A HIGHLY COST-EFFECTIVE ANALYTICAL DEVICE FOR PERFORMING IMMUNOASSAYS WITH ULTRA HIGH SENSITIVITY

• US 20060257862A1
• Filed: 07/28/2006
• Published: 11/16/2006
• Est. Priority Date: 02/09/2001
• Status: Active Grant

• Current Assignee: Council of Scientific & Industrial Research, Department of Biotechnology A Department of Govenment of India
• Original Assignee: Council of Scientific & Industrial Research, Department of Biotechnology A Department of Govenment of India
• Inventors: Dhar, Tarun K, Pal, Arindam
• Granted Patent: US 7,312,028 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se

Abstract

A simple easy to manufacture analytical device capable of performing membrane based immunoassays on batch of samples within 3 to 10 minutes wherein the method permits focused application of samples, costly labeled immunoassay and signal amplification reagents, said device includes an antibody-immobilized micro porous membrane, breadth corner layer of which is directly attached to a semi-rigid liquid-impervious body with water insoluble adhesive; absorbent body is provided separately and is not attached to analytical device during manufacture, absorbent body is wetted and is placed proximal to the lower surface of the membrane thereby forming networks of capillary channels with the absorbent body; flow of samples or reagents is always kept downwards and focused without application of any force to the absorbent body and the use of disposable adsorbent body permits stepwise addition of signal amplification reagents for ultra sensitive detection of diagnostically important molecules by visual examination of the membrane surface.

Claims

1-21. -21. (canceled)

22. An method for assembling an absorbent body of an analytical device comprising

a liquid-permeable porous reaction membrane having an upper and a lower surface,

an exposed area of the upper surface having immobilized thereupon an antibody or antigen capable of binding to the target analyte, wherein said immobilized antibody or antigen being concentrated in multiple spotted regions of the said upper surface, and a portion of the lower surface of the reaction membrane in breadth corner has no immobilized antigen or antibody;

a semi-rigid liquid impervious bottom support layer attached to the lower surface of the reaction membrane at the breadth corner by water insoluble adhesive or tape having glue on both sides; and

a body of absorbent material having an upper surface and a lower surface, capable of absorbing liquid, wherein the body of absorbent material is provided separately from the analytical device and in use, the absorbent material is pre-wetted with a liquid and is placed between and in contact with the reaction membrane, which is above the body of the absorbent material and the bottom support layer, which is below the body of the absorbent material and the body of absorbent material is capable of absorbing liquid and is larger is size than the reaction membrane, wherein the absorbent body is assembled by;

(a) soaking the absorbent body with liquid and assembled in such a way that upper surface of the absorbent body is in intimate contact with lower surface of the reaction membrane and upper surface over bottom support layer,

(b) removing the entrapped air in between lower and upper surface of reaction membrane and absorbent body by pressing the upper surface of the reaction membrane,

(c) the void volume of reaction membrane is saturated and the distance separating the reactive membrane and the absorbent body is such that networks of capillary channels is formed were the two members are in contact,

(d) the flow of applied sample or reagent is always downwards and focused without application of any force to the absorbent body, and

(e) the void volume of the wetted absorbent body is still sufficient to substantially fill the additional volume of fluid introduced during assay.

23. The method as claimed in claim 22 wherein, absorbent body is soaked in deionized distilled water, buffer and other similar materials.

24. The method as claimed in claim 22 wherein, the upper surface of the reaction membrane is pressed with small roller, rim-less small test-tube and other similar tools.

25. The method as claimed in claim 22 wherein, pre-wetted absorbent body saturates the void volume of reaction membrane, which thereby does not allow spread of sample or immunoassay reagents.

26. The method as claimed in claim 22 wherein, the costly-labeled reagents are used efficiently.

27. The method as claimed in claim 22 wherein, 10 to 100 μ

l of sample or labeled reagents are used.

28. The method as claimed in claim 22 wherein, multiple of 10 to 100 μ

l of sample or labeled reagents are applied.

29. The method as claimed in claim 22 wherein, the label reagent is premixed with standard or sample prior to addition to different areas of the membrane in the device or it can be added after standard or sample addition.

30. The method as claimed in claim 22 wherein, more than one specific antibody is immobilized in the same or different areas for simultaneous detection of multiple analytes in a sample with a single assay device.

31. The method as claimed in claim 22 wherein, after application of samples and reagents absorbent body is discarded.

32. The method as claimed in claim 22 wherein, the reaction membrane is washed directly over device with the help of wash bottle.

33. The method as claimed in claim 22 wherein, for high sensitivity elements of signal amplification are further added.

34. The method as claimed in claim 22 wherein, the signal amplification method like Super-CARD is applied.

35. The method as claimed in claim 22 wherein, biotinylated tyramine is added directly over reaction membrane in the device.

36. The method as claimed in claim 22 wherein, a fresh pre-wetted absorbent body is assembled and avidin-peroxidase conjugate is added.

37. The method as claimed in claim 36 wherein, the substrate solution added directly over reaction membrane produced color spots within well-defined area.

38. The method as claimed in claim 37 wherein, the exposed area of the reaction membrane is sufficiently greater to allow visualization of the intensity of the color spots.

39. The method as claimed in claim 38 wherein, visual comparison with known concentration in reference standard gives semi-quantitative estimate of the amount of antigen present in the sample.

40. The method as claimed in claim 36 wherein, the substrate solution is added directly without application of signal amplification.

41. The method as claimed in claim 36 wherein, signal amplification step is not necessary for that analyte which is present in high concentration.

42. The method as claimed in claim 22 wherein, the assay results is obtained within 3 to 10 minutes.

43. The method as claimed in claim 22 wherein, the analyte is selected from the group consisting of antigens, antibodies, haptens, drugs, hormones, macromolecules, toxins, bacteria, viruses, enzymes, tumor markers, environmental pollutants, nucleic acids and other natural receptors.