Production and Purification of Rabbit’s Polyclonal Antibody Against Factor VIII

Sohrabi, Simin; Akbarzadeh, Azim; Norouzian, Dariush; Farhangi, Ali Akbar; Mortazavi, Mohammad Seddiq; Mehrabi, Mohammad Reza; Chiani, Mohsen; Saffari, Zahra; Ghassemi, Soheil

doi:10.1007/s12291-011-0142-2

Cited by 3 publications

(4 citation statements)

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“…Development and uses of polyclonal antibodies in rabbits and hens have been widely described before with inherent variations depending on the nature of the antigen and the dose and route of administration [ 42 , 43 ]. Although only two animals (a pair of hens or rabbits) were used to produce antibodies against each antigen, the immune response of each animal was similar as demonstrated by Western blot analysis.…”

Section: Discussionmentioning

confidence: 99%

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens

et al. 2017

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BackgroundDetection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Methodology/Principal findingsPolyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody) and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.ConclusionsPolyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

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Section: Discussionmentioning

confidence: 99%

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens

et al. 2017

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“…Previously, several research groups described the procedure of anti-FVIII-antibodies generation. For example, Sohrabi et al (2011) used Al(OH) 3 as an adjuvant for rabbit immunization with FVIII. The authors shot 4000 µg of FVIII per each injection and obtained antibody titers between 1/1000 and 1/10,000, lower than the levels obtained with the use of much smaller amounts of antigen immobilized in gel, as described above.…”

Section: Discussionmentioning

confidence: 99%

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

Tykhomyrov¹,

Nedzvetsky²,

Shemet³

et al. 2017

Turk J Biol

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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.

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“…For instance, ammonium sulfate and caprylic acid precipitation, hydroxylapatitechromatography, DEAE ion exchange chromatography and immunoaffinity chromatography, to name a few. The produced antibody is dependent on employing techniques like Western blotting and ELISA [10][11][12]. Recombinant human EPO hormone that has been started to be marketed and used in medical care is purified by several methods that require antibody.…”

Section: Discussionmentioning

confidence: 99%

“…Their results have shown that all of these techniques can purify antigen-specific antibodies of rabbit sera. Sohrabi and colleagues have also used different techniques such as ionexchange, affinity chromatography, ammonium sulfate precipitation of IgG or caprylic acid methods to purify rabbit's polyclonal antibody against recombinant coagulating factor VIII [12,13]. In this research, after polyclonal antibody production in rabbit and purification by various methods, Western blot and ELISA tests were performed to confirm the presence and the levels anti-EPO antibody.…”

Section: Introductionmentioning

confidence: 99%

Purification of human anti-erythropoietin polyclonal antibodies by precipitation and chromatography as an optimized method with potential application in vaccine studies

Maboudi

Yaghoubi

Hadadian

2016

vacres

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Introduction: Polyclonal antibodies are required to be affinity purified. Improved purification methods of polyclonal antibody provide an opportunity to pick the most purified immunoglobulins as a primary or secondary antibody in immunoassays that are included in many vaccine studies. Two common techniques for purifying proteins is salt precipitation and chromatography purification. Our work focuses on purification of polyclonal antibodies against recombinant human erythropoietin (EPO) antigen using these techniques. Methods: A polyclonal antibody was produced by antigen injection with Freund's adjuvant into female albino rabbits. After separation of immunoglobulins using caprylic acid and ammonium sulfate precipitation, selected samples were analyzed by ion exchange chromatography for separation of polyclonal antibody from albumin. The purified proteins were analyzed by SDS-PAGE and antibody was detected by Western Blot analysis and ELISA. Results of immunodiffusion test detected polyclonal antibody production in rabbits. Results: Caprylic acid precipitation was shown to be a more effective purification method than ammonium sulfate. Analysis of protein by spectrophotometer showed 97.6% purity by caprylic acid and 77% purity by ammonium sulfate method. Western Blot and ELISA tests confirmed the presence of antibody against EPO. Conclusion: These findings suggest that caprylic acid can be used as a quality control method in a production facility with minimal cost. On the other hand, ion exchange chromatography is the most common purification method for proteins. Therefore, combination of these techniques may effectively reduce contaminations in antibody purification procedures which may positively affect the interpretations of vaccine efficacies.

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Production and Purification of Rabbit’s Polyclonal Antibody Against Factor VIII

Cited by 3 publications

References 19 publications

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens

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

Purification of human anti-erythropoietin polyclonal antibodies by precipitation and chromatography as an optimized method with potential application in vaccine studies

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