Application of water-dispersible hydrophobic dyes or pigments as labels in immunoassays

• US 4,373,932 A
• Filed: 01/02/1981
• Issued: 02/15/1983
• Est. Priority Date: 01/11/1980
• Status: Expired due to Term

• Current Assignee: Akzona Incorporated
• Original Assignee: Akzona Incorporated
• Inventors: Gribnau, Thomas C. J., Roeles, Frits, Leuvering, Johannes H. W.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US06/222,263
• Time in Patent Office: 774 Days
• Field of Search: 23/230 B, 23/915, 424/12, 424/3, 424/7, 424/8, 8/526, 8/527, 422/61, 252/408
• US Class Current: 436/501
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

Processes, reagents and test kits for the qualitative and/or quantitative determination of an immunochemically reactive component, in which one or more labelled components are used, that are obtained by direct or indirect coupling of such a component or components to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymer nuclei coated with such a dye or pigment.

During the reaction or after an adequate reaction time the nature and/or the quantity of the dye is determined in the test medium, or optionally after a separation of the bound and free labelled components in one of the fractions.

Claims

1. A process for the qualitative and/or quantitative determination of an immunochemically reactive component selected from the group consisting of a hapten, antigen, and antibody in an aqueous test medium using the immunochemical reactivity of said component comprising:

(1) providing a known amount of at least one labelled immunochemically reactive component obtained by the direct or the indirect attachment of an immunochemically reactive component to particles of an aqueous dispersion of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with said dye or pigment, said particles having a particle size of at least 5 nm;

(2) providing at least one non-labelled immunochemically reactive component;

(3) mixing said components (1) and (2) with a sample containing said test medium;

(4) allowing the immunochemical reaction to proceed to form free and bound labelled component(s);

(5) optionally separating the free and bound labelled component(s) in the test medium or in one of the fractions obtained after separation; and

(6) determining the nature and/or the quantity of the dye or pigment, said determination providing a qualitative and/or quantitative indication of the immunochemically reactive component(s) to be determined.

2. The process of claim 1, wherein the determination (6) takes place by the detection and determination of the physicochemical changes of a dye sol.

3. The process of claim 1 or claim 2, wherein two or more immunochemically reactive components are determined simultaneously, wherein for each component to be determined there is present a corresponding immunochemically reactive component that is labelled with a dye sol particle respective for that component, and further wherein respective chromophores are used that can clearly be distinguished from each other spectrally and/or visually.

4. The process of claim 1, further comprising obtaining the labelled component by adding to the dye or pigment dispersion a predetermined quantity of the immunochemically reactive component to be labelled, wherein the latter at least partially envelopes the dispersed particles and optionally supplementary coating said particles with a polar macromolecule that is immunochemically inert in the corresponding determination.

5. The process of claim 1, wherein the labelled component is obtained by adding to said dispersion of dye or pigment at least one macromolecule that is immunochemically inert in the corresponding determination and that coats the dispersion particles and thereafter attaching by adsorption, possibly bio-specific, or via covalent attachment the immunochemically reactive component to the coating material.

6. The process of claim 1, wherein the labelled component is obtained by placing a dye and/or organic pigment sol in a monomer environment and causing the latter to be polymerised in situ, resulting in envelopment of the sol particles, and subsequently absorbing or covalently attaching the immunochemically reactive component to the polymer material.

7. The process of claim 1, wherein the labelled component is obtained by covalently attaching the immunochemically reactive component to colloidal dye particles, either by prior chemical activation of suitable functional groups in the dye and/or the immunochemically reactive component, or by the use of conjugates of organic hydrophobic chromophores and reactive groups, known as reactive (dispersion) dyes.

8. The process of claim 5 wherein the dye and/or organic pigment sol particles are first protected by a hydrophilic macromolecule after which (co-)polymerisation takes place in the presence of an organic initiator.

9. A test kit for the determination of one or more immunochemically reactive components in an aqueous medium, comprising:

(a) a known amount of at least one labelled immunochemically reactive component obtained by the direct or indirect attachment of an unlabelled immunochemically reactive component to particles of an aqueous dispersion of (i) a hydrophobic dye or pigment or (ii) polymer nucleic coated with said dye or pigment, said particles having a particle size of at least 5 nm,

(b) other immunochemical reagents, and

(c) directions for use of said test kit.

10. An immunochemical reagent consisting of an aqueous dispersion of particles of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with said dye or pigment directly or indirectly attached to an immunochemically reactive component, said particles having a particle size of at least 5 nm.

11. A freeze-dried or spray dried reagent containing at least one labelled immunochemically reactive component obtained by direct or indirect attachment of an unlabelled immunochemically reactive component to particles of an aqueous dispersion of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with said dye or pigment, said labelled immunochemically reactive component being freeze-dried or spray dried and said particles having a particle size of at least 5 nm.

12. A process for the detection and/or determination of at least one immunochemically reactive component in an aqueous test medium comprising:

providing a known amount of at least one labelled immunochemically reactive component having attached thereto particles of an aqueous dispersion of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm;

providing an aqueous test medium containing at least one immunochemically reactive component to be detected or determined;

contacting said labelled immunochemically reactive component(s) with said immunochemically reactive component(s) to be detected or determined; and

measuring the presence or quantity of said hydrophobic dye or pigment to provide a measure of the immunochemically reactive component(s) to be detected or determined.

13. The process of claim 12 wherein the immunochemically reactive components are selected from the group consisting of an antigen, an antibody, and a hapten.

14. The process of claim 12 wherein said aqueous dispersion of a hydrophobic dye or pigment is a sol.

15. The process of claim 14 wherein said hydrophobic dye or pigment has a particle size of about 10 to about 500 nm.

16. An immunochemical reagent comprising an immunochemically reactive component having coupled thereto a member selected from the group consisting of particles of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm.

17. The immunochemical reagent of claim 16 which is freeze-dried.

18. The immunochemical reagent of claim 16 which is spray-dried.

19. The immunochemical reagent of claim 16 wherein said particles of a hydrophobic dye or pigment are provided by an aqueous dispersion of said particles.

20. The immunochemical reagent of claim 19 wherein said aqueous dispersion is a sol.

21. The immunochemical reagent of claim 20 wherein said particles have a size of about 10 to about 500 nm.

22. A test kit for the determination of at least one immunochemically reactive component in an aqueous medium, comprising:

(a) a known amount of at least one immunochemical reagent comprising an immunochemically reactive component having coupled thereto a member selected from the group consisting of particles of a hydrophobic dye or pigment and polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm,

(b) other immunochemical reagents; and

(c) directions for use of said test kit.

23. A process for the detection and/or determination of at least one component of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous test sample, by applying the known binding affinity of such said protein and said substance for one another, comprising:

(a) mixing a known amount of one or more labelled components, obtained by coupling directly or indirectly the desired component of said reaction with particles of an aqueous dispersion of a hydrophobic dye or pigment or polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm, and

(b) detecting and/or determining during the reaction or after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of said dye or pigment and/or a formed agglomerate containing said dispersed particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.

24. The process of claim 23, whereby the component or components of the reaction between a specific binding protein and the corresponding bindable substances are immunochemical components selected from the group consisting of haptens, antigens, and antibodies, comprising:

(a) employing a known amount of labelled components, obtained by coupling directly or indirectly the desired immunochemical component to particles of an aqueous dispersion of (i) a hydrophobic dye or pigment or (ii) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm, and

(b) detecting or determining after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of said dye or pigment and/or a formed agglomerate containing said dye or pigment particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the immunochemical component or components to be detected and/or determined.

25. A test kit, to be used for the determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous medium according to a predetermined protocol, comprising:

(a) a known amount of a hydrophobic dye or pigment-labelled component that has been obtained by coupling a component of said reaction to particles of an aqueous dispersion of a hydrophobic dye or pigment or polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm;

(b) other immunochemical reagents; and

(c) directions for the performance of said protocol.

26. A freeze-dried reagent for use in an immunoassay, containing a hydrophobic dye or pigment-labelled component, that has been obtained by coupling directly or indirectly the desired component to particles of an aqueous dispersion of a hydrophobic dye or pigment or polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm.

27. A method for the determination of a first component of an immunochemical reaction in an aqueous medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces an immunochemical component which is at least immunochemically equivalent to said first component to be determined, and (2) a known amount of an insolubilized second component capable of reacting with either said first component to be determined or said reagent;

(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component, and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and

(c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

28. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) an immunochemical component which is at least immunochemically equivalent to said first component to be determined and (ii) an immunochemically inert macromolecule, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent;

(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and

(c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

29. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical component which is at least immunochemically equivalent to said first component, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent;

(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and

(c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

30. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized second immunochemical component which is at least immunochemically equivalent to said first component and capable of reacting with said reagent;

(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and

(e) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

31. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent, said reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) a second immunochemical component capable of reacting with said first component to be determined and (ii) an immunochemically inert macromolecule, and (2) a known amount of an insolubilized second immunochemical component which is at least immunochemically equivalent to said first component to be determined and capable of reacting with said reagent;

(b) allowing sufficient time for the reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and

(c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

32. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) containing a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer of copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized third immunochemical component and capable of reacting with said reagent;

(b) allowing sufficient time for the reaction to take place, whereby a fraction of said first component to be determined bound to said insolubilized component and a fraction of said first component to be determined free from said insolubilized component are produced; and

(c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

33. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) binding a known amount of a second immunochemical component of said reaction capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier;

(b) contacting said bound second component with a sample containing the first component to be determined;

(c) allowing sufficient time for reaction to take place;

(d) contacting said bound reaction product of (c) with a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surface an immunochemical component capable of reacting with said first component to be determined;

(e) allowing sufficient time for reaction to take place, to bind a fraction of said reagent to that part of said bound second immunochemical component which has undergone the reaction step (c), leaving a remaining fraction of said reagent free and not bound; and

(f) determining the amount of the dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.

34. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) binding a known amount of a second immunochemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier;

(b) contacting said bound second component with a sample containing the first component to be determined;

(c) allowing sufficient time for reaction to take place;

(d) contacting said bound reaction product of step (c) with an immunochemical excess of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces (i) an immunochemical component capable of reacting with said first component to be determined and (ii) an immunochemically inert macromolecule;

(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and

(f) determining the amount of the dye or pigment dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said component to be determined in said sample.

35. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) binding a known amount of a second immunochemical component capble of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier;

(b) contacting said bound second component with a sample containing the first component to be determined;

(c) allowing sufficient time for reaction to take place;

(d) contacting said bound reaction product of step (c) with an immunochemical excess of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm and having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical component capable of reacting with said first component to be determined;

(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and

(f) determining the amount of the dye or pigment dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.

36. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substances, comprising:

(a) contacting a sample containing the first component to be determined with a known amount of a reagent, said reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces a second immunochemical component of said reaction capable of reacting with said first component to be determined;

(b) allowing sufficient time for reaction to take place; and

(c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

37. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces (i) a second immunochemical component of said reaction capable of reacting with said first component to be determined and (ii) an immunochemically inert macromolecule;

(b) allowing sufficient time for a reaction to take place; and

(c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

38. A method for the determination of a first component of an immunochemical reaction in an aqueous test medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:

(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined;

(b) allowing sufficient time for a reaction to take place; and

(c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

39. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample soution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Palanil Red BF dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Palanil Red BF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

40. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Resolin Brilliant Blue RRL dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Resolin Brilliant Blue RRL in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

41. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Samaron Brilliant Red H6GF dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separting the second solid phase from the second liquid phase; and

(h) determining the amount of Samaron Brilliant Red H6GF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

42. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;

(d) separating the first and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Samaron Brilliant Yellow HIOGF dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Samaron Brilliant Yellow HIOGF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

43. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially Palanil Luminous Red G dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Palanil Luminous Red G in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

44. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Palanil Luminous Yellow G dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG to said phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Palanil Luminous Yellow G in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

50. The method of claim 44 wherein said hydrophobic dye or pigment is Palanil Red FF.

45. A method for the immunochemical determination of hepatitis Surface B antigen (HBsAg), comprising:

(a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulin with a sample solution containing the HBsAg to be determined;

(c) allowing sufficient time for an immunological rection between the bound immunoglobulin and the HBsAg in the sample solution in step (b) to take place, to bind the HBsAg to be determined to the insolubilized sheep anti-HBsAg, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HBsAg therein with an immunochemical excess of particles of Palanil Red BF or Samuron Brilliant Red H6GF particles having anti-HBsAg immunoglobulin attached to their surfaces, said particles having a particle size of at least 5 nm;

(f) allowing sufficient time for a second immunochemical reaction to take place, to bind said reagent to that part of said HBsAg solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Palanil Red BF or Samuron Brilliant Red H6GF in the second liquid phase or the second solid phase, which is a measure of the amount of HBsAg in said sample solution.

46. A method for the immunochemical determination of human placental lactogen (HPL) in a liquid sample, comprising:

(a) binding a known amount of rabbit anti-HPL immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier;

(b) contacting said bound rabbit anti-HPL with a sample solution containing the HPL to be determined;

(c) allowing sufficient time for an immunological reaction between the bound rabbit anti-HPL immunoglobulin and the HPL in the sample solution in step (b) to take place to bind the HPL to be determined to the insolubilized rabbit anti-HPL, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HPL therein with a known amount of particles of a Palanil Red BF or Palanil Yellow 36 to form a particle-HPL conjugate, said particles having a particle size of at least 5 nm;

(f) allowing sufficient time for a second immunochemical reaction to take place to bind said reagent to that part of said solid phase bound rabbit anti-HPL immunoglobulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Palanil Red BF or Palanil Yellow 36 in the second solid phase or the second liquid phase, which is a measure of the amount of testosterone in said liquid sample.

47. A method for the immunochemical determination of human anti-Rubella sera, comprising:

(a) binding a known amount of Rubella viral antigen to the surface of a water-insoluble, water-insuspensible, solid carrier;

(b) contacting said bound Rubella viral antigen with a human serum sample containing the human anti-Rubella sera to be determined;

(c) allowing sufficient time for an immunological reaction between the bound Rubella viral antigen and the serum containing the human anti-Rubella sera to be determined to take place to bind the anti-Rubella sera to the insolubilized Rubella viral antigen, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound anti-Rubella titer therein with a known amount of a reagent, said reagent consisting essentially of dispersion particles of Palanil Red BF or Resolin Brilliant Blue RRL, said particles having a particle size of at least 5 nm and having sheep anti-human immunoglobulin attached to their surfaces to form a particle-sheep anti-human immunoglobulin conjugate;

(f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to the bound anti-Rubella titer solid phase, resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), to form a second solid and a second liquid phase;

(g) separating the second liquid and solid phases; and

(h) determining the amount of Palanil Red BF or Resolin Brilliant Blue RRL in the second solid phase or the second liquid phase, which is a measure of the human anti-Rubella serum to be determined in said sample.

48. A method for the immunochemical determination of human prolactin (PRL), comprising:

(a) binding a known amount of monoclonal (anti-PRL) immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound monoclonal (anti-PRL) immunoglobulin with a sample solution containing the PRL to be determined;

(c) allowing sufficient time for an immunological reaction between the monoclonal (anti-PRL) immunoglobulin and the PRL in the sample solution in step (b) to take place, to bind the PRL to be determined to the insolubilized monoclonal (anti-PRL) immunoglobulin, forming a first solid phase and leaving a first liquid phase;

(d) separating the first solid phase from the first liquid phase;

(e) contacting said first solid phase and bound PRL with a known amount of a reagent consisting essentially of dispersion particles of Palanil Luminous Red G or Palanil Luminous Yellow G, said particles having a particles size of at least 5 nm and having monoclonal (anti-PRL) immuno-globulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said PRL solid phase bound immunoglobuliln which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid and second liquid phases; and

(h) determining the amount of Palanil Luminous Red G or Palanil Luminous Yellow G in the second solid phase or the second liquid phase, which is a measure of the amount of the PRL to be determined in said sample solution.

49. A method for the immunochemical determination of human chorionic gonadtropin (HCG), comprising:

(a) contacting an aqueous sample containing the HCG to be determined with a known amount of a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm and having rabbit anti-HCG immunoglobulin attached to their surfaces;

(b) allowing sufficient time for an immunological reaction between said reagent and any HCG in the sample solution to take place; and

(c) determining the color of the sample solution, which is a measure of the amount of HCG to be determined in said sample solution.

51. A method for the simultaneous determination of human chorionic gonadotropin (HCG) and human placental lactogen (HPL), comprising:

(a) binding a known amount of rabbit anti-HCG immunoglobulin and a known amount of rabbit anti-HPL immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound immunoglobulins with a sample containing the HCG and the HPL to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulins and said HCG and said HPL in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG and to bind the HPL to be determined to the insolubilized rabbit anti-HPL, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound HCG therein with a known amount of a reagent, said reagent consisting essentially of Resolin Brilliant Blue RRL dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) contacting said first reacted solid phase and bound HPL therein with a known amount of a reagent, said reagent consisting essentially of Palanil Yellow 36 dispersion particles, said particles having rabbit anti-HPL immunoglobulin attached to their surfaces;

(g) allowing sufficient time for a second immunological reaction to take place, to bind the reagent containing Resolin Brilliant Blue RRL dispersion particles to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c) and to bind the reagent containing Palanil Yellow 36 dispersion particles to that part of said HPL solid phase bound immunoglobulin which has undergone the reaction in step (c);

(h) separating the second solid phase from the second liquid phase; and

(i) determining the presence of Resolin Brilliant Blue RRL and Palanil Yellow 36 in the second liquid phase or the second solid phase, which is an indicator or HCG and HPL respectively in the sample solution.

52. A method for the immunological determination of testosterone in a liquid sample, comprising:

(a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound rabbit anti-testosterone with a sample solution containing the testosterone to be determined;

(c) allowing sufficient time for an immunological reaction between the bound rabbit anti-testosterone immunoglobulin and the testosterone in the sample solution in step (b) to take place to bind the testosterone to be determined to the insolubilized rabbit anti-testosterone, forming a first solid phase and leaving a first liquid phase;

(d) separating the first liquid and solid phases;

(e) contacting said first reacted solid phase and bound testosterone therein with a known amount of particles of Palanil Red BF, said particles having a particle size of at least 5 nm and having testosterone 11 α

-hemisuccinyl bovine serum albumin attached to their surfaces to form a particle-testosterone-11 α

-hemisuccinyl bovine serum albumin conjugate;

(f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to that part of said solid phase rabbit anti-testosterone immunoglobulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of Pananil Red BF in the second solid phase, which is a measure of the amount of testosterone in said liquid sample.

53. A method for the immunological determination of testosterone in a liquid sample, comprising:

(a) contacting an aqueous sample containing the testosterone to be determined with (1) a known amount of a reagent consisting essentially of Samaron Brilliant Red H6GF or Samaron Brilliant Yellow H10GF particles, the particles having a particle size of at least 5 nm and having attached thereto rabbit anti-T¹¹ -bovine serum albumin immunoglobulin and (2) a known amount of T³ -bovine serum albumin;

(b) allowing sufficient time for a reaction to take place, whereby a first fraction of testosterone bound to said T³ -bovine serum albumin and a fraction of testosterone free of T³ -bovine serum albumin are produced; and

(c) determining the amount of Samaron Brilliant Red H6GF or Samaron Brilliant Yellow H10GF particles in one of said fractions, which is a measure of the amount of testosterone in said sample.

54. A method for the determination of human chorionic gonadtropin (HCG), comprising:

(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound receptor protein with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the insolubilized HCG receptor protein and the HCG to be determined;

(d) adding a known amount of reagent to said sample solution, said reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment and having HCG attached to their surfaces, said particles having a particle size of at least 5 nm;

(e) allowing sufficient time for a reaction to take place between (1) the dispersion reagent and (2) the insolubilized HCG receptor protein not bound to HCG, to form a solid phase containing a fraction of dispersion reagent bound to insolubilized HCG receptor protein, and a liquid phase of unbound and free dispersion reagent;

(f) separating the liquid and solid phases; and

(g) determining the amount of dye or pigment in either the solid or liquid phase, which is a measure of the amount of HCG in said sample solution.

55. A method for the immunochemical determination of human chorionic gonadtropin (HCG), comprising:

(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier;

(b) contacting said bound receptor protein with a sample solution containing the unknown HCG to be determined;

(c) allowing sufficient time for an immunochemical reaction between the bound receptor protein and the HCG in the sample solution to take place, to bind the HCG to be determined to the insolubilized HCG receptor protein, forming a first solid phase and leaving a first liquid phase;

(d) separating the first solid phase from the first liquid phase;

(e) contacting said first solid phase and insolubilized HCG therein with a known amount of a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said insolubilized HCG which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;

(g) separating the second solid phase from the second liquid phase; and

(h) determining the amount of dye or pigment in the second solid phase or the second liquid phase, which is a measure of the HCG to be determined in said sample solution.

56. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresponding bindable substance, according to a predetermined protocol, comprising:

(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component; and

(b) directions for the performance of said protocol.

57. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresponding bindable substance, according to a predetermined protocol, comprising:

(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule; and

(b) directions for the performance of said protocol.

58. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresonding bindable substance, according to a predetermined protocol, comprising:

(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first component; and

(b) directions for the performance of said protocol.

59. A freeze-dried reagent for use in an immunoassay, consisting essentially of dispersion particles of a hydrophobic dye or pigment having attached to their surfaces an immunochemical component, said particle having a particles size of at least 5 nm.

60. The reagent of claim 59 wherein said dye is Palanil Red BF and said immunochemical component is rabbit anti-HCG immunoglobulin.

61. A freeze-dried reagent for use in an immunoassay to determine a first immunochemical component, consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particles size of at least 5 nm and having attached to their surfaces (i) a component immunochemically equivalent to the first component to be determined and (ii) an immunochemically inert macromolecule.

62. A freeze-dried reagent for use in an immunoassay to determine a first immunochemical component, consisting essentially of dispersion particles of a hydrophobic dye or pigment having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component which is immunochemically similar to said first component, and said particles having a particle size of at least 5 nm.

63. A test kit, to be used for the detection and/or determination of at least one component of the reaction between a specific binding protein and a corresponding bindable substance thereto in an aqueous medium according to a predetermined protocol, comprising:

(a) a known amount of a dye- or pigment-labelled component obtained by coupling a component of said reaction to particles of (1) a hydrophobic dye or pigment or (2) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm;

(b) a known amount of at least one additional immunochemical reagent, at least one of said reagents being a ligand or an immobilized ligand, which ligand is selected from the group consisting of

(aa) a ligand capable of binding with the dye- or pigment-labelled component (a);

(bb) a ligand capable of binding with a binding partner of the dye- or pigment-labelled component (a);

(cc) a ligand capable binding with at least one of the component(s) to be determined; and

(dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detection and/or determination of at least one component of an immunochemical reaction in an aqueous medium between a specific binding protein and a corresponding bindable substance thereto.

64. A reagent for use in an immunoassay to determine a first immunochemical component, consisting essentially of dispersion particles of a hydrophobic dye or a pigment having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, said particles having a particle size of at least 5 nm.

65. The reagent of claim 64 wherein said dye is Palanil Red BF, the component (1) is rabbit (anti HCG) immunoglobulin, and said immunochemically inert macromolecule is bovine serum albumin.