Filter Paper Sampling Of Blood For The Detection .
Vet. World, 2012, Vol.5(6): 341-345RESEARCHFilter paper sampling of blood for the detection of antibodies toInfectious Bursal Disease virus using a commercial ELISA kitTofayel Ahmed1, Md Bashir Uddin2, Md Rafiqul Islam3, Syed Sayeem Uddin Ahmed4,5,Joyita Basu3, Mohammad Mejbah Uddin6,71. Veterinary Surgeon, Department of Livestock Services, Fulpur Upazilla, Mymensingh, Bangladesh2. Sylhet Agricultural University, Faculty of Veterinary and Animal Science, Department of Medicine andSurgery, Sylhet-3100, Bangladesh 3. Bangladesh Agricultural University, Faculty of Veterinary Science,Department of Pathology, Mymensingh-2202, Bangladesh 4. Chittagong Veterinary and Animal SciencesUniversity, Faculty of Veterinary Medicine, Department of Medicine and Surgery, Chittagong-4202,Bangladesh 5. University of Copenhagen, Faculty of Life Sciences, Department of Large Animal Sciences,Frederiksberg C, Denmark 6. Chittagong Veterinary and Animal Sciences University, Faculty of VeterinaryMedicine, Department of Anatomy and Histology, Chittagong-4202, Bangladesh 7. Kagoshima University,Faculty of Agriculture, Department of Veterinary Clinical Sciences, Kagoshima 890-0065, JapanCorresponding author: Mohammad Bashir Uddin, email: bashir vet@yahoo.comReceived: 14-11-2011, Accepted: 09-12-2011, Published Online: 10-03-2012doi: 10.5455/vetworld.2012.341-345AbstractAim: The present study was designed to investigate the feasibility of the filter paper sampling of blood for the detection ofIBDV 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 thecorresponding serum sample in a commercial ELISA was determined. Correlation between the filter paper extracts basedELISA and conventional serum based ELISA was also examined and the reproducibility of the filter paper extracts basedELISA was tested.Results: A very good correlation (r 0.9; n 15; p 0.01) was observed between the results obtained with filter paper extractsbased ELISA and serum based ELISA. The antibody titre determined by two systems were very close and did not exceed morethan a two-fold dilution with the exception of samples having very low antibody levels, where relatively higher backgroundreaction was observed with filter paper extracts based ELISA. The filter paper extracts based ELISA appeared to be quitereproducible 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-profilingchicken flocks for IBDV antibodies.Key Words: Filter paper extract based ELISA, IBD, Serum based ELISA, SeroprofilingTo cite this article: Ahmed T, Uddin MB, Islam MR, Ahmed SSU, Basu J, Uddin MM (2012) Filter paper sampling ofblood for the detection of antibodies to Infectious Bursal Disease virus using a commercial ELISA kit, Vet World5(6):341-345, doi: 10.5455/vetworld.2012.341-345IntroductionPoultry industry is a rising sector in Bangladeshbut the development of this sector is often interruptedby frequent outbreaks of a number of emerging andrecurrent diseases. One of the most important diseasesof commercial poultry is infectious bursal disease(IBD). It is an acute, highly contagious immunosuppressive viral infection of chickens [1,2,3]. Amongpoultry diseases in Bangladesh, IBD is the number onekiller causing up to 80% mortality in field outbreaks[4, 5, 6, 7].Vaccination is the only means for the preventionof IBD but vaccination failure is quite common due tovarious factors such maintenance, storage or inadequateimmune response following vaccination or neutrawww.veterinaryworld.orglization of vaccine virus by the maternal antibodiespresent in chicks. Therefore, maternal antibody levelsin chicks should be determined before vaccination.The enzyme-linked immunosorbent assay (ELISA) iscommonly used and considered to be the easiest methodfor measuring maternal antibody levels against IBDV[8]. The commercial IBDV ELISA kits are availablethrough an ELISA assay for detecting antibodies toIBDV also has recently been developed locally.Conventionally, serum obtained from blood isused in ELISA, but it is often difficult to collectsufficient blood from very young chicks withoutkilling the birds. As an alternative, a technique hasbeen developed for sampling blood on filter paper,where a small drop of blood is soaked on a filter paperVeterinary World, Vol.5 No.6 June 2012341
Filter paper sampling of Blood for the detection Antibodies to IBDV using a Commercial ELISA KitTable-1. Similarities between absorbance values of sera samples (1: 500 dilutions) and corresponding filter paper extracts (serial two-fold 2620.305Corresponding dilution ofFilter paper extractsexpected to viveabsorbance similar to that1:500 dilution of SerumFilter paper :256)Dilution Geometric mean(Log2) dilution 0822.31.81.83.91.6strip, which can be later extracted with PBS and usedin ELISA [9]. Filter paper extracts based ELISA hasbeen successfully used to detect antibodies to foot andmonth disease virus [10], rinderpest virus [11, 12],duck plague virus [13] and Pasteurella multocida[14].However, there is no study about filter paperbased ELISA for the detection of antibody to IBDVusing commercial kits. Therefore, the present studywas designed to investigate the feasibility of the filterpaper sampling of blood for the detection of IBDVantibodies in chicken sera using a commercial ELISA kit.Materials and MethodsSampling and sera collection: The study wasperformed during the period of January - June, 2004.Blood samples were obtained from chickens, whichwere reared in the Department of Pathology,Bangladesh Agricultural University. The chicks werevaccinated against infectious bursal disease virus witha commercial vaccine "Nobilis D-78" (Intervet, theNetherlands) at 14 days of age. Blood samples from 4birds were obtained at day old, then at 4, 6 and 8 weeksof age, respectively either from the jugular vein duringslaughter (day old chicks) or from the wing vein. The100 µl of blood samples were soaked scientifically andaseptically on commercially available Whatman filterpaper no.1( 2 cm x 5 cm x 0.3 mm ) and allowed to dryat room temperature away from direct sun light afterthat stored in screw-capped air tight vessels at – 200Cfor subsequent extraction in Dulbecco's phosphatebuffer saline and used in filter paper based ELISA.Blood samples were also collected in vials andallowed to clot at room temperature at slanting positionin order to obtain serum. The separated serum wascollected in fresh vials and clarified by low speedcentrifugation (3000 rpm for 15 minutes) then storedfrozen at -200C until used. Five discs cut out from eachblood soaked filter paper strips with a paper punchwww.veterinaryworld.org2.3were extracted with 300µl PBS for 60 minutes at roomtemperature.A commercial ELISA kit (Infectious BursalDisease Antibody Test Kit-IDEXX Laboratory, Inc.,Westbrook, Maine 04092, USA) was used. The testwas used and the test performed as described by themanufacturer. The frozen sera samples were thawed.Five hundred fold (1:500) dilution of sera sampleswere made in sample diluent. The Filter paper extractswere used immediately after extraction. Filter paperextracts were diluted either serially in two-folddilutions or at an appropriate fixed dilution (1: 5). Thiswas done in the same laboratory, BAU. The opticaldensity (OD) or absorbance values were determined at650 micron using an ELISA reader (SPECTERA max340 pc, Molecular Devices Inc., USA) at the Surgeryand Obstetrics Department of Bangladesh AgriculturalUniversity.Negative control mean, positive control mean,sample to positive (S/P) ratio and endpoint titres werecalculated. Sera samples with S/P ratios of less than orequal to 0.2 were considered negative and S/P ratiosgreater than 0.2 (titre greater than 396) were consideredpositive.Optimum dilution for filter paper extracts: Todetermine the optimum dilution for filter paperextracts, the ELISA was performed using five selectedsera samples at 1: 500 dilution and their correspondingfilter paper extracts at two-fold serial dilutions rangingfrom 1: 2 up to 1: 256.The absorbance values for the dilution of eachfilter paper extracts giving an absorbance value closeto that of 1: 500 dilution of the corresponding serumsample was determined (Table 1).Comparison of ELISA results based on sera andfilter paper extracts: A total of 15 sera and corres-ponding filter papers extracts were tested at dilutionsof 1:500 and 1:5 accordingly. The antibody titreVeterinary World, Vol.5 No.6 June 2012342
Filter paper sampling of Blood for the detection Antibodies to IBDV using a Commercial ELISA KitTable-2. The ELISA absorbance values and antibody titer of 15 sera samples (1: 500 dilution) andcorresponding filter paper extracts (1: 5 dilution)Sampling occasion SampleIIIIIIIV123123412341234Mean absorbanceSerum 1: 500Filter paper extract 1: gainst IBDV in these two systems were calculatedand compared by correlation-regression analysis.Inter-assay variability: For determining the interassay variability between ELISA performed using seraand filter paper extracts respectively, replicate filterpaper and blood samples were collected from each ofthe 25 week old birds (n 5) and tested at dilutionsexplained above. Inter-assay co-efficient of variation(CV) was calculated for each bird.ResultsIn this study, filter paper sampling of blood wasadopted for detection of IBDV antibodies in chickensera using a commercial ELISA kit.Mean predicted titreSerum 1: 500Filter paper extract 1: 0691475465760500 dilution, collected on 4 different occasions andtheir corresponding filter paper extracts at 1:5 dilution.The absorbance values and calculated titres arepresented in Table 2.The antibody titres determined by two systemswere very close and did not exceed more than a twofold dilution, with the exception of occasion II. Thecorrelation between the ELISA titres determined fromthe serum and filter paper extracts were analysed bycorrelation regression analysis. There was a verystrong positive correlation (r 0.9; n 15; p 0.01).The results are shown in Figure 1.Determination of optimum dilution for filterpaper extracts: After analysis it was found that, theabsorbance value of each serum sample at 1: 500dilution matched closely to the absorbance value (orOptical Density) of the filter paper extracts at adilution between 1: 2 and l: 8.The exact corresponding dilution of the filterpaper extracts expected to give an absorbance valueequal to that given by the 1: 500 dilution of the serumwas also calculated by correlation-regression analysis.The geometric mean of the calculated correspondingfilter paper extracts dilutions was found to be Log2 2.3,which is equal to 4.92. Therefore, a filter paper extractdilution of 1: 5 was considered to be equivalent to 1:500 dilution of the serum.Comparison of ELISA results based on sera andfilter paper extracts: For comparison of ELISAresults based on sera and filter paper extracts, theELISA was performed using 15 serum samples at 1 :www.veterinaryworld.orgFigure-1. Correlation between ELISA titres determined fromthe serum and filter paper extractInter-assay variability: Sera and filter paperextracts antibody titres, Mean SD and % CV areshown in Table 3. The coefficient of variation obtainedfrom this study was less than 10%.Veterinary World, Vol.5 No.6 June 2012343
Filter paper sampling of Blood for the detection Antibodies to IBDV using a Commercial ELISA KitTable-3. Percent coefficient of variation (CV) of ELISA titres determined from 5 replicate samples ofeach of 5 birdsSampleAntibody titreSerum (1:500)l2345125651378612093933713320Filter paper extracts (1:5)abcdeMean 615515162531020387701417116458 131015702 47511257 9708416 28114005 4497.963.038.623.333.21DiscussionThe whole blood dried on filter paper as analternative to serum has been used previously inELISA for detecting antibodies to foot and mouthdisease virus [10], Rinderpest virus [11, 12] Duckplague virus [13] anaplasma [14] Pasteurella multocida[15] and Schistosome [9]. Although no report isavailable on- the use of filter paper extract to detectantibodies-to IBDV by ELISA [16] used filter paperextract for detecting IBDV by antibodies by agar gelprecipitation test. The difference was significantbetween serum samples and filter-paper eluates inwhich supports the present findings.The filter paper extracts based assay used in thepresent study with a commercial ELISA kit that isroutinely used in between poultry industry formeasuring maternal antibody levels, in chicks topredict the optimum time for vaccination. The kits arevalidated for use on sera samples. However, it is oftenquite difficult to obtain sufficient blood from youngchicks due to their small size. The study proposes thefilter paper sampling technique and extracts usage asan alternative to bleeding relatively large volumes ofblood and sera testing in the commercial assay. Thefilter paper extracts can be used in the ELISA protocolrecommended by the kit manufacturer without anymodification except that the extracts should be furtherdiluted 1:5 in the sample diluent instead of the 1:500recommended for sera samples. This finding is inpartial agreement with that of Evengard and Linder [9]who observed that 1:5 dilution of filter paper extractswere equivalent to 1: 400 dilution of correspondingserum sample in bovine of schistosome-specificantibodies. The variation could be due to speciesdifferences or differences in the filter paper extractionprocedures.A very good correlation (r 0.9; n 15; p 0.01)was observed between the results obtained with thefilter paper extracts and serum based ELISA in thepresent study respectively. The antibody titrewww.veterinaryworld.orgdetermined by two systems were very close and didnot exceed more than a two-fold dilution, with theexception of one particular sampling occasion(Occasion II). In fact, at that particular occasion thebirds had very low levels of antibodies (having the titrebetween 78 to 107 with serum based ELISA).However, with filter paper based ELISA the antibodieswere between 617 and 1361. This discrepancy wouldsuggest that at very low antibody levels, the filterpaper extracts based ELISA might give relatively ahigh background reaction. Further studies are requiredin this regard. One possible option is to extract theblood soaked filter paper in sample diluent instead ofPBS to reduce nonspecific binding.Besides the drawback mentioned above, the filterpaper based ELISA appeared to be quite reproduciblewith a coefficient of variation less than 10%. Heller[11] also suggested that the accuracy and precision offilter paper extract based ELISA were excellent with asensitivity of 100% and specificity of 98.26%.The major limitation of the present study was thesmall sample size for determination of the optimumdilution of filter paper extracts and for comparison ofthe filter paper extracts based ELISA and serum basedELISA. This was due to the resource constraint as onlyone kit (5 antigen-coated plates) was available for thestudy.ConclusionFilter paper based ELISA could be a usefulalternative to serum dependant ELISA assays for seroprofiling chicken flocks for IBDV antibodies.AcknowledgementsThis research work was conducted during theperiod of January –June, 2004 as the part of MS. Thefirst author (as MS student) humbly desires to expresshis deepest gratitude to his MS supervisors. Theauthors would like to thanks to all respondents of thestudy areas.Veterinary World, Vol.5 No.6 June 2012344
Filter paper sampling of Blood for the detection Antibodies to IBDV using a Commercial ELISA KitCompeting interestsThe authors declare that they have no ert, P.D. & Saif, Y.M., (1997). "Infectious bursaldisease virus." In: Calnek BW, Barnes HJ, Beard CW,McDougald LR, Saif YM (eds.). Diseases of Poultry,Ames: Iowa State University Press, 721-738.McFerran, J.B., (1993). Infectious bursal disease. In:Virus. McFerran JB, McNulty MS (eds.). Infectionsof birds, Elsevier Science Publishers, Amsterdam,213-228.Muller, H., Islam, M.R. & Raue, R., (2003). Researchon infectious bursal disease the past, the present andthe future (Review). Veterinary Microbiology, 97:153-165.Chowdhury, E.H., Islam, M.R., Das, P.M., Dewan,M.L. & Khan, M.S.R., (1996) Acute infectious bursaldisease in chickens: pathological observation andvirus isolation. Asian-Aust. J. Anim. Sci, 9: 465-469.Islam, M.R., Das, P.M., Chowdhury, E.H. & Dewan,M.L., (1994a) Very virulent infectious bursal diseasevirus: a challenge for poultry industry in Bangladesh.Paper presented in the 12th Annual Conference ofBangladesh Society of Microbiologists, BARC,Dhaka, January 19 and February 11.Islam, M.R., Das, P.M., Chowdhury, E.H. & Dewan,M.L., (1994b) Some observations on infectious bursaldisease of chickens reproduced experimentally with ahighly virulent local isolate. Paper presented in the18th Bangladesh Science Conference, BangladeshAgricultural University, Mymensingh, 22-24 June.Rahman, M.M., Hossain, W.I.M.A., Rahman, M.M.,Mia, A.H. & Biswas, M.R.H., (1996) Isolation andIdentification of Infectious bursal disease virus inchickens in Bangladesh. Bang. Vet. J, 30:7-11.Snyder, D.B., Marquardt, W.W., Mallinson, E.T.,Russek-Cohen, E., Savage, P.K. & Allen, D.C., (1986)Rapid serological profiling by enzyme linkedimmunosorbant assay. IV. Association of Infectious10.11.12.13.14.15.16.bursal disease serology with broiler flockperformance. Avian Dis, 30: 139-148.Evengard, B., Hagi, H. & Linder, E., (1988) Afilter paper technique for the detection of IgG and IgMclass schistome-specific antibodies in an endemicarea. Ann. Trop. Med. Parasitol, 82: 307-309.Armstrong, R.M., Crowther, J.R. & Denyer, M.S.,(1991) The detection of antibodies against foot-andmouth disease virus (FMDV) in filter paper eluatesfrom pig sera or whole blood by ELISA. J. Virol.Methods, 34: 181-192.Heller, C., Stem, C., Wamwayi, H. & Grieve, A.,(1988) Development of a filter paper based ELISA forrinderpest antibodies. Vet. Rec, 142: 729.Ramadass, P., Ganesan, P.I., Meerarani, S. &Padmanaban, V.D., (1993) Usefulness of filter paperELISA for detection of rinderpest antibodies. IndianVet. J, 70: 795-797.Malmarugan, S., Sulochana, S., Verma, R., Sharma,N., Varma, T.K., Bagherwal, R.K. & Jaisal, T.N.,(2000) Comparison of whole blood dried on filterpaper and conventional serum for detection ofantibodies to duck plague virus by enzyme linkedimmunosorbant assay (ELISA). Advancementsin Veterinary Science. Indian Veterinary Congress,Izatnagar, India, 215-219. www.iaavr.org.Ssenyonga, G.S.Z., Montenegro, J.S., Kakoma, I. &Mansen, R., (1993). A comparative study of the use ofdried blood on filter papers and serum samples forserodiagnosis of anaplasmosis. Ug. J. Agric. Sci, 1:1-3.Natalia, L. & Priadi, A., (1998) The use of filter paperfor collecting samples for immunodiagnosis ofPasteurella multocida infection: analysis andcomparison of the protein composition of filter paperextracts and serum. Journal Ilmu Ternak danVeteriner, 3: 182-187.Roy, P., Nachimutu, K., Venugopalan, A.T.,Dorairajan, N., Purushothaman, V. & Koteeswaran,A., (1994) Filter paper technique for seromonitoringagainst Infectious bursal disease. Trop. Anim.
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 that stored in screw-capped air tight vessels at – 200C from 1: 2 up to 1: 256.
Sampling, Sampling Methods, Sampling Plans, Sampling Error, Sampling Distributions: Theory and Design of Sample Survey, Census Vs Sample Enumerations, Objectives and Principles of Sampling, Types of Sampling, Sampling and Non-Sampling Errors. UNIT-IV (Total Topics- 7 and Hrs- 10)
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TABLE OF CONTENTS 3 BLOOD CULTURE ESSENTIALS p. 2 1 What is a blood culture? p. 4 2 Why are blood cultures important? p. 4 3 When should a blood culture be performed? p. 5 4 What volume of blood should be collected? p. 6 5 How many blood culture sets should be collected? p. 8 6 Which media to use? p. 10 7 Timing of blood cultures p. 11 8 How to collect blood cultures p. 12
A blood can receive blood from a person with type A or type O. A person with type B blood can receive blood from a person with type B or type O. A person with type B blood can donate blood for persons with either type B or type AB blood. Actually, blood banking is more complicated than this simple description, with test run for other minor
1. Principle of Sampling Concept of sampling Sampling: The procedure used to draw and constitute a sample. The objective of these sampling procedures is to enable a representative sample to be obtained from a lot for analysis 3 Main factors affecting accuracy of results Sampling transport preparation determination QC Sampling is an
2.4. Data quality objectives 7 3. Environmental sampling considerations 9 3.1. Types of samples 9 4. Objectives of sampling programs 10 4.1. The process of assessing site contamination 10 4.2. Characterisation and validation 11 4.3. Sampling objectives 11 5. Sampling design 12 5.1. Probabilistic and judgmental sampling design 12 5.2. Sampling .
