Determination of antibody affinity by ELISA. Theory

Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation-and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6-10 residues) from the Aβ42 peptide associated with Alzheimer's disease into the complementarity determining regions of a domain (V H ) antibody generates antibody variants that recognize Aβ soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aβ motif ( 18 VFFA 21 ) are reactive with Aβ fibrils, whereas those displaying the amyloidogenic C terminus ( 34 LMVGGVVIA 42 ) are reactive with Aβ fibrils and oligomers (and weakly reactive with Aβ monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aβ conformers. Aβ gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aβ soluble oligomers and fibrils also neutralize the toxicity of each Aβ conformer. We expect that our antibody design strategy is not limited to Aβ and can be used to readily generate gammabodies against other toxic misfolded proteins.misfolding | beta-amyloid | protein engineering A hallmark of protein misfolding disorders is that polypeptides of unrelated sequence fold into similar oligomeric and fibrillar assemblies that are cytotoxic (1). The structures of these enigmatic conformers have captured the imagination of many investigators who have sought to explain the molecular basis of proteotoxicity in conformational disorders such as Alzheimer's disease. Because misfolded proteins are typically refractory to structural methods such as X-ray crystallography and solution NMR, few high-resolution structures of full-length misfolded proteins have been reported (ref. 2 and references therein). The structures of oligomeric intermediates have proven especially difficult to characterize because these conformers are labile, transient, and, in many cases, heterogeneous.Given the complexity of high-resolution structural analysis of misfolded proteins, alternative biochemical approaches are critical for understanding structure-function relationships of aggregated proteins. A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (3-13). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features ...

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“…We next measured the affinity of gammabodies for Aβ fibrils and oligomers using competitive ELISA analysis (33,34) (Fig. S5).…”

Section: Antibody Domains Displaying Amyloidogenic Aβ Motifs Recognizementioning

confidence: 99%

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Perchiacca

Ladiwala

Bhattacharya

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

128

134

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“…For estimating the avidity of MAbs and Fab fragments with E protein-containing TBE virus antigens, the dissociation constant in solution (K D ) was calculated using the data of the blocking ELISA according to a method described by Friguet et al (13) and modified by F. J. Stevens (50) and S. A. Bobrovnik (5). Briefly, the association constant in solution (K A ) was calculated by the use of the following equation: li ϫ (5), where li is the antigen concentration at each measurement point of the blocking ELISA, A 0 is the absorbance at 490 nm in the absence of blocking antigen, and A i is the absorbance at 490 nm in the presence of blocking antigen at each measurement point of the blocking ELISA.…”

Section: Vol 83 2009 Antigenic Structure Of Tbe Virus E Protein 8483mentioning

confidence: 99%

Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

Kiermayr

Stiasny

Heinz

2009

J Virol

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The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T‫1؍‬ icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.Flaviviruses form a genus in the family Flaviviridae (52) and comprise a number of important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses (30). They are small, enveloped viruses with only three structural proteins, designated C (capsid), M (membrane), and E (envelope). The E protein is oriented parallel to the viral membrane and forms a head-to-tail homodimeric complex ( Fig. 1A and B). The structure of the E ectodomain (soluble E [sE])-consisting of about 400 amino acids and lacking the 100 C-terminal amino acids (including the so-called stem and two transmembrane helices)-has been determined by X-ray crystallography for several flaviviruses (Fig. 1A) (25,34,36,38,44,55). Both of the essential entry functions-receptor-binding and membrane fusion after uptake by receptor-mediated endocytosis-are mediated by E, which is therefore the primary target for virusneutralizing antibodies (11,42,43,45).As revealed by cryo-electron microscopy (cryo-EM), mature infectious virions have smooth surfaces, comparable to a golf ball (27, 37). Their envelopes are icosahedrally symmetric and consist of a closed shell of 180 E monomers that are arranged in a herringbone-like pattern of 30 rafts of three dimers each (Fig. 1C) (27). On the other hand, capsid-lacking subviral particles, which can be produced in recombinant form by the coexpressio...

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“…following model derived from mass action and as described (49), with adjustment to take into account antibody bivalence (50): FRNT. Serially diluted antibody was mixed with an equal volume of diluted virus (30 focus-forming units per well) and incubated for 2 h at 37°C.…”

mentioning

confidence: 99%

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Tharakaraman¹,

Robinson²,

Hatas³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen-antibody interfaces to predict proteinprotein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1-3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein-protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies. (1). Engineering improved affinity and specificity of these compounds can augment their potency and safety while decreasing required dosages. Production of antibodies with binding properties of interest typically relies on methods involving screening large numbers of clones generated by the immune system or by mutant libraries (2, 3). Alternatively, computer-based design offers the potential to rationally mutate available antibodies for improved properties, including enhanced affinity and specificity to target antigens. Recently, several successful examples of antibody affinity improvement by computational methods using physical modeling with energy minimization have been described (4-6). However, such approaches require a 3D structure of the antibody-antigen complex and rarely result in affinity gains greater than 10-fold. Further, these approaches are sensitive to precise atomic coordinates, rendering them inapplicable to computer-generated models. More significantly, enhancement of affinity in the context of an antibody that recognizes multiple antigens (i.e., crossreactive) remains a particular challenge.Dengue is the most medically relevant arboviral disease in humans, with an estimated 3.6 billion people at risk for infection. More than 200 million infections of dengue virus (DV) are estimated to occur globally each year (7). The incidence, geographical outreach, and number of severe disease cases of dengue are increasing (8, 9), making DV of increasing concern as a human pathogen. The complex of DVs is composed of four distinct serotypes (designated DV1-4) (10), which vary from one another at the amino acid level by 25-40%. The sequence and antigenic variability of DVs have challenged efforts to develop an effective vaccine or therapeutic against a...

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Determination of antibody affinity by ELISA. Theory

Cited by 75 publications

References 7 publications

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

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