title: Heavy and light chain contributions to antigen binding in an anti-digoxin chain recombinant antibody produced by transfection of cloned anti-digoxin antibody genes.
abstract: We used immunoglobulin gene transfection to study the effect that substituting an homologous light (L) chain for a parental L chain has on antigen fine specificity and affinity. High-affinity monoclonal anti-digoxin antibodies 26-10 and 40-100 were selected for study because their L chains are 92% homologous (although the H chains differ), and their binding with digoxin and digoxin analogs show very different properties. In order to generate a recombinant transfectoma, the genes encoding the 26-10 H and L chains were cloned. After the sequenced clones had been shown to contain the V gene and the transcriptional control elements, the H and L chain V region genes were subcloned into different expression vectors. Both constructs were transfected into myeloma J558L, a lambda 1 chain producer, to verify that the genetic constructs expressed correctly. The recombined 26-10 antibody was identical to parental 26-10 antibody in fine specificity and affinity. The 26-10 L chain construct was then transfected into a cell line, CR-101, that expresses the 40-100 H chain and a lambda 1 chain. The transfectoma 1E6, secreting 40-100 H chain and 26-10 L chain, was selected. Appropriate gene expression in 1E6 was proven by polymerase chain reaction cloning and sequencing. The fine specificity properties of the 1E6 recombinant derive from both the 40-100 and 26-10 antibodies; however, the affinity of 1E6 is 130 times less than that of the parental antibodies. We conclude that, in 1E6, the H and L chains are codominant in their influence on antigen specificity and that homologous pairing of H and L chains is required for optimal affinity.
authors.affiliation: Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston.
authors.forename: R I
authors.initials: RI
authors.lastname: Near
authors.forename: S C
authors.initials: SC
authors.lastname: Ng
authors.forename: M
authors.initials: M
authors.lastname: Mudgett-Hunter
authors.forename: N W
authors.initials: NW
authors.lastname: Hudson
authors.forename: M N
authors.initials: MN
authors.lastname: Margolies
authors.forename: J G
authors.initials: JG
authors.lastname: Seidman
authors.forename: E
authors.initials: E
authors.lastname: Haber
authors.forename: M A
authors.initials: MA
authors.lastname: Jacobson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004077:Digoxin / Q000276:immunology*; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D000941:Antigens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D004077:Digoxin