F VIII Subunits: Purification and Antigenic Properties

1987 ◽  
Vol 58 (04) ◽  
pp. 1043-1048 ◽  
Author(s):  
Ole Nordfang ◽  
Mirella Ezban ◽  
Jan J Hansen
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Hemophilia A ◽  
Exchange Chromatography ◽  
Inhibition Assay ◽  
Cation Exchange Chromatography ◽  
Inhibitor Activity ◽  
Fviii Inhibitor ◽  
Antigenic Properties

SummaryFactor VIII-Light Chain (FVIII-LC) and FVIII-Heavy Chain (FVIII-HC) were purified from human plasma by the use of immunosorbents containing monoclonal antibodies or human inhibitor antibodies. FVIII-LC was subsequently isolated in essentially pure state by cation exchange chromatography. The preparations obtained contained 50 ng of protein for each unit of FVIII-LC antigen (FVIII-LC: Ag).Affinity purified FVIII-LC and FVIII-HC preparations containing less than 0.3% of the opposite subunit were added in FVIILC inhibition assay of hemophilia A inhibitor antibodies. FVIII-LC was able to fully block the inhibitor activity in 6 out of 7 hemophilia A plasmas and partially block the inhibitor activity of one plasma. FVIII-HC only blocked FVIILC inhibiting antibodies form the plasma that was not fully blocked by FVIII-LC. It is suggested that FVIII-LC can be used for immunotherapy of the patients whose FVIILC inhibiting antibodies are directed towards FVIII-LC.When FVIII-LC was coupled to Sepharose at a concentration of 4800 units of FVIII-LC: Ag per ml Sepharose, 0.2 ml of the immunosorbent was able to bind 900 Bethesda units from 100 ml hemophilia A inhibitor plasma. This opens the possibility to remove FVIII inhibitor antibodies from circulation by extracorporeal immunotherapy with FVIII-LC coupled to Sepharose.

IMMUNOASSAY FOR FACTOR VIII-HEAVY CHAIN. AN INDICATOR FOR IMMUNE COMPLEXES DURING HIGH DOSE FVIII INHIBITOR TREATMENT

1987 ◽  
Author(s):  
O Nordfang ◽  
M Ezban ◽  
J B Knudsen
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Test Sample ◽  
High Dose ◽  
Fviii Inhibitor ◽  
Normal Human

Specificity studies have shown that most hemophilia A inhibitor antibodies are directed towards the light chain of coagulation factor VIII (FVIII). Thus, conventional immunoassays for FVIII-antigen (FVIII:Ag) presumably have reactivity for FVIII-Light Chain (FVIII-LC) . Our sandwich FVIII :Ag assay has been shewn to be specific for only FVIII-LC. We have now developed a specific immunoassay for FVIII-Heavy Chain (FVIII-HC) . This has made it possible to investigate the FVIII-HC content in hemophilia A plasma, and to study the expression of FVIII-HC in culture medium frcm transfected cell lines.By adding purified FVIII-LC and FVIII-HC in coagulation inhibition assay, plasma frcm one of seven hemophilia A inhibitor patients was found to be reactive with both FVIII-LC and FVIII-HC. IgG frcm this plasma was used for a FVIII-HC specific inhibition radioimmunoassay. The polyspecific antibodies were coated to microplates with removable wells. The coated wells were incubated with test sample and with purified 125I-FVIII-HC. When normal human plasma pool contains 1 U/ml of FVIII-HC, the sensitivity of the assay was 0.004 U/ml.For normal plasma and plasma frcm non inhibitor hemophilia A patients, FVIII-HC measurements correlated with FVIII:C and FVIII-LC measurements. However, after FVIII injection hemophilia A inhibitor patients in high dose FVIII treatment showed a much higher FVIII-HC content (1-5 U/ml) than FVIII-LC and FVIII:C (< 0.05 U/ml). These patients have previously been shown to have antibodies towards FVIII-LC. Therefore the antigen measurements indicate that inhibitor patients in high dose FVIII treatment have FVIII/anti-FVIII-LC immune complexes. These circulating immune complexes may be the mediator of an antibody dependent immune tolerance, during the high dose FVIII treatment.

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

2019 ◽  
Vol 35 (5) ◽  
Author(s):  
Yumiko Masuda ◽  
Masashi Tsuda ◽  
Chie Hashikawa‐Muto ◽  
Yusuke Takahashi ◽  
Koichi Nonaka ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cation Exchange ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography

scholarly journals Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

1985 ◽  
Vol 101 (6) ◽  
pp. 2055-2062 ◽  
Author(s):  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Coated Vesicles ◽  
Cell Lysates ◽  
Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

scholarly journals The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies

2010 ◽  
Vol 19 (6) ◽  
pp. 1191-1204 ◽  
Author(s):  
Shuang Chen ◽  
Hollis Lau ◽  
Yan Brodsky ◽  
Gerd R. Kleemann ◽  
Ramil F. Latypov
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Cation Exchange ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2117-2122
Author(s):  
DP O'Brien ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Heavy Chains ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

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