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.

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.

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

scholarly journals Genetic and Immunological Properties of Phage-Displayed Human Anti-Rh(D) Antibodies: Implications for Rh(D) Epitope Topology

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 3066-3078 ◽  
Author(s):  
Tylis Y. Chang ◽  
Don L. Siegel
Keyword(s):  
Heavy Chain ◽  
Relevant Information ◽  
Peripheral Blood Lymphocytes ◽  
Antibody Binding ◽  
Light Chains ◽  
Chain Gene ◽  
Light Chain Gene ◽  
Heavy Chains ◽  
Epitope Specificity ◽  
Gene Usage

Understanding anti-Rh(D) antibodies on a molecular level would facilitate the genetic analysis of the human immune response to Rh(D), lead to the design of therapeutically useful reagents that modulate antibody binding, and provide relevant information regarding the structural organization of Rh(D) epitopes. Previously, we described a Fab/phage display-based method for producing a large array of anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single alloimmunized donor. In the current study, we present a detailed analysis of 83 randomly selected clones. Sequence analysis showed the presence of 28 unique γ1 heavy chain and 41 unique light chain gene segments. These paired to produce 53 unique Fabs that had specificity for at least half of the major Rh(D) epitopes. Surprisingly, despite this diversity, only 4 closely related heavy chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21). Similarly, nearly all Vκ light chains (15/18) were derived from one germline gene (DPK9). λ light chains showed a more diverse VL gene usage, but all (23/23) used the identical Jλ2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In particular, 2 such clones differed by only 3 residues, which resulted in a change from epD2 to epD3 specificity. These results suggest a model in which footprints of anti-Rh(D) antibodies are essentially identical to one another, and Rh(D) epitopes, as classically defined by panels of Rh(D) variant cells, are not discrete entities. Furthermore, these data imply that the epitope specificity of an anti-Rh(D) antibody can change during the course of somatic mutation. From a clinical perspective, this process, which we term epitope migration, has significance for the design of agents that modulate antibody production and for the creation of mimetics that block antibody binding in the settings of transfusion reactions and hemolytic disease of the newborn.

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