Applied biotechnology for production of immunoglobulin Y specific to hepatitis A virus

Abstract

A new protocol for producing polyclonal antibody against hepatitis A virus (HAV) is described. Twenty hens were immunized three times with a commercial HAV vaccine and HAV from a cell culture with three types of adjuvants: CpG oligodeoxynucleotides (CpG-ODN), incomplete Freund's adjuvant and an alum adjuvant. In each of the last two booster inoculations, blood from the birds was collected and tested for HAV antibodies. Egg yolk was separated from egg white and immunoglobulin Y (IgY) antibody was then purified by polyethylene glycol 6000. The mean yield of total protein in yolk was 22.62 mg/mL. Specific activity of the antibody was tested using commercial ELISA, Western blotting, and in vitro neutralization assay demonstrating that anti-HAV IgY bound specifically. After the first immunization, birds immunized with HAV from cell culture plus incomplete Freund's adjuvant with/without CpG-ODN showed highest levels of anti-HAV IgY in serum (p<0.05). Viral combination with CpG-ODN resulted in early response of anti-HAV serum in hens, reflecting the amount of IgY transferred to the egg yolk (p<0.05). The results suggest that egg yolk may be a large scale source of specific antibodies against hepatitis A virus. Further applications of this method have yet to be tested.

Production and Characterization of IgY against Canine IgG: Prospect of a New Tool for the Immunodiagnostic of Canine Diseases

“…The FCA in the first immunization in combination with FIA in subsequent inoculations is preferable to prevent the adverse effects and yet induce high levels of IgY (Kapoor et al 2000;Li et al 2006;Chalghoumi et al 2009). Even though not all yolk contained IgY could be isolated by polyethylene glycol (PEG) (Stalberg and Larsson 2001), the results displayed a good level of IgY production, similar to the levels described with PEG precipitation in other studies (Pauly et al 2009;de Paula et al 2011). However, more efficient methods of purification have been reported (Akita and Nakai 1993;Stalberg and Larsson 2001;Bizanov et al 2004), and the effectiveness of PEG precipitation is debatable.…”

“…In this study, there was no decline in egg laying capacity after the immunization process, which remained stable during four months, with an average of one egg per day. This has been described for chickens with respect to antibody production, since the chicken laying capacity is usually slightly affected by the antigen injection (Schade et al 2005;Witkowski et al 2009;de Paula et al 2011;Matheis and Schade 2011). However, some studies have reported a reduction, or even an interruption in egg laying capacity for the chickens injected with toxic antigens (Schniering et al 1996;Schade et al 2005;Levesque et al 2007), an occurrence not evident in the present study.…”

“…Furthermore, the Freund´s complete adjuvant (FCA) is directly responsible for the majority of granulomatous lesions in inoculation sites (Bollen and Hau 1999), only in the first inoculation, replaced by Freund´s incomplete adjuvant (FIA), which causes a less adverse effect in subsequent immunizations. The absence of adverse effects has been described in many studies with chickens as antibody donors, even with different types of immunizing antigens (Levesque et al 2007;Witkowski et al 2009;de Paula et al 2011). Contrastingly with the mammals, adverse effects and lesions are usually described in the immunization process (Leenaars et al 1999).…”

“…This was not observed in this study with FCA in association with FIA. In order to avoid the reduced egg laying capacity due to the immunization process, de Paula et al (2011) immunized the chickens before the start of the laying period, the first inoculation being administered on the fourteenth day of life and the last, thirty days prior to the start of laying, and no decrease in laying capacity, or side effects due to the immunization process were detected (de Paula et al 2011). In the present study, however, the first immunization was at twenty weeks of age, near the start of the laying period (approximately twenty-two weeks of age) and the remaining two weeks during the laying period, with laying capacity decrease neither during nor after the immunization process.…”

An immunoenzymatic assay for the diagnosis of hepatitis A utilising immunoglobulin Y

“…The preparation and characterisation of chicken anti-HAV IgY was performed in a previous study (de Paula et al 2010) according to a method established in our laboratory. The experimental protocol was reviewed and approved by the Ethical Commission for the Use of Animals (University Centre Serra dos �rg�os, proSerra dos �rg�os, pro-, protocol 0272/2009).…”