Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Das, Chandra; Mainwaring, Robin; Langone, John J.

doi:10.1016/0003-2697(85)90322-7

Cited by 10 publications

(4 citation statements)

References 12 publications

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“…This control experiment was possible if 4G8 was bound to immobilized OAβ in Step 1 and analyte SpA used to bind this IgG2 mouse subtype in Step 2. Previous studies have shown empirically that 1:1 Fc:SpA binding predominates at excess SpA [ 53 , 56 ]. Given the SpA mass of 42 kD and IgG mass of 150 kD, a Step2/Step1 signal ratio of 0.280 is expected if all binding sites available in solution remain available after 4G8 is immobilized.…”

Section: Resultsmentioning

confidence: 99%

“…The SNA flow cell therefore provides the concentration of IgG with at least one glycan exhibiting a surface accessible α2,6-galactose-sialic acid linkage, denoted here as [IgG] FC_SNA [ 38 ]. The SpA flow cell provides a measurement of >95% of human IgG, with the CFCA concentration denoted here as [IgG] FC_SpA [ 53 , 54 ]. The OAβ flow cell provides the concentration of OAβ + IgG, denoted here as [IgG] FC_OAβ .…”

Section: Methodsmentioning

confidence: 99%

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Anti-Aβ Oligomer IgG and Surface Sialic Acid in Intravenous Immunoglobulin: Measurement and Correlation with Clinical Outcomes in Alzheimer’s Disease Treatment

et al. 2015

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The fraction of IgG antibodies with anti-oligomeric Aβ affinity and surface sialic acid was compared between Octagam and Gammagard intravenous immunoglobulin (IVIG) using two complementary surface plasmon resonance methods. These comparisons were performed to identify if an elevated fraction existed in Gammagard, which reported small putative benefits in a recent Phase III clinical trial for Alzheimer’s Disease. The fraction of anti-oligomeric Aβ IgG was found to be higher in Octagam, for which no cognitive benefits were reported. The fraction and location of surface-accessible sialic acid in the Fab domain was found to be similar between Gammagard and Octagam. These findings indicate that anti-oligomeric Aβ IgG and total surface sialic acid alone cannot account for reported clinical differences in the two IVIG products. A combined analysis of sialic acid in anti-oligomeric Aβ IgG did reveal a notable finding that this subgroup exhibited a high degree of surface sialic acid lacking the conventional α2,6 linkage. These results demonstrate that the IVIG antibodies used to engage oligomeric Aβ in both Gammagard and Octagam clinical trials did not possess α2,6-linked surface sialic acid at the time of administration. Anti-oligomeric Aβ IgG with α2,6 linkages remains untested as an AD treatment.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Anti-Aβ Oligomer IgG and Surface Sialic Acid in Intravenous Immunoglobulin: Measurement and Correlation with Clinical Outcomes in Alzheimer’s Disease Treatment

et al. 2015

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“…reported that soluble natural SpA had little or no mitogenic activity in vitro , although after SpA was conjugated onto beads the increase of effective valency conveyed the capacity for inducing in vitro B-cell proliferation ( 65 ). In solution, the native SpA form takes on an extended conformation ( 49 ), and intravenous administration of SpA rapidly results in the in vivo formation of high molecular weight IgG-containing non-immune complexes, which persist for more than 24 h in the circulation ( 47 ). But these reports did not directly consider the contribution of the Fab- and Fc-binding sites on B cells.…”

Section: Discussionmentioning

confidence: 99%

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

2018

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Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4–5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.

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“…The complexes may be elongated structures. Earlier data suggested that the largest complexes in protein A -rabbit IgG mixtures had two protein A molecules and four IgG molecules complexed [21,22] and the four peaks may well contain four, three, two and one IgG molecules respectively. The smallest peak was sometimes asymmetric suggesting that the number of protein A molecules attached to the IgG molecule varied (theoretically from 0 to 2).…”

Section: Gel-filtration Of Ig Protein a Complexesmentioning

confidence: 99%

Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A

Ibrahim

1993

Scand J Immunol

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Protein A binds immunoglobulins and it has two target structures, one in Fc gamma (CH) and the other in selected VH regions. The protein has five homology regions (domains), A, B, C, D, and E. Fc-binding and VH-binding have been reported to be non-competitive, suggesting that different domains are responsible for the binding of the two ligands. On the other hand, all five domains have been reported to bind Fc. I studied binding of different immunoglobulins by protein A or its domain B (rBB). The results show that separate domains bind VH and Fc. If all five domains are capable of binding Fc, the ones that bind VH have low affinity for Fc. Furthermore, the number of Fc-binding domains varies depending on the type of the IgG being bound. Human IgG1 or IgG2 or rabbit IgG (Fc) seem to be bound by several domains (possibly four), and domain B is one of them. Mouse IgG1 or IgG2b are bound by fewer domains not including B. Murine IgG2a is also bound by fewer domains but B is one of them.

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Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Cited by 10 publications

References 12 publications

Anti-Aβ Oligomer IgG and Surface Sialic Acid in Intravenous Immunoglobulin: Measurement and Correlation with Clinical Outcomes in Alzheimer’s Disease Treatment

Anti-Aβ Oligomer IgG and Surface Sialic Acid in Intravenous Immunoglobulin: Measurement and Correlation with Clinical Outcomes in Alzheimer’s Disease Treatment

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A

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