title: Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture.
abstract: This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.
authors: La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.
authors: John
authors: J
authors: Sidney
authors: Scott
authors: S
authors: Southwood
authors: Carrie
authors: C
authors: Moore
authors: Carla
authors: C
authors: Oseroff
authors: Clemencia
authors: C
authors: Pinilla
authors: Howard M
authors: HM
authors: Grey
authors: Alessandro
authors: A
authors: Sette
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D002850:Chromatography, Gel; D006649:Histocompatibility Antigens / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D006649:Histocompatibility Antigens; D010446:Peptide Fragments
references: J Immunol. 1992 Sep 15;149(6):1896-904
references: 1517560
references: J Exp Med. 1995 Jun 1;181(6):2097-108
references: 7539039
references: Cell. 1993 Sep 10;74(5):929-37
references: 8104103
references: Immunology. 2010 Jul;130(3):319-28
references: 20408898
references: Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2242-6
references: 1549590
references: Science. 1988 Nov 18;242(4881):1045-7
references: 3194755
references: Immunogenetics. 2009 Jan;61(1):1-13
references: 19002680
references: J Immunol. 1992 Oct 15;149(8):2634-40
references: 1401902
references: Cell. 1994 Jan 28;76(2):287-99
references: 8293464
references: BMC Bioinformatics. 2010 Nov 22;11:568
references: 21092157
references: J Immunol. 1991 Oct 15;147(8):2663-9
references: 1717570
references: Cell. 1990 Aug 10;62(3):563-7
references: 2199065
references: J Mol Biol. 1997 Apr 18;267(5):1258-67
references: 9150410
references: J Immunol. 2007 Jun 15;178(12):7890-901
references: 17548627
references: Immunome Res. 2011 May;7(2):
references: 28747991
references: Immunogenetics. 2011 Jun;63(6):325-35
references: 21305276
references: PLoS Comput Biol. 2008 Apr 04;4(4):e1000048
references: 18389056
references: J Virol. 2008 Dec;82(24):12241-51
references: 18842709
references: Biochemistry. 2005 Sep 20;44(37):12491-507
references: 16156661
references: Curr Opin Immunol. 1995 Apr;7(2):263-9
references: 7546387
references: BMC Immunol. 2008 Mar 16;9:8
references: 18366636
references: J Immunol. 2010 Oct 1;185(7):4189-98
references: 20810981
references: Nat Biotechnol. 2006 Jul;24(7):817-9
references: 16767078
references: Immunogenetics. 2005 Jun;57(5):304-14
references: 15868141
references: Annu Rev Immunol. 1994;12:181-207
references: 7516668
references: Structure. 1994 Apr 15;2(4):245-51
references: 8087551
references: J Immunol. 1990 Sep 15;145(6):1809-13
references: 1975260
references: J Immunol. 2008 Jun 1;180(11):7193-202
references: 18490718
references: Curr Opin Immunol. 1994 Feb;6(1):52-6
references: 7513526
references: Nature. 1990 Aug 2;346(6283):476-80
references: 2198471
references: PLoS Pathog. 2009 Dec;5(12):e1000695
references: 20019801
references: Curr Opin Immunol. 1994 Feb;6(1):24-31
references: 7513524
references: Annu Rev Immunol. 1995;13:587-622
references: 7612235
references: Tissue Antigens. 2001 May;57(5):405-14
references: 11556965
references: Eur J Immunol. 1989 Dec;19(12):2237-42
references: 2481588
references: J Immunol Methods. 2011 Nov 30;374(1-2):5-12
references: 21044632
references: Immunity. 1994 Dec;1(9):751-61
references: 7895164
references: J Immunol. 1994 Mar 15;152(6):2890-8
references: 8144889
references: Annu Rev Immunol. 1993;11:403-50
references: 8476568
references: Immunogenetics. 2000 Aug;51(10):816-28
references: 10970096
references: Immunome Res. 2010 Nov 13;6:9
references: 21073747
references: Nature. 1991 Jul 4;352(6330):67-70
references: 2062379
references: Eur J Immunol. 1996 Aug;26(8):1911-8
references: 8765039
references: Nucleic Acids Res. 2010 Jan;38(Database issue):D854-62
references: 19906713
references: J Immunol. 2005 Oct 15;175(8):5504-15
references: 16210659
references: Protein Sci. 2003 Mar;12(3):551-9
references: 12592025
references: Methods Mol Biol. 2009;524:361-7
references: 19377958
references: Eur J Immunol. 1994 Feb;24(2):385-92
references: 8299688
references: J Immunol. 2011 Oct 15;187(8):4268-79
references: 21918184
references: J Biol Chem. 1993 Jul 25;268(21):15425-34
references: 8393442
references: J Immunol. 2010 Mar 1;184(5):2492-503
references: 20139279
references: Biochemistry. 2004 Nov 23;43(46):14852-63
references: 15544356
references: Nature. 1989 Jun 1;339(6223):392-4
references: 2786148
references: BMC Bioinformatics. 2008 Dec 12;9 Suppl 12:S22
references: 19091022
references: J Immunol. 1994 May 1;152(9):4516-25
references: 7512598
references: J Immunol. 1984 Feb;132(2):662-7
references: 6228596
references: PLoS Comput Biol. 2006 Jun 9;2(6):e65
references: 16789818
references: J Immunol. 1995 Jan 1;154(1):247-59
references: 7527812
references: Science. 1992 Mar 6;255(5049):1261-3
references: 1546328
references: J Immunol. 1992 Feb 1;148(3):844-51
references: 1730877
references: Tissue Antigens. 1983 Aug;22(2):107-17
references: 6194571
references: J Immunol. 1990 Sep 15;145(6):1799-808
references: 2167910
references: J Immunol. 2010 Jul 15;185(2):943-55
references: 20554959
references: Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13980-5
references: 16172378
references: Hum Immunol. 1996 Feb;45(2):79-93
references: 8882405
references: J Immunol. 1994 Jan 1;152(1):163-75
references: 8254189
references: J Exp Med. 1994 Dec 1;180(6):2353-8
references: 7964508
references: Biochim Biophys Acta. 1995 Apr 13;1243(3):453-60
references: 7537104
references: Cell. 1990 Jul 27;62(2):285-95
references: 2196996
references: J Immunol Methods. 2001 Sep 1;255(1-2):57-66
references: 11470286
references: Immunome Res. 2010 May 17;6:4
references: 20478058
references: Immunome Res. 2009 May 05;5:2
references: 19416502
references: Nature. 1988 Dec 22-29;336(6201):778-80
references: 2462673
references: Immunogenetics. 1997;45(4):249-58
references: 9002445
references: J Immunol. 1993 Dec 1;151(11):5966-74
references: 8245441
references: EMBO J. 1992 Aug;11(8):2829-39
references: 1379172
references: Biochem J. 1963 Oct;89:114-23
references: 14097352
references: J Immunol. 1994 Jun 15;152(12):5742-8
references: 8207204
references: Immunogenetics. 1999 Nov;50(3-4):213-9
references: 10602881
references: J Immunol. 1990 Mar 1;144(5):1849-56
references: 2307844
references: J Immunol. 1990 Apr 1;144(7):2493-8
references: 2319129
references: J Immunol. 1995 Nov 1;155(9):4307-12
references: 7594589
references: Immunogenetics. 2007 Sep;59(9):745-51
references: 17701407
references: Nature. 1991 May 23;351(6324):290-6
references: 1709722
references: J Immunol. 1993 Sep 15;151(6):3163-70
references: 7690794
references: J Immunol. 1994 May 15;152(10):4946-57
references: 8176213
references: Cell. 1986 Dec 26;47(6):1071-7
references: 3490919
references: J Immunol. 1996 Oct 15;157(8):3480-90
references: 8871647
references: Immunogenetics. 1995;41(4):178-228
references: 7890324
references: J Virol. 2007 May;81(10):4928-40
references: 17329346
references: J Immunol. 2012 May 15;188(10):5020-31
references: 22504645
references: Eur J Immunol. 1991 Sep;21(9):2069-75
references: 1889458
references: J Immunol. 2009 Jun 15;182(12):7763-75
references: 19494300
references: Biochem Pharmacol. 1973 Dec 1;22(23):3099-108
references: 4202581
references: Nature. 1990 Nov 15;348(6298):213-6
references: 1700303
references: Immunome Res. 2008 Jan 25;4:2
references: 18221540
references: J Biomol Screen. 2009 Feb;14(2):173-80
references: 19196700
references: Mol Immunol. 1994 Aug;31(11):813-22
references: 8047072
references: Tissue Antigens. 2002 Apr;59(4):251-8
references: 12135423
references: Curr Biol. 1994 Jul 1;4(7):653-5
references: 7953548
references: Science. 1987 Mar 13;235(4794):1353-8
references: 2435001
references: Med Res Rev. 2002 Mar;22(2):168-203
references: 11857638
references: Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7202-6
references: 8692969
references: J Immunol. 1995 Jan 15;154(2):685-93
references: 7529283
references: Annu Rev Cell Biol. 1993;9:163-206
references: 7506551
references: Int Immunol. 1990;2(5):443-51
references: 1707655
references: BMC Immunol. 2008 Jan 22;9:1
references: 18211710
references: J Immunol. 1991 Jul 1;147(1):189-97
references: 1711073
references: Tissue Antigens. 2004 May;63(5):395-400
references: 15104671
references: J Immunol. 1998 Apr 1;160(7):3363-73
references: 9531296
references: J Immunol. 2003 Dec 1;171(11):5964-74
references: 14634108
references: J Immunol. 1998 Jun 15;160(12):6062-71
references: 9637523
references: Cell. 1993 Jul 16;74(1):197-203
references: 8334703
references: Science. 1994 Apr 29;264(5159):716-9
references: 7513441
references: Curr Opin Immunol. 1994 Feb;6(1):45-51
references: 7513525
references: Biotechniques. 1992 Dec;13(6):901-5
references: 1476743
references: Curr Opin Immunol. 1992 Feb;4(1):79-86
references: 1596372