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 - Gödöllő. Hungary
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[Biochemistry] |
[Immunology] |
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.
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[Biochemistry] |
[Immunology] |
