivery of an oral fluid from an oral mucosa to the lateral flow chromatography strip in the shortest possible time.

In a particularly preferred embodiment, the lateral flow chromatography strip is a nitrocellulose strip (eg, Syntron QuickScan 6, Avitar Visualine II, Avitar Technologies, Canton, Massachusetts). A typical chromatographic strip is a Millipore SRHF nitrocellulose membrane (Millipore Corp., Bedford, Massachusetts) having dimensions of 4mm x 50mm. A porous matrix material measures 7mm x 52mm [0.159cm (0.0625in) thick] and overlaps the nitrocellulose membrane by approximately 64mm2. In another embodiment, the capillary matrix is paddle-shaped having a total surface area of approximately 720mm.2 and overlaps the chromatographic strip by approximately 8 mm2. The small overlap is particularly well suited in embodiments containing a conjugate pad, which serves to effectively channel oral fluid through the conjugate pad.

ES 2 264 580 T3

Side Flow Chromatography Strip

The assay device of this invention can utilize almost any lateral flow chromatography strip for the detection and / or quantification of the analyte (s). Lateral flow chromatography analyzes are known to those skilled in the art (see, eg, US Patents 5,569,608; 5,120,643; 5,656,503; 4,855,240; 5,591,645; UK Patent GB 2204398A and European Patent EP 0323605 B1), and such assays are commercially available at retail or from original equipment manufacturers for numerous analytes.

Lateral flow immunoassays usually involve the application of a liquid sample suspected of containing an analyte to be detected in an application site of a lateral flow (immunochromatographic) test strip. The strip is composed of a matrix material (eg, paper, nitrocellulose, etc., see, eg, US Patent No. 5569608) through which the fluid sample and the suspended or dissolved analyte therein they can flow by capillarity from the application zone to the detection zone in which a visible signal, or the absence of such signal, reveals the presence or absence of the analyte.

Typically, the strip will include a means to immunospecifically bind the analyte to be detected with its specific binding partner (eg, when the analyte is an antigen, the binding partner is an antibody or antibody fragment, and vice versa) bearing a detectable label. In a scheme such as that disclosed in US Patent No. 4,446,232, the strip contains a labeled enzyme, a mobile binding partner for the analyte that is in a zone downstream of the sample application zone. If the analyte is present in the sample, it will combine with its labeled binding partner to form a complex that will flow along the strip to a detection zone that contains a substrate for the enzyme label capable of providing a signal (e.g. g., a colored response) in the presence of the enzyme tag.

The strip commonly contains an area where the analyte becomes immobilized, so that the labeled binding partner that does not combine with the analyte, due to the absence of analyte in the sample, will be captured, thereby inhibiting its ability to reach the detection zone. Various modifications of this technique have been published, many of which assume some specific competitive binding system in which the presence or absence of the analyte in the sample is determined by the detection or lack of a labeled binding partner in the region of detection. In US Patent No. 4,868,108, a similar scheme is disclosed with the addition of an immobilized capture reagent for the enzyme-labeled binding partner in the detection zone to concentrate the enzyme tag and increase its ability to react with the enzyme tag. enzyme substrate, and thereby make the analysis more sensitive.

Not all schemes for immunochromatography rely on an enzyme-labeled binding partner / enzyme substrate to provide the signal for analyte detection. In US Patent No. 4,806,311, a multi-zone test device is disclosed for the determination by a specific binding assay of an analyte and an immobilized binding partner, therefore, together with a detection zone for receive the labeled reagent that migrates into it from the reagent zone. The detection zone contains an immobilized form of a binding substance for the labeled reagent. The labeled reagent carries a detectable chemical group that has a detectable physical property that is detectable based on its own physical properties, so it does not require a chemical reaction with another substance. Examples of such groups are fluorescent colored species, phosphorescent molecules, radioisotopes, and electroactive moieties. US Patent No. 4,313,734 describes the use of gold soles as labels for antibodies that are detectable without a chemical change.

Many lateral flow immunochromatography systems use particulate (microparticulate) markers (eg, gelatin, stained latex, or colloidal gold) that are labeled with a binding partner (eg, an antibody or antigen) that binds to the analyte of interest.

Microparticles or other detectable moieties attached to an analyte-binding moiety (eg, an antibody or an antigen) are dried on (or otherwise located on) either a lateral flow chromatographic strip or on a short bed of sample application (typically a fiberglass) that is in turn attached to one end of a strip of chromatographic medium such as nitrocellulose. Other material that binds to the analyte of interest is attached to the chromatographic medium at or near the end opposite the end that has the short application bed.

The liquid sample to be analyzed is placed in the short bed, causing suspension of the microparticles in the liquid and allowing any analyte in the liquid sample to bind to the analyte-binding material bound to the microparticles. The liquid sample leaves the short application bed by diffusion and capillary action, and begins to migrate along the nitrocellulose strip transporting the microparticles down the strip together with the liquid. When the liquid containing the suspended microparticles reaches the region of the chromatographic strip that carries the second binding material, the analyte (if present in the original sample) will form a molecular bond between the analyte binding material that is on the microparticles and analyte-binding material that is attached to the strip, which results in the immobilization of the microparticles at the point on the strip where the analyte-binding material is fixed. This immobilization of the microparticles results in a visible signal (eg, a colored strip or not) at this point on the strip. If the analyte is not present in the sample, the microparticles will continue to pass through this point on the chromatographic strip, without producing any visible signal.

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It will be understood that it is possible to use other labels (eg, fluorescent labels) in addition to microparticles. Additionally, a single chromatographic strip can contain reagents to detect or quantify a number of different analytes.

It will also be understood that the lateral flow strip can use analyte detection that does not involve the use of an antibody-antigen recognition system. Thus, for example, the strip can be impregnated in a detection zone with a chemical that reacts with the analyte itself to produce a signal.

The devices of this invention can be readily assembled with any of a variety of commercially available lateral flow chromatography assays (eg, Syntron QuickScan 6, Avitar Visualine II, Avitar Technologies, Guangzhou, Massachusetts, etc).

Optional lock strip

The analysis devices of this invention can optionally include a blocking strip between the porous matrix and the lateral flow chromatography strip. The blocking strip may be impregnated with buffers to adjust the pH (eg, up to pH 7.5) of the oral fluid to be compatible with lateral flow chromatography analysis. The blocking strip can also include one or more blocking reagents that reduce the nonspecific binding of the analyte and / or test device reagents, thereby reducing the occurrence of false positives. Suitable blocking reagents include, but are not limited to, bovine serum albumin (ASB), deoxycholate, and n-lauroylsarcosine. In a particularly preferred embodiment, the blocking solution includes: 4% polyvinyl alcohol (MW: 10 kd (Aldrich 36,062-2); 4% sodium n-lauroylsarcosine (Sigma L5777), 2% polyvinyl pyrrolidone (MW: 10 kd, Sigma P2263) in 60 X Tris EDTA (Sigma T9285) or 7.5% sodium n-lauroylsarcosine (Sigma L5777), 2.5% tectronic 1307 (BASF), 7.5% polyethylene glycol compound (Sigma P2263) in 100x Tris EDTA buffer (Sigma T9285).

The blocking short bed can be composed of a wide variety of materials as long as they do not impede the flow of oral fluid from the capillary matrix to the lateral flow chromatography strip. Such materials include, but are not limited to, paper, cellulose, nitrocellulose, and the like. Particularly preferred materials will be selected to reduce or eliminate reflux of reagents or oral fluid from the chromatographic test strip into the capillary matrix. In a preferred embodiment, when present, the blocking short bed is a # 470 cellulose filter from Schleicher & Schuell (Schleicher & Schuell, GMBH, Germany).

The optional conjugate strip

In one embodiment, the lateral flow chromatography reagents (eg, antibody-labeled gold particles, etc.) are arranged on or on the lateral flow chromatographic strip itself. However, in another embodiment, there may be one or more chromatographic reagents arranged on a conjugate strip (G), e.g. eg to facilitate manufacturing. The conjugate strip is placed juxtaposed to the lateral flow chromatography strip such that oral fluid must pass through or through the conjugate strip in order to migrate up the chromatographic strip. Alternatively, the conjugate strip may be woven into the lateral flow chromatography strip or it may be aligned, in the same plane, as the lateral flow chromatography strip. As with the blocking strip, the conjugate strip can be formed of any convenient material (eg, nitrocellulose, fiberglass, polyester, etc.) that is compatible with the test and does not substantially impede the flow of the oral fluid. and the reagents. In a preferred embodiment, the conjugate pad is a 4mm x 4mm glass fiber short pad or a polyester short pad (eg, Ahlstrom Remay # 2033).

Assembling the analysis device

The components (eg, capillary matrix, lateral flow chromatography strip, optional blocking strip) can be assembled by any of a wide variety of means known to those skilled in the art. Thus, for example, the components can be welded or glued and the like.

In a preferred embodiment, the components are simply pressed and held in place by a housing (H). The housing (H) can be of any desired construction. For example, in a prototypical construction, soda straws were used that provided both adequate structure and visibility to measure the accuracy of the test. Acrylic tubing has also been used. It will be recognized that other standard pipe materials can be used, or that specially designed housings can be custom molded or extruded.

The components were assembled such that the lateral flow chromatography strip was arranged longitudinally in the straw, being fixed at one end to one end of the capillary matrix (W). The H-shell thus provided a convenient handle for the introduction of the capillary matrix into the oral cavity of a mammal (eg, a human).

Figures 1, 2 and 3 illustrate various embodiments of the analysis device of this invention. The immunochromatography or lateral flow chromatography strip (C) is arranged longitudinally in the housing (H). One end of the chromatographic strip is in direct contact, or via the blocking short bed (B), with a part of the capillary matrix (W). The capillary matrix (W) protrudes from the housing (H) in which it has a face (3) that acts as an absorbent surface to absorb the oral fluid. Oral fluid migrates through the matrix (W) and through the bed

ES 2 264 580 T3 blocking short (B), if present, where it is finally delivered to a receiving zone (R) on the lateral flow chromatography strip.

The oral fluid then migrates along the lateral flow chromatography strip where it interacts with various reagents (eg, antibody- or antigen-labeled binding partners (eg, antibodies or antigens)) that are deposited on the chromatographic strip and / or on the optional conjugate short bed (G) which, when present, is in contact with the chromatographic strip.

The oral fluid / reagent combination continues to migrate along the chromatographic strip until it reaches one or more indicator zones (20). If an analyte is present, the indicator zone immobilizes the bound labeled analyte or, if not, produces a detectable signal. One or more of the indicator areas may also indicate a sufficient amount of sample (eg, with a color change) when the required sample volume and / or required analyte concentration is reached. Indicators of a sufficient amount of sample are known to those skilled in the art, and in co-pending application USSN 08 / 456,459, as well as US Patent No. 5,479,937, indicators are particularly described. advantageous of the sufficient quantity of sample.

Indicator areas can be read (eg, by visual inspection or by other means) through viewing windows (18). The device is optionally equipped with a blocking short bed (B). This short bed can be impregnated with a blocking reagent (eg, n-lauroylsarcosine) and / or one or more buffers to adjust the pH of the sample. In addition, the blocking pad can be formed of a semi-permeable material that allows oral fluid to flow into the chromatographic strip, but prevents backflow of oral fluid and / or reagents into the capillary matrix. The other end (the opposite side of the capillary matrix) of the chromatographic strip may have an absorbent short pad attached which acts as a reservoir to receive the oral fluid, thereby preventing backflow into the capillary matrix.

It will be understood that the devices can be assembled in a wide variety of ways. In Figure 3, a preferred embodiment is illustrated. In this embodiment, the lateral flow chromatography strip (C) is arranged in a housing (H) that acts as a handle for handling the device. The housing / handle (H) is provided with viewing windows (18) to view the sufficient amount of sample and the results of the analysis. The capillary matrix protrudes from the housing to provide a planar surface to be inserted into the oral cavity where the face (3) of the capillary matrix can contact the oral mucosa.

For convenience, the analysis device can be optimally equipped with a cover (22) for protecting the surface of the capillary matrix before and after use. This prevents contamination of the testing device and facilitates sanitary disposal of used devices.

The reader will understand that with the interaction of the hydrophilic capillary matrix W and the lateral chromatographic strip C, an extremely simple construction is possible. Also, the end product works in a similar way to a conventional thermometer. The test is quick, showing the prototypes the full test results in about 2 minutes. Furthermore, the test easily leads to the stimulation of saliva itself, such as by placing slightly acidic components at the end of the wick.

Most importantly, the test requires only minimal volumes of fluid from the oral cavity to be performed. For example, in the designs shown, the analyzes used only 100 to 200 µl of oral fluid. This is an improvement of at least a factor of 4 over the sample volume requirements of the previous analyzes.

Using the analysis device

In use, the testing device of this invention is inserted into the oral cavity of a mammal (eg, a human) such that the handle in which the chromatographic strip is disposed is outside the mouthpiece. The face of the capillary matrix preferably comes into contact with the oral mucosa and, in a particularly preferred embodiment, is pressed into the oral mucosa, at the gingival ridge, eg, pressed between the cheek and the gums.

The testing device is held in place, preferably without chewing, until a sufficient amount of sample has been collected. This can be for a predetermined time interval or until the capillary matrix achieves a characteristic tactile quality, or until an indicator of a sufficient amount of sample (eg, a color change) indicates a suitable sample.

At the recommended time (after completion of immunochromatography analysis), the device is read (e.g., by visual inspection of the indicator area (s) through the observation window (s), to determine if the subject is positive or negative for the analyte (s) of interest or the amount of analyte (s).

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Equipment for the detection of analytes in oral fluids

In another embodiment, this invention provides kits for the detection of one or more analytes in oral fluids. The kits include one or more of the analysis devices described herein. In addition, kits may include informational materials that contain instructions (ie, protocols) for practicing the testing procedures of this invention. Although informational materials typically comprise written or printed materials, they are not limited thereto. In this invention, any medium capable of storing such instructions and communicating them to the end user is contemplated. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD-ROM), and the like. Such media may include addresses of Internet sites that provide such informational materials.

Kits can optionally contain any of the buffers, reagents, detection reagents, etc., that are useful in practicing the methods of this invention.

30 members in 10 offices

This recordMember, by publication datePriority claim

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Priority claims
Document	Office	Kind	Date
19980079958P	United States of America	–	1998-03-30
7995898	United States of America	P	1998-03-30
7995898	United States of America	P	1998-03-30
79958P99914312	–	–	–
US19980079958P	–	–	–

Members30

Family members, oldest first
Document	Office	Kind
CA2326392A1	Canada	A1	1999-10-07
WO9950656A1	World Intellectual Property Organization (WIPO)	A1	1999-10-07
AU3219099A	Australia	A	1999-10-18
EP1086372A1	European Patent Office (EPO)	A1	2001-03-28
US6303081B1	United States of America	B1	2001-10-16
JP2002510048A	Japan	A	2002-04-02
EP1086372A4	European Patent Office (EPO)	A4	2002-09-18
US2002155029A1	United States of America	A1	2002-10-24
US2002192839A1	United States of America	A1	2002-12-19
EP1086372B1	European Patent Office (EPO)	B1	2006-05-31
AT328278T	Austria	T	2006-06-15
ATE328278T1	Austria	T1	2006-06-15
DE69931632D1	Germany	D1	2006-07-06
EP1696236A2	European Patent Office (EPO)	A2	2006-08-30
DK1086372T3	Denmark	T3	2006-09-18
EP1696236A3	European Patent Office (EPO)	A3	2006-09-20
DE69931632T2	Germany	T2	2006-12-07
ES2264580T3This record	Spain	T3	2007-01-01
US7192555B2	United States of America	B2	2007-03-20
US2007116597A1	United States of America	A1	2007-05-24
US7541194B2	United States of America	B2	2009-06-02
US2009170072A1	United States of America	A1	2009-07-02
EP1696236A8	European Patent Office (EPO)	A8	2010-06-02
US2010239458A1	United States of America	A1	2010-09-23
JP4623536B2	Japan	B2	2011-02-02
US8062908B2	United States of America	B2	2011-11-22
US2012149124A1	United States of America	A1	2012-06-14
EP1696236B1	European Patent Office (EPO)	B1	2014-07-16
DK1696236T3	Denmark	T3	2014-08-18
ES2501241T3	Spain	T3	2014-10-01

Device for assay of antibodies in oral fluids

Abstract

Term

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DESCRIPTION

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TABLE 1

TABLE 2

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Priority claims5

Members30

Numbers

Titles2

Classification