Filter paper sampling of blood for the detection of antibodies to Infectious Bursal Disease

Aim: The present study was designed to investigate the feasibility of the filter paper sampling of blood for the detection of IBDV antibodies in chicken sera using a commercial ELISA kit. Materials and Methods: Optimum dilution for filter paper extracts that would give a result equivalent to that of the corresponding serum sample in a commercial ELISA was determined. Correlation between the filter paper extracts based ELISA and conventional serum based ELISA was also examined and the reproducibility of the filter paper extracts based ELISA was tested. Results: A very good correlation (r = 0.9; n = 15; p<0.01) was observed between the results obtained with filter paper extracts based ELISA and serum based ELISA. The antibody titre determined by two systems were very close and did not exceed more than a two-fold dilution with the exception of samples having very low antibody levels, where relatively higher background reaction was observed with filter paper extracts based ELISA. The filter paper extracts based ELISA appeared to be quite reproducible with a coefficient of variation less than 10%. Conclusion: Filter paper based ELISA could be a useful alternative to serum dependant ELISA assays for sero-profiling chicken flocks for IBDV antibodies.


Introduction
lization of vaccine virus by the maternal antibodies present in chicks.Therefore, maternal antibody levels Poultry industry is a rising sector in Bangladesh in chicks should be determined before vaccination.but the development of this sector is often interrupted The enzyme-linked immunosorbent assay (ELISA) is by frequent outbreaks of a number of emerging and commonly used and considered to be the easiest method recurrent diseases.One of the most important diseases for measuring maternal antibody levels against IBDV of commercial poultry is infectious bursal disease [8].The commercial IBDV ELISA kits are available (IBD).It is an acute, highly contagious immunothrough an ELISA assay for detecting antibodies to suppressive viral infection of chickens [1,2,3].Among IBDV also has recently been developed locally.poultry diseases in Bangladesh, IBD is the number one Conventionally, serum obtained from blood is killer causing up to 80% mortality in field outbreaks used in ELISA, but it is often difficult to collect [4,5,6,7].
sufficient blood from very young chicks without Vaccination is the only means for the prevention killing the birds.As an alternative, a technique has of IBD but vaccination failure is quite common due to been developed for sampling blood on filter paper, various factors such maintenance, storage or inadequate where a small drop of blood is soaked on a filter paper immune response following vaccination or neutra-strip, which can be later extracted with PBS and used were extracted with 300µl PBS for 60 minutes at room in ELISA [9] manufacturer.The frozen sera samples were thawed.However, there is no study about filter paper Five hundred fold (1:500) dilution of sera samples based ELISA for the detection of antibody to IBDV were made in sample diluent.The Filter paper extracts using commercial kits.Therefore, the present study were used immediately after extraction.Filter paper was designed to investigate the feasibility of the filter extracts were diluted either serially in two-fold paper sampling of blood for the detection of IBDV dilutions or at an appropriate fixed dilution (1: 5).This antibodies in chicken sera using a commercial ELISA kit.
was done in the same laboratory, BAU.The optical

Materials and Methods
density (OD) or absorbance values were determined at 650 micron using an ELISA reader (SPECTERA max Sampling and sera collection: The study was 340 pc, Molecular Devices Inc., USA) at the Surgery performed during the period of January -June, 2004.
and Obstetrics Department of Bangladesh Agricultural Blood samples were obtained from chickens, which University. were reared in the Department of Pathology, Negative control mean, positive control mean, Bangladesh Agricultural University.The chicks were sample to positive (S/P) ratio and endpoint titres were vaccinated against infectious bursal disease virus with calculated.Sera samples with S/P ratios of less than or a commercial vaccine "Nobilis D-78" (Intervet, the equal to 0.2 were considered negative and S/P ratios Netherlands) at 14 days of age.Blood samples from 4 greater than 0.2 (titre greater than 396) were considered birds were obtained at day old, then at 4, 6 and 8 weeks positive.of age, respectively either from the jugular vein during slaughter (day old chicks) or from the wing vein.The Optimum dilution for filter paper extracts: To 100 µl of blood samples were soaked scientifically and determine the optimum dilution for filter paper aseptically on commercially available Whatman filter extracts, the ELISA was performed using five selected paper no.1( 2 cm x 5 cm x 0.3 mm ) and allowed to dry sera samples at 1: 500 dilution and their corresponding at room temperature away from direct sun light after filter paper extracts at two-fold serial dilutions ranging 0 that stored in screw-capped air tight vessels at -20 C from 1: 2 up to 1: 256.for subsequent extraction in Dulbecco's phosphate The absorbance values for the dilution of each buffer saline and used in filter paper based ELISA.
filter paper extracts giving an absorbance value close Blood samples were also collected in vials and to that of 1: 500 dilution of the corresponding serum allowed to clot at room temperature at slanting position sample was determined (Table 1). in order to obtain serum.The separated serum was Comparison of ELISA results based on sera and collected in fresh vials and clarified by low speed filter paper extracts: A total of 15 sera and correscentrifugation (3000 rpm for 15 minutes) then stored 0 ponding filter papers extracts were tested at dilutions frozen at -20 C until used.Five discs cut out from each of 1:500 and 1:5 accordingly.The antibody titre blood soaked filter paper strips with a paper punch Corresponding dilution of against IBDV in these two systems were calculated 500 dilution, collected on 4 different occasions and and compared by correlation-regression analysis.their corresponding filter paper extracts at 1:5 dilution.
The absorbance values and calculated titres are Inter-assay variability: For determining the interpresented in Table 2. assay variability between ELISA performed using sera The antibody titres determined by two systems and filter paper extracts respectively, replicate filter were very close and did not exceed more than a twopaper and blood samples were collected from each of fold dilution, with the exception of occasion II.The the 25 week old birds (n=5) and tested at dilutions correlation between the ELISA titres determined from explained above.Inter-assay co-efficient of variation the serum and filter paper extracts were analysed by (CV) was calculated for each bird.correlation regression analysis.There was a very strong positive correlation (r = 0.9; n = 15; p<0.01).

Results
The results are shown in Figure 1.In this study, filter paper sampling of blood was adopted for detection of IBDV antibodies in chicken sera using a commercial ELISA kit.

Determination of optimum dilution for filter
paper extracts: After analysis it was found that, the absorbance value of each serum sample at 1: 500 dilution matched closely to the absorbance value (or Optical Density) of the filter paper extracts at a dilution between 1: 2 and l: 8.
The exact corresponding dilution of the filter paper extracts expected to give an absorbance value equal to that given by the 1: 500 dilution of the serum was also calculated by correlation-regression analysis.The geometric mean of the calculated corresponding filter paper extracts dilutions was found to be Log 2.3, 2 which is equal to 4.92.Therefore, a filter paper extract dilution of 1: 5 was considered to be equivalent to 1: shown in Table 3.The coefficient of variation obtained results based on sera and filter paper extracts, the from this study was less than 10%.ELISA was performed using 15 serum samples at 1 :

Figure- 1 .
Correlation between ELISA titres determined from the serum and filter paper extract 500 dilution of the serum.Inter-assay variability: Sera and filter paper Comparison of ELISA results based on sera and extracts antibody titres, Mean ± SD and % CV are filter paper extracts: For comparison of ELISA