Diagnostic test

• US 4,208,187 A
• Filed: 10/06/1978
• Issued: 06/17/1980
• Est. Priority Date: 12/19/1977
• Status: Expired due to Term

• Current Assignee: American Home Products Corporation (Pfizer Inc.)
• Original Assignee: American Home Products Corporation (Pfizer Inc.)
• Inventors: Givner, Morris L.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US05/949,174
• Time in Patent Office: 620 Days
• Field of Search: 23/230 B, 23/917, 424/12, 210/22 R, 210/23 F, 422/59, 422/69, 422/101
• US Class Current: 436/521
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/814 Pregnancy
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

A simple, sensitive, reliable and safe method and device for detecting a polypeptide or glycoprotein antigen. The test involves concentration in the presence of polyvinylpyrrolidone by ultrafiltration of a sample of urine or serum from a subject; followed by determining the presence of said polypeptide or glycoprotein antigen or a particular subunit thereof in the concentrated sample.

Claims

1. In a diagnostic test method suitable for the detection of physiological conditions characterized by the presence of a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone in urine including:

(a) clarifying a sample of the urine to be tested;

(b) subjecting about 5 to 50 ml of the clarified urine sample to absorbent induced ultrafiltration through an ultrafiltration membrane backed by a layer of absorbent capable of sorbing urine serum passing through the membrane, and said membrane having a molecular weight with cut-off from about 5,000 to about 200,000, whereby said polypeptide or glycoprotein antigen is retained in the sample;

(c) continuing the ultrafiltration until the retentate sample is one-tenth to one-five hundredth its original volume;

(d) diluting the retentate with sufficient water to provide a retentate sample of about 0.5 ml; and

(e) contacting the retentate sample with the appropriate amount of an immunologic reagent for detecting the presence of said polypeptide or glycoprotein antigen in the retentate sample, with the proviso that when said antigen is human chorionic gonadotropin, then the immunologic reagent comprises other than pyruvic aldehyde stabilized erythrocytes sensitized to human chorionic gonadotropin with a bifunctional molecule;

the improvement which comprises contacting said membrane with a dilute solution of polyvinylpyrrolidone prior to or concurrent with ultrafiltration, wherein said polyvinylpyrrolidone has an average molecular weight greater than about 9,000 and less than about 40,000.

5. The method of claim 1, 2 or 3 suitable for the detection of human chorionic gonadotropin of pregnancy or cancer in urine at a concentration as low as about 40 m.I.U./ml of urine and capable of detecting pregnancy as early as day 26 of a regular 28 day menstrual cycle wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 0.5% to about 4.0%, weight by volume.

6. The method of claim 1, 2 or 3 for the detection of human chorionic gonadotropin wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000 and the concentration of the dilute solution of polyvinylpyrrolidone is from about 1.0% to about 2.0%, weight by volume.

7. The method of claim 1, 2 or 3 suitable for the detection of human chorionic gonadotropin of pregnancy or cancer in urine at a concentration as low as about 40 m.I.U/ml of urine and capable of detecting pregnancy as early as day 26 of a regular 28 day menstrual cycle wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 0.5% to about 4.0%, weight by volume.

8. The method of claim 1, 2 or 3 for the detection of human chorionic gonadotropin wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 1.0% to about 2.0%, weight by volume.

11. The method of claim 1, 2 or 3 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 0.5% to about 4.0%, weight by volume.

12. The method of claim 1, 2 or 3 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 1.0% to about 2.0%, weight by volume.

13. The method of claim 1, 2 or 3 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 0.5% to about 4.0%, weight by volume.

14. The method of claim 1, 2 or 3 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of the dilute solution of polyvinylpyrrolidone is from about 1.0% to about 2.0%, weight by volume.

17. The method of claim 1, 2, 3 or 4 suitable for the detection of physiological conditions characterized by the presence of a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone in urine wherein said immunologic reagent comprises:

(1) pyruvic aldehyde stabilized erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone, said stabilized erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinialdehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; and

(2) an antiserum to detect said polypeptide or glycoprotein antigen.

18. The test method of claim 1, 2 3 or 4 suitable for the detection of physiological conditions characterized by the presence of a polypeptide of glycoprotein antigen selected from chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone in urine wherein said membrane has a molecular weight cut-off from about 10,000 is about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the immunologic reagent comprises;

(1) pyruvic aldehyde stabilized erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone, said stabilized erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinialdehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(33-dimethylaminopropyl)carbodiimide; and

(2) an antiserum to detect said polypeptide or glycoprotein antigen.

19. The method of claim 1, 2 or 3 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000;

the concentration of the dilute solution of polyvinylpyrrolidone is from about 1.0% to about 2.0%, weight by volume; and

the immunologic reagents comprises;

(1) pyruvic aldehyde stabilized erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from human chorionic gonadotropin, luteinizing hormone, follicle stimulating hormone, and human menopausal gonadotropin, said stabilized erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinialdehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; and

(2) an antiserum to detect said polypeptide or glycoprotein antigen.

2. In a diagnostic test method suitable for the detection of physiological conditions characterized by the presence of a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone in urine including:

(a) clarifying a sample of the urine to be tested;

(b) subjecting about 5 to 50 ml of the clarified urine sample to absorbent induced ultrafiltration through an ultrafiltration membrane backed by a layer of absorbent capable or sorbing urine passing through the membrane, and said membrane having a molecular weight cut-off from about 5,000 to about 50,000, whereby said polypeptide or glycoprotein antigen is retained in the sample;

(c) continuing the ultrafiltration until the retentate sample is one-tenth to one-five hundredth its original volume

(d) diluting the retentate with sufficient water to provide a retentate sample of about 0.5 ml; and

(e) contacting the retentate sample with the appropriate amount of an immunologic reagent for detecting the presence of said polypeptide or glycoprotein antigen in the retentate sample, with the proviso that when said antigen is human chorionic gonadotropin, then the immunologic reagent comprises other than pyruvic aldehyde stabilized erythrocytes sensitized to human chorionic gonadotropin with a bifunctional molecule;

the improvement which comprises pre-washing said membrane with about 0.5% to about 4.0% solution of polyvinylpyrrolidone prior to ultrafiltration, wherein said polyvinylpyrrolidone has an average molecular weight greater than about 9,000 and less than about 40,000.

3. In a diagnostic test method suitable for the detection of physiological conditions characterized by the presence of a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyroid stimulating hormone in urine including:

(a) clarifying a sample of the urine to be tested through a filter;

(b) subjecting about 5 to 50 ml of the clarified urine sample to absorbent induced ultrafiltration through an ultrafiltration membrane backed by a layer of absorbent capable of sorbing urine serum passing through the membrane, and said membrane having a molecular weight cut-off from about 5,000 to about 50,000, whereby said polypeptide or glycoprotein antigen is retained the sample;

(c) continuing the ultrafiltration until the retentate sample is one-tenth to one-five hundredth its original volume;

(d) diluting the retentate with sufficient water to provide a retentate sample of about 0.5 ml; and

(e) contacting the retentate sample with the appropriate amount of an immunologic reagent for detecting the presence of said polypeptide or glycoprotein antigen in the retentate sample, with the proviso that when said antigen is human chorionic gonadotropin, then the immunologic reagent comprises other than pyruvic aldehyde stabilized erythrocytes sensitized to human chorionic gonadotropin with a bifunctional molecule;

the improvement which comprises pretreating said filter with about a 0.5% to about 4.0% solution of polyvinylpyrrolidone prior to ultrafiltration, wherein said polyvinylpyrrolidone has an average molecular weight greater than about 9,000 and less than about 40,000.

4. In a diagnostic test method suitable for the detection of physiological conditions characterized by the presence of a polypeptide or glycoprotein antigen selected from chorionic gonadotropin, human chorionic gonadotropin, pregnant mare'"'"'s serum gonadotropin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin and thyriod stimulating hormone in urine including:

(a) clarifying a sample of the urine to be tested through a filter;

(b) subjecting about 5 to 50 ml of the clarified urine sample to absorbent induced ultrafiltration through an ultrafiltration membrane backed by a layer of absorbent capable of sorbing urine serum passing through the membrane and said membrane having a molecular weight cut-off from about 5,000 to about 50,000, whereby said polypeptide or glycoprotein antigen is retained in the sample;

(c) continuing the ultrafiltration until the retentate sample is one-tength to one-five hundredth is original volume;

(d) diluting the retentate with sufficient water to provide a retenate sample of about 0.5 ml; and

(e) contacting the retentate sample with the appropriate amount of an immunologic reagent for detecting the presence of said polypeptide or glycoprotein antigen in the retentate sample, with the proviso that when said antigen is human chorionic gonadotropin, then the immunologic reagent comprises other than pyruvic aldehyde stabilized erythrocytes sensitized to human chorionic gonadotropin with a bifunctional molecule;

the improvement which comprises contacting said urine sample before of after clarification with a sufficient amount of polyvinylpyrrolidone to give a concentration of the polyvinylpyrrolidone in the urine of from about 0.01% to about 2.0%, wherein said polyvinylpyrrolidone has an average molecular weight greater than about 9,000 and less than about 40,000.

9. The method of claim 4 suitable for the detection of human chorionic gonadotropin of pregnancy or cancer in urine at a concentration as low as about 40 m.I.U./ml of urine and capable of detecting pregnancy as early as day 26 of a regular 28 day menstrual cycle wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the concentration of polyvinylpyrrolidone in the urine is from about 0.1% to about 1.0%, weight by volume.

10. The method of claim 4 for the detection of human chorionic gonadotropin wherein said membrane has a molecular weight cut-off from about 15,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of the polyvinylpyrrolidone in the urine is from about 0.1% to about 1.0%, weight by volume.

15. The method of claim 4 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human monopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000; and

the concentration of polyvinylpyrrolidone in the urine is from about 0.1% to aobut 1.0%, weight by volume.

16. The method of claim 4 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is about 33,000; and

the concentration of polyvinylpyrrolidone in the urine is from about 0.1% to about 1.0%, weight by volume.

20. The method of claim 4 suitable for the detection of luteinizing hormone, follicle stimulating hormone or human menopausal gonadotropin in urine wherein said membrane has a molecular weight cut-off from about 10,000 to about 35,000;

the average molecular weight of said polyvinylpyrrolidone is from about 28,000 to about 35,000;

the concentration of polyvinylpyrrolidone in the urine is from about 0.1% to about 1.0%, weight by volume; and

the immunologic reagent comprises;

(1) pyruvic aldehyde stabilized erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from human chorionic gonadotropin, luteinizing hormone, follicle stimulating hormone, and human menopausal gonadotropin, said stabilized erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinialdehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; and

(2) an antiserum to detect said polypeptide or glycoprotein antigen.