Abdul Samad, Norani

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1998: Volume 2EVALuATION OF FILTER PAPER SERO-ASSAY(FIPSA) TO DETECT THREE VIRUSESNorani Abdul-SamadFaculty of Science and Environmental StudiesUniversiti Putra Malaysia, 43400 UPM, Serdang, Selangor,MalaysiaKeywords: FiPSA, CYVV, HCRV, ToMV.IntroductionThe application of serological detection methods forthe plantviruses requires the development of rapid, simple and sensi-tive immunoassays that employ the specific relationship be-tween an antigen and its homologous antibody. The adapta-tion and improvement of rapid immunofilter assay has stead-ily been developed and could easily be used for virus diagno-sis with considerable high sensitivity. The filter paper sero-assay (FiPSA) protocol is analogous to the indirect ELISA(dot-immunobinding assay) of Hawkes et al. (1982) thatutilised filter paper instead of nitrocellulose membrane.Haber and Knapen (1989) had demonstrated the usefulnessof the modified technique for the detection of several viruses.In this project, the sensitivity of FiPSA is examined, testedand compared in three different plant viruses; Clitoria yellowvein virus (CYVV), Hibiscus chlorotic ringspot virus(HCRV) and tomato mosaic virus (ToMV).Materials and MethodsThe CYVV, HCRV and ToMV isolates and the antisera wereas described by Abdul-Samad (1994), Abdul-Samad and Mat(1995) and Abdul-Samad et al. (1996), respectively.Whatrnan No. 1 filter paper disks (7 mm diameter) wereheated at 150° C for 1 hr and stored in the dessicator untilused. Albumin from rabbit normal serum was used as block-ing solution. The dried disks were soaked for 15 min in theblocking solution, suction-dried and stored in the dessicator.The purified viruses were diluted to a series of dilution inTris-buffered saline (TBS). Leaf extract from infected plantswere prepared simply as crude plant materials which werefree of particulate matter and a serial dilution was preparedin spotting buffer. Two microlitres of samplewere spotted inthe centre of each disk and transferred to individual cylindri-cal vial and dried at 40° C for 10 min. Antisera were diluted75 x in TBS; normal serum diluted similarly as control.Three hundred microlitres of each serum was applied to thedisk and shaken gently for 60 min after which the serum wasaspirated from the vial. The disks were washed in 1 ml ofTBS for 10 min with shaking and aspirated. The disk wasthen immersed in 300 f.11protein A-peroxidase at 2 f.1g/mlin82TBS and incubated withshaking (60 rpm) for 30 min. Thedisks were removed and washed again as above. They wereincubated in the substrate [5 parts TBSIl part 4-chloro-l-napthol (3 mg/ml in methanol)/0.018 parts 3% hydrogen per-oxide] for 1 min in the dark in a volume just sufficient to en-sure immersion. They were then separated from the substrateand immersed in water for 10 min for further colour devel-opment and air-dried at 40° C.Results and DiscussionPositive reactions appeared as violet-blue centres againstwhite or light backgrounds. The green colour sap on thespots did not interfere with the results. False positives were ,controlled by using antigen-free samples, as well as by incu-bating duplicates of samples with normal or non-specific se-rum. From the results obtained, the use of blocking solutionto preblock the disks was an important factor in increasingthe sensitivity of detection to reduce nonspecific binding toacceptable amounts (Haber and Knapen, 1989).FiPSA was sufficiently sensitive to detect purified virusesand sap extracts of CYVV, HCRV and ToMV present in lowconcentration. For CYVV, the colour intensity could be de-tected up to 3.0 ng/ml for purified virus and 1:10,000 dilutionfor sap extract; for HCRV, 15 ng/ml for purified virus and1:4,800 dilution for sap extract; and for ToMV, up to 5.0ng/ml and 1:8,000 dilution for purified virus and sap extract,respectively by using a densitometer. However, by using aquick visual detection the values were 50 ng/ml and 1:6,000,130 ng/ml and 1:1,200 and 90 nglml and 1:3,500 for purifiedand sap extract of CYVV, HCRV and ToMV, respectively.ConclusionsUltimately, it is hoped that this rapid immunoassay will beused as a tool for quick virus detection rather than for accu-rate quantification.ReferencesAbdul-Sarnad, N. 1994. Natural occurrence of Clitoria yellow veinvirus in Calopogonium mucunoides in Malaysia. Plant Disease.78: 1123.Abdul-Sarnad, N. and Mat, M. 1995. Hibiscus chlorotic ringspot vi-'rus in Malaysian hibiscus. Plant Disease. 79: 967.Abdul-Sarnad, N., Singh, M. and Yusoff, K. 1996. First report oftomato mosaic tobarnovirus from Malaysia. Pertanika J. Trop.Agric. Sci. 19: 1-6.Haber, S. and Knapen, H. 1989. Filter paper sero-assay (FiPSA) : Arapid, sensitive technique for serodiagnosis of plant viruses.Can-J. Plant PathoL 11: 109-113.Hawkes, R, Niday, E. and Gordon, I. 1982. A dot-immunobindingassay for monoclonal and other antibodies. AnaL Biochem-119:142-147.Supported by IRPA Grant 01-02-04-0234

Evaluation of filter paper sero-assay (FIPSA) to detect three viruses

http://psasir.upm.edu.my/20115/1/Evaluation%20of%20filter%20paper%20sero-assay%20%28FIPSA%29%20to%20detect%20three%20viruses.pdf

Evaluation of filter paper sero-assay (FIPSA) to detect three viruses

Abstract

Similar works

Full text

Available Versions

Universiti Putra Malaysia Institutional Repository