Chromatographic immunoassay device and method utilizing particle valency for quantification

• US 6,121,008 A
• Filed: 03/20/1997
• Issued: 09/19/2000
• Est. Priority Date: 03/20/1996
• Status: Expired due to Term

• Current Assignee: Serex Incorporated (Nymox Pharmaceutical Corporation)
• Original Assignee: Serex Incorporated (Nymox Pharmaceutical Corporation)
• Inventors: Fitzpatrick, Judith, Lenda, Regina B.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/821,086
• Time in Patent Office: 1,279 Days
• Field of Search: 422/55, 422/56, 422/57, 422/58, 422/60, 435/7.2, 435/7.9, 435/7.93, 435/183, 435/188, 435/805, 435/968, 435/969, 435/970, 435/971, 435/973, 435/975, 435/287.1, 435/287.2, 435/287.7, 435/810, 436/512, 436/518, 436/525, 436/534, 436/810, 436/514
• US Class Current: 435/7.9
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/969 Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A method and device are provided for the semi-quantitative and quantitative determination of an analyte in a sample. A non-competitive trap which can bind unreacted labelled receptor to analyte but has virtually no binding capabilities to receptor in the presence of analyte is used. Labelled receptor:analyte complex is trapped in a second trap. The relative amounts of unbound receptor in the non-competitive trap versus the amount of receptor:analyte complex in the second trap is a measure of the amount of analyte in the sample.

Claims

1. A chromatographic device for determining the presence or amount of analyte in a sample comprising:

a) a sample receiving site,

b) a mobilization zone in liquid communication with and spatially distinct from the sample receiving site,

wherein the mobilization zone comprises liquid-mobilizable labelled multivalent receptors,

wherein the liquid-mobilizable labelled multivalent receptors have at least one binding site which binds to the analyte in the sample to form receptor;

analyte complexes, and

c) at least two separate trap zones downstream of and in liquid communication with the mobilization zone,

wherein each trap zone comprises at least one different immobilized ligand which binds to an analyte binding site of the liquid-mobilizable labelled multivalent receptors with an avidity or affinity different than the avidity or affinity of the immobilized ligand to an analyte binding site of the liquid-mobilizable labelled multivalent receptors in the other trap zone,

wherein the avidity or affinity of the immobilized ligand increases in each trap zone further downstream of the mobilization zone, and

wherein the presence or amount of the analyte in the sample is determined from the distance over which the receptor;

analyte complexes migrate through the trap zones.

2. The device of claim 1 wherein the immobilized ligands of at least one of the trap zones comprise relands which non-competitively bind to the liquid-mobilizable labelled multivalent receptor in the presence of analyte under conditions in which the sample is reacted with the liquid-mobilizable labelled multivalent receptor, and

wherein the immobilized ligand in monomeric form and the liquid-mobilizable labelled multivalent receptor bind with an association constant between 10³ M^-1 and 10⁵ M^-1.

3. The device of claim 1 wherein at least one of the immobilized ligands can compete with the analyte for binding to the liquid-mobilizable labelled multivalent receptor.

4. The device of claim 1 further comprising an affinity trap zone containing immobilized antibodies binding to the liquid-mobilizable labelled multivalent receptor.

5. The device of claim 4 wherein the immobilized antibodies are immunoreactive with heavy or light chains of antibody.

6. The device of claim 1 wherein the immobilized ligands are in different trap zones of the device which are in liquid communication with the sample receiving zone and mobilization zone, wherein a receptor:

analyte complex moves to the first trap zone with immobilized ligand binding to the receptor;

analyte complex having the lowest number of analyte molecules bound to the liquid-mobilizable labelled multivalent receptor, then moves to a second trap zone with immobilized ligand binding to a receptor;

analyte complex having a higher number of analyte molecules bound to the liquid-mobilizable labelled multivalent receptor.

7. The device of claim 6 comprising multiple trap zones having the same immobilized ligand therein at different densities.

8. The device of claim 1 wherein each immobilized ligand is selected from the group consisting of relands, molecules which do not effectively compete with analyte for binding to the liquid-mobilizable labelled multivalent receptor, competitive ligands, and immunogens.

9. The device of claim 1 wherein the sample receiving site, the mobilization zone and the trap zones are on a membrane and the membrane is at least partially enclosed in a laminat, except for the sample receiving site, to control flow of reactants through the trap zones.

10. The device of claim 9 including proximal and distal ends, wherein the sample receiving site is adjacent the proximal end and the mobilization zone is between the distal end and the sample receiving site, wherein the device further comprises a wicking material attached at the proximal end for absorbing or filtering the sample.

11. The device of claim 1 wherein the liquid-mobilizable labelled multivalent receptors are labeled with a label or labels selected from the group consisting of colored or fluorescent dyes, chromogens, colloidal gold, enzymes, and latex.

12. The device of claim 1 further comprising a means for correlating the amount or the presence of the trapped analyte:

multivalent receptor;

complexes, the trapped unbound liquid-mobilizable labelled multivalent receptor, or both, to the presence of or the amount of analyte in the sample.

13. The device of claim 1 wherein multiple analyte binding domains are bound to a particle to form the liquid mobilizable multivalent receptors.

14. The device of claim 1 wherein the analyte binding domains of the liquid-mobilizable labelled multivalent receptor on any one particle are antibodies which are all reactive with the same epitope of analyte.

15. The device of claim 1 wherein the immobilized ligands are in different trap zones which are in liquid communication with the sample receiving zone and mobilization zone, wherein the affinity of the different trap zones is increased by increasing the affinity of the immobilized ligands for the receptors.

16. The device of claim 1 wherein at least one of the immobilized ligands is multivalent.

17. The device of claim 16 wherein the affinity of the trap zone is increased by increasing the valency of the immobilized ligand.

18. The device of claim 1 further comprising a built-in control.

19. The device of claim 1 wherein the mobilization zone is of sufficient length that analyte in the sample complexes with the liquid-mobilizable labelled multivalent receptors prior to reaching a first trap zone.

20. A chromatographic device for detecting an analyte in a sample comprising:

a) a sample receiving site,

b) a mobilization zone downstream of and in liquid communication with the sample receiving site,

the mobilization zone comprising liquid-mobilizeable labelled receptors binding to analyte in the sample to form receptor;

analyte complexes, and

c) at least one trap zone downstream of and in liquid communication with the mobilization zone

wherein at least one trap zone comprises an immobilized ligand which is non-competitive with the analyte bound by the receptor under the conditions used to interact the sample with the immobilized ligands, the association constant of the non-competitive ligand in monomeric form and the receptor being between 10³ M^-1 and 10⁵ M^-1 ,

whereby when analyte present in the sample forms receptor;

analyte complexes, the non-competitive ligand in the trap does not bind receptor;

analyte complex and the receptor;

analyte complexes migrate through the trap zone, but wherein the non-competitive ligand does bind to the liquid-mobilizable labelled receptor not bound to analyte the liquid-mobilizable labelled receptor does not migrate through the trap zone, and

d) a means for correlating the amount of the receptor;

analyte complex or the trapped unbound liquid-mobilizeable labeled receptor or both to the amount of analyte in the sample.

21. The device of claim 20 wherein when there is more than one trap zone, at least one of the trap zones which bind receptor:

analyte complex comprises antibody immunoreactive with the liquid-mobilizeable labelled receptor.

22. The device of claim 21 further comprising a trap zone containing immobilized ligands competitive with analyte for receptor.

23. The device of claim 20 wherein the sample receiving site, the mobilization zone, the non-competitive ligand trap zone, and the detection zone are present in a unitary continuous strip.

24. The device of claim 20 wherein the trap zones binding receptor:

analyte complex are selected from the group consisting of traps containing ligands competitive with analyte for receptor, immunogen ligand affinity traps, and traps containing ligands for binding antibody.

25. The device of claim 20 wherein the sample receiving site further comprises a hydrophilic material for absorbing or filtering the sample.

26. The device of claim 20 wherein the reland is formed by substituting neutral groups for ionic groups in analyte.