Enzyme-free diagnostic binding reagents

• US 4,719,176 A
• Filed: 10/31/1983
• Issued: 01/12/1988
• Est. Priority Date: 10/31/1983
• Status: Expired due to Fees

• Current Assignee: Irving M. Klotz
• Original Assignee: Irving M. Klotz
• Inventors: Klotz, Irving M.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/547,232
• Time in Patent Office: 1,534 Days
• Field of Search: 422/61, 435/6, 435/810, 436/34, 436/501, 436/524, 436/531, 436/537, 436/544, 436/547, 808/809, 808/904, 530/387, 530/402, 536/27
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

An enzyme-free diagnostic reagent, methods of its use and diagnostic systems containing that reagent are disclosed. The enzyme-free diagnostic reagent comprises an enzyme-free catalyst coupled by a linking group to a first binding agent, binds in aqueous medium to a second binding moiety to form a binding complex, and indicates the amount of second binding moiety present in the complex by means of its catalytic reactivity with co-reactant molecules.

Claims

1. An enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said catalyst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hydrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed in homogenous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entity needed for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed.

2. The enzyme-free diagnostic reagent of claim 1 wherein said enzyme-free hydrolytic catalyst has a molecular weight of about 150 to 1000 daltons, and prior to being coupled to said first binding agent has a structure corresponding to a formula selected from the group consisting of ##STR9## wherein R¹ is hydrogen, C₁ -C₁₂ alkyl or Z, or R¹ and Z taken together form a 5- or 6-membered ring that contains a linking moiety;

R² is hydrogen or C₁ -C₄ alkyl;

R³ is C₁ -C₄ alkyl, and R⁴ is alkyl or substituted arylalkyl containing 1 to about 18 carbon atoms, or two of R³, R⁴ and Z taken together form a 5- or 6-membered ring containing a linking moiety, or R³, R⁴ and Z taken together form a bicyclic ring compound containing a linking moiety and wherein each of the rings of the bicyclic ring compound contains 5- or 6-membered ring that contains a linking moiety; and

Z is a linking group including a linking moiety prior to linking of the catalyst to the first binding agent.

3. The enzyme-free diagnostic reagent of claim 1 wherein an average of about 1 to about 10 enzyme-free catalysts are coupled per molecule of said enzyme-free diagnostic reagent.

4. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent is a receptor.

5. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent comprises a receptor that is an antibody or an idiotype-containing polyamide portion of an antibody.

6. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent comprises biotin.

7. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent comprises a polynucleic acid.

8. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent comprises a ligand.

9. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent is a ligand.

10. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent includes avidin bound thereto.

11. The enzyme-free diagnostic reagent of claim 1 wherein said first binding agent comprises a polynucleic acid.

12. The enzyme-free diagnostic reagent of claim 1 wherein said enzyme-free catalyst is coupled adjacent to an epitopic region of a first binding agent that is an antigen having a molecular weight of less than about 6000 daltons, and an average per 1 to about 3 coupled catalysts are present per molecule of said antigen.

13. The enzyme-free diagnostic reagent of claim 1 wherein said enzyme-free catalyst is coupled adjacent to the idiotypic region of a receptor as first binding agent, said receptor being an antibody or the idotype-containing polyamide portion of an antibody, and an average of about 1 to about 3 coupled catalysts are present per molecule of said receptor.

14. The enzyme-free diagnostic reagent of claim 1 wherein said linking group includes a multivalent radical bonded to a plurality of catalysts.

15. The enzyme-free diagnostic reagent of claim 1 wherein said multivalent radical is selected from the group consisting of a polymeric polyamine and s-triazinyl.

16. The enzyme-free diagnostic reagent of claim 1 wherein said catalyst is additionally capable of retaining catalytic reactivity in the absence of both said coupled first binding agent and solvent, after heating anaerobically to a temperature of about 100 degrees C. for a period of about one minute.

17. A diagnostic kit comprising at least one package containing an aqueous solution including a known amount of an enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay, said diagnostic reagent comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said catalyst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hyrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed in homogeneous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entitity needed for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed.

18. The diagnostic kit of claim 17 further including at least one additional package that contains a solution of a second known concentration of hydrolyzable co-reactant molecules for said enzyme-free catalyst.

19. A method of carrying out a binding assay comprising the steps of

(a) providing an enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said catalyst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hydrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed. in homogeneous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entitity needed for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed;

(b) providing a first aqueous composition containing said second binding moiety;

(c) admixing in an aqueous medium a known, predetermined amount of said diagnostic reagent and a known, predetermined amount of said first aqueous composition to form a binding complex between said diagnostic reagent and said second binding moiety;

(d) admixing a plurality of hydrolyzable co-reactant molecules with the admixture of step (c); and

(e) measuring the reaction between said enzyme-free catalyst and said co-reactant molecules.

20. The method of claim 19 wherein said second binding moiety is the entity to be assayed.

21. The method of claim 19 wherein said second binding moiety is needed for the assay and the entity to be assayed is provided by admixing a composition to be assayed with said binding complex-containing aqueous medium prior to said admixture of co-reactant molecules.

22. The method of claim 19 wherein said enzyme-free hydrolytic catalyst has a molecular weight of about 150 to about 1000 daltons, and prior to being coupled to said first binding agent has a structure corresponding to a formula selected from the group consisting of ##STR10## wherein R¹ is hydrogen, C₁ -C₁₂ alkyl or Z, or R¹ and Z taken together form a 5- or 6- membered ring that contains a linking moiety;

R² is hydrogen or C₁ -C₄ alkyl;

R³ is C₁ -C₄ alkyl, and R⁴ is alkyl or substituted arylalkyl containing 1 to about 18 carbon atoms, or two of R³, R⁴ and Z taken together form a 5- or 6-membered ring containing a linking moiety, or R³, R⁴ and Z taken together form a bicyclic ring compound containing a linking moiety and wherein each of the rings of the bicyclic ring compound contains 5- or 6-membered ring that contains a linking moiety; and

Z is a linking group including a linking moiety prior to linking of the catalyst to the first binding agent.

23. The method of claim 19 wherein said diagnostic reagent contains an average of about 1 to about 3 catalysts per molecule.

24. The method of claim 23 wherein

(a) said diagnostic reagent comprises said enzyme-free catalyst coupled adjacent to the epitopic region of an antigen having a molecular weight of less than about 6000;

(b) said diagnostic reagent is admixed with a composition containing

(i) a predetermined, known amount of receptors that bind to both the epitope of said diagnostic reagent and to a homologous epitope of an entity to be assayed; and

(ii) a first aqueous composition to be assayed for the presence of the entity to be assayed;

(c) the rate of reaction between said catalyst of said diagnostic reagent in the admixture so formed and said co-reactant molecules is measured; and

(d) said measured rate is compared to the rate of reaction of an aqueous medium containing a known, predetermined amount of said diagnostic reagent and co-reactant molecules and free from a substance that binds to said diagnostic reagent.

25. The method of claim 23 wherein

(a) said diagnostic reagent comprises said enzyme-free catalyst coupled to an antigen;

(b) said second binding moiety comprises a receptor that binds to and forms a dissociatable binding complex with sid diagnostic reagent;

(c) a predetermined known amount of said dissociatable binding complex is formed in an aqueous medium between said diagnostic reagent and said second binding moiety;

(d) prior to admixture of said co-reactant molecules, a predetermined, known amount of a composition to be assayed that contains the same or a homologous epitope-containing antigen as the assayed entity is admixed with said dissociatable complex, said admixture dissociating said first-named complex, forming a second binding complex between the assayed entity and said receptor, and freeing at least some of said diagnostic reagent previously bound to said receptor in said dissociatable complex; and

(e) said co-reactant molecules are thereafter admixed, and said reaction rate is measured and compared to the reaction rate exhibited by said catalyst in said dissociatable complex.

26. The method of claim 23 wherein

(a) said diagnostic reagent comprises as the first binding agent a receptor that binds to said second binding moiety, and said catalyst is coupled adjacent to the idiotypic region of said receptor;

(b) said second binding moiety is a protein ligand having a molecular weight of at least about 20,000 daltons; and

(c) said measured rate is compared to the rate of reaction of an aqueous medium containing a known, predetermined amount of said diagnostic regent and co-reactant molecules and free from a substance that is bound by said antibody first binding agent.

27. The method of claim 26 wherein said receptor is an idiotype-containing polyamide.

28. An enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said enzyme-free catalyst prior to being coupled to said first binding agent having a structure corresponding to a formula selected from the group consisting of ##STR11## wherein R¹ is hydrogen, C₁ -C₁₂ alkyl or Z, or R¹ and Z taken together form a 5- or 6-membered ring that contains a linking moiety;

R² is hydrogen or C₁ -C₄ alkyl;

R³ is C₁ -C₄ alkyl, and R⁴ is alkyl or substituted arylalkyl containing 1 to about 18 carbon atoms, or two of R³, R⁴ and Z taken together form a 5- or 6- membered ring containing a linking moiety, or R³, R⁴ and Z taken together form a bicyclic ring compound containing a linking moiety and wherein each of the rings of the bicyclic ring compound contains 5- or 6-membered ring that contains a linking moiety; and

Z is a linking group including a linking moiety prior to linking of the catalyst to the first binding agent;

said catalyst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hydrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed in homogeneous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entity needed for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed.

29. The enzyme-free diagnostic reagent of claim 28 wherein said first binding agent is a ligand.

30. The enzyme-free diagnostic reagent of claim 28 wherein said first binding agent is a receptor.

31. The enzyme-free diagnostic reagent of claim 28 wherein said first binding agent includes avidin bound thereto.

32. The enzyme-free diagnostic reagent of claim 28 wherein said first binding agent comprises a polynucleic acid.

33. A diagnostic kit comprising at least one package containing an aqueous solution including a known amount of an enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay, said diagnostic reagent comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said enzyme-free hydrolytic catalyst prior to being coupled to said first binding agent has a structure corresponding to a formula selected from the group consisting of ##STR12## wherein R¹ is hydrogen, C₁ -C₁₂ alkyl or Z, or R¹ and Z taken together form a 5- or 6- members;

R² is hydrogen or C.sub. -C₄ alkyl;

R³ is C₁ -C₄ alkyl, and R⁴ is alkyl or substituted arylalkyl containing 1 to about 18 carbon atoms, or two of R³, R⁴ and Z taken together form a 5- or 6- membered ring containing a linking moiety, or R³, R⁴ and Z taken together form a bicyclic ring compound containing a linking moiety and wherein each of the rings of the bicyclic ring compounds contains 5- or 6-membered ring that contains a linking moiety; and

Z is a linking group including a linking moiety prior to linking of the catalyst to the first binding agent;

said catalyst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hydrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed in homogeneous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entity for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed.

34. The diagnostic kit of claim 33 wherein said first binding agent is a ligand.

35. The diagnostic kit of claim 33 wherein said first binding agent is a receptor.

36. The diagnostic kit of claim 33 wherein said first binding agent includes avidin bound thereto.

37. The diagnostic kit of claim 33 wherein said first binding agent comprises a polynucleic acid.

38. A method of carrying out a binding assay comprising the steps of

(a) providing an enzyme-free diagnostic reagent for determining the presence of an entity to be assayed in a homogeneous assay comprising an enzyme-free hydrolytic catalyst coupled by a linking group to a first binding agent, said enzyme-free hydrolytic catalyst prior to being coupled to said first binding agent has a structure corresponding to a formula selected from the group consisting of ##STR13## wherein R¹ is hydrogen, C₁ -C₁₂ alkyl or Z, or R¹ and Z taken form a 5- or 6-membered ring that contains a linking moiety;

R² is hydrogen or C₁ -C₄ alkyl;

R³ is C.sub. -C₄ alkyl, and R⁴ is alkyl or substituted arylalkyl containing 1 to about 18 carbon atoms, or two of R³, R⁴ and Z taken together form a 5- or 6-membered ring containing a linking moiety, or R³, R⁴ and Z taken together form a bicyclic ring compound containing a linking moiety and wherein each of the rings of the bicyclic ring compound contains 5- or 6-membered ring that contains a linking moiety; and

Z is a linking group including a linking moiety prior to linking of the catalyst to the first binding agent.

said cataylst having a molecular weight together with the linking group of about 75 to about 2000 daltons, wherein said catalyst catalyzes a hydrolytic chemical reaction with successive preselected hydrolyzable co-reactant molecules when said co-reactant molecules and said catalyst are admixed in homogeneous solution and said catalyst is coupled to said first binding agent as said diagnostic reagent;

said diagnostic reagent specifically binding in aqueous medium to a second binding moiety to form a binding complex, said second binding moiety being (a) the entity to be assayed in a direct assay, or (b) an entity needed for the assay in an indirect assay, said diagnostic reagent indicating the amount of said second binding moiety present in said binding complex by means of the catalytic reactivity of said catalyst with said co-reactant molecules, said catalytic reactivity being changed when said binding complex is formed;

(b) providing a first aqueous composition containing said second binding moiety;

(c) admixing in an aqueous medium a known, predetermined amount of said diagnostic reagent and a known, predetermined amount of said first aqueous composition to form a binding complex between said diagnostic reagent and said second binding moiety;

(d) admixing a plurality of hydrolyzable co-reactant molecules with the admixture of step (c); and

(e) measuring the reaction between said enzyme-free catalyst and said co-reactant molecules.

39. The method of claim 38 wherein said first binding agent is a ligand.

40. The method of claim 38 wherein said first binding agent is a receptor.

41. The method of claim 38 wherein said first binding agent includes avidin bound thereto.

42. The method of claim 38 wherein said first binding agent comprises a polynucleic acid.