Method of detecting cytopenia that is mediated by drug-dependent antibody binding to blood cells

• US 5,585,243 A
• Filed: 09/15/1993
• Issued: 12/17/1996
• Est. Priority Date: 09/15/1993
• Status: Expired due to Fees

• Current Assignee: The Blood Center of Southeastern Wisconsin Incorporated
• Original Assignee: The Blood Center of Southeastern Wisconsin Incorporated
• Inventors: Curtis, Brian R., Aster, Richard H.
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/120,837
• Time in Patent Office: 1,189 Days
• Field of Search: 435/7.21, 435/7.2, 435/7.24, 435/7.92, 435/7.95, 435/7.25, 435/7.9, 436/519, 436/520
• US Class Current: 435/7.21
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/80 involving blood groups or blood types or red blood cells white blood cells G01N33/56972

Abstract

Drug-dependent antibodies that bind to granulocytes, erythrocytes, platelets or membrane proteins derived from these cells, in the presence of a drug, but not in its absence, can be detected using a sensitive assay. Detection of the drug-dependent antibodies permits diagnosis of cytopenia mediated by the drug.

Claims

1. A method of detecting drug-dependent antibodies in a patient, wherein a drug or a metabolite of said drug is suspected of inducing thrombocytopenia, said method comprising:

(i) separately incubating human platelets or platelet membrane protein derived from said human platelets with a serum or plasma sample from said patient, and a reagent drug or drug metabolite solution or a buffer solution, said drug or said metabolite of said drug being solubilized in said buffer solution to form said reagent drug or drug metabolite solution, said incubation being affected at a temperature and for a time sufficient to form complexes of (1) said drug or said metabolite, (2) said platelets or said platelet membrane protein, and (3) any of said drug-dependent antibodies in said serum or plasma, to form respective test and control reaction solutions; and

,

(ii) immunologically determining the amount of antibody bound to said platelets or said platelet membrane protein in said test and control reaction solutions;

wherein said drug-dependent antibodies are present when the amount of bound antibody is greater in the test reaction solution than in the control reaction solution; and

wherein said drug or said metabolite is selected from the group consisting of acetazolamide, actinomycin, allopurinol, allylisopropylacetylurea, allylisopropylbarbiturate, alpha methyldopa, amiodarone, amphotericin B, ampicillin, amrinon, antazoline, antipyrine, apalcillin, benoxaprofen, bleomycin, butabarbitone, butoconazole, captopril, carbamazepine, carbenicillin, ceftazidine, ceftriazone, centalum, cephalexin, cephalothin, cephamandole, chenodeoxycholic acid, chlordiazepoxide, chloroquine, chlorothiazide, chlorpheniramine, chlorpropamide, chlorthalidone, cimetidine, clarithromycin, clometacine, clonazepam, clopamide, cloxacillin, cocaine, cyclophosphamide, cyclosporine A, danazol, desferrioxamine, diatrizoate, diazepam, diazoxide, diclofenac, didanosine, difluoromethylornithine, digitalis, digitoxin, digoxin, diltiazem, disulfiram, doxepin, ethambutol, etretinate, famotidine, fenoprofen, flavone-8-acetic acid, fluconazole, fluphenazine, furosemide, fusidic acid, gentamicin, glibenclamide, glymidine, haloperidol, heroin, hydrochlorothiazide, hydroxychloroquine, hydroxyquinoline, ibuprofen, imipramine, indomethacin, interferon, iocetamic acid, iopanoic acid, isoniazid, isotretinoin (vitamin A), L-dopa aminoglutethimide, levamisole, levodopa, lidocaine, lincomycin, lithium salts, meclofenamate, meprobamate, methazolamide, methicillin, methotrimeprazine, methylchlorothiazide, methylphenidate, mexiletine, mezlocillin, mianserin, minoxidil, morphine, moxalactam, N-acetylsulfanilamide sodium, nalidizic acid, naproxen, nifedipine, nimesulfide, nitrofurantoin, nitroglycerin, nomifensine, noramiopyrine, norfloxacin, novobiocin, octreotide acetate, odansetron, oxprenolol, oxyphenbutazone, oxytetracycline, p-aminosalicylate, paramethadione, penicillamine, pentagastrin, pentamidine, pentosan polysulfate, a phenothiazine, phenylbutazone, phenytoin, phthalazinol, phthalysulfathiazole, piperacillin, piperazine, piroxicam, plicamycin, polyurethane, primidone, procainamide, prochlorperazine, propranolol, propylthiouracil, ranitidine, simvastatin, sodium salicylate, spiramycin, spironolactone, stibogluconate, streptomycin, succinylsufathiazole, sulfadimethoxine, sulfaguanidine, sulfamerazine, sulfamethizole, sulfamethoxypyridazine, sulfanilamide, sulfasalazine, sulfathiazole, sulindac, suramin, thioguanin, thiomerazol, thiouracil, ticlopidine, tienilic acid, tobramycin, tolbutamide, tolmetin, trimethoprim, valproic acid and metabolites thereof.

4. The method of any one of claims 1-3, wherein said determining comprises binding a fluorescently labelled-anti-immunoglobulin specific binding partner to said bound antibodies and measuring bound fluorescence intensity by flow cytometry.

5. The method of claim 4, wherein said flow cytometry comprises measuring a ratio of the mean fluorescence intensity of said test reaction solution to the mean fluorescence intensity of said control reaction solution, and wherein a ratio of 1.5 or greater indicates the presence of said drug-dependent antibodies.

6. The method of any one of claims 1-3, wherein the concentration of said drug or said metabolite in said reagent drug or said metabolite solution is about 0.1 mM to about 1 mM.

7. The method of claim 6, wherein said drug or said metabolite is sparingly soluble in water and wherein said solution further comprises a solubilizing agent in an amount effective to solubilize said drug or said metabolite.

8. The method of claim 7, wherein said solubilizing agent is albumin or dimethyl sulfoxide.

9. The method of claim 8, wherein the effective amount of solubilizing agent is about 0.1 to 5% by weight of said reagent drug solution.

10. The method of claim 8, wherein said albumin is selected from the group consisting of ovalbumin, bovine serum albumin, equine serum albumin, ovine serum albumin, porcine serum albumin, subhuman primate serum albumin and human serum albumin.

11. The method of any one of claims 1-3, wherein said reagent drug solution containing said drug is used.

12. The method of any one of claims 1-3, wherein said drug metabolite solution containing said metabolite is used.

13. The method of claim 12, wherein said metabolite is provided as a urine sample, urine extract or serum sample from a normal person who has ingested said drug.

14. The method of any one of claims 1-3, wherein said determining is carried out in the presence of said drug or said drug metabolite.

15. The method of any one of claims 1-3, wherein said determining comprises, reacting said bound antibody with a labeled anti-immunoglobulin antibody or complement component.

16. The method of claim 15, wherein said determining is carried out in the presence of said drug or said metabolite.

17. The method of claim 1, wherein

(a) said drug is sparingly soluble in water;

(b) the concentration of said drug in said reagent drug solution is about 0.1 mM to about 1.0 mM;

(c) said solution further comprises a solubilizing agent in an amount effective to solubilize said drug at said concentration; and

,

(d) said drug is selected from the group consisting of acetazolamide, carbamazepine, chlorothiazide, chlorpropamide, chlorthalidone, danazol, diazepam, diazoxide, diclofenac, digitalis, digitoxin, digoxin, diltiazem, etretinate, fenoprofen, fluconazole, furosemide, hydrochlorothiazide, ibuprofen, indomethacin, isotretinoin, levodopa, methylchlorothiazide, nalidixic acid, naproxen, nifedipine, norfloxacin, phenylbutazone, phenytoin, piroxicam, spiramycin, spironolactone, sulfadimethoxine, sulfamerazine, sulfamethizole, sulfanilamide, sulfasalazine, sulindac, thiazide, tolbutamide, tolmetin and trimethoprim.

2. A method of detecting drug-dependent antibodies in a patient, wherein a drug or a metabolite of said drug is suspected of inducing hemolytic anemia, said method comprising:

(i) separately incubating human erythrocytes or erythrocyte membrane protein derived from said human erythrocytes with a serum or plasma sample from said patient, and a reagent drug or drug metabolite solution or a buffer solution, said drug or said metabolite of said drug being solubilized in said buffer solution to form said reagent drug or drug metabolite solution, said incubation being affected at a temperature and for a time sufficient to form complexes of (1) said drug or said metabolite, (2) said erythrocytes or said erythrocyte membrane protein, and (3) any of said drug-dependent antibodies in said serum or plasma, to form respective test and control reaction solutions; and

,

(ii) immunologically determining the amount of antibody bound to said erythrocytes or erythrocyte membrane protein in said test and control reaction solutions;

wherein said drug-dependent antibodies are present when the amount of bound antibody is greater in the test reaction solution than in the control reaction solution; and

wherein said drug or said metabolite is selected from the group consisting of acetaminophen, antazoline, carbromal, chlorpropamide, diethylstilbestrol, erythromycin, 5-fluorouracil, ibuprofen, insulin, isoniazid, melphalan, mesantoin, nalidixic acid, p-aminosalicylic acid, phenacetin, a phenothiazine, probenecid, propanil, rifampicin, streptomycin, sulfonylurea, sulindac, tetracycline, thiazides, thiopental, tolbutamide, tolmetin, triamterene, warfarin and metabolites thereof.

18. The method of claim 2 wherein

(a) said drug is sparingly soluble in water;

(b) the concentration of said drug in said reagent drug solution is about 0.1 mM to about 1 mM;

(c) said solution further comprises a solubilizing agent in an amount effective to solubilize said drug at said concentration; and

,

(d) said drug is selected from the group consisting of chlorothiazide, chlorpropamide, 5-fluorouracil, ibuprofen, probenecid, sulindac, tolbutamide, tolmetin and triamterene.

3. A method of detecting drug-dependent antibodies in a patient, wherein a drug or a metabolite of said drug is suspected of inducing granulocytopenia, said method comprising:

(i) separately incubating human granulocytes or granulocyte membrane protein derived from said human granulocytes with a serum or plasma sample from said patient, and a reagent drug or drug metabolite solution or a buffer solution, said drug or said metabolite of said drug being solubilized in said buffer solution to form said reagent drug or drug metabolite solution, said incubation being affected at a temperature and for a time sufficient to form complexes of (1) said drug or said metabolite, (2) said granulocytes or said granulocyte membrane protein, and (3) any of said drug-dependent antibodies in said serum or plasma, to form respective test and control reaction solutions; and

,

(ii) immunologically determining the amount of antibody bound to said granulocytes or granulocyte membrane protein in said test and control reaction solutions;

wherein said drug-dependent antibodies are present when the amount of bound antibody is greater in the test reaction solution than in the control reaction solution; and

wherein said drug or said metabolite is selected from the group consisting of acetaminophen, acetazolamide, allopurinol, aminopyrine, amitriptyline, amoxapine, captopril, carbamazepine, carbimazole, cephalosporin, chloramphenicol, chlordiazepoxide, tolbutamide, chloroquine, chlorothiazide, chlorthalidone, cimetidine, clindamycin, clozapine, dapsone, desipramine, dipyrone, disopyramide, doxepin, ethacrynic acid, gentamicin, hydralazine, hydrochlorothiazide, imipramine, indomethacin, isoniazid, levamisole, mephenytoin, meprobamate, metamizole, methimazole, methyldopa, noramidopyrine, oxyphenbutazone, para-aminosalicylic acid, penicillamine, pentazocine, phenacetin, a phenothiazine, phenylbutazone, phenytoin, procainamide, propranolol, propylthiouracil, pyrimethamine, quinidine, ranitidine, rifampin, streptomycin, sulfonamides, tetracycline, ticlopidine, tocainide, trimethoprin-sulfamethooxazole, vancomycin and metabolites thereof.

19. The method of claim 3, wherein

(a) said drug is sparingly soluble in water;

(b) the concentration of said drug in said reagent drug solution is about 0.1 mM to about 1 mM;

(c) said solution further comprises a solubilizing agent in an amount effective to solubilize said drug at said concentration; and

,

(d) said drug is selected from the group consisting of acetazolamide, carbamazepine, chlorothiazide, chlorthalidone, clozapine, dapsone, hydrochlorothiazide, indomethacin, pentazocine, phenylbutazone, sulfamethooxazole and tolbutamide.