US 5,750,333 A · Reversible flow chromatographic binding assay

Abstract

Disclosed are devices and methods for performing an assay which determines the presence or quantity of an analyte in a fluid sample by detecting binding of the analyte to at least one immobilized analyte capture reagent and washing unbound material from the immobilized analyte capture reagent. The devices and methods an elongated solid phase flow matrix, including capillary channels capable of driving capillary fluid movement, and further including (i) a first region adapted for receipt of the fluid sample, (ii) a second region at which the analyte capture reagent is immobilized, (iii) a third region for application of a wash reagent capable of removing unbound substances from the second region; and (iv) an absorbent reservoir of high volume capacity. The second region is positioned intermediate to the first region and the third region and intermediate to the absorbent reservoir and the third region. The device also includes means to detect analyte bound at the second region. The flow matrix and the regions thereof are sized and positioned to cause the fluid sample to flow initially along the elongated flow matrix in one direction toward and through the second region, and subsequently, the liquid reagent to flow along the elongated flow matrix in a second direction opposite the first direction, through the second region, and into the absorbent reservoir, drawing unbound substances with it.

Claims24

Dependent claims

A device for performing an assay which determines the presence or quantity of an analyte in a fluid sample suspected of containing the analyte by capturing analyte with an analyte capturing reagent immobilized at a detection region, and detecting captured analyte in the detection region with an analyte binding disclosure reagent conjugate which, when bound to captured analyte provides a detectable reaction in the presence of a detecting reagent, the assay comprising washing unbound analyte binding disclosure reagent from the detection region, the device comprising:

(a) an elongated solid phase flow matrix capable of driving fluid movement by capillary force, the flow matrix further comprising,
(i) a first matrix region for receipt of analyte-containing fluid,
(ii) a second matrix region at which the analyte capture reagent is immobilized, the second matrix region being positioned in between the first matrix region (i) and a matrix third region (iii),
(iii) the third matrix region being for receipt of the detecting reagent,
(b) an absorbent reservoir of excess absorbing capacity, positioned for contact with an absorbent contacting area of the matrix, the absorbent contacting area being between the first matrix region and the second matrix region,
(c) at least one sealed container containing the detecting reagent,
(d) means for releasing and transporting the detecting reagent from the sealed container such that detecting reagent is transported by capillary flow to said third matrix region,
(e) a supply of the analyte binding disclosure reagent conjugate,
whereby said flow matrix and said regions thereof are sized and positioned to cause said analyte-containing fluid to flow initially along said flow matrix in one direction toward and through said second region, and subsequently, upon introduction of detecting reagent into the third region of said flow matrix, detecting reagent flows along said elongated flow matrix in a second direction opposite said first direction, through said second region and into said absorbent reservoir.

6 more elements

A device for performing an assay which determines the presence or quantity of an analyte in a fluid sample suspected of containing the analyte by capturing analyte with an analyte capturing reagent immobilized at a detecting region, and detecting captured analyte in the detection region with an analyte binding disclosure reagent conjugate, which, when bound to captured analyte provides a detectable reaction in the presence of a detecting reagent, the assay comprising washing unbound analyte binding disclosure reagent from the detection region, the device comprising:

(a) an elongated solid phase flow matrix capable of driving fluid movement by capillary force, the flow matrix further comprising,
(i) a first matrix region for receipt of analyte-containing fluid,
(ii) a second matrix region at which the analyte capture reagent is immobilized, the second matrix region being positioned in between the first matrix region (i) and a matrix third region (iii),
(iii) the third matrix region being for receipt of a wash reagent capable of removing from the second matrix region substances that are not bound to the analyte capture reagent,
(iv) a fourth matrix region for receipt of the detecting reagent, said device further comprising an absorbent reservoir, whereby the third and fourth regions and absorbent reservoir are sized and positioned to cause wash reagent to flow by capillary force into said second matrix region in front of said detection reagent, and whereby both wash and detection reagents flow by capillary force into said absorbent reservoir;
(b) the absorbent reservoir having excess absorbing capacity, and being positioned for contact with a contacting area of the matrix between the first matrix region and the second matrix region,
(c) at least two sealed containers, said first container containing the detecting reagent, and said second container containing the wash reagent, said containers being positioned to introduced their contents at the fourth and third matrix regions, respectively,
(d) means for releasing and transporting wash and detecting reagents from said sealed containers such that wash and detecting reagents are transported by capillary flow to said third and fourth regions, respectively, and
(e) a supply of analyte binding disclosure reagent conjugate;
whereby said flow matrix and said regions thereof are sized and positioned to cause said fluid sample to flow initially along said flow matrix in one direction toward and through said second region, and subsequently, upon introduction of said wash and detector reagents into the third region of said flow matrix, said wash and detector reagents to flow along said flow matrix in a second direction opposite said first direction, through said second region, and into said absorbent reservoir, thereby drawing unbound reagents with it, and causing analyte and conjugate to pass twice over said second region, thus increasing the probability of formation of analyte:analyte capture reagent complexes.

7 more elements

A device for performing an assay which determines the presence or quantity of an analyte in a fluid sample suspected of containing said analyte, utilizing (1) at least one immobilized analyte capture reagent, (2) analyte binding disclosure reagent conjugate capable of binding analyte, said analyte binding disclosure reagent conjugate comprising a detectable label;

and (3) liquid detecting reagent capable of detecting the detectable label on the analyte binding disclosure reagent, and capable of removing unbound sample and reagents from said immobilized capture reagent complex, said device comprising:
(a) an elongated solid phase flow matrix, capable of driving fluid movement, said flow matrix further comprising,
(i) a first region positioned for receipt of the fluid sample,
(ii) a second region capable of being brought into fluid contact with an absorbent reservoir (b);
(iii) a third at which said analyte capture reagent is immobilized;
(iv) a fourth region positioned for receipt of said liquid detecting reagent, said third (iii) region being positioned between said first (i) and fourth (iv) regions, said third (iii) region also being positioned between said second (ii) and said fourth (iv) regions;
(b) an absorbent reservoir of excess fluid-holding capacity;
(c) at least one sealed container of said liquid detecting reagent positioned to be introduced at said fourth region of said matrix;
(d) means for releasing detecting reagent from said sealed container and transporting by capillary force, said detecting reagent from said sealed container to said fourth region,
(e) a supply of said analyte binding disclosure reagent conjugate,
whereby said flow matrix and said regions thereof are sized and positioned to cause said fluid sample to be introduced into said flow matrix and subsequently, upon introduction of said detecting reagent into the fourth region of said elongated flow matrix, causing said fluid sample to move toward said second region and into said absorbent reservoir, thereby drawing unbound reagent with it.

8 more elements

and (3) a liquid detecting reagent capable of detecting the detectable label on the analyte binding disclosure reagent and (4) a washing reagent capable of removing fluid sample and unbound reagents, said device comprising:
(a) an elongated solid phase flow matrix, capable of driving fluid movement, said flow matrix further comprising,
(i) a first region adapted for receipt of said fluid sample,
(ii) a second region capable of being brought into fluid contact with an absorbent reservoir,
(iii) a third region at which said analyte capture reagent is immobilized,
(iv) a fourth region adapted for receipt of wash reagent capable of removing substances not bound to analyte capture reagent,
(v) a fifth region positioned for receipt of said detecting reagent whereby wash reagent flows through said third region in front of said detecting reagent and whereby both wash and detecting reagents flow through said second and third regions into said absorbent reservoir;
(b) said absorbent reservoir of excess fluid-holding capacity,
(c) at least two sealed containers, one of said containers containing said wash reagent and the other of said containers containing said detecting reagent, said containers positioned to introduce fluid at said fourth and fifth matrix regions, respectively,
(d) means for releasing wash and detecting reagents from the sealed containers such that wash and detecting reagents are transported by capillary flow to said fourth and fifth regions, respectively,
whereby said flow matrix and said regions thereof are sized and positioned to cause said fluid sample to be introduced into said flow matrix at said first region and to flow through said third region, and subsequently, upon establishing fluid contact between said second region and said absorbent reservoir and upon introduction of said wash reagent into said fourth region and detection reagent into said fifth region, causing said fluid sample to pass again through said third region toward said second region and into said absorbent reservoir, followed sequentially by said wash reagent and said detection reagent, thereby drawing unbound reagents to said absorbent reservoir.

8 more elements

Dependencies are inferred from the claim text.

Reversible flow chromatographic binding assay

Life of this patent

Reversible flow chromatographic binding assay

Life of this patent

Abstract

Claims24