On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins

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On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins. / Akerström, B; Nilson, B H; Hoogenboom, H R; Björck, L.

In: Journal of Immunological Methods, Vol. 177, No. 1-2, 28.12.1994, p. 151-63.

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T1 - On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins

AU - Akerström, B

AU - Nilson, B H

AU - Hoogenboom, H R

AU - Björck, L

PY - 1994/12/28

Y1 - 1994/12/28

N2 - Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for 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 shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, 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-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.

AB - Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for 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 shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, 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-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.

KW - Amino Acid Sequence

KW - Bacterial Proteins

KW - Blotting, Western

KW - Carrier Proteins

KW - Chromatography, Affinity

KW - Humans

KW - Immunoglobulin Fragments

KW - In Vitro Techniques

KW - Membrane Proteins

KW - Molecular Sequence Data

KW - Protein Binding

KW - Recombinant Proteins

KW - Staphylococcal Protein A

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/0022-1759(94)90152-X

DO - 10.1016/0022-1759(94)90152-X

M3 - Article

C2 - 7822821

VL - 177

SP - 151

EP - 163

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 1872-7905

IS - 1-2

ER -