Competitive immunoassay device

• US 5,648,274 A
• Filed: 06/02/1995
• Issued: 07/15/1997
• Est. Priority Date: 05/29/1991
• Status: Expired due to Term

• Current Assignee: Smithkline Diagnostics, Inc. (GSK plc)
• Original Assignee: Smithkline Diagnostics, Inc. (GSK plc)
• Inventors: Chandler, Howard M.
• Primary Examiner(s): Hutzell, Paula K.
• Assistant Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US08/459,466
• Time in Patent Office: 774 Days
• Field of Search: 422/56, 422/58, 422/61, 422/59, 422/68.1, 422/70, 435/7.1, 435/7.9, 435/7.92, 435/7.93, 435/7.95, 435/975, 435/287.1, 435/287.2, 435/287.9, 435/810, 436/518, 436/514, 436/527, 436/538, 436/808, 436/169, 436/805, 436/810
• US Class Current: 436/514
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01L 2300/0825 Test strips
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/5055 Hinged, e.g. opposable surfaces
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/30 of temperature
• CPC Class Codes: G01N 30/90 Plate chromatography, e.g. thin layer or paper chromatography
• 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 33/54388 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 435/975 Kit
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A chromatographic assay device for detection and/or determination of an analyte in a competitive immunoassay gives a semiquantitative or quantitative indication of analyte concentration in a single assay device while also giving a positive indication that flow has occurred properly through the device. In one form, the device comprises: (1) a first opposable component including a sample preparation zone and an absorber; and (2) a second opposable component including a first chromatographic medium with capture and detection zones, a second chromatographic medium with a comparison zone, and a comparison label. In another form, the second opposable component includes one chromatographic medium with capture, detection, and control zones. Test kits incorporating the devices and methods for their use are also disclosed.

Claims

1. A competitive immunoassay device comprising:

(a) a first opposable component including;

(i) a sample preparation zone including a labeled specific binding partner for an analyte conjugated to a first member of an auxiliary specific binding pair in resolubilizable form; and

(ii) an absorber for absorbing fluid therein separated from the sample preparation zone on the first opposable component; and

(b) a second opposable component hingedly attached to the first opposable component including;

(i) a first chromatographic medium having first and second ends and including thereon;

(A) a first zone of immobilized analyte or analog thereof located closer to the first end of the chromatographic medium than to the second end of the chromatographic medium; and

(B) a second zone of an immobilized molecule which is a second member of the auxiliary specific binding pair with specific affinity for the first member located closer to the second end of the first chromatographic medium than to the first end of the first chromatographic medium;

(ii) a second chromatographic medium having a first end and a second end and including thereon a comparison zone containing a known quantity of the analyte or analog thereof immobilized to the comparison zone, the second chromatographic medium being separated from the first chromatographic medium on the first opposable component; and

(iii) a comparison label zone distinct from the comparison zone and separated from the first and second chromatographic medium including therein a labeled specific binding partner to the analyte or analog thereof in resolubilizable form in operable contact with the second chromatographic medium;

wherein when the first and second opposable components are brought into opposition from a position in which they are not in opposition, the sample preparation zone comes into operable contact with the first end of the first chromatographic medium to apply the sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to the first chromatographic medium, and the absorber comes into operable contact with the second end of the first chromatographic medium and the second end of the second chromatographic medium to draw fluid through the first and second chromatographic medium from their first end to their second end so that the device gives a detectable comparative indication of the presence of an analyte.

2. The immunoassay device of claim 1 wherein the sample preparation zone on the first opposable component further includes a second labeled specific binding partner that binds a molecule not cross-reactive with the analyte in resolubilizable form, and the first chromatographic medium further includes a flow control indicator located near the second end of the first chromatographic medium including an immobilized molecule binding the second labeled specific binding partner so that the flow control indicator gives a positive indication that flow has occurred through the first chromatographic medium.

3. The immunoassay device of claim 1 wherein the label of both the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the labeled specific binding partner to the analyte in the comparison label zone are visibly detectable labels.

4. The immunoassay device of claim 3 wherein the labeled specific binding partner to the analyte conjugated to the first member of the auxiliary specific binding pair at the sample preparation zone and the labeled specific binding partner to the analyte at the comparison label zone are the same label.

5. The immunoassay device of claim 1 wherein the first member of the auxiliary specific binding pair is biotin.

6. The immunoassay device of claim 5 wherein the second member of the auxiliary specific binding pair is streptavidin.

7. The immunoassay device of claim 6 wherein the analyte is a β

-lactam antibiotic, the first zone of immobilized analyte or analog thereof is 7-aminocephalosporanic acid conjugated to immunoglobulin, and the labeled specific binding partner for the analyte conjugated to biotin is biotinylated penicillin binding protein.

8. The immunoassay device of claim 6 wherein the analyte is an antibiotic selected from the group consisting of gentamycin, sulfamethazine, and tetracycline, the first zone of immobilized analyte or analog thereof is analyte covalently conjugated to immunoglobulin, and the labeled specific binding partner for the analyte conjugated to biotin is a biotinylated anti-analyte antibody.

9. A test kit for detection and/or determination of an analyte comprising, packaged in separate containers:

(a) the immunoassay device of claim 1; and

(b) a liquid for resolubilizing the labeled specific binding partner to the analyte or analog thereof in the comparison label zone.

10. A method for detecting and/or determining an analyte in a test sample by a competitive immunoassay comprising the steps of:

(a) adding a test sample to the sample preparation zone of the competitive immunoassay device of claim 1;

(b) allowing the test sample to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair;

(c) adding a liquid to the comparison label zone to resolubilize the labeled specific binding partner to the analyte or analog thereof;

(d) bringing the first and second opposable components into opposition from a position in which they are not in opposition to bring the absorber on the first opposable component into operable contact with the second end of the first chromatographic medium and the second end of the second chromatographic medium and to bring the sample preparation zone into operable contact with the first end of the first chromatographic medium;

(e) allowing the sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to flow through the first chromatographic medium and allowing the labeled specific binding partner to the analyte or analog thereof to flow through the second chromatographic medium so that, if analyte is present in the test sample, the labeled specific binding partner to the analyte conjugated to the first member of the auxiliary specific binding pair binds to the second zone containing the immobilized molecule that is the second member of the auxiliary specific binding pair on the first chromatographic medium and so that the labeled specific binding partner to the analyte or analog thereof binds to the comparison zone on the second chromatographic medium; and

(f) comparing the intensity of label at the second zone on the first chromatographic medium containing the immobilized molecule that is the second member of the auxiliary specific binding pair and at the comparison zone on the second chromatographic medium to detect and/or determine the analyte in the test sample.

11. A competitive immunoassay device comprising:

(a) a first opposable component including;

(i) a sample preparation zone including;

(A) a labeled specific binding partner for an analyte conjugated to a first member of an auxiliary specific binding pair in resolubilizable form; and

(B) a predetermined quantity of the analyte or an analog thereof covalently bound to a labeled specific binding partner for a molecule that does not cross-react with the analyte in resolubilizable form; and

(ii) an absorber separated from the sample preparation zone; and

(b) a second opposable component including a chromatographic medium with first and second ends, the chromatographic medium including thereon in discrete, nonoverlapping zones;

(i) a first capture zone of immobilized analyte or analog thereof;

(ii) a second detection zone, including an immobilized molecule that is a second member of the auxiliary specific binding pair with specific affinity for the first member; and

(iii) a third control zone, including an immobilized molecule capable of being specifically bound to the labeled specific binding partner covalently conjugated to the analyte or analog thereof located in the sample preparation zone on the first opposable component, the first, capture zone being located closer to the first end of the chromatographic medium than to the second end of the chromatographic medium, the third, control zone being located closer to the second end of the chromatographic medium than to the first end of the chromatographic medium, and the second, detection zone being located between the first capture zone and the third control zone;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition from a position in which they are not in opposition causes the absorber to come into operable contact with the second end of the chromatographic medium and causes the sample preparation zone to come into contact with the first end of the chromatographic medium so that the test sample, the resolubilized labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair, and the predetermined quantity of the analyte or analog thereof covalently bound to the labeled specific binding partner flow through the chromatographic medium from the first end to the second end so that the device gives a detectable comparative indication of the presence of the analyte.

12. The immunoassay device of claim 11 wherein the label of the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the label of the labeled specific binding partner for the molecule that does not cross-react with the analyte are both visually detectable labels.

13. The immunoassay device of claim 11 wherein the first member of the auxiliary specific binding pair is biotin.

14. The immunoassay device of claim 13 wherein the second member of the auxiliary specific binding pair is streptavidin.

15. The immunoassay device of claim 14 wherein the analyte is a β

-lactam antibiotic, the labeled specific binding partner for the analyte conjugated to biotin is a penicillin binding protein, the immobilized analyte or analog thereof bound at the capture zone on the chromatographic medium is 7-aminocephalosporanic acid covalently conjugated to immunoglobulin G, the labeled specific binding partner for a molecule that does not cross-react with the analyte is an anti-rabbit immunoglobulin G antibody, and the immobilized molecule bound at the control zone on the chromatographic medium is rabbit immunoglobulin G.

16. The immunoassay device of claim 14 wherein the analyte is an antibiotic selected from the group consisting of gentamycin, sulfamethazine, and tetracycline, the labeled specific binding partner for the analyte conjugated to biotin is an anti-analyte antibody, the labeled specific binding partner for a molecule that does not cross-react with the analyte is anti-rabbit immunoglobulin G, the immobilized analyte or analog thereof bound at the capture zone on the chromatographic medium is the analyte covalently bound to immunoglobulin G of a species other than the species other than rabbit, and the immobilized molecule bound at the control zone is rabbit immunoglobulin G.

17. A method for detecting and/or determining an analyte in a test sample by a competitive immunoassay comprising the steps of:

(a) applying the sample to the sample preparation zone of the competitive immunoassay device of claim 11;

(b) allowing the sample applied to the sample preparation zone to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the predetermined quantity of the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not cross-react with the analyte;

(c) bringing the first and second opposable components into opposition from a position in which they are not in opposition so that the absorber is brought into operable contact with the second end of the chromatographic medium and so that the sample preparation zone is brought into operable contact with the first end of the chromatographic medium;

(d) allowing the sample, the labeled specific binding partner for the sample, and the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not cross-react with the analyte to migrate through at least a portion of the chromatographic medium including the detection zone and the control zone; and

(e) detecting and/or determining the concentration of analyte in the test sample by comparing the intensities of label at the detection zone and the control zone of the chromatographic medium.

18. A competitive immunoassay device comprising:

(a) a first opposable component including;

(i) a plurality of sample preparation zones, each sample preparation zone including a labeled specific binding partner for an analyte conjugated to a first member of an auxiliary specific binding pair in resolubilizable form; and

(ii) an absorber for absorbing fluid therein separated from the sample preparation zones on the first opposable component; and

(b) a second opposable component hingedly attachable to the first opposable component including;

(i) a plurality of first chromatographic media, one for each sample preparation zone, each first chromatographic medium having first and second ends and including thereon;

(A) a first zone of an immobilized analyte or analog thereof located closer to the first end of the first chromatographic medium than to the second end of the first chromatographic medium;

(B) a second zone of an immobilized molecule that is a second member of the auxiliary specific binding pair with specific affinity for the first member located closer to the second end of the first chromatographic medium than to the first end of the first chromatographic medium;

(ii) a plurality of second chromatographic media, separated from the first chromatographic media on the second opposable component, one for each sample preparation zone, each second chromatographic medium having a first end and a second end and including thereon a comparison zone containing a known quantity of an analyte or analog thereof immobilized to the comparison zone; and

(iii) a plurality of comparison labeled zones, distinct from the comparison zones and separated from the first and second chromatographic media, one for each second chromatographic medium, each comparison labeled zone including therein a labeled specific binding partner to the analyte or analog thereof in resolubilizable form in operable contact with the second chromatographic medium;

wherein, when the first and second opposable components are brought into opposition from a position in which they are not in opposition, the sample preparation zones come into operable contact with the first end of each first chromatographic medium to apply the sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to each first chromatographic medium, and the absorber comes into operable contact with the second end of each first chromatographic medium and the second end of each second chromatographic medium to draw fluid through the first and second chromatographic media from their first ends to their second ends so that the device gives a detectable comparative indication of the presence of an analyte in at least one first and second chromatographic medium.

19. The immunoassay device of claim 18 wherein the sample preparation zones on the first opposable component further each include a second labeled specific binding partner that binds a molecule not cross-reactive with the analyte in resolubilizable form, and each first chromatographic medium further includes a flow control indicator including a molecule binding each second labeled specific binding partner so that each flow control indicator gives a positive indication that flow has occurred through each first chromatographic medium.

20. The immunoassay device of claim 18 wherein the first member of the auxiliary specific binding pair is biotin.

21. The immunoassay device of claim 18 wherein the second member of the auxiliary specific binding pair is streptavidin.

22. A test kit for detection and/or determination of at least one analyte comprising, packaged in separate containers:

(a) the immunoassay device of claim 18; and

(b) a liquid for resolubilizing the labeled specific binding partner to the analyte or analog thereof in each comparison label zone.

23. A method for detecting and/or determining at least one analyte in at least one test sample by a competitive immunoassay comprising the steps of:

(a) adding a test sample to at least one of the sample preparation zones of the competitive immunoassay device of claim 18;

(b) allowing the at least one test sample to resolubilize the labeled specific binding partners for the analyte conjugated to the first member of the auxiliary specific binding partner;

(c) adding a liquid to at least one of the comparison label zones to resolubilize the labeled specific binding partner to the analyte or analog thereof;

(d) bringing the first and second opposable components into opposition from a position in which they are not in opposition to bring the absorber on the first opposable component into operable contact with the second end of the first chromatographic media and the second end of the second chromatographic media and to bring the sample preparation zones into operable contact with the first ends of the first chromatographic media;

(e) allowing the at least one sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to flow through the first chromatographic media and the labeled specific binding partner to the analyte or analog thereof to flow through the second chromatographic media so that, if analyte is present in any test sample, the labeled specific binding partner to the analyte conjugated to biotin binds to the detection zone on the corresponding first chromatographic medium and so that the labeled specific binding partner binds to the comparison zone on the corresponding second chromatographic medium; and

(f) comparing the intensity of label at the detection zone on each first chromatographic medium and at the comparison zone on each second chromatographic medium to detect and/or determine the analyte in each test sample.

24. A competitive immunoassay device comprising:

(a) a first opposable component including;

(i) a plurality of sample preparation zones, each sample preparation zone including;

(A) a labeled specific binding partner for an analyte conjugated to a first member of an auxiliary specific binding pair in resolubilizable form; and

(B) a predetermined quantity of an analyte or analog thereof covalently bound to a labeled specific binding partner for a molecule that does not cross-react with the analyte in resolubilizable form; and

(ii) an absorber separated from the sample preparation zone; and

(b) a second opposable component including a plurality of chromatographic media each with first and second ends, each chromatographic medium including thereon in discrete, nonoverlapping zones;

(i) a first capture zone of immobilized analyte or analog thereof;

(ii) a second detection zone, including an immobilized molecule that is a second member of the auxiliary specific binding pair with specific affinity for the first member; and

(iii) a third control zone including an immobilized molecule capable of being specifically bound to the labeled specific binding partner covalently conjugated to the analyte or analog thereof, the first capture zone being located closer to the first end of the chromatographic medium than to the second end of the chromatographic medium, the third, control zone, being located closer to the second end of the chromatographic medium than to the first end of the chromatographic medium, and the second detection zone being located between the first and third zones;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition from a position in which they are not in opposition causes the absorber to come into operable contact with the second end of each chromatographic medium and causes the sample preparation zones to come into contact with the first end of each chromatographic medium so that the test samples, the resolubilized labeled specific binding partners for the analyte conjugated to the first member of the auxiliary specific binding pair, and the predetermined quantities of the analyte or analog thereof covalently bound to a labeled specific binding partner flow through each chromatographic medium from the first end to the second end so that the device gives a detectable comparative indication of the presence of an analyte in at least one chromatographic medium.

25. The immunoassay device of claim 24 wherein the label of the labeled specific binding partner for each analyte conjugated to the first member of the auxiliary specific binding pair and the label of each labeled specific binding partner for the molecule that does not cross-react with the analyte are both visually detectable labels.

26. The immunoassay device of claim 24 wherein the first member of the auxiliary specific binding pair is biotin.

27. The immunoassay device of claim 26 wherein the second member of the auxiliary specific binding pair is avidin.

28. A method for detecting and/or determining at least one analyte in at least one test sample by a competitive immunoassay comprising the steps of:

(a) applying a sample to at least one of the sample preparation zones of the competitive immunoassay device of claim 24;

(b) allowing the at least one sample applied to the sample preparation zones to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the predetermined quantity of the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not cross-react with the analyte;

(c) bringing the first and second opposable components into opposition from a position in which they are not in opposition so that the absorber is brought into operable contact with the second end of each chromatographic medium and so that each sample preparation zone is brought into operable contact with the first end of each chromatographic medium;

(d) allowing the at least one sample, the labeled specific binding partner for the sample, and the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not cross-react with the analyte to migrate through at least a portion of each chromatographic medium including the detection zone and the control zone; and

(e) detecting and/or determining the concentration of analyte in each test sample by comparing the intensities of label at the detection zone and the control zone of each chromatographic medium.