Compositions and methods for generating antibodies
View Patent ↗The compositions and methods of the present invention comprise the efficient and effective presentation of antigens to the appropriate components of the immune system resulting in the production of species-specific antibodies in vitro. In general, these compositions comprise one or more antigenic components together with a colloidal metal, optionally combined with derivatized PEG (polyethylene glycol) or other agents. The invention also comprises novel cytokine cocktails for generating desired antibodies.
1. A method for producing species-specific antibodies comprising
co-culturing B cells and monocytes in the presence of a denatured antigen,
generating activated B cells by adding a stimulation media comprising CD40 ligand (CD40L), or an anti-CD40 antibody and one of the following cytokine stimuli: (a) interleukin-2(IL-2) and interleukin-21(IL-21); (b) interleukin-5(IL-5), interleukin-10(IL-10), and transforming growth factor-beta (TGF-β; or (c) interleukin-4(IL-4), interleukin-13(IL-13), or both IL-4 and IL-13;
detecting the production of a species-specific antibody; and
generating a hybridoma from the activated B-cells.
2. The method of claim 1 , wherein the B cells and monocytes are co-cultured at a ratio of 1:5.
3. The method of claim 2 , wherein the denatured antigen is denatured by application of external stress, by application of heat, by the addition of an acid, by the addition of a base by exposure to a chaotropic agent, or by binding a colloidal metal nanoparticle.
4. The method of claim 1 , wherein the monocytes are isolated peripheral blood monocytes.
5. The method of claim 3 , wherein the antigen is heat denatured by the application of heat.
6. The method of claim 3 , wherein the at least one antigen is denatured by binding to a colloidal metal.
7. The method of claim 5 , wherein the denatured antigen is bound to a colloidal metal.
8. The method of claim 6 , wherein the colloidal metal is colloidal gold.
9. The method of claim 6 , wherein the colloidal metal is pegylated with a derivatized polyethylene glycol (PEG).
10. The method of claim 9 , wherein the derivatized PEG is a thiolated PEG derivative.
11. The method of claim 1 , wherein the antigen is derived from toxins, bacteria, viruses, protozoa, nucleic acids, tumor antigens, foreign blood cells, or the cells of transplanted organs.
12. The method of claim 1 , wherein the antigen is a cytokine
13. The method of claim 12 , wherein the cytokine is a lymphokine, monokine, chemokine, or interleukin.
14. The method of claim 12 , wherein the cytokine is selected from the group consisting of: interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-15, interleukin-16, interleukin-17, interleukin-18, Type I interferon, Type II interferon, tumor necrosis factor-alpha, or lymphotoxin.
15. The method of claim 1 , wherein the antigen is tumor necrosis factor-alpha.
16. The method of claim 1 , wherein the antigen is a growth factor.
17. The method of claim 16 , wherein the growth factor is selected from the group consisting of: fibroblast growth factor, kerotinocyte growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, epidermal growth factor, vascular endothelial growth factor, nerve growth factor, transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), Schwann-cell derived growth factor, nerve growth factor, platelet-derived growth factor, insulin like growth factor 1, insulin like growth factor 2, and glial growth factor.
18. The method of claim 17 , wherein the growth factor is epidermal growth factor.
19. The method of claim 1 , wherein the antigen is C. difficiles toxin A or B.
20. The method of claim 1 , wherein the antigen is a interleukin-2 receptor alpha or beta subunit.
21. The method of claim 7 , wherein the colloidal metal is pegylated with a derivatized PEG.
22. The method of claim 21 , wherein the derivatized PEG is a thiol PEG derivative.
23. The method of claim 1 , wherein the stimulation media comprises IL-2, CD40L, and IL-21.
24. The method of claim 23 , wherein the species-specific antibody is an IgG species-specific antibody.
25. The method of claim 1 , wherein the stimulation media comprises anti-CD40, IL-5, IL-10, and TGF-β.
26. The method of claim 25 , wherein the species-specific antibody is an IgA species-specific antibody.
27. The method of claim 1 , wherein the stimulation media comprises CD40L and IL-4, or IL-13, or both.
28. The method of claim 27 , wherein the species-specific antibody is a IgE species-specific antibody.
29. The method of claim 1 , wherein the species-specific antibody is a human species-specific antibody.
30. The method of claim 1 , wherein the stimulation media further comprises interleukin-6(IL-6), or granulocyte macrophage colony-stimulating factor GM-CSF, or both.
31. The method of claim 1 , wherein the stimulation media further comprises TNF-α.