Interactions Outside the Antigen-Combining Site

Nezlin, Roald

doi:10.1016/b978-012517970-6/50006-0

Cited by 4 publications

(6 citation statements)

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“…The purity of IgG3 in the elution fractions was lower when using a high wash buffer conductivity, which we attributed to the elimination of the charged and polar contacts (Hey and Zhang, 2012) primarily responsible for IgG3-protein G interactions in the Fc region (Nezlin and Nezlin, 1998). In contrast, hydrophobic interactions dominate antibody binding to protein A (Nezlin and Nezlin, 1998). A high wash buffer pH (8.0) moderately reduced the elution purity of IgG3 compared to pH 4.0, probably reflecting the weaker interaction between IgG3 and protein G under these conditions.…”

Section: Igg3 Purification From Plant Extracts By Protein G Chromatographymentioning

confidence: 84%

“…In a DoE approach, we found that the pH and conductivity of the wash and elution buffers significantly affected IgG3 purity, with the highest purities (close to 100%) achieved with a low wash buffer pH (4.0), a low elution buffer pH (2.0), and a low wash buffer conductivity (5 mS cm −1 ) combined with a high elution buffer conductivity (25 mS cm −1 ) (Figure 3, Supplementary Table S4). The purity of IgG3 in the elution fractions was lower when using a high wash buffer conductivity, which we attributed to the elimination of the charged and polar contacts (Hey and Zhang, 2012) primarily responsible for IgG3-protein G interactions in the Fc region (Nezlin and Nezlin, 1998). In contrast, hydrophobic interactions dominate antibody binding to protein A (Nezlin and Nezlin, 1998).…”

Section: Igg3 Purification From Plant Extracts By Protein G Chromatographymentioning

confidence: 86%

“…Protein L cannot be used in place of protein A because protein L binds kappa light chains rather than the lambda light chains used here. However, protein G binds to a broader range of antibodies than protein A, including all four human IgG subclasses (Nezlin and Nezlin, 1998). We therefore used the Streptococcus spp.…”

Section: Igg3 Purification From Plant Extracts By Protein G Chromatographymentioning

confidence: 99%

“…The production of antibodies in plants is a promising strategy to meet the growing demand for diagnostics and therapeutics (Schillberg et al, 2005;Capell et al, 2020) because high biomass yields of ∼500,000 kg ha −1 y −1 (Huebbers and Buyel, 2021) can be combined with high yields of recombinant antibodies per unit biomass [∼2 g kg −1 (Zischewski et al, 2016)] to favor the broad application of antibodies against S. aureus. However, IgG3 subclass antibodies are preferable for this purpose because all other IgG subclasses are recognized by S. aureus protein A (Nezlin and Nezlin, 1998). Importantly, although the inability of protein A to bind IgG3 is an asset for therapeutic activity, it prevents the use of protein A as an affinity ligand for antibody purification (Hober et al, 2007) and detection by surface plasmon resonance (SPR) spectroscopy using off-the-shelf sensor chips.…”

Section: Introductionmentioning

confidence: 99%

“…group C/G is a promising candidate (Higgins et al, 1995). Whereas protein A binds the antibody Fc region mainly via hydrophobic interactions, protein G binds the same region mostly via charged and polar interactions (Nezlin and Nezlin, 1998). Therefore, elution from protein G resins typically requires high rather than low salt concentrations to disrupt ionic interactions (Hey and Zhang, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

2021

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Immunoglobulin subclass IgG1 is bound and neutralized effectively by Staphylococcus aureus protein A, allowing the bacterium to evade the host’s adaptive immune response. In contrast, the IgG3 subclass is not bound by protein A and can be used to treat S. aureus infections, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). However, the yields of recombinant IgG3 are generally low because this subclass is prone to degradation, and recovery is hindered by the inability to use protein A as an affinity ligand for antibody purification. Here, we investigated plants (Nicotiana spp.) as an alternative to microbes and mammalian cell cultures for the production of an IgG3 antibody specific for the S. aureus alpha toxin. We targeted recombinant IgG3 to different subcellular compartments and tested different chromatography conditions to improve recovery and purification. Finally, we tested the antigen-binding capacity of the purified antibodies. The highest IgG3 levels in planta (>130 mg kg−1 wet biomass) were achieved by targeting the endoplasmic reticulum or apoplast. Although the purity of IgG3 exceeded 95% following protein G chromatography, product recovery requires further improvement. Importantly, the binding affinity of the purified antibodies was in the nanomolar range and thus comparable to previous studies using murine hybridoma cells as the production system.

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Section: Igg3 Purification From Plant Extracts By Protein G Chromatographymentioning

confidence: 84%