Immunoassay product and process

• US 20070243628A1
• Filed: 10/18/2006
• Published: 10/18/2007
• Est. Priority Date: 11/03/2005
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Millipore Corporation (Merck & Co., Inc.)
• Inventors: Greenizen, Kurt, Mabuchi, Masaharu, Clark, Phillip, Emerick, Marc, Kimura, Hiroko
• Granted Patent: US 8,557,600 B2
• Time in Patent Office: Days
• US Class Current: 436/183
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/50255 Multi-well filtration
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A rapid, efficient and convenient method to detect one or more biological entities on a blotting membrane is provided. The detection can relate to the position, nature or amount of the biological substance on one or more membranes. The invention method involves a pressure assisted regiment (such as vacuum or positive gas pressure) for the supply and removal of reagents and permits washing of the contaminants from substances embedded in the membrane to be detected using very low volumes of liquid. This method enables completion of the blocking, washing and antibody binding steps in about 30 minutes without comprising blot quality. In another aspect, the invention is directed to an apparatus useful in conducting the method of the invention. The device is comprised of several layers including a porous support layer below the blotting membrane(s), a flow distributor above the blotting membrane(s) and a well on the flow distributor to contain the liquid to the desired area and to allow for lower starting volumes of such liquid. Preferably, the flow distributor is a non-binding or low binding hydrophilic porous membrane such as a 0.22 micron membrane.

Claims

1. A device for conducting immunoassays comprising a porous support, a flow distributor placed on top of the porous support and one or more reagent wells mounted on top of the flow distributor.

5. The device of claim 1 wherein the flow distributor and the one or more reagent wells are an integral unit.

6. The device of claim 1 wherein the flow distributor is a membrane and the flow distributor and one or more reagent wells are an integral unit.

14. The device of claim 1 further comprising a collection tray below the holder for the recovery of reagents.

15. The device of claim 1 further comprising a collection tray below the holder for the recovery of reagents and wherein the tray is subdivided into two or more separate subtrays.

2. A device for conducting vacuum assisted immunoassays comprising a vacuum manifold, a porous support, a flow distributor placed on top of the porous support and one or more reagent wells mounted on top of the flow distributor.

3. A device for conducting vacuum assisted immunoassays comprising a porous support, one or more membranes containing one or more biological entities to be assayed, the one or more membranes being mounted on top of the porous support, a flow distributor on top of the one or more membranes and one or more reagent wells mounted on top of the flow distributor.

4. A device for conducting pressure assisted immunoassays comprising a collection manifold, a porous support, one or more membranes containing one or more biological entities to be assayed, the one or more membranes being located on top of the porous support, a flow distributor placed on top of the one or more membranes, one or more reagent wells mounted on top of the flow distributor, a pressure cap removably sealed on top of the one or more reagent wells, the cap having an inlet to its interior, the inlet being connected to a source of positive gas pressure.

7. A process for conducting vacuum assisted immunoassays comprising the steps of:

a. providing a vacuum manifold, a porous support placed on the vacuum manifold, one or more membranes containing one or more biological entities to be assayed, the one or more membranes being placed on the porous support, a flow distributor placed on top of the one or more membranes and one or more wells placed on top of the flow distributor,

b. adding one or more reagents to the one or more wells and applying a vacuum to pull the reagents into the one or membranes, and

c. adding one or more washing agents to the one or more wells and applying a vacuum to pull the washing agents and any unbound reagents through the flow distributor, one or more membranes and porous support and into the vacuum manifold.

8. The process of claim 7 wherein steps (b) and (c) are repeated one or more times.

9. The process of claim 7 further comprising a step (d) of detecting the one or more biological entities.

10. The process of claim 7 further comprising a step (d) of detecting the one or more biological entities by a detection mechanism selected from the group consisting of radiological entities and calorimetric entities.

11. The process of claim 7 wherein in step (b) the reagent is an antibody and is allowed to incubate on/in the one or more blotting membranes without applying the vacuum for a set period of time.

12. A process of passing a wash or reagent-containing liquid through one or more blotting membranes containing one or more biological entities, at least one of which is to be detected wherein the process comprises:

a. providing a vacuum manifold, a porous support placed on the vacuum manifold, a flow distributor and one or more wells placed on top of the flow distributor,

b. placing the one or more blotting membranes containing the one or more biological entities on the porous support,

c. placing the flow distributor on top of the one or more blotting membranes, adding a liquid to the well of the flow distributor, and

d. applying a vacuum to draw the liquid through the one or more blotting membranes.

13. A process of passing a wash or reagent-containing liquid through a blotting membrane containing one or more biological entities, at least one of which is to be detected wherein the process comprises:

a. providing a device comprised of a collection manifold, a porous support, a flow distributor placed on top of the support, one or more wells placed on top of the flow distributor and a pressure cap removably attached to the top of the one or more wells, the cap having an inlet to its interior which inlet is connected to a supply of positive gas pressure,

b. placing one or more layers of blotting membrane containing the one or more biological entities on the porous support,

c. placing the flow distributor on top of the one or more layers of blotting membrane, adding a liquid to the one or more wells of the flow distributor, and

d. applying a positive pressure through the inlet to the cap to move the liquid from the wells through the one or more layers of blotting membrane and into the manifold.