Year 2017, Volume 41 , Issue 6, Pages 857 - 867 2017-12-01

Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII

ARTEM TYKHOMYROV [1] , VICTOR NEDZVETSKY [2] , SERGIY SHEMET [3] , CAN ALİ AĞCA [4]


Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potential biomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund's adjuvant or with antigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbent assay (ELISA). IgG purification was performed by affine chromatography on protein A-sepharose. Immune sera and IgG were tested by immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercial plasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed for immunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with PAAG-trapped antigen induced more potent immune response compared to the standard immunization procedure with Freund's adjuvant. The lowest working amount of immune IgG, measured by ELISA, was ~50 ng. Immunoblotting demonstrated that anti-BDDrFVIII antibodies effectively recognize the whole FVIII molecule (320 kDa), as well as different truncated polypeptides thereof, and are suitable for immunocytochemical analysis of FVIII-producing cells. An optimized procedure for the production of polyclonal antibodies against FVIII with the use of PAAG-immobilized BDDrFVIII (moroctocog alpha) was proposed and successfully validated. The produced antibodies are suitable for detecting and measuring FVIII-related antigens and may have various biomedical applications.
B-Domain deleted recombinant factor VIII (moroctocog alpha), antibody production, plasma-derived FVIII products, platelets, hemophilia A
Journal Section Articles
Authors

Author: ARTEM TYKHOMYROV

Author: VICTOR NEDZVETSKY

Author: SERGIY SHEMET

Author: CAN ALİ AĞCA

Dates

Publication Date : December 1, 2017

Bibtex @ { tbtkbiology386328, journal = {Turkish Journal of Biology}, issn = {1300-0152}, eissn = {1303-6092}, address = {}, publisher = {TUBITAK}, year = {2017}, volume = {41}, pages = {857 - 867}, doi = {}, title = {Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII}, key = {cite}, author = {TYKHOMYROV, ARTEM and NEDZVETSKY, VICTOR and SHEMET, SERGIY and AĞCA, CAN ALİ} }
APA TYKHOMYROV, A , NEDZVETSKY, V , SHEMET, S , AĞCA, C . (2017). Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII. Turkish Journal of Biology , 41 (6) , 857-867 . Retrieved from https://dergipark.org.tr/en/pub/tbtkbiology/issue/34855/386328
MLA TYKHOMYROV, A , NEDZVETSKY, V , SHEMET, S , AĞCA, C . "Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII". Turkish Journal of Biology 41 (2017 ): 857-867 <https://dergipark.org.tr/en/pub/tbtkbiology/issue/34855/386328>
Chicago TYKHOMYROV, A , NEDZVETSKY, V , SHEMET, S , AĞCA, C . "Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII". Turkish Journal of Biology 41 (2017 ): 857-867
RIS TY - JOUR T1 - Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII AU - ARTEM TYKHOMYROV , VICTOR NEDZVETSKY , SERGIY SHEMET , CAN ALİ AĞCA Y1 - 2017 PY - 2017 N1 - DO - T2 - Turkish Journal of Biology JF - Journal JO - JOR SP - 857 EP - 867 VL - 41 IS - 6 SN - 1300-0152-1303-6092 M3 - UR - Y2 - 2019 ER -
EndNote %0 Turkish Journal of Biology Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII %A ARTEM TYKHOMYROV , VICTOR NEDZVETSKY , SERGIY SHEMET , CAN ALİ AĞCA %T Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII %D 2017 %J Turkish Journal of Biology %P 1300-0152-1303-6092 %V 41 %N 6 %R %U
ISNAD TYKHOMYROV, ARTEM , NEDZVETSKY, VICTOR , SHEMET, SERGIY , AĞCA, CAN ALİ . "Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII". Turkish Journal of Biology 41 / 6 (December 2017): 857-867 .
AMA TYKHOMYROV A , NEDZVETSKY V , SHEMET S , AĞCA C . Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII. Turkish Journal of Biology. 2017; 41(6): 857-867.
Vancouver TYKHOMYROV A , NEDZVETSKY V , SHEMET S , AĞCA C . Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII. Turkish Journal of Biology. 2017; 41(6): 867-857.