Visualization polymers and their application to diagnostic medicine

• US 4,687,732 A
• Filed: 06/10/1983
• Issued: 08/18/1987
• Est. Priority Date: 06/10/1983
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Brigati, David J., Leary, Jeffry J., Ward, David C.
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/503,298
• Time in Patent Office: 1,530 Days
• Field of Search: 436/537, 436/801, 436/803, 436/827, 436/501, 436/504, 436/545, 436/546, 436/800, 436/804, 436/808, 435/6, 435/7, 435/14, 435/21, 435/25, 435/28, 435/188, 435/810
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• 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 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/801 Electron dense compounds, e.g. ferritin
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/827 Lectins

Abstract

A method for detecting a minute quantity of an inorganic or organic target molecule by combining it with a composition of a detecting agent for the target molecule which carries, by direct or indirect means, a visualization polymer. The visualization polymer is composed of multiple units of a visualization monomer which are covalently linked together directly or indirectly covalently linked together by coupling agents which bond to chemical groups of the monomer. The monomer may be an enzyme, a tagged polypeptide, a tagged polyol, a tagged polyolefin or a tagged carbohydrate. The detecting agent may be an antibody, an enzyme, a lectin, strand of a DNA receptor protein, avidin, streptavidin and the like. The visualization polymer produces a high degree of amplification for the detection of the target molecule.

Claims

1. A visualization polymer comprising essentially identical multiple visualization units each having at least one visualization site;

said visualization units being directly bonded together though chemical groups or backbone moieties of adjacent said visualization units, or linked by a coupling agent covalently bonded to chemical groups or backbone moieties of adjacent said visualization units,

said visualization unit being an enzyme, wherein the enzymatic site is the visualization site, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged polycarbohydrate wherein the tag of the tagged polypeptide, tagged polyol, tagged polyolefin or tagged polycarbohydrate is the visualization site thereof;

said chemical group being an amine group, an oxidized 1,2-diol group, a carboxyl group, a mercaptan group, a hydroxy group or a carbon-hydrogen bond;

said backbone moiety being an amide bond, a carbon-carbon bond, a carbon-oxygen bond or a carbon-hydrogen bond; and

said chemical group or backbone moiety being located at a position which is at least one atom away from the visualization site of said visualization unit;

said coupling agent being derived from a bi- or multi-(homo- or hetero-) functional organic cross-linking reagent of the formula ##STR4## wherein A, B and E are independently selected from the group consisting of hydrogen, a carboxylic acid group, an acid halide group, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylacarbonyl group, a hydrazine group, an acylhydrazide group, an azide group and an N-maleimide group;

at least two of A, B and E are other than hydrogen; and

R¹ is an aliphatic group of at least two carbons or an aromatic group of at least six carbons; and

the tag of said tagged polypeptide, tagged polyol, tagged polyolefin or tagged carbohydrate is a fluorescent group, a dye, a radioactive group, a photon emitter or an electron dense group.

2. A visualization polymer according to claim 1 wherein the visualization unit is an enzyme selected from the group consisting of peroxidase, galactosidase, luciferinase, glucose oxidase, acid phosphatase or alkaline phosphatase.

3. A visualization polymer according to claim 1 wherein the cross-linking reagent is an aliphatic-1, omega-diacyloxy disuccinimide having from 4 to 20 carbons in the aliphatic group.

4. A visualization polymer according to claim 1 wherein said visualization units are directly bonded together through oxidative cross-linking using an oxidative enzyme.

5. A visualization polymer according to claim 4 wherein the oxidative enzyme is horseradish peroxidase.

6. A visualization polymer according to claim 1 wherein the visualization unit is horseradish peroxidase or alkaline phosphatase.

7. A visualization polymer according to claim 6 wherein the visualization units are linked by a coupling agent derived from disuccinimidyl suberate which bonds with epsilon amine groups.

8. A visualization polymer according to claim 6 wherein the visualization unit is peroxidase and the visualization units are coupled by a coupling agent derived from tri(lysyl)lysine which bonds with oxidized 1,2-diol groups.

9. A detection-visualization composition comprising a detecting agent carrying a visualization polymer as described according to claim 1, said detecting agent being an antibody, a lectin, avidin, streptavidin, a DNA repressor protein, a hormone, a stereo-specific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

10. A detection-visualization composition according to claim 9 wherein said detecting agent carries said visualization polymer through an intermediate ligand binding complex comprising a first ligand covalently bonded to said detecting agent, a ligand binding compound and a second ligand covalently bonded to said visualization polymer;

said detecting agent, said ligand binding compound and said visualization polymer forming a complex with said first and second ligands functioning as ligands to said ligand binding compound.

11. A composition according to claim 9 wherein said detecting agent is an antibody, a lectin or a complementary polynucleotide sequence, said ligand binding compound is avidin or streptavidin, and said first and second ligands are independently selected from N-(omega acyl) amido(biotin or iminobiotin), said omega acyl group being of from about 2 to 20 carbons in length;

or an N-(omega-oligomer)amido(biotin or imido biotin) wherein the omega-oligomer is a polyol, a polyamide or a polyvinyl group of from about 2 to 30 units in length;

or N-(omega alkenyl)amido (biotin or iminobiotin), said omega alkenyl group being about 3 to 20 carbons in length.

12. A detection kit for analysis of target molecules in a biological material comprising a metered quantity of a detection agent-visualization polymer composition as described according to claim 9.

13. A detection-visualization composition according to claim 9 wherein said visualization polymer is bonded or complexed directly with said detecting agent.

14. A composition according to claim 13 wherein said detecting agent is covalently bonded to said visualization polymer with a linking group derived from a bi- or multi-(homo or hetero) functional organic cross-linking reagent.

15. A visualization polymer complex comprising a visualization polymer according to claim 1 covalently bonded to a ligand, and a ligand binding compound associated with said ligand.

16. A visualization polymer complex according to claim 15 wherein the ligand binding compound is a lectin, avidin, streptavidin, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

17. A visualization polymer complex according to claim 15 wherein the ligand binding compound is a lectin, avidin or streptavidin.

18. A visualization polymer complex according to claim 17 wherein the ligand binding compound is lectin and the ligand is a sugar bound to the visualization through a bi- or multi-(homo or hetero) functional organic cross-linking reagent.

19. A visualization polymer complex according to claim 17 wherein the ligand binding compound is avidin or streptavidin and the ligand is an N-(omega acyl)amino(biotin or iminbiotin), said omega acyl group being of from about 2 to 20 carbons in length;

or an N-(omega-oligomer)amido(biotin or iminobiotin);

said omega-oligomer being a polyol, a polyamide or a polyvinyl group of from about 2 to 30 units in length.

20. A visualization polymer complex according to claim 19 wherein the ligand is N-(omega acyl)amido(biotin or iminobiotin).

21. A detection kit for analysis of target molecules in a biological material comprising metered quantities of a buffered, aqueous solution of a detection agent component and a buffered, aqueous solution of a visualization polymer complex component and a standardized quantity of an aqueous mixture of said visualization polymer, said detection agent component and said visualization polymer complex component being capable of associating when combined,

said visualization polymer complex component comprising a visualization polymer covalently bonded to a ligand, and a ligand binding compound associated with said ligand;

said visualization polymer comprising essentially identical multiple visualization units each having at least one visualization site;

said visualization units being directly bonded together through chemical groups or backbone moieties of said visualization units, or linked by a coupling agent covalently bonded to chemical groups or backbone moieties of said visualization units;

said visualization unit being an enzyme, wherein the enzymatic site is the visualization site, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged polycarbohydrate wherein the tag of the tagged polypeptide, tagged polyol, tagged polyolefin or tagged polycarbohydrate is the visualization site thereof,

said chemical group being an amine group, an oxidized 1,2-diol group, a carboxy group, a mercaptan group, a hydroxy group or a carbon-hydrogen bond;

said backbone moiety being an amide bond, a carbon-carbon bond, a carbon-oxygen bond or a carbon-hydrogen bond;

said chemical group or backbone moiety being located at a position which is at least one atom away from the visualization site of said visualization unit;

said coupling agent being derived from a bi- or multi-(homo-or hetero-) functional organic cross-linking reagent of the formula ##STR5## wherein A, B and E are independently selected from the group consisting of hydrogen, a carboxylic acid group, an acid halide group, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylcarbonyl group, a hydrazine group, an acylhydrazide group, an azide group and an N-maleimide group;

at least two of A, B and E are other than hydrogen; and

R¹ is an aliphatic group of at least two carbons or an aromatic group of at least six carbons; and

the tag of said tagged polypeptide, tagged polyol, tagged polyolefin or tagged carbohydrate is a fluorescent group, a dye, a radioactive group, a photon emitter or an electron dense group.

22. A kit according to claim 21 wherein said detecting agent is an antibody, a lectin, avidin, streptavidin, a DNA repressor protein, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

23. A method for visualizing the presence of an inorganic or organic target molecule in a biological material, which comprises:

combining said target with a detecting agent with carries a visualization polymer comprising essentially identical multiple visualization units directly bonded together or linked by a coupling agent through chemical groups or backbone moieties of said visualization units;

said visualization unit having at least one visualization site and said visualization unit being an enzyme wherein the enzymatic site is the visualization site, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged polycarbohydrate wherein the tag of the tagged polypeptide, the tagged polyol, the tagged polyolefin or the tagged polycarbohydrate is the visualization site thereof,

said chemical group being an amine group, an oxidized 1,2-diol group, a carboxy group, a mercaptan group, a hydroxy group or a carbon-hydrogen bond;

said chemical group or backbone moiety being located within said visualization unit at a position which is at least one atom away from the visualization site of said visualization unit;

said coupling agent being derived from a reactive bi- or multi- homo- or hetero-functional organic cross-linking reagent of the formula ##STR6## wherein A, B and E are independently selected from the group consisting of hydrogen, a carboxylic acid group, an acid halide group, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylcarbonyl group, a hydrazine group, an acylhydrazide group, an azide group and an N-maleimide group;

at least two of A, B and E are other than hydrogen; and

R¹ is an aliphatic group of at least two carbons or an aromatic group of at least six carbons; and

the tag of the tagged polypeptide, tagged polyol, tagged polyolefin or tagged carbohydrate is a fluorescent chemical group, a dye, a radioactive group, a photon emitter or an electron dense moiety.

24. A method according to claim 23 wherein the chemical group is a mercaptan and the cross-linking reagent forms sulfide bonds using an alphahalomethylcarbonyl group or an N-maleimide group.

25. A method according to claim 23 wherein said detecting agent is an antibody, a lectin, a DNA repressor protein, avidin, streptavidin, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein, or a complementary polynucleotide sequence.

26. A method according to claim 23 wherein said molecule target is produced by or has an effect upon a biological organism.

27. A method according to claim 26 wherein said molecule target is a protein, a lipid, a carbohydrate, a hormone, a steroid, a polynucleotide, a nucleic acid, a nucleoprotein, a nucleoside, a nucleotide, a purine or pyrimidine base, a drug, a sugar, a carcinogen, an antibiotic, a haptene, an antigen, a heavy metal, an organometallic compound, a metal-protein complex or a toxic inorganic salt.

28. A method according to claim 23 wherein the chemical group is a 1,2-diol group which is oxidized to a dialdehyde group and the cross-linking reagent forms hydrazone, amide, acylhydrazide or imine bonds with said dialdehyde group.

29. A method according to claim 28 wherein the chemical groups are epsilon or primary amino groups which form amide bonds with the cross-linking reagent.

30. A method according to claim 29 wherein said visualization unit is peroxidase, luciferase, glucose oxidase, galactoxidase, acid phosphatase or alkaline phosphatase.

31. A method according to claim 23 wherein the visualization polymer is bonded to the detecting agent by an organic bivalent linking group.

32. A method according to claim 31 wherein the organic bivalent linking group is dervied from a bi- or multi- homo- or hetero-functional organic cross-linking reagent.

33. A method according to claim 31 wherein the detecting agent is an antibody, lectin, avidin, steptavidin, a DNA repressor protein, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

34. A method according to claim 23 wherein said detecting agent carries said visualization polymer through an intermediate ligand binding complex comprising a first ligand covalently bonded to said detecting agent, a second ligand covalently bonded to said visualization polymer and a ligand binding compound wherein said first and second ligands are complexed with said ligand binding compound.

35. A method according to claim 34 wherein said ligand binding compound is an antibody, lectin, avidin, streptavidin, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

36. A method according to claim 34 wherein the detecting agent is a lectin, the ligand binding compound is avidin or streptavidin, and the first and second ligands are independently selected from N-(omega acyl)amido(biotin or iminobiotin), said omega acyl group being of 4 to 20 carbons;

or an N-(omega-oligomer) amido(biotin or iminobiotin) wherein the omega-oligomer is a polyol, a polyamide or a polyvinyl group of from 2 to 30 units.

37. A method according to claim 36 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.

38. A method according to claim 34 wherein the detecting agent is an antibody, the ligand binding compound is avidin or streptavidin, and the first and second ligands are independently selected from N-(omega acyl)amido(biotin or iminobiotin), said omega acyl group being of from 4 to 20 carbons;

or an N-(omega-oligomer)amido(biotin or iminobiotin) wherein the omega-oligomer is a polyol, a polyamide or polyvinyl group of 2 to 30 units in length.

39. A method according to claim 38 wherein a first target-specific antibody is used to detect said target molecule and form a target/first antibody conjugate, a second antibody having said biotin label ligand and being general for said first antibody is incubated with said conjugate and said visualization polymer is complexed with said second antibody through said first and second ligands and said ligand binding compound.

40. A method according to claim 38 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.

41. A method according to claim 34 wherein said detecting agent is a complementary polynucleotide sequence.

42. A method according to claim 41 wherein said compound is an antibody, lectin, avidin, streptavidin, or a high affinity enzyme.

43. A method according to claim 41 wherein said compound is lectin and said first and second ligands are binding sugars.

44. A method according to claim 41 wherein said compound is avidin or streptavidin and said first and second ligands are independently selected from N-(omega acyl)amido(biotin or iminobiotin), said omega acyl group being of from 4 to 20 carbons;

or an N-(omega-oligomer)amido(biotin or iminobiotin) wherein the omega-oligomer is a polyol or polyamide or polyvinyl group of 2 to 30 units in length, or N-(alkenyl)amido(biotin or iminobiotin) having from 3 to 20 carbons in the alkenyl group.

45. A method according to claim 44 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.