Assay of blood or other biologic samples for target analytes

• US 5,776,710 A
• Filed: 12/23/1996
• Issued: 07/07/1998
• 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: Terstappen, Leon W. M. M., Lovell, Stephen J., Malick, Adrien P., Ozinskas, Alvydas J., Dhanesar, Subhash, Manion, Kristen L., Wardlaw, Stephen C., Levine, Robert A., Rodriguez, Rodolfo R.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/771,507
• Time in Patent Office: 561 Days
• Field of Search: 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, 73/61.51, 73/61.65
• 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 an anticoagulated whole blood sample in a transparent tube, said method comprising the steps of:

a) adding a group of capture bodies to the sample, which capture bodies have a specific gravity which ensures that said capture bodies will settle into a predetermined location in the sample in the tube, each capture body in said group being coupled with a binding material to form capture body couples which are specific to the target analyte;

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

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

d) centrifuging the sample so as to aggregate the capture bodies into a distinct location in the tube; and

e) determining if the aggregated capture bodies exhibit the presence of the labeled antibodies or other binding material, and therefore the presence of the target analyte.

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

a) adding a group of capture bodies to the sample, which capture bodies have a predetermined specific gravity, each capture body in said group being coupled with a binding material to form capture body couples which are specific to the target analyte;

b) adding to said sample labeled antibodies or other binding material specific to said target analyte;

c) intimately admixing said sample and said labeled binding material so as to form a capture body/labeled binding material sample mixture;

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

e) densimetrically aggregating the capture bodies into at least one distinct band; and

f) determining if any band exhibits the presence of the labeled binding material, and therefore the presence of the target analyte.

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

a) positioning a capture body in the sample, said capture body being coupled with a binding material to form a capture body couple which is specific to a first epitope or other binding site on the target analyte;

b) admixing said sample with a label which can bind to said target analyte so as to form a capture body/label sample mixture;

c) incubating the capture body/label sample mixture;

d) centrifuging the sample mixture; and

e) determining if the capture body exhibits the presence of the label, and therefore the presence of the target analyte in the sample.

4. The method of claim 3 wherein said capture body is a cylindrical plastic insert, and wherein said binding material comprises a layer of antibodies coated onto said insert.

5. The method of claim 3 wherein said centrifuging step involves centrifuging the capture body first in one direction in the tube, and then in the opposite direction in the tube so that the insert is displaced back and forth through the tube a number of times.

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

a) positioning a capture body in the sample, said capture body being coupled with a binding material to form a capture body couple which is specific to a first epitope or other binding site on the target analyte cell;

b) admixing said sample with a labeled binding material which will bind to and label said target analyte cell so as to form a capture body/labeled binding material sample mixture;

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

d) centrifuging the sample mixture; and

e) determining if the capture body exhibits the presence of the labeled binding material, and therefore the presence of the target analyte cell in the sample.

7. A method of assaying a s ample of anticoagulated whole blood in a transparent tube for the presence or absence of CD4 or CD34 blood cells, said method comprising the steps of:

a) positioning a capture body in the sample, said capture body being coupled with a binding material which is specific to an epitope or other binding site on CD4 or CD34 blood cells, said capture body having a specific gravity which is greater than the specific gravity of reticulate cells in the blood sample;

b) admixing said blood sample with a label material which will bind with CD4 or CD34 blood cells so as to form a capture body/label material/blood sample mixture;

c) incubating the capture body/label material/blood sample mixture;

d) centrifuging the sample mixture; and

e) determining if the capture body exhibits the presence or absence of labeled CD4 or CD34 blood cells.

8. The method of claim 7 wherein said label material is a fluorescent stain which stains genetic material in CD4 or CD34 blood cells.

9. The method of claim 8 further comprising the steps of:

including a reference zone in said tube which provides a predetermined intensity level of fluorescence corresponding to a known cell count;

measuring the fluorescent intensity level of said capture body;

measuring the fluorescent intensity level of said reference zone; and

comparing the measured fluorescent intensity levels of said capture body with said reference zone to calculate the CD4 or CD34 cell count as a percentage of the reference zone fluorescence as compared with the capture body fluorescence.

10. The method of claim 9 wherein said reference zone is provided by a fluorescently labeled cell layer in said blood sample mixture.

11. The method of claim 9 wherein skid reference zone is provided by a fluorescent body in the sample mixture.

12. The method of claim 7 wherein said label material is an antibody specific to the CD4 epitope found on certain blood cells.

13. The method of claim 7 wherein said label material is an antibody specific to the CD34 epitope found on certain blood cells.