ALLERGY TEST BASED ON FLOW CYTOMETRIC ANALYSIS

• US 20100221756A1
• Filed: 09/11/2008
• Published: 09/02/2010
• Est. Priority Date: 09/11/2007
• Status: Active Grant

• Current Assignee: Buhlmann Laboratories AG
• Original Assignee: Buhlmann Laboratories AG
• Inventors: Schneider, Michael, Weber, Jakob, Sainte-Laudy, Jean
• Granted Patent: US 8,563,258 B2
• Time in Patent Office: Days
• US Class Current: 435/7.240
• CPC Class Codes: G01N 2800/24 Immunology or allergic disorders
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

The invention pertains to a method for the determination of basophil activation induced by a test substance by flow cytometric measurement of the changes of the mean or median fluorescence intensities (WI) of the basophilic F_cεRI receptor present on the cell surface of basophils (MFI-F_cεRI) and/or the IgE antibodies bound to the F_cεRI receptor (MFI-IgE), and the CD63 antigen exposed on the cell surface of basophils after their activation (MFI-CD63), by means of a mixture of anti-CD63, anti-F_cεRI or anti-IgE, and anti-CCR3 antibodies each labelled with a distinct fluorophore, of which at least one antibody acts as a basophil selection marker and at least two antibodies act as basophil activation markers, and bringing the mean fluorescence intensities of the activation markers in correlation to obtain an Activation Index. These methods combining the measurement of an early (such as IgE, F_cεRI or CD203c) and a late basophil activation marker (such as CD63), respectively, provide a markedly improved clinical sensitivity in allergy diagnosis over existing methods which consider only one activation marker, such as CD203c or CD63, expressed in percentage of basophil activation. It is also an aspect of the present invention to provide an ex-vivo allergy provocation test comprising the above-mentioned flow cytometric measurement and analysis of the results as well as a test kit for carrying out the test in-vitro.

Claims

1. A method for the determination of basophil activation induced by a test substance by flow cytometric measurement of the changes of the mean or median fluorescence intensities (MFI) of the basophilic F_cε

RI receptor present on the cell surface of basophils (MFI-F_cε

RI) and/or the IgE antibodies bound to said F_cε

RI receptor (MFI-IgE), and the CD63 antigen exposed on the cell surface of basophils after their activation (MFI-CD63), by means of a mixture of anti-CD63, anti-F_cε

RI or anti-IgE, and anti-CCR3 antibodies each labelled with a distinct fluorophore, of which at least one antibody acts as a basophil selection marker and at least two antibodies act as basophil activation markers, wherein at least one antibody may function both as selection and activation marker, and bringing the changes of the mean fluorescence intensities of said activation markers in correlation to obtain an Activation Index.

2. A method according to claim 1, wherein the Activation Index is defined as the product of the values either related to MFI-CD63 (A) or to MFI-IgE (B) or to MFI-F_cε

RI (B) expressed as

$y=\frac{a-d}{\left(1+{\left(\frac{x}{c}\right)}^b\right)}+d$

wherein

i) a is defined as the minimum value of the function curve,

ii) b is defined as the slope of the curve,

iii) c is defined as the turning point of the curve,

iv) d is defined as the maximum asymptotic value of the curve,

v) x is defined as 1 minus the ratio between (A) of the negative control and (A) of the test substance or, alternatively, (A) of the positive control (MFI-CD63 of negative control divided by MFI-CD63 of test substance or, alternatively, of positive control) or 1 minus the ratio between (B) of the test substance or, alternatively, (B) of the positive control and (B) of the negative control (MFI-IgE or MFI-F_cε

RI of test substance or, alternatively, of positive control divided by MFI-IgE or MFI-F_cε

RI of negative control),

vi) y is defined as the Activation Index of (A) or (B), and

vii) a combined Activation Index is calculated by addition of y(A) and y(B) or by multiplying y(A) with y(B).

3. Method according to claims 1, wherein said mixture comprises as labelled antibodies any combination of

i) anti-CCR3, anti-IgE, anti-F_cε

RI, anti-CD45, anti-CD203c, anti-CD 123 and anti-HLA-DR, anti-CRTH2 and anti-CD3, basophil specific monoclonal Ba103 antibody, and basophil specific monoclonal 212H6 antibody for basophil selection (“

selection marker”

), and

ii) anti-CD63, anti-IgE, anti-F_cε

RI, anti-CD203c, anti-CCR3, anti-CD45, anti-CD13, anti-CD69, anti-CD164, and anti-CD107a for basophil activation (“

activation marker”

)

4. Method according to claim 3, wherein the combination of the labelled antibodies is selected from

I. anti-IgE or anti-F_cε

RI as both selection and activation marker and anti-CD63 as activation marker,

II. anti-CCR3 as both selection and activation marker and anti-CD63 as activation marker,

III. anti-CCR3 as selection marker and both anti-CD63 and anti-IgE or anti-F_cε

RI as activation markers,

IV. anti-CCR3 as selection marker and both anti-CD203c and anti-CD63 as activation markers, and

V. anti-CD203c as both selection and activation marker and anti-CD63 as activation marker.

5. Method according to claim 1, wherein the test substance is a mitogen, an antigen, an allergen, a protein or peptide, a protein or peptide allergen, a group or mixture of protein and/or peptide allergens, a non-proteinaceous allergen, a low molecular weight allergen, a low molecular weight drug substance or a hapten.

6. Method according to claim 1, wherein the antigen or allergen is a low molecular weight substance below 1000 Da.

7. Method according to claim 5, wherein the allergen is a non-proteinaceous allergen, a paucivalent allergen or a true hapten.

8. Method according to claim 5, wherein the allergen is a low molecular weight drug substance.

9. Method according to claim 8, wherein the drug substance is selected from the group consisting of antibiotics, such as penicillins and β

-lactams (preferably Penicillin G, Penicillin V, PPL, MDM, Amoxicillin, Ampicillin), cephalosporins (preferably Cefazolin, Cefamandole, Cefaclor, Cefuroxime, Ceftriaxone, Cefotaxime, Ceftazidime, Cefepime), carbapenems, monobactams, β

-lactamase inhibitors (preferably Clavulanic acid), macrolides, aminoglycosides, rifamycins, glycopetides, polypeptides, tetracyclins, imidazoles, quinolones, pyrazolones, sulfonamides (preferably Sulfomethoxazole), streptogramins, nitrofurans, is oniazides, pentamidines and any other class of antiobiotics;

antiseptics (preferably Chlorhexidine);

fungicides;

antiviral agents;

anti-malarial agents;

analgesics, COX-2 inhibitors and non-steroidal anti-inflammatory drugs (preferably Aspirin, Lys-Aspirin, Ibuprofen, Ketoprofen, Diclofenac, Naproxen, Paracetamol (Acetaminophen), Dipyrone (Metamizol), Indomethacine, Mefenamic acid, Phenylbutazone and Propyphenazone);

neuromuscular blocking agents (preferably Suxamethonium, Atracurium, Cis-atracurium, Mivacurium, Pancuronium, Rocuronium, Vecuronium, and Succinylcholine);

hypnotics and local anesthetics (preferably Midazolam, Propofol, Thiopental, Fentanyl and Lidocaine);

tranquilizers;

opioids;

radio contrast media such as ionic iodinated contrast agents, non-ionic iodinated contrast agents, Isosulfan Blue, Patent Blue and Methylene Blue;

proton pumping inhibitors;

anticonvulsants and neuroleptics (preferably Carbamazepine, Phenytoin and Valproic acid);

anti-psychotic agents;

antidepressants;

dopamins;

anti-histamines;

cortico steroids and glucocorticoids;

chemotherapeutic and immunosuppressive agents;

diuretics;

anticoagulants;

vasoconstrictors and vasodilatators;

cardiac drugs such as statins, ACE inhibitors, alpha-receptor blockers, beta-receptor blockers calcium antagonists and anti-hypertonic agents;

(anti-)ulcer drugs;

(anti-)thyroid drugs;

estrogens;

heparins and derivatives;

insulins;

streptokinases and urokinases; and

any other drug causing adverse drug reactions or drug hypersensitivities.

10. Method according to claim 5, wherein the allergen is a colloid, plasma expander or auxiliary agent.

11. Method according to claim 10, wherein the colloid, plasma expander or auxiliary agent is selected from the group consisting of albumine, dextrane, gelatine, hetastarch, pentastarch, sinistrin, polydocanol 600 (ethoxysclerol), lactose, carboxymethylcellulose, hydroxypropylcellulo se, protamine and aprotinine.

12. Method according to claim 5, wherein the allergen is a food additive.

13. Method according to claim 12, wherein the food additive is selected from the group consisting of food preservatives, food colorants, food finishers, anti-oxidants, and emulsifiers.

14. Method according to claim 5, wherein the allergen is an occupational, environmental or pollutant agent.

15. Method according to claim 14, wherein the occupational, environmental or pollutant agent is selected from the group consisting of isocyanates, isothiazolinones, formaldehyde, ethylene oxide, phthalic anhydride, chloramine T, DMSO, latex, and enzymes used in the baking, food processing and washing industry.

16. Method according to claim 5, wherein the test substance is a single allergen or a mixture of any combination of allergens.

17. Method according to claim 1, wherein the test substance is a protein or peptide allergen or a mixture of protein allergens and/or peptide allergens.

18. An in-vitro allergy test comprising the steps of

a) incubating either

i. an anti-coagulated human or animal whole blood sample, or

ii. an anti-coagulated and subsequently purified human or animal blood sample

with a test substance for 5 to 120 minutes, preferably 10-30 minutes, at 30 to 40°

C., preferably at 35-38°

C., to activate the basophils in said sample,

b) stopping said activation by

i) adding a stop solution containing EDTA to said sample, and/or

ii) putting said sample immediately at 0 to 5°

C.,

c) adding to said sample a mixture of antibodies each labelled with a distinct fluorophore, wherein said mixture comprises as labelled antibodies any combination of

i) anti-CCR3, anti-IgE, anti-Fcε

RI, anti-CD45, anti-CD203c, anti-CD123 and anti-HLA-DR, anti-CRTH2 and anti-CD3, basophil specific monoclonal Ba103 antibody, and basophil specific monoclonal 212H6 antibody for basophil selection (selection marker) and,

ii) anti-CD63, anti-IgE, anti-Fcε

RI, anti-CD203c, anti-CCR3, anti-CD45, anti-CD13, anti-CD69, anti-CD164, and anti-CD107a for basophil activation (activation marker)

and incubating said sample mixture for 5 to 45 minutes, preferably 10 to 20 minutes, at 2 to 8°

C.,

d) lysing remaining erythrocytes in said sample mixture and analysing it on a flow cytometer,

e) measuring the basophil activation, as expresssed in changes of the MFI, in said sample mixture induced by said test substance,

f) calculating the basophil Activation Index according to claim 2 by correlating the MFI changes.

19. Test according to claim 18, wherein the combination of the labelled antibodies is selected from

I. anti-IgE or anti-Fcε

RI as both selection and activation marker and anti-CD63 as activation marker,

II. anti-CCR3 as both selection and activation marker and anti-CD63 as activation marker,

III. anti-CCR3 as selection marker and both anti-CD63 and anti-IgE or anti-Fcε

RI as activation markers,

IV. anti-CCR3 as selection marker and both anti-CD203c and anti-CD63 as activation markers, and

V. anti-CD203c as both selection and activation marker and anti-CD63 as activation marker.

20. Test according to claim 18, wherein the concentration of the test substance in the sample is in the range of 0.1 ng/ml to 10 mg/ml.

21. Test according to claim 18, wherein the test substance is a substance.

22. Test according to claim 18, wherein the whole blood sample or purified blood sample preferably comprises EDTA as anticoagulant.

23. Test according to claim 18, wherein the whole blood sample or purified blood sample has a volume between 10 and 200 μ

l, preferably 20 to 50 μ

l.

24. Test according to claim 18, wherein before step a) interleukin-3 is added to the whole blood sample or purified blood sample.

25. Test according to claim 24, wherein said interleukine-3 is added in an amount resulting in a range of concentration of 0.2 to 20 ng/ml, preferably 2 ng/ml.

26. Test according to claim 18, wherein steps a) and c) are carried out simultaneously for 5 to 120 minutes, preferably 10-30 minutes, at 30 to 40°

C., preferably at 35 to 38°

C.

27. Test according to claim 18, wherein lysis step d) is omitted and said sample mixture is directly measured on a flow cytometer.

28. A test kit for an in-vitro allergy test according to claim 18, wherein said kit comprises per 100 tests

a) 1 to 5 ml anti-IgE or anti-Fcε

RI,

b) 1 to 5 ml anti-CD63, and

c) 1 to 5 ml anti-CCR3.

29. A test kit for an in-vitro allergy test according to claim 18, wherein said kit comprises per 100 tests

a) 1 to 5 ml anti-CD63,

b) 1 to 5 ml anti-CD203c, and

c) 1 to 5 ml anti-CCR3.

30. A test kit according to claim 28 further comprising 1 to 5 ml of a mixture of antibodies, wherein the mixture comprises as labelled antibodies any combination of

i) anti-CCR3, anti-IgE, anti-Fcε

RI, anti-CD45, anti-CD203c, anti-CD123 and anti-HLA-DR, anti-CRTH2 and anti-CD3, basophil specific monoclonal Ba103 antibody, and basophil specific monoclonal 212H6 antibody for basophil selection, and

ii) anti-CD63, anti-IgE, anti-Fcε

RI, anti-CD203c, anti-CCR3, anti-CD45, anti-CD13, anti-CD69, anti-CD164, and anti-CD107a for basophil activation, preferably

I. anti-IgE or anti-F_cε

RI as both selection and activation marker and anti-CD63 as activation marker,

II. anti-CCR3 as both selection and activation marker and anti-CD63 as activation marker,

III. anti-CCR3 as selection marker and both anti-CD63 and anti-IgE or anti-F_cε

RI as activation markers,

IV. anti-CCR3 as selection marker and both anti-CD203c and anti-CD63 as activation markers, and

V. anti-CD203c as both selection and activation marker and anti-CD63 as activation marker.

31. Test kit according to claim 28, wherein the test kit comprises additionally up to 200 ml of a cellular incubation buffer.

32. Test kit according to claim 28, wherein the cellular incubation buffer contains additionally 0.2 to 20 ng/ml of interleukine-3, preferably 2 ng/ml.

33. Test kit according to claim 28, wherein the cellular incubation buffer contains additionally 0.5 to 10 mmol/l of calcium ions.

34. Test kit according to claim 28, wherein the cellular incubation buffer contains additionally 0.01 to 0.5% (w/v) of heparin, preferably 0.05% (w/v).

35. Test kit according to claim 28, wherein the test kit comprises additionally up to 5 ml of a Stimulation Control, selected from the groups of anti-IgE antibody, anti-Fcε

RI antibody, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), complement factor 5a (C5a), platelet activation factor (PAF), toll-like receptor (TLR) agonists, ionomycin, calcium ionophore A23187, lipopolysaccharides (LPS) and phytohaemagglutinins (PHA), preferably

36. Test kit according to claim 28, wherein the test kit comprises additionally up to 5 ml of a Stimulation Control selected from an anti-Fcε

RI antibody or fMLP or a mixture thereof.

37. Test kit according to claim 28, wherein the test kit comprises additionally an unlimited number of vials containing 0.1 ng to 10 mg of a test substance.

38. Test kit according to claim 28, wherein the test kit comprises additionally up to 500 ml of a blocking buffer.

39. Test kit according to claim 28, wherein the blocking buffer contains additionally 0.5 to 10 mmol/l of EDTA, preferably 2 mmol/1.

40. Test kit according to claim 28, wherein the test kit comprises additionally up to 500 ml of an erythrocyte lysing reagent.

41. Test kit according to claim 28, wherein the test kit comprises additionally up to 200 ml of a wash buffer.

42. Test kit according to claim 28, wherein the test kit comprises additionally a software package for data processing, calculation of the basophil Activation Index, and interpretation of the individual test results on the basis of pre-defined positivity thresholds as given by the corresponding receiver operating characteristics (ROC) curves for each test substance.

43. Method for measuring allergic and drug hypersensitivity or the response to a hyposensitization therapy of said allergic or drug hypersensitivity, wherein the changes of the mean or median fluorescence intensities (MFI) of the basophilic F_cε

RI receptor present on the cell surface of basophils (MFI-F_cε

RI) and/or the IgE antibodies bound to the F_cε

RI receptor (MFI-IgE), and the CD63 antigen exposed on the cell surface of basophils after their activation (MFI-CD63) are determined in an ex-vivo sample obtained from a hypersensitive patient by means of a method according to claim 1 using at least one test substance, a negative control sample, preferably cellular incubation buffer, and a positive control sample, and the presence or absence of an allergic or drug hypersensitivity or the success or failure of a hypo sensitization therapy is determined by comparing the Activation Index values.

44. A method according to claim 1, wherein said method is for diagnosing allergic and drug hypersensitivity.

45. A test kit according to claim 28, wherein said test kit is for diagnosing allergic and drug hypersensitivity.

46. A method according to claim 1, wherein said method monitoring monitors the response to a hypo sensitization therapy.

47. A test kit according to claim 28, wherein said test kit is for monitoring the response to a hypo sensitization therapy.

48. A test kit according to claim 18, wherein said test kit is for monitoring the response to a hyposensitization therapy, wherein interleukine 3 is omitted.

49. A test kit according to claim 28, wherein said test kit is for monitoring the response to a hyposensitization therapy, wherein interleukine-3 in the cellular incubation buffer is omitted.