scholarly journals The utility of photo-affinity labels as ‘mapping’ reagents. A study of sub-populations of a specific rabbit antibody by using structurally related photo-affinity reagents

1974 ◽  
Vol 141 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Richard A. G. Smith ◽  
Jeremy R. Knowles
Keyword(s):  
Binding Site ◽  
Antibody Binding ◽  
The Other ◽  
Structural Isomer ◽  
Rabbit Antibody ◽  
Affinity Reagents ◽  
Affinity Labels ◽  
Antibody Binding Site ◽  
Antibody Complex ◽  
Specific Rabbit

A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other.

scholarly journals The antibody binding site. Labelling of a specific antibody against the photo-precursor of an aryl nitrene

1972 ◽  
Vol 128 (3) ◽  
pp. 499-508 ◽  
Author(s):  
G. W. J. Fleet ◽  
J. R. Knowles ◽  
R. R. Porter
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Association Constant ◽  
Hypervariable Region ◽  
Antibody Binding ◽  
Light Chains ◽  
Small Peptides ◽  
Group 4 ◽  
Antibody Binding Site ◽  
Specific Rabbit

The isolation of specific rabbit antibodies for the haptenic group 4-azido-2-nitrophenyl, is described. These antibodies bind 1.8–2.0mol of hapten [∈-(4-azido-2-nitrophenyl)-l-lysine]/mol with an association constant of nearly 107m-1 at 4°C. On photolysis of the antibody–hapten complex, resulting in the formation of an aryl nitrene at the binding site, hapten was covalently bound to the antibody, and the antibody binding site was blocked. The ratio of labelling of heavy- and light-chains was 2.5:1. Two small peptides were isolated from digests of labelled heavy-chain, indicating that some 13% of the label in the antibody was attached to cysteine-92 and to alanine-93. These residues are adjacent to the major hypervariable region in rabbit heavy-chain (residues 95–105).

scholarly journals Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

1974 ◽  
Vol 139 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Christopher E. Fisher ◽  
Elizabeth M. Press
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Rabbit Antibody ◽  
Heavy Chains ◽  
Group 4 ◽  
Hypervariable Regions ◽  
Affinity Labelling ◽  
Antibody Complex

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.

Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

1998 ◽  
Vol 4 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Ari Hemminki ◽  
Seija Niemi ◽  
Lasse Hautoniemi ◽  
Hans Söderlund ◽  
Kristiina Takkinen
Keyword(s):  
Binding Site ◽  
Antibody Binding ◽  
Fine Tuning ◽  
Antibody Binding Site
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