Poster
# 47
Poster |
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6th Internet World Congress for Biomedical Sciences |
Sándor Losonczy(1), Csaba Szabó(2), Zsuzsnna Kiss(3), László Bárdos(4)
(1)(2)(3)(4)Animal Physiology and Health - Gödöllõ. Hungary
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[Biochemistry] |
[Immunology] |
The main immunoglobulin fraction of fowls is called IgY, in order to distinguish it from the mammalian IgG. There are some crucial points of difference between IgG and IgY. Avian IgY is more resistant to heat, low pH and/or ion strength of environment than IgG. IgY has no reactivity to mammalian auto-antibodies, Fc-receptors and red blood cells fig.1. In assays, these cross-reactions can be eliminated using avian-originated antibodies (ABs). Serum IgY is identical to yolk AB, as it has been proven in comparative studies. The isolation and purification for further different uses of AB has great potentials. The extraction and purification of immunoglobulin fraction (IgY) from egg yolk of domestic hens and Japanese quail hens is described earlier (4,5,8).
According to the literature (1,4) and our experience (5), advantages of the IgY extracted from egg yolk are the following: 1. birds (chicken, quail etc.) produce ABs against highly conservative mammalian protein, too; 2. the amount of antigen needed for immune response is very low; 3. collecting and storing eggs are non-invasive and not expensive in contrast to bleeding animals and deep frozen storage of sera; 4. AB isolation and purification of yolks by combined methods are quick and inexpensive, 5. the avian AB is acid- and heat resistant, for these reasons it might be used for oral immunotherapy as well.
With regard to animal welfare (i.e. blood sampling), ABs, purified from egg yolks of immunized chickens can play an increasing role as an alternative to classic (mammalian) ABs. They can be used in immunodiagnostics (1,6) and therapy as well (9,10).
In the field of immunology the Japanese quail as a model animal could have an increasing importance. But it should be mentioned that specific anti quail antibodies (ABs) are not aviable on the market, although the production of AB in Japanese quail has special benefits. The quail’s advantages are smaller body weigh, less food intake and smaller room in the batteries. Sexual maturity of the female quail is 35-40 days in contrast with that of the domestic hen, which starts laying at the age of 130-150 days.
The development of a sensitive ELISA for the measurement of quail IgY (QIgY) was the main purpose of our study.
Preparation of quail’s IgY
The QIgY was prepared by combined water dilution (WD) and dextrane sulfate (DS) precipitation, based on the methods developed by Kokko et al.(4). The purification was followed by gel filtration and ion-exchange chromatography (5,8).
Production of antibody
Two New Zealand white female rabbits were immunized. Animals were injected intradermaly into their inrerscapular region by 200 microgram antigen (QIgY) in 250 microlitre complete Freund adjuvant (Sigma, St. Louis). The booster immunizations were administrated with 100 g antigen dissolved in incomplete Freund adjuvant three times with two weeks intervals. The blood samples were taken from the marginal ear vein into heparinized tubes.
The AB (anti-quail-IgY = aQIgY) content of sera was tested against QIgY as antigen by immundiffusion technique (7). The samples containing antibodies were collected and the pool was refrigerated.
Immunoaffinity chromatography
For the purification of AB a HIgY-Sepharose 4B resign was prepared by using CN-Br Sepharosre 4B (Pharmacia Biotech, Uppsala). The prepared immunoaffiny column (vol.: 10 mL) was used and treated according to the manual of Pharmacia Biotech.
ELISA The purified antibody was conjugated with horseradish peroxidase (HRP)(Sigma, St. Louis) by two step glutaraldehyde reaction (2).
The ELISA technique was carried out on standard 96-well flat bottom microtiter plates (Greiner, Germany). Both HIgY and QIgY were used as capture antigens. Wells were blocked for nonspecific binding with normal rabbit serum. The labeled AB (aHQIgY-HRP) was used in 1:20 000 to 1: 40 000 dilution. The coating (0.05 M carbonate pH 9.6), dilution and washing (PBS) buffers, the incubation times, and the color developing techniques were the same as the usual direct ELISA, which uses HRP as enzyme conjugate (10).
The suitable AB was produced in rabbits. As a result of the immunization protocol, high titre of anti-QIgY was produced in the rabbits. In the immundiffusion tests an unambiguous cross-reaction was detectable between anti-QIgY and HIgY. Both quail IgY (QIgY) and hen IgY (HIgY) were precipitated by this developed AB. For this reason it was marked as anti-hen-quail-IgY (a-HQIgY). Therefore, it is possible that these (quail and hen) IgYs contain homologue sequences, having good immunogen character.
The pooled rabbit serum (6.1 mL) containing anti IgY AB and 5 mL NaHCO3 buffer (pH 8.3) was taken up to the CN-Br-Sepharose 4B-HIgY immunaffinity column. After an overnight incubation (20 oC) the column was eluted by 0.1 M Glycine-HCl buffer (pH 2.5). The effluent was monitored at 280 nm and the fractions having higher density than 0.5 OD280 were collected into tubes containing 0.5 mL 1 M Tris buffer (pH 8.0) (3). The specific antibody content of these pooled fraction (12 mL) was 7.05 mg (i.e.:1.16 mg/mL)fig. 2.
The purified AB was conjugated with horseradish peroxidase (aHQIgY-HRP) and a sensitive direct ELISA was developed, based on this labeled AB.
The optimal concentrations were determined for the ELISA in the case of both capture antigens (HIgY and QIgY). The investigations were carried out with 16-2000 ng antigens. The antibody (aHQIgY-HRP) dilutions were 20, 30 and 40 thousand times respectively. It was found that there was a linear binding region (LBR) in the range of 125-250 ng/well antigen concentrations in both cases of capture antigens )fig. 3.
The quality control parameters of the assay using five parallel samples were the following: sensitivity 1 ng, range of measurement 1-20 ng, reproducibility > 95%.
The IgY concentrations of quail’s serum and diluted egg yolk samples were compared with the standard QIgY dilutions by ELISA titration )fig. 4.The dilutions ranged from 1:5000 up to 1: 160 000. The character of lines indicates that the antigen binding potential of aHQIgY-HRP conjugate represents the same nature in our ELISA system, apart from the source of antigen.
The prepared aHQIgY AB, which was used in this developed ELISA method was suitable for the measurement of total and specific IgY concentration in both domestic hen (Gallus domesticus) and Japanese quail (Coturnix coturnix japonica).
As a result of our experiments it is very likely that there are identical sequences of IgYs of both species. This part of IgY has a good antigen character at the same time. Probably, this phenomenon has an occurrence in other Galliform species, too. Further investigations will be carried out in this field.
The authors thank Miss K. Karchesz for her excellent assistance. This work was supported by Hungarian grant FKFP No 0473.
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[Biochemistry] |
[Immunology] |
