Immunoassay for determination of cells

• US 5,374,531 A
• Filed: 03/22/1993
• Issued: 12/20/1994
• Est. Priority Date: 03/22/1993
• Status: Expired due to Fees

• Current Assignee: Zynaxis, Inc. (China Fruits Corp.)
• Original Assignee: Zynaxis, Inc. (China Fruits Corp.)
• Inventors: Jensen, Bruce D.
• Primary Examiner(s): Saunders, David
• Assistant Examiner(s): GRUN, JAMES LESLIE
• Application Number:
  US08/034,138
• Time in Patent Office: 638 Days
• Field of Search: 435/7.24, 435/7.2, 435/34, 435/39, 435/975, 435/7.92, 435/7.32, 435/7.31, 436/526, 436/523, 436/533, 436/534, 436/503, 436/518, 436/548
• US Class Current: 435/7.24
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Methods and test kits are provided for the quantitative or qualitative determination of selected analytes, e.g., cell subsets in a mixed cell population, using a particulate separation reagent and a particulate detection reagent. The invention enables cell monitoring of AIDS patients in a efficient and reliable manner.

Claims

1. A method for determining the presence or concentration of particulate analyte in a sample, the particles comprising said analyte having at least one characteristic determinant on the surfaces thereof, the density of said determinant on any one analyte particle in said sample being the same as or different from the density of said determinant on any other said analyte particle, each said analyte particle having a multiplicity of said at least one characteristic determinant at spaced apart locations on the surfaces thereof, said method comprising the steps of:

a. adding to said sample, substantially simultaneously, a separation reagent and a detection reagent, said separation reagent comprising a particulate support having affixed thereto a specific binding substance that binds specifically to a characteristic determinant of said analyte, said particulate support facilitating separation of said analyte particles attached to said separation reagent from said sample, said detection reagent comprising a particulate support including a detectable label and having affixed thereto a specific binding substance that binds specifically to a characteristic determinant of said analyte, said detection reagent, when unbound, being separable from both said separation reagent and said detection reagent bound to said analyte; and

said specific binding substance of said separation reagent and said specific binding substance of said detection reagent binding specifically to the same characteristic determinant, the diameter of said particulate supports of said separation and said detection reagents being larger than the average distance between said spaced apart characteristic determinants, the amounts of said added separation and detection reagents being sufficient to substantially completely cover the surfaces of said analyte particles, thereby forming rosettes, the ratio of said added separation reagent to said added detection reagent being such as to effect separation of a constant fraction of said rosettes and render said separated rosettes detectable;

b. subjecting said sample to conditions promoting rosette formation between said separation and detection reagents and said analyte particles;

c. separating said rosettes from unbound detection reagent; and

d. measuring the label in said separated rosettes or in said separated unbound detection reagent, said measurement being determinative of the presence or concentration of said particulate analyte in said sample.

2. A method as claimed in claim 1, wherein the concentration of said particulate analyte is determined directly as a function of measured label.

3. A method as claimed in claim 1, wherein said particulate analyte comprises human cells and said at least one characteristic determinant comprises at least one cell surface antigen.

4. A method as claimed in claim 3, wherein said human cells are lymphocytes and said at least one characteristic antigen is selected from the group consisting of CD2, CD3, CD4, CD8, CD16, CD19, CD34, and CD56.

5. A method as claimed in claim 3, wherein said separation reagent comprises a magnetic particulate support, to which is affixed antibody that binds specifically to said at least one cell surface antigen.

6. A method as claimed in claim 5, wherein said antibody comprises monoclonal antibody.

7. A method as claimed in claim 5, wherein the diameter of said separation reagent is in the range of about 0.1 to about 10 microns.

8. A method as claimed in claim 3, wherein said detection reagent comprises a particulate polymer support bearing a fluorescent substance as said detectable label, to which is affixed antibody that binds specifically to said at least one cell surface antigen.

9. A method as claimed in claim 8, wherein said antibody comprises monoclonal antibody.

10. A method as claimed in claim 8, wherein the diameter of said detection reagent is in the range of about 0.1 to about 10 microns.

11. A method as claimed in claim 1, wherein the particulate supports of said separation and said detection reagents are of uniform size.

12. A method for determining the presence or concentration of a subset of lymphocytes in a blood sample, said lymphocytes having on the surfaces thereof at least one antigen selected from the group consisting of CD4, CD8, CD3, CD2, CD16, CD19, CD34, and CD56 antigens, the density of said at least one antigen on any one lymphocyte in said blood sample being the same as or different from the density of said at least one antigen on any other of said lymphocytes, the concentration of said lymphocyte subset sought to be determined being independent of said antigen density, said method comprising the steps of:

a. adding to said blood sample a reagent mixture comprising a separation reagent and a detection reagent;

said separation reagent comprising finely divided, magnetically responsive particles, to which are affixed monoclonal antibody that binds specifically to one antigen of said group of antigens;

said detection reagent comprising finely divided non-magnetic particles that bear a detectable fluorescent substance and to which are affixed monoclonal antibody that binds specifically to said one antigen of said group of antigens;

the diameter of said magnetically responsive particles and the diameter of said fluorescent substance-bearing particles being at least 0.1 microns and being larger than an expected average distance between molecules comprising said one antigen of said group of antigens on the surfaces of said lymphocytes; and

the amounts of said added separation and detection reagents being sufficient to substantially completely cover the surfaces of said lymphocytes, thereby forming rosettes, said detection reagent comprising about 30 to about 70% and said separation reagent comprising the remaining about 30 to about 70% by particle count, of said reagent mixture;

b. incubating the sample from step a. at a temperature in the range of about 4°

C. to about 37°

C. for a time sufficient to cause rosette formation between said separation and detection reagents an said lymphocytes;

c. magnetically separating the resultant rosettes from the non-magnetic components of said sample;

d. washing said separated rosettes to remove therefrom unbound detection reagent; and

e. measuring the label in said washed rosettes, said measurement being determinative of the presence or concentration of said lymphocites in said blood sample.

13. A method as claimed in claim 12 wherein the antibody of said separation reagent and the antibody of said detection reagent bind specifically to CD4 antigen.

14. A method as claimed in claim 12, wherein the antibody of said separation reagent and the antibody of said detection reagent bind specifically to CD8 antigen.