Naslov
Assay of alpha and gamma interferon mRNA in chicken blood by competitive nucleic acid hybridization in microtitre plates

Autori
Novak, Renata ; Ester, Katja ; Ragland, W. L.

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
COST action 839 - Working group 2 founding meeting / Dren, Csaba ; ter Huurne Agnes - Budimpešta, 1999

Skup
COST action 839 - Working group 2 founding meeting

Mjesto i datum
Budimpešta, Mađarska, 27.05.1999. - 30.05.1999

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
interferon; chicken; mRNA; immune status

Sažetak
Nucleic acid probes for chicken alpha and gamma interferon were prepared from plasmids provided by Drs. M. J. Sekellick and J. Lowenthal, respectively. Probes were immobilized on nitrocellulose discs and placed in microtitre plate wells. Either known amounts of interferon DNA (for standard curves) or total RNA extracted from buffy coats with proteinase K- phenol procedure were allowed to bind to capture probe in hybridization medium. Wells were washed and biotinylated probe in excess of the capture probe allowed to bind to capture probe. An avidin-biotin-alkaline phosphatase detection system was used to measure the amount of labelled probe bound to the discs. Signal was inversely related to amount of competing nucleic acid. Standard curves had r2 from 0.97 to 0.99. Dabas White Leghorn SPF chickens (Charles River Farms, Budapest) were vaccinated at one day of age with inactivated Newcastle disease vaccine (Pestical, Pliva d.d., Zagreb), and vaccinated again at 3 wk. Heart blood (1 ml) was collected into heparin 0, 4, 24, 48, 72 and 168 hr later. Blood samples were collected from naďve chickens also. The samples were sedimented at 250 g for 30 min and buffy coat collected by pipette along with enough underlaying red cells to assure complete recovery of lymphocytes (total volume 250 ml). Fifty microliters were used for counting lymphocytes whilst total RNA was extracted from the remaining 200 ml. Proteins and DNA were removed by phenol-chloroform, and total RNA precipitated from the aqueous phase by addition of absolute ethanol and sodium acetate. Precipitated RNA was taken up in 100 ml DEPC-treated Tris-EDTA buffer, and 25 ml aliquots assayed, in duplicate. Levels of both interferons remained constant in naďve chickens. The mRNA for both alpha and gamma interferon increased rapidly in response to secondary vaccination. They approached maximal levels in four hours, peaked at 48 hours, and declined to normal levels in seven days. Curve fitting has indicated we should be able to sample chickens two hours after antigenic stimulation. Signal per volume of blood was concordant with signal per number of lymphocytes, indicating the test probably can be done simply with a constant volume of blood.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA