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6th Internet World Congress for Biomedical Sciences

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ELISA for the measurement of IgY concentrations of hen’s and quail’s serum and yolk

Sándor Losonczy(1), Csaba Szabó(2), Zsuzsnna Kiss(3), László Bárdos(4)
(1)(2)(3)(4)Animal Physiology and Health - Gödöllő. Hungary

[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS & DISCUSSION] [FIGURES] [CONCLUSIONS] [ACKNOWLEDGEMENTS] [BIBLIOGRAPHY] [Discussion Board]
INTRODUCTION Previous: ALKALINE PHOSPHATASE ISOENZYMES IN THE SERUM AND BRONCHOALVEOLAR LAVAGE OF PATIENTS WITH BRONCOPULMONARY PATHOLOGY. RESULTS & DISCUSSION
[Biochemistry]
Next: Differences In The Catalytic Effects Of Related Metallo <font face="Symbol">b</font>- Lactamases Metal Ions On The <font face="Symbol">b</font>- Lactam Antibiotics Methanolysis		 [Immunology]
Next: Monoclonal antibody reacting with human middle ear cholesteatomas

MATERIAL & METHODS

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).

Discussion Board
Discussion Board

Any Comment to this presentation?

[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS & DISCUSSION] [FIGURES] [CONCLUSIONS] [ACKNOWLEDGEMENTS] [BIBLIOGRAPHY] [Discussion Board]
INTRODUCTION Previous: ALKALINE PHOSPHATASE ISOENZYMES IN THE SERUM AND BRONCHOALVEOLAR LAVAGE OF PATIENTS WITH BRONCOPULMONARY PATHOLOGY. RESULTS & DISCUSSION
[Biochemistry]
Next: Differences In The Catalytic Effects Of Related Metallo <font face="Symbol">b</font>- Lactamases Metal Ions On The <font face="Symbol">b</font>- Lactam Antibiotics Methanolysis		 [Immunology]
Next: Monoclonal antibody reacting with human middle ear cholesteatomas
Sándor Losonczy, Csaba Szabó, Zsuzsnna Kiss, László Bárdos
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Last update: 13/01/00