Claims
- 1. A method of assaying a peptide for binding to an individual class I molecule, the method comprising the steps of:
providing a peptide of interest; providing individual soluble class I molecule-endogenous peptide complexes in which individual soluble class I molecules have endogenous peptides loaded therein; mixing the peptide of interest with the individual soluble class I molecules; and identifying individual soluble class I molecule-peptide of interest complexes, wherein the individual soluble class I molecules have the peptide of interest loaded therein.
- 2. The method of claim 1 further comprising the step of treating the individual soluble class I molecule-endogenous peptide complexes under conditions that cause the individual soluble class I molecules to release the endogenous peptides prior to mixing the peptide of interest with the individual soluble class I molecules.
- 3. The method of claim 2 wherein the step of treating the individual soluble class I molecules-endogenous peptide complexes involves heating the individual soluble class I molecule-endogenous peptide complexes to cause the individual soluble class I molecules to release the endogenous peptides.
- 4. The method of claim 1 wherein, in the steps of providing the peptide of interest and identifying individual soluble class I molecule-peptide of interest complexes, the peptide of interest is labeled to allow identification of individual soluble class I molecule-peptide of interest complexes from unbound peptide of interest.
- 5. The method of claim 4 wherein the peptide of interest is labeled with a radiolabel or a fluorescent label.
- 6. The method of claim 5 wherein, in the step of identifying individual soluble class I molecule-peptide of interest complexes, the peptide of interest is labeled with a fluorescent label, and the individual soluble class I molecule-peptide of interest complexes are identified by fluorescence polarization.
- 7. The method of claim 1 wherein, in the method of providing individual soluble class I molecule-endogenous peptide complexes, the individual soluble class I molecule-endogenous peptide complexes are produced by the method comprising the steps of:
obtaining genomic DNA or cDNA encoding at least one class I molecule; identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA; PCR amplifying the allele encoding the individual class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule; cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble class I molecule; transfecting the construct into a cell line to provide a cell line containing a construct that encodes an individual soluble class I molecule, the cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules; culturing the cell line under conditions which allow for expression of the individual soluble class I molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell; and isolating the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto.
- 8. The method of claim 7 wherein the construct further encodes a tag which is attached to the individual soluble class I molecule and aids in isolating the individual soluble class I molecule.
- 9. The method of claim 8 wherein the tag is selected from the group consisting of a HIS tail and a FLAG tail.
- 10. The method of claim 1 wherein, in the step of providing a peptide of interest, the peptide of interest is identified by a method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell, the method comprising the steps of:
providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules; infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line; culturing the uninfected cell line and the infected cell line under conditions which allow for expression of individual soluble class I molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell; isolating the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line; separating the endogenously loaded peptide ligands from the individual soluble class I molecules from the uninfected cell line and separating the endogenously loaded peptide ligands from the individual soluble class I molecules from the infected cell line; isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line; comparing the endogenously loaded peptide ligands isolated from the infected cell line to the endogenously loaded peptide ligands isolated from the uninfected cell line; and identifying at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line that is not presented by the individual soluble class I molecule on the uninfected cell line.
- 11. The method of claim 10 further comprising the step of identifying a source protein from which the at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line and not presented by the individual soluble class I molecule on the uninfected cell line is obtained.
- 12. The method of claim 10 wherein, in the step of identifying at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line but not on the uninfected cell line, the at least one endogenously loaded peptide ligand is obtained from a protein encoded by at least one of the microorganism, the gene from a microorganism or the tumor gene with which the cell line was infected to form the infected cell line.
- 13. The method of claim 10 wherein, in the step of identifying at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line but not on the uninfected cell line, the at least one endogenously loaded peptide ligand is obtained from a protein encoded by the uninfected cell line.
- 14. The method of claim 13, wherein the protein encoded by the uninfected cell line from which the at least one endogenously loaded peptide ligand is obtained has increased expression in a tumor cell line.
- 15. The method of claim 10 wherein, in the step of infecting a portion of the uninfected cell line, the portion of the uninfected cell line is infected with HIV.
- 16. The method of claim 1 wherein, in the step of providing a peptide of interest, the peptide of interest is identified by a method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell, the method comprising the steps of:
providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules; infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line; culturing the uninfected cell line and the infected cell line under conditions which allow for expression of the individual soluble class I molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell; isolating the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line; separating the endogenously loaded peptide ligands from the individual soluble class I molecules from the uninfected cell line and separating the endogenously loaded peptide ligands from the individual soluble class I molecules from the infected cell line; isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line; comparing the endogenously loaded peptide ligands isolated from the uninfected cell line to the endogenously loaded peptide ligands isolated from the infected cell line; and identifying at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the uninfected cell line that is not presented by the individual soluble class I molecule on the infected cell line.
- 17. The method of claim 16 further comprising the step of identifying a source protein from which the at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the uninfected cell line and not presented by the individual soluble class I molecule on the infected cell line is obtained.
- 18. The method of claim 17 wherein, in the step of infecting a portion of the uninfected cell line, the portion of the uninfected cell line is infected with HIV.
- 19. The method of claim 1 wherein, in the step of identifying individual soluble class I molecule peptide of interest complexes, the complexes are identified using an antibody that recognizes the individual soluble class I molecule having a peptide loaded therein.
- 20. A method of assaying a peptide for binding to an individual class I molecule, the method comprising the steps of:
providing a peptide of interest; labeling the peptide of interest; providing individual soluble class I molecules; mixing the labeled peptide of interest with the individual soluble class I molecules; and identifying individual soluble class I molecule-labeled peptide of interest complexes, wherein the individual soluble class I molecules have the labeled peptide of interest loaded therein.
- 21. The method of claim 20 wherein, in the step of labeling the peptide of interest, the peptide of interest is labeled with a radiolabel or a fluorescent label.
- 22. The method of claim 21 wherein, in the steps of labeling the peptide of interest and identifying individual soluble class I molecule-labeled peptide of interest complexes, the peptide of interest is labeled with a fluorescent label, and the individual soluble class I molecule-labeled peptide of interest complexes are identified by fluorescence polarization.
- 23. The method of claim 21 wherein, in the steps of labeling the peptide of interest and identifying individual soluble class I molecule-labeled peptide of interest complexes, the peptide of interest is labeled with a radiolabel, and the individual soluble class I molecule-radiolabeled peptide of interest complexes are isolated from unbound radiolabeled peptide of interest.
- 24. The method of claim 23 wherein the individual soluble class I molecule-radiolabeled peptide of interest complexes are isolated from unbound radiolabeled peptide of interest by gel filtration.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. §119(e) of provisional U.S. Serial No. 60/274,605, filed Mar. 9, 2001, entitled “HLA PROTEIN PRODUCTION FROM CDNA”, and provisional Attorney Docket No. 6680.044, filed Mar. 7, 2002, entitled “EPITOPE TESTING USING SOLUBLE HLA”, the contents of which are hereby expressly incorporated in their entirety by reference. This application is also a continuation-in-part of U.S. Ser. No. 09/974,366, filed Oct. 10, 2001, entitled “COMPARATIVE LIGAND MAPPING FROM MHC CLASS I POSITIVE CELLS;” and is also a continuation-in-part of U.S. Ser. No. 10/022,066, filed Dec. 18, 2001, entitled “METHOD AND APPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF,” the contents of which are also hereby expressly incorporated in their entirety by reference.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60274605 |
Mar 2001 |
US |
|
60362799 |
Mar 2002 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
09974366 |
Oct 2001 |
US |
Child |
10095818 |
Mar 2002 |
US |
Parent |
10022066 |
Dec 2001 |
US |
Child |
10095818 |
Mar 2002 |
US |