Affinity column and process for detection of low molecular weight toxic substances

• US 4,859,611 A
• Filed: 01/21/1986
• Issued: 08/22/1989
• Est. Priority Date: 02/28/1985
• Status: Expired due to Term

• Current Assignee: Boston University, Massachusetts Institute of Technology
• Original Assignee: Boston University, Massachusetts Institute of Technology
• Inventors: Groopman, John D., Ferrari, Christopher, Bargoot, Frederick G., Wogan, Gerald N.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Saunders, David A.
• Application Number:
  US06/820,388
• Time in Patent Office: 1,309 Days
• Field of Search: 422/69, 422/70, 436/513, 436/518, 436/529, 436/807, 436/815, 436/824, 436/825, 436/172, 436/548, 530/413
• US Class Current: 436/518
• CPC Class Codes: C07K 16/14 against material from fungi, algea or lichens
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/56961 Plant cells or fungi
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

An affinity matrix and a method for the detection of low molecular weight compositions such as aflatoxins are provided utilizing specific monoclonal IgM antibody having an affinity constant not less than about 1×10⁹ liters per mole. Methods for the preparation and use of such affinity matrices are also given. The detection is rapid, accurate, reproducible, and allows for quantitative recovery of the composition of interest.

Claims

1. A method for detecting toxins in a test sample, said method comprising:

preparing a sample believed to contain a toxin;

placing said sample on an affinity column comprised of a solid phase sorbant material and, immobilized thereon, a monoclonal antibody specific for said toxin;

eluting said column with a solvent to recover a first eluent, whereby said toxin is retained on said column by said monoclonal antibody and separated from all other ingredients of said sample;

eluting said column with a releasing agent to recover a second eluent, whereby said toxin is released from said antibody and recovered in said second eluent; and

subjecting said second eluent to fluorescence measurement by exposing said second eluent to UV light for detection of the presence of said toxin, with the proviso that said second eluent is not subjected to high pressure liquid chromatography or other purification subsequent to recovery from said column.

2. A method according to claim 1, wherein said releasing agent comprises a not less than 50% solution of an aprotic solvent.

3. A method according to claim 2, wherein said aprotic solvent is dimethyl sulfoxide, dimethyl formamide or dimethyl acetamide.

4. A method according to claim 1, wherein said releasing agent comprises a 50-100% solution of a lipophilic solvent.

5. A method according to claim 4, wherein said solvent comprises 100% methanol or 100% ethanol.

6. A method according to claim 5, wherein said toxin is aflatoxin B₁ or aflatoxin M₁ and said antibody is specific for said aflatoxins with an affinity of at least about 1×

10⁸ liters per mole.

7. A method according to claim 6, wherein said second eluent is exposed to UV light at 365 nm for visual detection of the presence of aflatoxin.

8. A method according to claim 6, wherein said second eluent is subjected to quantitative analysis for determining the level of aflatoxin in said sample.

9. A method according to claim 1, wherein said toxin is a mycotoxin having a molecular weight of not more than about 1,000 daltons.

10. A method according to claim 9, wherein said toxin is a member selected from the group consisting of aflatoxins, fluoranthene, nitropyrene, nitrosopyrene, nitrofluoranthene, nitrochrysene, aminobiphenyl, and their respective conjugates and derivatives.

11. A method according to claim 1, wherein said sample is serum, urine or food product.

12. A method according to claim 11, wherein said food product is milk, corn or peanut products.

13. A method for detecting aflatoxins in a test sample, said method comprising:

placing a sample believed to contain aflatoxin on an affinity column comprised of a solid phase sorbant material and, immobilized thereon, a monoclonal antibody for said aflatoxin;

eluting said column with a solvent to recover a first eluent, whereby said aflatoxin is retained on said column by said monoclonal antibody and separated from all other ingredients of said sample;

eluting said column with a releasing agent to recover a second eluent, whereby said aflatoxin is released from said monoclonal antibody and recovered in said second eluent; and

subjecting said second eluent to fluorescence measurement by exposing said second eluent to UV light for detection of the presence of said aflatoxin, with the proviso that said second eluent is not subjected to high pressure liquid chromatography or other purification subsequent to recovery from said column.

14. A method according to claim 13, wherein said releasing agent comprises a not less than 50% solution of an aprotic solvent.

15. A method according to claim 14, wherein said aprotic solvent is dimethyl sulfoxide, dimethyl formamide or dimethyl acetamide.

16. A method according to claim 13, wherein said releasing agent comprises a 50-100% solution of a lipophilic solvent.

17. A method according to claim 16, wherein said solvent comprises 100% methanol or 100% ethanol.

18. A method according to claim 13, wherein said antibody has an affinity of at least about 1×

10⁸ liter per mole for the aflatoxin of interest.

19. A method according to claim 13, wherein said sample comprises serum, urine or food product.

20. A method according to claim 19, wherein said food product is milk, corn or peanut products.

21. A method according to claim 13, wherein said second eluent is exposed to UV light at 365 nm.

22. A method according to claim 13, wherein said second eluent is subjected to quantitative analysis for determining the level of aflatoxin in said sample.

23. A method for detecting aflatoxins in a test sample, said method comprising:

placing a sample believed to contain aflatoxin on an affinity column comprised of a solid phase sorbant material and, immobilized thereon, a monoclonal antibody for said aflatoxin;

eluting said column with a solvent to recover a first eluent, whereby said aflatoxins are retained on said column by said antibody and separated from all other ingredients of said sample;

eluting said column with a releasing agent comprised of ethanol or methanol to recover a second eluent, whereby said aflatoxins are released from said antibody and recovered in said second eluent; and

subjecting said second eluent to fluorescence measurement by exposing said second eluent to UV light at 365 nm for detection of the presence of aflatoxins, with the proviso that said second eluent is not subjected to high pressure liquid chromatography or other purification subsequent to recovery form said column.

24. A method according to claim 23, wherein said antibody has an affinity of at least about 1×

10⁸ liters per mole to said aflatoxins.

25. A method according to claim 23, wherein said sample comprises serum, urine or food product.

26. A method according to claim 25, wherein said food product is milk, corn or peanut products.