ELISA kit for IBD manufactured by IDEXX laboratory, Inc. West Brook, Maine 04092, USA was used in this study.
Rearing and vaccination of chickens: Total 100 chickens of 1 day old were collected from a commercial hatchery and they were divided into two groups named as treatment group (flock-1) and control group (flock2). Each Flock consists of 50 chickens. All the chickens were the progeny from the parent stock that had the history of vaccination. Both flocks were supplied with recommended standard feed. These chickens were reared for 6 weeks maintaining all the hygienic measures in a well-ventilated poultry house AHRD, BLRI, Savar, Dhaka. At the age of days 16 and 21 chickens of treatment group were vaccinated with Gumboro live vaccine while chickens of control group kept without vaccination.
Sample collection and preparation of sera: During the experiment, blood samples were collected on day 1, 8 & 16 before vaccination from both treatment and control groups. At the age of days 28 blood samples were again collected at 28 days from both treatment and control groups. The presence or absence of antibody to IBD was determined by relating the (A650) value of the unknown to the Positive Control mean. The positive control was standardized and represented significant antibody levels to IBD in chicken serum. The relative level of antibody in the unknown was determined by calculating the sample to positive (S/P) ratio. Endpoint titles were calculated using the equation described in. the calculation section. One ml or 2.5 ml sterile disposable syringes were used to collect blood samples aseptically directly from the heart or wing vein. Soon after the collection of blood, the syringes with blood were kept at 4-8 °C overnight for clotting of blood in one side of the syringe. Clotted blood was removed carefully with sterile needle and sera were transferred into sterilized eppendorf tubes. Separate needles were used for each syringe. The sera in eppendorf tubes were subjected to centrifugation at 1000 rpm for 10 min for clarification. Finally, the clarified sera were stored at –20 °C until tested. This serum was used as a test sample for the detection of IBDV specific antibody level in the chicken using ELISA.
ELISA test for IBD: ELISA at a single dilution (1: 500) of serum was applied for the detection of the IBDV specific antibody. ELISA kit for IBD manufactured by IDEXX laboratory, Inc. West brook, Maine 04092, USA was used in this study. Serum was diluted 500 folds (1: 500) with sample diluent, provided in the ELISA kit for IBD, prior to assay (i.e. by diluting 1 µl of the sample with 500 µl of sample diluent). In a 96 well plate, pre-coated with IBDV antigen, the wells with the numbers A1 and A2 were selected and used for the negative control serum and well A3 and A4 for positive control serum. The remaining 92 wells were used for 46 samples (one sample in two well). 100 µl of negative control serum and 100 µl of positive control serum (without dilution) were taken in to each selected wells A1, A2 and A3, A4 respectively. Then 100 µl of diluted 46 test samples was taken in to appropriate wells. The plate was incubated at room temperature for 30 minutes and then washed with deionized distilled water four times and each time 200 µl deionized distilled water in to each well. 100 µl of conjugate was taken in to each well and was incubated at room temperature for 30 minutes. The plate was washed again with deionized distilled water four times and each time 200 µl deionized distilled water in to each well. 100 µl of substrate was added in to each well and was kept for 15 minutes. In each well, 100 µl of stopping solution was added. Reading was taken by ELISA reader, using 650 nm filters. The rest of the test samples were tested following the same procedure (IDEXXUSA).
Calculation: The presence or absence of antibody IBDV was determined by relating the A (650) value unknown to the positive control mean. The positive control been standardized and represents significant antibody levels IBD in chicken serum. The relative level of antibody unknown can be determined by calculating the sample to (S/P) ratio. The equation of calculation provided in ELISA kit was used the calculation of antibody titer.
a) Negative Control Mean (NCX) = Well A 1(A650) + Well A2 (A650) / 2
b) Positive Control Mean (PCX) = Well A3 (A650) + Well A4 (A650) /2
c) S/P Ratio = (SAMPLE MEAN – NCX) / (PCX - NCX)
d) Titer relates S/P at a 1: 500 dilution to an end point titer: Log Titer = 1.09(Log S/P) + 3.36
Interpretation of results: Serum samples with S/P ratios of less than or equal to 0.2 should be considered negative. S/P ratios greater than 0.2 (titers greater than 396) should be considered positive and indicates either vaccination or exposure to IBDV (IDEXX laboratory, Inc. West brook, Maine 04092, USA.