![]() ![]() For others, only some groups played a significant role, e.g., the Cdelta group of Ile278 or the Cbeta group of Phe280. For some residues, e.g., Tyr279, most groups of the side chain contributed to the interaction. Four hydrophobic residues of P11, Val276, Ile278, Tyr279, and Phe280, were predominant in the interaction. The changes of the P11 residues into progressively shorter residues, the comparison of changes into Pro and Ala, and the study of double mutants showed the following. The affinities between mAb164 and the MalE-P11 hybrids were measured by competition enzyme-linked immunosorbent assay (ELISA). P11 was fused with a carrier protein, MalE, to facilitate its manipulation. It recognizes a synthetic peptide, P11, constituted of residues 273-HGRVGIYFGMK-283 of TrpB with high affinity. Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. We have analyzed the recognition between an antigenic undecapeptide and a monoclonal antibody through a mutational approach. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. This was confirmed by testing the binding of mAb 4D12 to HLA-B*3501 mutant molecules at residue 171 carrying these peptides. On the other hand, 4D12 bound only to HLA-B*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B*3501 molecules. Analysis using an HLA-B*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. The 4D12 mAb bound only to HLA-B*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis of the binding of mAb 4D12 to HLA-B*3501 and -B*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. Real-time analysis of antibody binding by surface plasmon resonance is a powerful method for studying such changes in the time domain of a few seconds to a few minutes.The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B*3501 and HLA-B*5101 molecules carrying self-peptides. A simple model that provides a quantitative description of this process could not be found. Slow conformational changes of p24 seem to be the most probable explanation. Intermediate steps with faster time constants must be involved. Experiments suggest a reversible change of binding properties in the epitope region with an overall time constant of about 100 s at room temperature. Biphasic association was also found in solution. shortly after dissociation of Fab-antigen complex the fast association phase is enhanced. At high concentration of CB-4/1 Fab the association of the antigen-antibody complex proceeds in two phases, while dissociation is mono-exponential. Recombinant p24 was immobilised in a hydrophilic carboxymethyldextran matrix. The interaction between HIV core protein p24 and the murine monoclonal antibody CB-4/1 or its Fab fragment showed unusual kinetics. ![]()
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