title: Blot-sequencing of antibodies: application to analysis of V gene usage among anti-steroid monoclonal antibodies.
abstract: Automated gas-phase protein sequencing has been used to characterize variable regions of antibody heavy and light chains separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto Immobilon polyvinylidene difluoride membranes ('blot-sequencing'). Starting from 100 micrograms of antibody, 20 or more residues of N-terminal VH and VL sequences can regularly be obtained, which is often sufficient to assign the V region to a known family or subgroup. We have applied the blot-sequencing method to analysis of VH and VL usage among a panel of monoclonal anti-steroid antibodies, namely anti-progesterone, anti-pregnanediol, anti-estrone and anti-testosterone. The results demonstrate restricted, repetitive usage of VL subgroups and VH families related to anti-steroid specificities. VL regions of the VK1 group were particularly associated with anti-progesterone, VK21 with anti-estrone, and VK8 and VK9 with anti-pregnanediol. VH regions of anti-progesterone antibodies were all derived from the VHVGAM3.8 family; anti-estrone and anti-pregnanediol antibodies were derived from the VH7183 and VH36-60 families. The latter two families appear to characterize antibodies raised against steroids conjugated to proteins via a sugar bridge. Differences in VH/VL combination were associated with diversity of antibody specificity. In order to extend the sequence data obtained by this technique and confirm family assignments, we have shown that internal V-region sequences can be obtained by limited chemical cleavage of whole antibody with cyanogen bromide, followed by separation of individual fragments by SDS-PAGE and blot-sequencing.
authors.affiliation: Department of Immunology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
authors.forename: M J
authors.initials: MJ
authors.lastname: Taussig
authors.forename: P R
authors.initials: PR
authors.lastname: Symington
authors.forename: M
authors.initials: M
authors.lastname: Woods
authors.forename: M J
authors.initials: MJ
authors.lastname: Sims
authors.forename: D
authors.initials: D
authors.lastname: Beale
authors.forename: A S
authors.initials: AS
authors.lastname: Humphreys
authors.forename: A J
authors.initials: AJ
authors.lastname: Northrop
authors.forename: P J
authors.initials: PJ
authors.lastname: Barker
authors.forename: N S
authors.initials: NS
authors.lastname: Huskisson
authors.forename: J
authors.initials: J
authors.lastname: Coley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000918:Antibody Specificity; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005803:Genes, Immunoglobulin / Q000276:immunology*; D012739:Gonadal Steroid Hormones / Q000276:immunology*; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D008969:Molecular Sequence Data; D011276:Pregnanediol / Q000031:analogs & derivatives* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D012739:Gonadal Steroid Hormones; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C022270:pregnanediol-3 alpha-glucuronide; D011276:Pregnanediol; D003488:Cyanogen Bromide