Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization.
نویسندگان
چکیده
Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light-chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.
منابع مشابه
Bacterial Expression and Purification of C1C2 Domain of Human Factor VIII
With the aim of the production of human factor VIII antigen and its corresponding antibody an epitope coding fragment of the light-chain of hFVIII, fused to a His6-tag, was isolated and over-expressed in Escherichia coli. The over-expressed hFVIII-epitope containing peptide was confirmed by its reaction with a rabbit serum directed against native hFVIII as well as antiHis6-tag antibody. An expr...
متن کاملProduction and Characterization of Murine Monoclonal Antibodies Recognizing Conformational and Linear Epitopes Localized on Human IgA2 Molecules
Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies (MAbs) specific for conserved conformat...
متن کاملA Sensitive Neutralization Assay for Influenza C Viruses Based on the Acetylesterase Activity HEF Glycoprotein
Influenza C virus possesses specific neuraminate-O-acetylesterase as a receptor-destroying function. This enzymatic activity of the viral glycoprotein HEF (Hemagglutinin, esterase activity and fusion factor) can be visualized in situ by the use of distinct color substrates. Hereby the localization, as well as the quantity of synthesized HEF protein is detectable. We further developed the estera...
متن کاملIdentification of Two Epitopes on the Outer Surface Protein A of the Lyme Disease Spirochete Borrelia burgdorferi
A murine IgM monoclonal antibody (MA-2C6) with κ-light chains directed against an antigenic determinant of outer surface protein A (OspA) of the Lyme disease spirochete, Borreliaburgdorferi, is produced. This antibody could bind specifically to OspA antigen of several isolates of B. burgdorferi, but not to the non-Lyme disease bacteria such as T. pallidum and B. hermsii. Antibody MA-2C6 was pur...
متن کاملEvaluation of inhibitor antibodies in haemophilia A population
Background: Inhibitory antibody to exogenous Factor VIII (FVIII) is a major complication of hemophilia treatment. This study was conducted to determine the prevalence of inhibitor antibody directed against FVIII.Methods: From May 2010 to May 2011, 52 patients with severe hemophilia A admitted in Amirkola Children’s Hospital were evaluated. Those who had abnormal mixing study, antibody against F...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Blood
دوره 74 5 شماره
صفحات -
تاریخ انتشار 1989