Comparison of analytical methods for the evaluation of antibody responses against epitopes of polymorphic protein antigens

Helg, A.; Mueller, Markus; Joss, Andrea; Pöltl-Frank, Friederike; Stuart, Fiona; Robinson, John A.; Pluschke, Gerd

doi:10.1016/s0022-1759(03)00075-9

Cited by 26 publications

(15 citation statements)

References 30 publications

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“…It is therefore not unlikely that removing a flexible hydrophobic moiety at the apex of the 2F5 antigen binding site would cause artifacts in apparent binding affinity for the gp41 epitope bound to the solid phase. In contrast, in a competitive ELISA, the free antibody available for binding to the adsorbed antigen is determined by the binding constant of the antibody to the competitor in solution (20). This allows for a more accurate comparison of the relative binding affinities of antibodies.…”

Section: Discussionmentioning

confidence: 99%

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Julien¹,

Huarte

Maeso

et al. 2010

J Virol

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The identification and characterization of broadly neutralizing antibodies (bnAbs) against HIV-1 has formed a major research focus, with the ultimate goal to help in the design of an effective AIDS vaccine. One of these bnAbs, 2F5, has been extensively characterized, and residues at the apex of its unusually long complementaritydetermining region (CDR) H3 loop have been shown to be crucial for neutralization. Structural studies, however, have revealed that the 100 TLFGVPI 100F apex residues of the CDR H3 loop do not interact directly with residues of its core gp41 epitope. In an attempt to gain better insight into the functional role of this element, we have recombinantly expressed native 2F5 Fab and two mutants in which either the apical Phe100B(H) residue was changed to an alanine or the CDR H3 residues 100 TLFGVPI 100F were replaced by a Ser-Gly dipeptide linker. Isothermal titration calorimetry (ITC) and competitive-binding enzyme-linked immunosorbent assays (ELISAs) rendered strikingly similar affinity constants (K d [dissociation constant] of ϳ20 nM) for linear peptide epitope binding by 2F5 Fabs, independent of the presence or absence of the apex residues. Ablation of the CDR H3 apex residues, however, abolished the cell-cell fusion inhibition and pseudovirus neutralization capacities of 2F5 Fab. We report competitive ELISA data that suggest a role of 2F5 CDR H3 apex residues in mediating weak hydrophobic interactions with residues located at the C terminus of the gp41 membrane proximal external region and/or membrane components in the context of core epitope binding. The present data therefore imply an extended 2F5 paratope that includes weak secondary interactions that are crucial for neutralization of Env-mediated fusion.

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Section: Discussionmentioning

confidence: 99%

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Julien¹,

Huarte

Maeso

et al. 2010

J Virol

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“…After place exchange, the resulting particles were characterized by NMR to determine the degree of functionalization. Place exchange involving a 5:1 tiopronin to incoming peptide ratio resulted in approximate final average compositions of Au 616 Tiopronin 183 Ebola-l 12 [24][25][26] This study relied upon a QCM to quantitatively determine the binding between the monoclonal antibody and our nanocluster mimic. QCM operates via an oscillating quartz crystal that measures mass adsorption due to a change in oscillation frequency.…”

Section: Resultsmentioning

confidence: 99%

Design and synthesis of an antigenic mimic of the Ebola glycoprotein

et al. 2008

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An antigenic mimic of the Ebola glycoprotein was synthesized and tested for its ability to be recognized by an anti-Ebola glycoprotein antibody. Epitope-mapping procedures yielded a suitable epitope that, when presented on the surface of a nanoparticle, forms a structure that is recognized by an antibody specific for the native protein. This mimic-antibody interaction has been quantitated through ELISA and QCM-based methods and yielded an affinity (K d = 12 × 10 −6 M) within two orders of magnitude of the reported affinity of the native Ebola glycoprotein for the same antibody. These results suggest that the rational design approach described herein is a suitable method for the further development of protein-based antigenic mimics with potential applications in vaccine development and sensor technology.

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“…However, the rate of anti-STARP seropositivity in patients with acute falciparum malaria seems to be higher than that of anti-CSP seropositivity because only 16% of P. falciparum-infected patients gave positive results based on enzyme-linked immunosorbent assay and immunofluorescent antibody test (IFA) (Tapchaisri et al 1983). Although different analytical methods could result in different rates of immunoreactivity to malarial polymorphic epitopes, both IFA and Western blotting analysis seem not to be compromised by antibody binding avidity to the variant sequences (Helg et al 2003). Therefore, it is unlikely that the non-reactive sera to recombinant STARP in this study would be due to the presence of STARP variants in P. falciparum population.…”

Section: Discussionmentioning

confidence: 99%

Naturally acquired IgG antibodies against the C-terminal part of Plasmodium falciparum sporozoite threonine–asparagine-rich protein in a low endemic area

et al. 2011

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Plasmodium falciparum sporozoite threonine-asparagine-rich protein (STARP), a 78-kDa surface protein, is considered a potential vaccine candidate. The C-terminal part of STARP has been evolved under positive selection, suggesting the presence of immunodominant epitopes. However, little is known about the immune responses against STARP among individuals upon natural malaria exposure. In this study, we have cloned and expressed in Escherichia coli the recombinant C-terminal part of STARP spanning 118 amino acids in order to examine the humoral immune response against this protein. Blood samples were randomly collected from 74 individuals living in a malaria endemic area of Thailand who were acutely infected with P. falciparum (n = 54) and with Plasmodium vivax (n = 20). Malaria-negative blood samples were also obtained from 27 individuals living in the same endemic area who had experienced prior infection with P. falciparum 6 months to 1 year before sample collection and 20 healthy subjects without history of malaria exposure. Western blot analysis revealed that IgG antibodies against this recombinant peptide were found in 23 of 54 serum samples (42.6%) from P. falciparum-infected individuals. All serum samples from P. vivax-infected cases, non-infected individuals, and those who experienced prior infection with P. falciparum gave negative results, indicating that naturally acquired IgG antibodies against the C-terminal part of STARP are species-specific and short-lived. Provided that antibodies against STARP could confer protection, it is likely that malaria vaccine derived from the C-terminal part of STARP could probably be boosted upon natural exposure to P. falciparum.

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Comparison of analytical methods for the evaluation of antibody responses against epitopes of polymorphic protein antigens

Cited by 26 publications

References 30 publications

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Design and synthesis of an antigenic mimic of the Ebola glycoprotein

Naturally acquired IgG antibodies against the C-terminal part of Plasmodium falciparum sporozoite threonine–asparagine-rich protein in a low endemic area

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