ISSN:0952-3499    

DOI:10.1002/jmr.300060202

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractThe kinetics of antibody–antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti‐fluorescein antibodies are featured in the review as model proteins to explore fluorescence‐based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti‐fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k1) and dissociation (k2). The near diffusion‐limited k1rates and the prolonged k2rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti‐metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformationa

ISSN:0952-3499    

DOI:10.1002/jmr.300060203

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractRecent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran‐modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three‐dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kdvalues for antigenic peptide/class I interactions at 25°C are in the range of ca 10−4–10−6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid‐phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/pept

ISSN:0952-3499    

DOI:10.1002/jmr.300060204

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractThe fine modulation of peptide–antibody interactions was investigated with anti‐peptide monoclonal antibodies recognizing peptide 125–136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcoreTM, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope results in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide–antibody inter

ISSN:0952-3499    

DOI:10.1002/jmr.300060205

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAn ideal peptide vaccine should contain both B‐ and T‐cell epitopes. Recognition of antigen by B cells is highly dependent on the three‐dimensional conformation of the antigen whereas T cells recognize antigen only after it has been processed to release a peptide fragment which is bound to the major histocompatibility complex (MHC) class II molecule. However, T cells provide ‘help’ to B cells displaying the same processed, MHC‐restricted from of the antigen, demonstrating that the T‐cell response to a protein antigen is under genetic control. Thus, strategies for co‐inclusion of T cell ‘helper’ epitopes with the B‐cell determinant elicit immune responses that are in most cases genetically restricted to only one or a few alleles of the MHC with limited activity across divergent MHC class II haplotypes. This genetically restricted T cell stimulatory activity of peptides is a serious obstacle and consequently such constructs would be of limited practical value as a vaccine targeted to a majority of an outbred population. In the study described here, we have engineered tow peptides to encompass the sequences from the universally immunogenic tetanus toxoid (TT) epitope and the contraceptive vaccine candidate lactate dehydrogenase C4(LDH‐C4). We demonstrate the feasibility of using ‘promiscuous’ T‐Cell epitopes colinearly constructed with a defined B‐cell epitope to induce high titer antipeptide IgG antibodies specific for native protein antigen LDH‐C4in several inbred strains of mice, outbred mice and rabbits. There appears to be a strong correlation between the capacity for the hybrid peptides to be stimulatory for the corresponding T cells in C57BL/6 (H‐2b) and C3H/HeJ (H‐2k) mice and their ability to be immunogenic. This correlation, however, appears to break down in H‐2dstrains of mice since no antibodies were detected in BALB/c and barely detectable levels of antibodies in B10.D2 although activated T cells were detectable. Conversely, high titers of antipeptide antibodies are elicited in some strains (B10.BR) (H‐2k); C57BL/10 (H‐2k) without detectable IL‐2 responses. Finally, we show that a determinant which was previously restricted to H‐2kcan be rendered immunogenic in H‐2bwith the ‘promiscuous’ TT epitope. Thus, certain haplotype‐restricted immune responses can be bypassed, setting forth the ground work for the design of a universal vaccine by broadening the effective response in a larger number of individuals typica

ISSN:0952-3499    

DOI:10.1002/jmr.300060206

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA complete series of configurationally isomers (L‐L,L‐D,D‐LANDD‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz1H NMR, theL‐DandD‐Lisomers, but not hteL‐LandD‐Disomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH2and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH2, the NOE enhancement observed between Leu‐δCH3and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated thatL‐DandD‐Lisomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, theL‐Lisomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and theD‐Lisomer was strongest withKiof 2.2 × 10−5M. It was found that theD‐Lisomer was only slowly hydrolysed but theL(orD)‐Disomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (Ki= 6.3 × 10−6M). Since the free acid analogue of theD‐Lisomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S1site of chymotrypsin

ISSN:0952-3499    

DOI:10.1002/jmr.300060207

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

ISSN:0952-3499    

DOI:10.1002/jmr.300060201

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY