A small domain (6.5 kDa) of bacterial protein G inhibits C3 covalent binding to the Fc region of IgG immune complexes

Abstract Background: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient’s serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient’s serum revealed an abnormal vitamin-B12-binding protein. SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient’s eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

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Inhibition of C3 covalent binding to IgG immune aggregates by recombinant protein G (domain III)

Immunology Letters ◽

10.1016/s0165-2478(97)87272-0 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 109

Author(s):

F Vivanco

Keyword(s):

Recombinant Protein ◽

Covalent Binding ◽

Protein G ◽

Immune Aggregates ◽

Domain Iii

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Presentation of Antigen in Immune Complexes Is Boosted by Soluble Bacterial Immunoglobulin Binding Proteins

Journal of Experimental Medicine ◽

10.1084/jem.189.8.1217 ◽

1999 ◽

Vol 189 (8) ◽

pp. 1217-1228 ◽

Cited By ~ 10

Author(s):

Michel Léonetti ◽

Jérome Galon ◽

Robert Thai ◽

Catherine Sautès-Fridman ◽

Gervaise Moine ◽

...

Keyword(s):

B Cells ◽

Immune Complexes ◽

Binding Proteins ◽

Protein A ◽

Infection Process ◽

Protein G ◽

Nonspecific Immune Response ◽

Immunoglobulin Binding ◽

Snake Toxin ◽

Lines Of Evidence

Using a snake toxin as a proteic antigen (Ag), two murine toxin–specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag–specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20–100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP–Ab–Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS® analyses showed that an Ag–Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag–Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor–containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.

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Novel UV-activated biofunctionalization of up-converting nanocrystals for detection of proteins

Journal of Nanostructure in Chemistry ◽

10.1007/s40097-021-00404-x ◽

2021 ◽

Author(s):

Małgorzata Misiak ◽

Maciej Gawłowski ◽

Agnieszka Kowalczyk ◽

Michał Skowicki ◽

Katarzyna Prorok ◽

...

Keyword(s):

Lower Energy ◽

Water Solubility ◽

Protein G ◽

Light Activation ◽

Bacterial Protein ◽

Nonspecific Binding ◽

Modified Surface ◽

Initial Coating ◽

Biological Environment ◽

Direct Attachment

AbstractLanthanide doped nanocrystals capable to emit higher energy photons under excitation with lower energy radiation are promising for a broad range of applications including biodetection, biosensing, and bioimaging. However, the adaptation of these nanoparticles to the biological environment that requires good water-solubility, stability and ease of further functionalization still remains a challenge. The application of nanoparticles for biodetection or in various assays encountered many difficulties arising mainly from the strong tendency of nanoparticles for aggregation or nonspecific binding. Here we present a new method to obtain soluble and stable in water-based buffers NaYF4:Yb3+Er3+ nanocrystals with modified surface ready for further conjugation with biomolecules. In the presented approach polyvinylpyrrolidone/vinyl alcohol copolymer (PVP/VA) with photo-activatable linker (N-5-Azido-2-nitrobenzoyl group—ANB-NOS) was used for initial coating due to its high non-covalent affinity to nanoparticles surface. Subsequent coating with aminated dextran by ultraviolet light activation of ANB-NOS was carried out. This step has a significant impact on nanocrystals stability in the physiological buffer as well as on protein conjugation. Conjugation of biomolecules was possible by employing another photo-linker—sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA). Bacterial Protein G has been selected to create a universal immune-imaging agent due to its ability to bind Fc fragment of most mammalian immunoglobulins. Moreover, the direct attachment of antibodies to nanoparticles was also examined. The activity of biofunctionalized nanocrystals was tested in immunoblot test, that confirmed preserved activity of attached molecules and lack of undesired nonspecific binding or precipitation on the assay membrane. Graphic abstract

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Irreversible Blockade of Factor VIII Antibodies.

Blood ◽

10.1182/blood.v104.11.1728.1728 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1728-1728

Author(s):

Stephanie Planque ◽

Miguel A. Escobar ◽

Hiroaki Taguchi ◽

Sangeeta Karle ◽

Yasuhiro Nishiyama ◽

...

Keyword(s):

Factor Viii ◽

Immune Complexes ◽

Binding Sites ◽

High Titer ◽

Covalent Binding ◽

Sodium Dodecyl ◽

Inhibitory Effect ◽

Binding Activity ◽

Antigen Binding

Abstract High titer antibodies (Abs) to Factor VIII (FVIII) can render FVIII replacement therapy ineffective in severe hemophilia A patients. An antigen capable of specific and covalent binding is predicted to reverse the effects of pathogenic Abs potently compared to conventional noncovalently binding antigens, as dissociation of the former type of antigen from the immune complexes is precluded. Recently, we described the apparently universal presence of enzyme-like nucleophiles in the antigen binding sites of Abs (Planque et al, J Biol Chem, 2003). Here, we report a covalently reactive antigen analog (CRA) of FVIII that inactivates anti-FVIII Abs specifically. Recombinant FVIII was derivitized at Lys side chains with phosphonate diester groups, positioning these electrophiles within the antigenic epitopes expressed by FVIII. Comparable binding of FVIII-CRA and underivitized FVIII by a monoclonal Ab and polyclonal Abs from 5 hemophilia patients was evident, suggesting maintenance of the FVIII epitope structure necessary for noncovalent Ab recognition. FVIII-CRA formed immune complexes with polyclonal anti-FVIII Abs from hemophilia patients that were not dissociated by sodium dodecyl sulfate (2%), a detergent that disrupts noncovalent interactions. Removal of free FVIII-CRA from the reaction mixtures by affinity chromatography on Protein G did not restore the FVIII-binding activity of the Abs, confirming irreversible occupancy of the Ab binding sites. No binding of the Abs by an irrelevant peptidyl CRAs was observed, suggesting the role of noncovalent antigen binding in imparting specificity to the covalent reaction. Ab inhibition of the functional role of FVIII in coagulation was studied by the chromogenic assay. Treatment of the Abs with the FVIII-CRA followed by removal of free FVIII-CRA relieved the inhibitory effect of the Abs on FVIII mediated activation of FIXa/FX. An irrelevant peptidyl CRA did not relieve the Ab inhibitory effect. Compared to FVIII, the FVIII-CRA activated FIXa/FX poorly, suggesting that the integrity of the FIXa binding site had been disrupted. These observations encourage the development of electrophilic CRA analogs of FVIII for treatment of patients with inhibitory Abs to FVIII.

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Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115572 ◽

1975 ◽

Vol 33 ◽

pp. 390-391

Author(s):

Wm. J. Arnold ◽

J. Russo ◽

H. D. Soule ◽

M. A. Rich

Keyword(s):

Horseradish Peroxidase ◽

Mammary Tumor ◽

Covalent Binding ◽

Petri Dish ◽

Gamma Chain ◽

Mammary Tumor Virus ◽

Mammary Tumor Cells ◽

Murine Mammary Tumor ◽

Tumor Virus ◽

Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

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A Sensitive On-Grid Immunoelectron Microscopic Procedure for Diagnosis of Rotavirus Infection

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s143192760000221x ◽

1980 ◽

Vol 38 ◽

pp. 506-507

Author(s):

M. F. Miller ◽

A. R. Rubenstein

Keyword(s):

Electron Microscopy ◽

Specific Antibody ◽

Immune Complexes ◽

Acute Gastroenteritis ◽

Plant Viruses ◽

Aggregation Process ◽

Microscopic Technique ◽

Fecal Material ◽

Present Communication ◽

Number Of Particles

Studies of rotavirus particles in humans, monkeys and various non-primates with acute gastroenteritis have involved detection of virus in fecal material by electron microscopy. The EM techniques most commonly employed have been the conventional negative staining (Fig. 1) and immune aggregation (Fig. 2) procedures. Both methods are somewhat insensitive and can most reliably be applied to samples containing large quantities of virus either naturaLly or as a result of concentration by ultracentrifugation. The formation of immune complexes by specific antibody in the immune aggregation procedures confirms the rotavirus diagnosis, but the number of particles per given microscope field is effectively reduced by the aggregation process. In the present communication, we describe use of an on-grid immunoelectron microscopic technique in which rotavirus particles are mounted onto microscope grids that were pre-coated with specific antibody. The technique is a modification of a method originalLy introduced by Derrick (1) for studies of plant viruses.

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Localization of Immune Complexes in the Basement Membrane of the Ductuli Efferentes of the Rhesus Monkey

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600001963 ◽

1980 ◽

Vol 38 ◽

pp. 456-457

Author(s):

D. Marsh

Keyword(s):

Basement Membrane ◽

Fluorescence Microscopy ◽

Rhesus Monkey ◽

Immune Complex ◽

Immune Complexes ◽

Vas Deferens ◽

Frozen Sections ◽

Efferent Ducts ◽

Complex Deposition ◽

Sperm Antibodies

As a result of vasectomy, spermatozoa are confined to the epididymis and vas deferens, where they degenerate, releasing antigens that enter the circulation or are engulfed by macrophages. Multiple antigens of the sperm can elicit production of autoantibodies; circulating anti-sperm antibodies are found in a large percentage of vasectomized men, indicating the immunogenicity of the sperm. The increased prevalence of macrophages in the liomen of the rhesus monkey testicular efferent ducts after vasectomy led to further study of this region. Frozen sections were used for evaluation of immunopathological status by fluorescence microscopy with fluorescein-conjugated antibody. Subsequent granular deposits of immune complexes were revealed by positive immunofluorescence staining for complement. The immune complex deposition in the basement membrane surrounding the efferent ducts implies that this region is involved in antigen leakage (Fig. 1).

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