Assay of blood or other biologic samples for target analytes

• US 5,834,217 A
• Filed: 12/11/1996
• Issued: 11/10/1998
• Est. Priority Date: 10/30/1992
• Status: Expired due to Term

• Current Assignee: QBC Diagnostics, Inc. (Madison Medical Diagnostics LLC)
• Original Assignee: Becton, Dickinson & Co
• Inventors: Wardlaw, Stephen C., Levine, Robert A., Terstappen, Leon W. M. M., Lovell, Stephen J., Rodriguez, Rodolfo R., Malick, Adrien P., Ozinskas, Alvydas J., Dhanesar, Subhash, Manion, Kristen L.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/763,858
• Time in Patent Office: 699 Days
• Field of Search: 73/61.51, 73/61.65, 422/57, 422/58, 422/72, 422/73, 422/101, 435/7.24, 435/7.25, 435/287.1, 435/287.2, 435/971, 435/973, 436/45, 436/70, 436/164, 436/165, 436/514, 436/518, 436/523, 436/528, 436/531, 436/534, 436/538, 436/541, 436/805, 436/810, 436/824, 436/829
• US Class Current: 435/7.24
• CPC Class Codes: B01L 3/5021 Test tubes specially adapted for centrifugation purposes centrifuges B04B5/04
• CPC Class Codes: G01N 33/491 by separating the blood components G01N15/05 takes precedence
• CPC Class Codes: G01N 33/5375 by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• 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 436/805 Optical property
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10T 436/111666 Utilizing a centrifuge or compartmented rotor

Abstract

A patient'"'"'s health may be diagnosed by centrifuging blood samples in a transparent tube, which tube contains one or more bodies or groups of bodies such as floats, inserts, liposomes, or plastic beads of different densities. Each density-defined body carries analyte-capture binding materials such as antigens or antibodies, which are specific to an epitope, or other specific high affinity binding site on a target analyte which target analyte may be in the blood or other sample being tested; and the level of which analyte is indicative of the patient'"'"'s health. At least one labeled binding material which is also specific to an epitope, or other specific high affinity binding site on the target analyte is added to the sample so as to form labeled binding material/analyte/body complexes in the sample. Upon centrifugation, the complexes will settle out in different areas in the tube according to the respective density of the body or bodies; and the degree of label emission of the complex layers can enable qualitative and/or quantitative analyses of the sample to be made. Unbound labeled binding materials will be separated from the complexed layers by the washing action of ascending or descending components of the sample during the centrifugation step. Unbound labeled binding material will thus not interfere with the analysis.

Claims

1. A method for detecting a target analyte in a biologic fluid sample in a transparent tube, said method comprising the steps of:

a) adding one or more capture bodies to the sample, which capture bodies have a predetermined specific gravity, each capture body being coupled with a binding material to form one or more capture body couples which are specific to a binding site on the target analyte;

b) adding to said sample, labeled binding material moieties which are specific to a binding site on said target analyte to form a capture body binding material couple/labeled binding material moiety sample mixture;

c) incubating the capture body binding material couple/labeled binding material moiety sample mixture;

d) densimetrically displacing the capture bodies into a predictable region in the sample mixture; and

e) determining if any capture bodies in the sample exhibit the presence of the labeled binding material, and therefore the presence of the target analyte in the sample.

2. The method of claim 1 further comprising the step of displacing unbound labeled antibodies from the capture bodies during the step of densimetric separation.

3. The method of claim 2 wherein the fluid sample is anticoagulated whole blood, and wherein the capture bodies have a specific gravity that is less than the specific gravity of the heaviest of any red cells in the blood sample.

4. The method of claim 3 wherein the capture bodies have a specific gravity which is greater than the specific gravity of the heaviest of the any red cells.

5. The method of claim 3 wherein said step of displacing is performed by the cells in the whole blood sample.

6. The method of claim 1 wherein the fluid sample is an aqueous based biological sample.

7. The method of claim 6 further comprising the step of displacing unbound labeled antibodies from the capture bodies during the step of densimetric separation.

8. The method of claim 7 wherein said step of displacing is performed by providing a label-immiscible density gradient material in the tube, into which density gradient material the density-markers settle.

9. The method of claim 1 wherein the density-markers are densimetrically separated into a part of the tube having an internal sample-occupying portion which is less in cross sectional area than the cross sectional area of the remainder of the tube.

10. The method of claim 9 wherein said portion of the tube is formed by positioning an axially elongated insert in the tube.

11. The method of claim 9 wherein said portion of the tube is formed by a localized constriction in the tube.

12. A method for detecting one or more different target analytes in a biologic fluid sample in a transparent tube, said method comprising the steps of:

a) adding capture bodies to the sample, there being one group of capture bodies for each target analyte suspected to be in the sample, each group of capture bodies having a different specific gravity from each other group of capture bodies, and each capture body in each group thereof being coupled with a binding material which is specific to a first epitope or other binding site on one of the target analytes, whereby each of the different capture body/binding material couple groups is specific to a different one of the suspected target analytes;

b) adding to said sample labeled antibodies or other binding material which is specific to another epitope or other binding site on each of said analytes to form a capture body/binding material couple and labeled binding material sample mixture;

c) incubating the capture body/binding material and labeled binding material sample mixture;

d) densimetrically separating the capture bodies into one or more distinct bands in the sample mixture; and

e) determining which, if any of said bands exhibit the presence of a labeled antibody or other binding material, and therefore the presence of a target analyte.

13. The method of claim 12 further comprising the step of displacing unbound labeled antibodies or other labeled binding material from the capture bodies during the step of densimetric separation.

14. The method of claim 13 wherein the fluid sample is anticoagulated whole blood, and wherein the capture bodies have a specific gravity that is greater than the specific gravity of the heaviest of red cells in the blood sample.

15. The method of claim 14 wherein the capture bodies have a specific gravity which is less than the specific gravity of the heaviest of the red cells.

16. The method of claim 14 wherein said step of displacing is performed by cells in the whole blood sample.

17. The method of claim 12 wherein the fluid sample is an aqueous based biological sample.

18. The method of claim 17 further comprising the step of displacing unbound labeled antibodies or other binding material from the capture bodies during the step of densimetric separation.

19. The method of claim 18 wherein said step of displacing is performed by providing a label-immiscible density gradient material in the tube, into which density gradient material the capture bodies settle.

20. The method of claim 12 wherein the capture bodies are densimetrically separated into a part of the tube having an internal sample-occupying portion which is less in cross sectional area than the cross sectional area of the remainder of the tube.

21. The method of claim 20 wherein said portion of the tube is formed by positioning an axially elongated insert in the tube.

22. The method of claim 20 wherein said portion of the tube is formed by a localized constriction in the tube.

23. A method for detecting one or more different target analytes in an anticoagulated whole blood sample in a transparent tube, said method comprising the steps of:

a) adding capture bodies to the sample, there being one group of capture bodies for each target analyte suspected to be in the sample, each group of capture bodies having a different specific gravity from each other group of capture bodies, with all of the capture bodies having a specific gravity that will ensure that said capture bodies will settle into the red cell layer of the blood upon centrifugation of the sample in the tube, and each capture body in each group thereof being coupled with a binding material which is specific to one of the target analytes, whereby each of the different capture body/binding material couple groups is specific to a different one of the suspected target analytes;

b) adding to said sample labeled antibodies or other binding material specific to said target analytes so as to form a capture body/binding material and labeled binding material sample mixture;

c) incubating the capture body/binding material and labeled binding material sample mixture;

d) centrifuging the sample so as to aggregate the capture bodies into one or more distinct bands; and

e) determining which, if any of the bands exhibit the presence of a labeled antibody or other binding material, and therefore the presence of a target analyte.