PRODUCTION, ISOLATION AND CHARACTERIZATION OF IgY ANTIBODIES TO CANINE PARVOVIRUS
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Date
2016-12
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
The overall goal of this study was to develop practical, natural, and efficient
antimicrobials from egg. The existence of an IgG-like molecule in avian eggs,
referred to as IgY, has been well documented, and extensive research has been carried
out on its characterization, production and purification. Although it is the functional
equivalent of mammalian IgG, the major serum antibody found in mammals, IgY is
structurally different, and has been found to exhibit several important differences
when compared to mammalian antibodies, including its physico-chemical properties
and immunological capabilities. Recently, considerable research has focused on the
use of IgY as an alternative to mammalian antibodies in several applications,
including immune therapeutic applications, especially for the oral passive
immunization against various bacteria and viruses. Much research has also been
carried out on the use of IgY as a replacement for IgG in various immunodiagnostic
and immune affinity purification purposes. The use of IgY offers several advantages
over polyclonal antibodies produced in mammals, including providing a much more
hygienic, cost efficient, convenient, humane and plentiful source of antigen-specific
antibodies.
Chicken immunoglobulin Y (IgY) may provide a new modality in the therapy
of various infectious animal diseases. This study presents evidence of its efficacy for
canine parvovirus (CPV), which is a highly infectious, fatal viral disease in dogs.
Initially IgY antibody production and separation was standardized using BSA as
antigen. BSA 0.2 mg/ml was used to immunize three 21weeks old white leghorn
chicken. Additional booster doses were administered weekly intervals up to 6 weeks
following the first injection. Sera from immunized chicken were collected on 21st day
to confirm the immune response to BSA. Among three BSA immunized birds, one
birds did not show any response to BSA antigen. Initially BSA specific IgY
antibodies were separated from eggs laid by immunized chicken using Ammonium
sulphate method. The specificity of IgY antibody raised against BSA was determined
by agar gel immunodiffusion test. A clear precipitation line was observed between
BSA and anti BSA IgY antibody precipitated from egg yolk, which shows specificity
of immune response to BSA. Anti BSA IgY antibody levels were monitored up to 120
days by indirect ELISA and found that the titres were maintained up to 120 days of
immunization with the highest titre on 75th day.
The present study was conducted as a preliminary step to monitor the in vitro
efficacy of IgY as a passive immunotherapeutic agent to control Canine parvovirus
infection in dogs. Canine parvovirus vaccine containing 103viral particles/ml was
used as antigen to immunize 21 weeks old white Leghorn chicken. The eggs from
immunized chicken were collected from 1st day to 135th day. Sera from immunized
chicken were also collected on 21st day to confirm the immune response to canine
parvovirus. Presence of antibodies against canine parvovirus was checked using
hemagglutination inhibition assay (HI). The HI titres of the serum collected from
immunized hens was found to be 256 HI units. Water soluble fraction was isolated
from eggs collected after the immunization and estimated the protein content and the
maximum protein concentration was found (35.20 ± 1.32a mg/ml) in the water soluble
fraction collected from egg yolk on 75th day of post immunization. The IgY was
separated from the WSF by using Ammonium sulphate method, Sodium chloride
method, Sodium sulphate method and PEG method. Among the four methods used for
IgY separation, Sodium chloride method and Sodium sulphate methods were found to
yield high protein content (6.70 to 6.71mg/ml) compared with Ammonium sulphate
method and polyethylene glycol method.
IgY was purified using DEAE cellulose column chromatography. Highest
protein concentration was observed in the 4th and 5th fractions. The purity of the
immunoglobulin present in the 4th and 5th fractions of DEAE cellulose column elute
was checked by determining the molecular weight of the protein. A single protein
band showing molecular weight 180 KDa was recorded. The titre of the purified
canine parvovirus specific IgY antibody was found to be 2048 HI units. Anti canine
parvovirus IgY antibody levels were monitored up to 135 days by indirect ELISA. It
was found that the titres were be maintained up to 135th day with peak titre (1.186) on
day 75th after immunization. Stability of canine parvovirus IgY antibody was studied
by exposing to different temperatures and different pH using HI assay. The HI titre
gradually decreased when the temperature increased to 50ºC and above. At 25ºC and
37ºC the IgY antibody was found to be stable when exposed to 10, 20 and 30 minutes.
Purified IgY antibody when subjected to pH 7 for 8 hours its HI titre was 1024. The
HI titre gradually decreased when the pH decreased below 7 and also when pH
increased above 7.
The stability of purified anti canine parvovirus antibody was completely lost
when it is exposed to pH 3 in the presence of pepsin as reflected by complete loss of
HI activity. HI titre of the IgY antibody was found to be 32 to 64 when exposed to pH
4.0 and pH 5.0 respectively in the presence of pepsin. The results indicated that the
activity of immunoglobulins reduced in the presence of pepsin. Hence, there is a need
to protect IgY immunoglobulins against the action of pepsin in the stomach of
puppies. Present study was helpful for production of desired IgY antibodies, which
can be obtained in large quantities against specific pathogen (canine parvovirus).
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