Early diagnosis of sepsis utilizing antigen-antibody interactions amplified by whole blood chemiluminescence

• US 5,804,370 A
• Filed: 11/02/1995
• Issued: 09/08/1998
• Est. Priority Date: 06/08/1994
• Status: Expired due to Term

• Current Assignee: Spectral Diagnstics, Inc.
• Original Assignee: Critichem Medical Products Limited
• Inventors: Romaschin, Alex D., Walker, Paul M.
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/552,145
• Time in Patent Office: 1,041 Days
• Field of Search: 435/5, 435/7.1, 435/7.2, 435/7.24, 435/7.31-7.37, 435/25, 435/34, 435/38, 435/962, 435/968, 435/973, 435/975, 436/513, 436/518, 436/536, 436/539, 436/808, 436/811
• US Class Current: 435/5
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/5768 Hepatitis A
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6869 Interleukin
• 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 435/962 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 435/975 Kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

The invention relates to a method for determining the extent of sepsis and/or an infection in a human or animal patient by detecting the amount of an antigen indicative of such infection. The amount of the antigen is detected in a patient blood derived test sample containing blood cell fractions. The method comprises:

i) incubating the test sample with an amount of test antibodies specific to the antigen indicative of sepsis and/or infection to form antibody/antigen complexes;

ii) allowing the antibody/antigen complexes to interact with the white blood cell fractions which results in the production of oxidants;

iii) introducing to either steps i) or ii) a chemiluminescent compound to the test sample;

iv) allowing the oxidants to react with the chemiluminescent compounds to emit luminescent light from the test sample;

v) measuring the amount of emitted light over a predetermined period; and

vi) correlating extent of sepsis and/or infection by comparison of the measured amount of emitted light of the test sample with measured amount of light emitted by a control sample which is treated the same as the test sample for steps i) to v) except that in step i) control antibodies are used which are of the same class as the test antibodies but are non-specific to the antigens indicative of infection.

Claims

1. A method for determining the presence or extent of an infection in a human or animal patient by determining the amount of a preselected antigen indicative of said infection in a sample of said patient'"'"'s blood, said sample comprising plasma and white blood cells, said method sequentially comprising:

i) providing first and second aliquots of equal volume of said sample;

ii) reacting the first aliquot of said sample with an amount of test antibody sufficient to form an antigen/antibody complex with said antigen, wherein said test antibody specifically binds to said antigen, to provide a test sample;

iii) reacting the second aliquot of said sample with an equal amount of a control antibody wherein said control antibody (a) does not specifically bind said antigen and (b) is of the same class and species of origin as the test antibody, to provide a control sample;

iv) incubating the test and control samples for a time sufficient for the antigen/antibody complex to react with the white blood cells and the complement proteins in the plasma to produce oxidants;

v) contacting a chemiluminescent compound which reacts with said oxidants to generate luminscent light with either the test and control samples of steps ii) and iii) or with the test and control samples of step iv);

vi) measuring light emission over a predetermined time period; and

vii) correlating differences in light emission between the test and control samples to the presence or amount of said antigen in said sample and thereby to the presence or extent of the infection in the patient.

2. The method of claim 1 wherein said sample is whole blood.

3. The method of claim 1 wherein said white blood cells are selected from the group consisting of neutrophils, lymphocytes, monocytes and combinations thereof.

4. The method of claim 1 wherein sufficient zymosan is contacted with said test and control samples before step vi) to stimulate said production of oxidants.

5. The method of claim 1 wherein sufficient opsonized zymosan is contacted with said test and control samples before step vi) to stimulate said production of oxidants.

6. The method of claim 1 wherein sufficient opsonized latex beads are contacted with said test and control samples before step vi) to stimulate said production of oxidants.

7. The method claim 1 wherein said chemiluminescent compound is selected from the group consisting of luminol, lucigenin and pholasin.

8. The method of claim 7 wherein the chemiluminescent compound is luminol.

9. The method of claim 8 wherein sufficient zymosan is contacted with said test and control samples before step vi) to stimulate said production of oxidants.

10. The method of claim 8 wherein sufficient opsonized zymosan is contacted with said test and control samples before step vi) to stimulate said production of oxidants.

11. The method of claim 1, wherein the test sample and control sample in step i) are incubated with three dilutions of said test and control antibodies, said dilutions being 1:

10, 1;

100 and 1;

1000, and wherein said correlating step further correlates said differences in light emission between said test samples and said control samples to the quantity of said antigen with the dilution which provides the greatest difference in light emissions.

12. The method of claim 11 wherein said test and control antibodies are IgM or IgG class monoclonal antibodies.

13. The method of claim 1 wherein the antigen is present on, released by or secreted by gram-negative bacteria, gram-positive bacteria, virus or fungus.

14. The method of claim 13, wherein the antigen is a Hepatitis A virus antigen.

15. The method of claim 13 wherein the antigen is gram-negative bacterial endotoxin lipid A.

16. The method of claim 15 wherein sufficient opsonized latex beads are contacted with said test and control samples before steps vi) to stimulate said production of oxidants.

17. A diagnostic kit for use in determining the extent of an infection in a human of animal patient by detecting the amount of a preselected antigen indicative of said infection in a sample of blood from said patient, said sample comprising plasma and white blood cells, said kit comprising:

i) a first container of test antibody which specifically binds to said antigen,

ii) a second container of chemiluminescent compound which reacts with oxidants produced by said white blood cells to generate luminescent light, and

iii) a third container of control antibody which does not specifically bind said antigen and which is of the same class and species of origin as the test antibody.

18. The diagnostic kit of claim 17 wherein said antibodies are of the IgM class.

19. The diagnostic kit of claim 18 wherein said IgM test antibody specifically binds to a gram-negative bacterial endotoxin Lipid A.

20. The diagnostic kit of claim 17 wherein said antibodies are of the IgG class.

21. The diagnostic kit of claim 20 wherein said IgG test antibody specifically binds to Hepatitis A virus.

22. The diagnostic kit of claim 17 wherein said chemiluminescent compound is selected from the group consisting of luminol, lucigenin and pholasin.

23. The diagnostic kit of claim 17 further comprising a fourth container of zymosan or opsonized zymosan.

24. A method for determining the presence or extent of sepsis in a human or animal patient by determining the amount of a preselected sepsis marker in a sample of said patient'"'"'s blood, said sample comprising plasma and white blood cells, said method sequentially comprising:

i) providing first and second aliquots of equal volume of said sample;

ii) reacting the first aliquot of said sample with an amount of test antibody sufficient to form an antigen/antibody complex with said marker, wherein said test antibody specifically binds to said marker, to provide a test sample;

iii) reacting the second aliquot of said sample with an equal amount of a control antibody wherein said control antibody (a) does not specifically bind said marker and (b) is of the same isotype as the test antibody, to provide a control sample;

iv) incubating the test and control samples for a time sufficient for the antigen/antibody complex to react with the white blood cells and the complement proteins in the plasma to produce oxidants;

v) contacting a chemiluminescent compound which reacts with said oxidants to generate luminescen light with either the test and control samples of steps ii) and iii) or with the test and control samples of step iv);

vi) measuring light emission over a predetermined period; and

vii) correlating differences in light emission between the test and control samples to the presence or amount of said marker in said sample and thereby to the presence or extent of the sepsis in the patient.

25. A method of claim 24 wherein said sample is whole blood.

26. The method of claim 24 wherein said white blood cells are selected from the group consisting of neutrophils, lymphocytes, monocytes and combinations thereof.

27. The method of claim 24 wherein the test sample and control sample in step i) are incubated with three dilutions of said test and control antibodies, said dilutions being 1:

10, 1;

100 and 1;

1000, and wherein said correlating step further correlates said differences in light emission between said test samples and said control samples to the extent of said sepsis with the dilution which provides the greatest difference in light emissions.

28. The method of claim 24 wherein said test and control antibodies are IgM or IgG class monoclonal antibodies.

29. The method of claim 24 wherein the sepsis marker in an inflammatory mediator.

30. The method of claim 29 wherein the inflammatory mediator is selected from the group consisting of tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8, interferon and transforming growth factor β

.

31. The method of claim 30 wherein in the sample is whole blood.

32. A diagnostic kit for use in determining the presence or extent of sepsis in a human or animal patient by detecting the amount of a preselected sepsis marker in a sample of blood from said patient, said sample comprising plasma and white blood cells, said kit comprising:

i) a first container of test antibody which specifically binds to said marker,

ii) a second container of chemiluminescent compound which reacts with oxidants produced by said white blood cells to generate chemiluminescent light, and

ii) a third container of control antibody which does not specifically bind to said marker and which is of the same class and species of origin as the test antibody.

33. The diagnostic kit of claim 32 wherein said marker is an inflammatory mediator.

34. The diagnostic kit of claim 33 wherein the inflammatory mediator is selected from the group consisting of tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8, interferon and transforming growth factor β

.

35. The diagnostic kit of claim 32 wherein said antibodies are of the IgM class.

36. The diagnostic kit of claim 32 wherein said antibodies are of the IgG class.

37. The diagnostic kit of claim 32 wherein said chemiluminescent compound is selected from the group consisting of luminol, lucigenin and pholasin.

38. The diagnostic kit of claim 32 further comprising a fourth container of zymosan or opsonized zymosan.