[23] Immunochemical techniques for studying coagulation proteins

Jenny, Richard J.; Messier, Terri L.; Ouellette, L A; Church, William R.

doi:10.1016/0076-6879(93)22026-c

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…Mice were injected with the hybridoma cell lines and antibody‐rich ascites was produced. MAbs were purified from ascites using standard gel filtration and ion‐exchange chromatography techniques (Jenny et al , 1993). On average, 1 mg of MAb was recovered per 1 ml of mouse ascites.…”

Section: Methodsmentioning

confidence: 99%

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

Stephenson

Novák³

et al. 2002

Molecular Microbiology

121

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Section: Methodsmentioning

confidence: 99%

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

Stephenson

Novák³

et al. 2002

Molecular Microbiology

121

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“…Anti-FIX antibody from the hybridoma, BC2, was produced from ascites tumors in mice and purified by conventional means 20 and stored in buffer containing 0.02 mol/L tris, 0.15 mol/L NaCl, pH 7.40. An MLA Electra 800 Automatic Coagulation Timer (Medical Laboratory Automation, Inc) was used to measure aPTT.…”

Section: Bc2 Production and Assaymentioning

confidence: 99%

Antithrombotic Efficacy of a Novel Murine Antihuman Factor IX Antibody in Rats

Feuerstein¹,

Patel²,

Toomey³

et al. 1999

ATVB

Self Cite

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Abstract-A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg ⅐ kg Ϫ1 ⅐ h Ϫ1 ), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl 3 -patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U ⅐ kg Ϫ1 ⅐ min Ϫ1 ), whereas the latter rendered the blood incoagulable (aPTTϾ1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18Ϯ0.49 to 1.80Ϯ0.3 mg, PϽ0.001), increasing vessel patency time (from 14.9Ϯ0.9 minutes to 58.3Ϯ1.7 minutes, PϽ0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle-versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (PϽ0.01), extended vessel patency time (PϽ0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8Ϯ0.7 to 3.3Ϯ0.6 mL (PϽ0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.

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“…We utilized a common purification scheme to purify FIX from transfected HEK 293 cells that of immunoaffinity chromatography [6] followed by hydroxyapatite chromatography, as described by Camire et al. [3].…”

Section: Discussionmentioning

confidence: 99%

Galectin 3‐binding protein is a potential contaminant of recombinantly produced factor IX

Blostein¹,

Cuerquis²,

Galipeau³

2007

Haemophilia

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Haemophilia B, or factor IX (FIX) deficiency, represents 15% of the hereditary haemophilias. The serious morbidity from the transmission of infectious agents in plasma-derived material has mandated a need for the production of recombinant product. The rate-limiting step for the production of recombinant FIX is gamma-carboxylation, a post-translational modification carried out only in mammalian cells. To test the carboxylation efficiency of recombinantly produced FIX in vitro and to improve the isolation of the pure active product, we produced FIX in a transfected human cell line (293 human embryonic kidney cells) and isolated material by immunoaffinity chromatography followed by hydroxyapatite chromatography. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified FIX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry (MS) sequencing revealed this protein to be galectin-3-binding protein (G3BP). The above results raise an important note of caution regarding the production of recombinant FIX and, indeed, other proteins produced recombinantly in mammalian cells.

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[23] Immunochemical techniques for studying coagulation proteins

Cited by 6 publications

References 10 publications

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

Antithrombotic Efficacy of a Novel Murine Antihuman Factor IX Antibody in Rats

Galectin 3‐binding protein is a potential contaminant of recombinantly produced factor IX

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