African Cassava Mosaic Virus (ACMV): Stability of Purified Virus and Improved Conditions for its Detection in Cassava Leaves by ELISA

Abstract

African Cassava Mosaic Virus (ACMV) was purified by a method which allowed the separation of monomer from dimcr virus particles. Optimal conditions for storing purified virus to be used for immunization were determined by ELISA and inoculation on Nicotiana benthamiana. Purified virus could be stored without loss of infectivity and serological activity for more than 145 days at 4 °C or frozen at –20 °C, but not longer than 40 days in the presence of 50 % redistilled glycerol. Rabbit and chicken immunoglobulins were used to detect ACMV in cassava leaves by direct and indirect ELISA. To obtain the same absorbance values, it was necessary to use longer incubation times with the indirect method, but the virus detection end‐point m sap from infected plants was the same for the two methods (1/512). Conditions for improving virus detection tn cassava samples were determined. The virus was better detected when leaves from diseased plants were ground in 100 mM Tris‐HCl containing 1 % polyvinylpyrrolidone at pH 8.5 than in phosphate buffer. Plant inhibitors were the restricting factor in the detection of virus by ELISA, but this difficulty was avoided when leaves to be tested were harvested from the top of the cassava plants.

Detection of geminiviruses from tropical countries by a double monoclonal antibody ELISA using antibodies to African cassava mosaic virus

“…In ACP-ELISA procedure the microtitre plates (Falcon, Oxnard, CA; Ref 3912) were coated with a purified preparation of ACMV (0.35 μg/ml) in 0.05 M carbonate buffer pH 9.6 (coating buffer) for 2 h. The DAS-ELISA tests were carried out using rabbit IgG raised against ACMV as a first antibody, at 1 μg/ml (2 h), in coating buffer, followed by incubation with the antigen at 0.35 μg/ml in phosphatebuffered saline, pH 7.4, containing 0.05% Tween 20 (PBS-T) overnight at 4°C. The rabbit IgG used in this test has been described previously (Kounounguissa et al, 1989 Labelling of reagent For biotinylating mAbs, N-hydroxysuccinimidobiotin (Sigma) (biotin) dissolved in distilled dimethylformamide (0.2 mg/ml) was added to mAbs (ascitic fluid) diluted at around 1.5 mg/ml, in a 1:5 biotin/mAbs ratio (w/w); after incubating the mixture at 25°C for 2 h, the reaction was stopped by addition of 1 M NH 4 Cl (Zrein et al, 1986 To detect ACMV in extracts from infected leaves of N benthamiana, mAbs were used at various concentrations: ascitic fluids were diluted from 10 -3 to 10 -4 for mAb 7 and from 5 x 10 -4 to 10 -5 for mAb 11.…”

“…These relationships reflect the degree of amino-acid sequence identity (about 70% or more) among the coat proteins of different members (Hamilton et al, 1984;Howarth et al, 1985). Among whitefly-transmitted geminiviruses, the particles of ACMV (Thomas et al, 1986), Indian cassava mosaic (ICMV; Aiton and Harrison, 1989) and okra leaf curl (OLCV; Swanson and Harrison, 1993) (Thomas et al, 1986;Harrison et al, 1991b;Muniyappa et al, 1991;Macintosh et al, 1992;Swanson et al, 1992aSwanson et al, , 1992b (Kounounguissa et al, 1989) were maintained in manually inoculated N benthamiana in an insect-proof glasshouse under controlled conditions (18-27°C, 16 h light/day).…”

“…Particles of ACMV TMS 30211 were purified according to Kounounguissa et al (1989). The purified preparations were stored frozen at -20°C after addition of 0.03% NaN 3 .…”