Simplified preparation of rabbit fab fragments

Coulter, A.R.; Harris, R.D.

doi:10.1016/0022-1759(83)90031-5

Cited by 63 publications

(24 citation statements)

References 12 publications

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“…A protein G column was used to prepare solutions containing 250 μM IgG purified from rabbit immune sera raised to either PNAG CP8, CP5 or from NRS. These preparations were dialyzed against MEM or PBS and added as titrants into calorimetry sample cells containing 5 μM of mouse mAb to CP8; human mAb to PNAG; Fab fragments purified from the human mAb to PNAG, prepared using papain conjugated to agarose beads (Sigma-Aldrich) as described (40); or MEM buffer. The heat of the reaction (microcalories per second) per injection of 10-μl volumes of titrant was determined by integration of the peak areas using Origin Version 5.0 software (OriginLab) with background dilution heats subtracted.…”

Section: Figurementioning

confidence: 99%

Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

Skurnik¹,

Merighi²,

Grout³

et al. 2010

J. Clin. Invest.

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New prophylactic approaches are needed to control infection with the Gram-positive bacterium Staphylococcus aureus, which is a major cause of nosocomial and community-acquired infections. To develop these, greater understanding of protective immunity against S. aureus infection is needed. Human immunity to extracellular Gram-positive bacterial pathogens is primarily mediated by opsonic killing (OPK) via antibodies specific for surface polysaccharides. S. aureus expresses two such antigens, capsular polysaccharide (CP) and poly-N-acetyl glucosamine (PNAG). Here, we have shown that immunization-induced polyclonal animal antisera and monoclonal antibodies specific for either CP or PNAG antigens have excellent in vitro OPK activity in human blood but that when mixed together they show potent interference in OPK activity. In addition, reductions in antibody binding to the bacterial surface, complement deposition, and passive protection were seen in two mouse models of S. aureus infection. Electron microscopy, isothermal calorimetry, and surface plasmon resonance indicated that antibodies to CP and PNAG bound together via an apparent idiotype-anti-idiotype interaction. This interaction was also found in sera from humans with S. aureus bacteremia. These findings suggest that the lack of effective immunity to S. aureus infections in humans could be due, in part, to interference in OPK when antibodies to CP and PNAG antigens are both present. This information could be used to better design S. aureus vaccine components.

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Section: Figurementioning

confidence: 99%

Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

Skurnik¹,

Merighi²,

Grout³

et al. 2010

J. Clin. Invest.

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“…Anti-LC3 antibody (SK2-6) was purified on an immobilized GST-LC3-glutathioneSepharose column. Preparation of Fab fragment was performed as previously described (Coulter and Harris, 1983). Immobilized Papain (PIERCE) was used to digest purified anti-LC3 antibody.…”

Section: Antibodiesmentioning

confidence: 99%

Dynein-dependent Movement of Autophagosomes Mediates Efficient Encounters with Lysosomes

Kimura

Noda

Yoshimori

2008

Cell Struct. Funct.

374

289

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ABSTRACT. Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule-and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.

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“…Antibodies were eluted in 0.2 M glycine, pH 2.5, and dialyzed in three changes of PBS. Monovalent Fab fragments of rabbit anti-Syn7#2 IgG were prepared according to Coulter and Harris (1983) and then affinity purified like the intact antibodies. Antibodies specific to Vamp 8 were purified similarly.…”

Section: Antibodiesmentioning

confidence: 99%

Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

Mullock

Smith

Ihrke

et al. 2000

MBoC

150

138

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Protein traffic from the cell surface or thetrans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, α and γ SNAP, and a Rab GTPase based on inhibition by Rab GDI. InSaccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

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Simplified preparation of rabbit fab fragments

Cited by 63 publications

References 12 publications

Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

Dynein-dependent Movement of Autophagosomes Mediates Efficient Encounters with Lysosomes

Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

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