Carrier bound immunosorbent

• US 4,478,946 A
• Filed: 06/25/1982
• Issued: 10/23/1984
• Est. Priority Date: 07/02/1981
• Status: Expired due to Fees

• Current Assignee: South African Inventions Development Corp.
• Original Assignee: South African Inventions Development Corp.
• Inventors: Polson, Alfred, Van der Merwe, Kirsten J.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Moskowitz, M.
• Application Number:
  US06/392,111
• Time in Patent Office: 851 Days
• Field of Search: 436/518, 436/523-535, 436/823
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals

Abstract

The invention provides an immuno-reactant, being either an antigen or an antibody covalently bonded to a cross-linked film, more particularly composed of cross-linked protein or peptides, enveloping a carrier body, e.g. a solid, non-porous glass bead of 6 mm diameter. The immuno-logically active parts of the immuno-reactant are present on the surface in a form in which they are available for immunosorption. If the immuno-reactant is a protein, e.g. an antibody, it can provide all or part of the film-forming material. In a particular embodiment the film carries antibodies against a second type of antibody which is captured immunosorptively to form a "double layer" carrier-bound immunosorbent, the antigen capturing sites of the second type of antibody providing the immunosorption sites of the product. The immunosorbents are used, for example, for RIA or ELISA assays.

Claims

1. A carrier-bound immunosorbent comprising a solid carrier body carrying an immobilized immuno-reactant, wherein the immunoreactant, being either an antigen or an antibody, is covalently bonded to a crosslinked proteinaceous film enveloping the carrier body in such a manner that immunologically active parts of the immunoreactant are present on its surface in a form in which they are available for immunosorption, wherein said film consists essentially of protein, partially hydrolysed protein, peptide or a combination thereof, crosslinked in situ with a crosslinking agent on the surface of the carrier body into a coherent film enveloping the surface and clinging thereto.

2. An immunosorbent according to claim 1, wherein immunologically active parts of the immuno reactant are exposed and project from the film.

3. An immunosorbent according to claim 1, wherein the film is predominantly cross-linked protein molecules.

4. An immunosorbent according to claim 3 wherein the immuno reactant itself provides the protein molecules or peptide portions or at least a substantial part thereof.

5. An immunosorbent according to claim 1, wherein the film includes a cross-linked additional protein or peptide film-forming substance other than the immuno reactant.

6. An immunosorbent according to claim 1 wherein the immuno-reactant has a molecular or particle mass of from 300 to 8×

10⁶ and higher in the case of bacteria and is bonded to the film in an amount of from 0.6 to 500 mg/m² of surface area of the carrier body.

7. An immunosorbent according to claim 6 wherein the film contains between 30 and 1000% by weight of protein or peptide extender based on the weight of immuno-reactant.

8. An immunosorbent according to claim 1, wherein the carrier body takes the form of a rod or bead.

9. An immunosorbent according to claim 8 wherein the carrier body is a solid bead having a density higher than 1 g/cc.

10. An immunosorbent according to claim 1, wherein protein A or concanavillin A is covalently bonded to the film and in its turn carries selectively captured antibody molecules, held by the tail ends of the molecules.

11. An immunosorbent according to claim 1, wherein the film carries covalently bonded thereto a first immuno reactant being one member of an antigen/antibody pair, the first immuno reactant holding by antigen/antibody interaction the second member of the pair, this second member projecting from the surface with a region thereof which itself in turn has a desired immuno reactivity for entering into an immunosorbent reaction with a third immuno reactant.

18. A diagnostic or analytical reagent, comprising an immunosorbent according to claim 1.

19. A reagent according to claim 18, adapted to be used for radio immuno assays or enzyme-linked immuno assays.

34. A method of selectively removing an immunoreactive substance from a mixture in which it is present, which comprises contacting said mixture with an immunosorbent as claimed in claim 1 which carries exposed on its surface an immobilised immunoreactant specific to immunogenic determinants present on said immunoreactive substance and not present on other substances in the mixture, thereby selectively immunosorbing said immunoreactive substance, and removing the immunosorbent from contact with the mixture.

35. In a radio immuno assay (RIA) comprising the step of selectively removing an immunoreactive substance from a mixture in which it is present, the improvement wherein said substance is removed using a method according to claim 34.

36. In an enzyme-linked immuno assay (ELISA), comprising the step of selectively removing an immunoreactive substance from a mixture in which it is present, the improvement wherein said substance is removed using a method according to claim 34.

37. A method according to claim 34 which comprises, after removing the immunosorbent from contact with the mixture, desorbing the first substance to release it from the immunosorbent in purified form.

38. A method according to claim 34, wherein in said immunosorbent, said film carries covalently bonded thereto a first immuno reactant being one member of an antigen/antibody pair, the first immuno reactant holding by antigen/antibody interaction the second member of the pair, this second member projecting from the surface with a region thereof which itself in turn has a desired immuno reactivity for entering into an immunosorbent reaction with a third immuno reactant.

39. A method according to claim 34 which comprises intimately contacting the immunosorbent, having exposed on its surface a standard amount of a first immunoreactant specific to a complementary second immunoreactant of an antigen/antibody pair with a solution of the second immuno-reactant, and determining the amount of the second immuno-reagent in the solution which becomes attached to the immunosorbent.

50. An immunosorbent according to claim 3, wherein said protein molecules are partially hydrolysed.

12. In a carrier-bound immunosorbent comprising a carrier body carrying an immobilized immunoreactant, the improvement wherein at least the surface of the carrier body is non-porous;

wherein a film coated thereon on the surface of the carrier carries covalently bonded thereto a first immunoreactant being one member of an antigen/antibody pair, the first immunoreactant holding by antigen/antibody interaction the second member of the pair, this second member projecting from the surface of said immunosorbent with a region thereof which itself in turn has a desired immunoreactivity for entering into an immunosorbent reaction with a third immunoreactant; and

wherein the first immunoreactant forms a covalently bonded part of a film consisting essentially of protein, partially hydrolysed protein, peptide, or a combination thereof, crosslinked in situ with a crosslinking agent on the surface of the carrier body into a coherent film clinging to said surface.

13. An immunosorbent according to claim 12 comprising a first layer including covalently bonded and cross-linked antibodies of a first species of animal elicited against antibodies of a second species of animal and a second layer immunosorbed on the first layer of specific antibodies elicited against a specific antigen in the second species of animal.

14. A carrier-bound immunosorbent which is specific to a specific antigen comprising a carrier body carrying an immobilised immuno reactant, comprising on a solid substrate a first layer including covalently bonded and cross-linked antibodies of a first species of animal elicited against antibodies of a second species of animal and a second layer immunosorbed on the first layer, of specific antibodies elicited against the specific antigen in the second species of animal;

and wherein said solid substrate is non-porous, or said first layer covers the substrate substantially completely.

15. An immunosorbent according to claim 14, wherein the antibodies of the first layer are randomly orientated and the antibodies of the second layer are predominantly radially outwardly orientated with their antigen capturing regions facing outwardly.

16. An immunosorbent according to claim 14, wherein the second species is a fowl.

17. An immunosorbent according to claim 16 wherein the antibodies elicited in the fowl have been recovered from the egg yolk of an egg or eggs of such fowl.

20. A process for preparing a carrier-bound immunosorbent, which comprises applying a film of cross-linkable protein, partially hydrolysed protein, or peptide material to the surface of a solid carrier body to envelop the body, including a desired immunoreactant incorporated in or on the surface of the film, and crosslinking the film in situ while on the surface of the carrier body, the crosslinked film thereby formed clinging to the carrier surface, and covalently bonding the immunoreactant to the film.

21. A process according to claim 20, wherein the immunoreactant itself provides wholly or partly the material of which the film is formed.

22. A process according to claim 20, wherein the immuno reactant is supplemented with an additional protein or peptide type of film-forming material.

23. A process according to claim 20, wherein an additional film-forming material is used to form the film proper, the immuno reactant being covalently bonded to the film.

24. A process according to claim 20, wherein a cross-linking or bifunctional linking reagent is used for cross-linking the film and/or for bonding the immuno reactant to the film.

25. A process according to claim 24, wherein said cross-linking or bifunctional linking reagent has in its molecule two or more functional groups, each of which is independently an aldehyde, imide, amino, cyano, halogen, carboxyl, activated carboxyl, acid anhydride or maleimide group.

26. A process according to claim 24, wherein the linking reagent is first reacted with one of the two partners to be grafted together to form a reactive intermediate which is then reacted with the other partner.

27. A process according to claim 25, wherein the immuno reactant lacks a reactive group suitable for the covalent bonding reaction with the bifunctional linking compound;

and wherein a reactive derivative is formed by introducing said suitable reactive group by reaction with a different bifunctional linking compound containing said suitable reactive group.

28. A process according to claim 20, wherein a free-radical intermediate is formed of the immunoreactant and the free radical intermediate reacts to become covalently bonded to the film.

29. A process according to claim 28, wherein the free-radical intermediate is produced by radiation.

30. A process according to claim 20, werein a first layer is formed having bonded to the film antibodies against antibodies for a second layer and wherein the carrier body including the first layer is exposed to a solution of the second layer antibodies until the required amount of the second layer antibodies has been immunosorbed by the first layer.

31. A process according to claim 30, wherein the number of said second layer antibody molecules available for capturing by the first layer is at least twice the number of exposed antibody sites in the first layer.

32. A process according to claim 30, wherein the exposure to the solution of said second layer antibodies takes place between 0°

C. and the denaturing temperature of the antibodies, at least part of the exposure being carried out at a low temperature at which a change occurs which results in a strong retention of the second layer by the first layer.

33. A process according to claim 32, wherein the exposure takes place first at a relatively higher temperature where the immunosorption is accelerated, followed by further exposure at the low temperature.

40. In a carrier-bound immunosorbent comprising a carrier body carrying an immobilised immuno reactant, the improvement wherein the surface of the carrier body or a film coated thereon carries covalently bonded thereto a double layer comprising a first layer of a first immuno reactant being one member of an antigen/antibody pair, the first layer holding by antigen/antibody interaction a second layer comprising the second member of the pair, this second layer in turn carrying on its surface in a form available for immunosorption molecular regions having a desired immuno reactivity for entering into an immunosorbent reaction with a third immuno reactant, at least one of the members of the pair being IgY.

41. An immunosorbent according to claim 40, wherein the second layer of the double layer comprises IgY.

42. A carrier-bound immunosorbent which is specific to a specific antigen, comprising a solid carrier surface carrying an immobilised immuno reactant, the immuno reactant being present in a double layer composed of a first layer comprising IgG specific against IgY and a second layer immunosorbed by the IgG comprising IgY which in turn is specific to the specific antigen.

43. An immunosorbent according to claim 42, wherein the first layer of the double layer is covalently bonded to the solid carrier surface.

44. An immunosorbent according to claim 43, wherein the solid carrier surface is non-porous.

45. A carrier-bound immunosorbent of high specificity, comprising a carrier body having a solid surface carrying a permanently adhering layer of protein, partially hydrolysed protein, peptide or a combination thereof, the layer comprising a covalently bonded and thereby immobilized immunoreactant, being either an antigen or an antibody, in such a manner that immunologically active parts of the immunoreactant are present exposed in a form in which they are available for immunosorption, and being essentially free of accessible pores and other nonspecific adsorption sites.

46. An immunosorbent according to claim 45 wherein the carrier body is solid.

47. An immunosorbent according to claim 45, wherein the layer is crosslinked.

48. An immunosorbent according to claim 47, wherein the layer is in the form of a coating crosslinked after application to the surface, consisting essentially of protein, partially hydrolysed protein, peptide or a combination thereof, and crosslinking moieties of a crosslinking agent rendering the film coherent and clinging to the surface.

49. An immunosorbent according to claim 45 wherein immunologically active parts of the immunoreactant project from the layer or coating.