Assay of monokines

• US 5,128,270 A
• Filed: 01/06/1989
• Issued: 07/07/1992
• Est. Priority Date: 05/19/1987
• Status: Expired due to Fees

• Current Assignee: Medgenix Diagnositcs SA
• Original Assignee: Ire-Medgenix SA
• Inventors: De Groote, Donat, Dehart, Isabelle, Franchimont, Paul, Reuter, Aimee, Gysen, Philippe, Delacroix, Dominique
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Bidwell, Carol E.
• Application Number:
  US07/299,803
• Time in Patent Office: 1,278 Days
• Field of Search: 435/7.24, 435/172.2, 435/240.67, 435/962, 435/968, 435/174, 436/503, 436/504, 436/518, 436/524, 436/527, 436/531, 436/547, 436/548, 424/1.1, 424/85.8, 424/85.2, 424/85.1, 530/351, 530/387
• US Class Current: 436/518
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• CPC Class Codes: G01N 33/6869 Interleukin
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The invention relates to assays of a monokine in a biological fluid, in particular in a blood sample, in particular of TNF or of IL-1. According to the present invention, the monokine in the serum or plasma obtained from whole blood stimulated with a mitogenic agent is assayed prior to any separation between the plasma (or serum) and the blood cells. According to another subject of the invention, the monokine is assayed in the case of an immunometric assay employing an oligoclonal system, consisting of several different monoclonal antibodies adsorbed on a solid phase and a non-adsorbed labeled antibody.

The invention also relates to immunoassay kits.

Claims

1. A method for determining a monokine in a blood sample which comprises the following steps:

1) stimulating a blood sample by incubating said sample for from about 2 to 4 hrs with a mitogenic agent;

2) directly separating blood cells from said stimulated sample obtained from step (1) to give plasma containing said mitogenic agent; and

3) determining the presence of said monokine in said plasma containing said mitogenic agent.

2. A method according to claim 1, including the additional step of combining said blood with an anticoagulant prior to said stimulating.

3. A method according to claim 2, wherein said anticoagulant is ethylenediamine tetraccetic acid, heparin or citrate.

4. A method according to claim 2, wherein said mitogenic agent is phytohemagglutinin (PHA) or a lipopolysaccharide (LPS).

5. A method according to claim 1, wherein said mitogenic agent is phytohemagglutinin (PHA) or a lipopolysaccharide (LPS).

6. A method according to claim 5, wherein said PHA is present in from about 0.02 to 50 μ

g/ml or said LPS is present in from about 10 to 100 μ

g/ml.

7. A method according to claim 6, wherein said PHA is present in from about 1 to 10 μ

g/ml or said LPS is present in from about 15 to 75 μ

g/ml.

8. A method for detecting the presence of the monokine TNFα

or IL-1β

in a blood sample, said method comprising;

(a) contacting a blood sample with an anticoagulant;

(b) incubating said blood sample with a mitogenic agent for from about 2 to 4 hrs at a temperature in the range of about 4°

C. to 40°

C.;

(c) directly separating said blood sample to remove blood cells and provide a plasma sample containing said mitogenic agent for assaying; and

(d) assaying said separated plasma sample to detect the presence of said monokine.

9. A method according to claim 8, wherein said anticoagulant is ethylenediamine tetraacetic acid, heparin or citrate;

and said mitogenic agent is PHA at a concentration in the range of about 0.02 to 50 μ

g/ml or LPS in the range of about 10 to 200 μ

g/ml.

10. A method according to claim 8, wherein said assaying is a displacement radioimmunoassay (RIA) assay.

11. A method according to claim 10, wherein said assay is performed with a rabbit polyclonal anti-TNFα

or IL-1β

antiserum lacking cross-reactivity with lymphokines or other monokines.

12. A method according to claim 8, wherein said assaying is an IRMA assay.

13. A method according to claim 12, wherein at least two monoclonal antibodies recognizing different epitopes of said monokine adsorbed on a solid phase are used to bind said monokine.

14. A method according to claim 13, wherein a first adsorbed antibody is selected for sensitivity to about 10 pg/ml of monokine and a second adsorbed antibody is selected for recovery selectivity of about 100% when known values for said monokine are assayed.

15. A method according to claim 8, wherein said anticoagulant is ethylenediamine tetraacetic acid.

16. A method according to claim 8, wherein said assaying employs monoclonal antibodies with no cross-reactivity with lymphokines or other monokines.