ON THE INTERACTION BETWEEN SINGLE-CHAIN FV ANTIBODIES AND BACTERIAL IMMUNOGLOBULIN-BINDING PROTEINS

Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv an tibodies, expressed in E. coil and with known specificity and sequence . Several of the single chain Fv antibodies showed affinity for staphy lococcal protein A and peptostreptococcal protein L, but not for the s treptococcal proteins G or H. The affinity of the binding was higher f or protein L (4.5 and 1.4 x 10(9) M(-1)) than for protein A (7.7 and 6 .7 x 10(8) M(-1)), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was show n to be specific by Western blotting, and the single chain Fv antibodi es could be purified from crude bacterial culture media by affinity ch romatography on protein L- or A-Sepharose. Protein A, which has affini ty for the V-H domain of the scFv antibodies, was tested against scFv antibodies containing V(H)1, V(H)3, V(H)4 and V(H)5 domains, and its b inding was restricted to approximately half of the scFv antibodies wit h a V(H)3 domain. Protein L, which has affinity for the V-L domain, wa s tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 dom ains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-bind ing V-L domains demonstrated that amino acid residues crucial to the b inding of protein L were distributed over a large area outside the hyp ervariable antigen-binding regions.