To determine the optimal amount of protein required for conjugation, a CG solution (1 ml) with pH range (6.0 – 9.0) was added to aqueous IgG solution (immunoglobulin concentrations ranging between 0 and 80 ?g/ml were used), and the mixture was stirred and incubated for 10 min at room temperature. Then a 10% solution of NaCl (0.1 ml) was added to each sample; the resulting mixtures were stirred, further incubated for 10 min, and then the OD was measured at 580 nm. The suspensions were observed for blue color as evidence of aggregation and red as the indication of optimum amount of IgG required for conjugation. The optimum IgG concentration was selected based on the data obtained.
To obtain the IgG-CG conjugate, the antibodies were dialyzed against 1000 volumes of 2 mM Borate-HCl buffer (pH 9.0) for overnight at 4°C. The pH of the CG solution was adjusted to 9.0 with 0.2 M solution of potassium carbonate, and then the CG solution was added to the IgG solution at the chosen concentration. The mixture was stirred for 30 min at room temperature, and BSA was added to a final concentration of 0.25%. CG particles with immobilized IgG molecules were separated by centrifugation for 60 min at 10000 rpm @ 4 °C. After discarding the supernatant, the gold pellets were suspended in PBS containing 0.25% BSA and 1.0% trehalose. For long term storage of conjugate preparations, sodium azide was added to a final concentration of 0.01% (w/v) before storage at 4 °C.
The LFIA strips were assembled in polypropylene cassettes (5 cm x 1 cm) with sample and observation windows. The cassettes encased four overlaying components, viz. sample application pad, conjugate release matrix, high protein binding nitrocellulose membrane (0.8 ?) andabsorbent pad. The polypropylene cassettes, all types of overlaying components and the necessary instrument (IgG printer and stripcutter) were procuredfrom Advanced Microdevices Pvt Ltd, Ambala Cantt., India. The LFIA devices were assembledas per the manufacturer’s instructions. Briefly, the test (T) and control (C) lines of anti-BBrMV IgG (0.75 mg/mL) and goat anti rabbit IgG (0.5mg/mL), respectively were printed on nitrocellulose (NC) membrane using automated antibody printing machine. At one end of the NC membrane, the conjugate release matrix soaked with the colloidal gold- anti-BBrMV IgG conjugates was placed. The sample pad was placed in such a way that it overlapped the conjugate release matrix. The absorbent pad was placed at the opposite end of the sample pad to collect excess reagents. After fabrication ofthese components assay strips were cut to a size of 4 x 0.4 cm (l x b) by using a programmablestrip cutter and encased. The entire LFIA cassette fabrication procedure was carried out under dust-free conditions. Assembled test strips were housed in plastic cases and stored at 4 °C under desiccated conditions until required. LFIA assay was performed at room temperature. The test strip was dipped into a sample analyzed in an upright position for 90 s, then taken out and placed on a horizontal surface. The qualitative result of immunochromatographic assay was estimated by eye after 5-10 min.
To evaluate the cross-reactivity of the LFIA test strip, BBrMV and two other common banana infecting viruses (BBTV, the banana bunchy top virus; CMV, the cucumber mosaic virus) from strongly positive field samples, were tested with the test strip. BBrMV-infected leaveswere used as a positive control, and virus extraction buffer (PBS-T) was used as a negative control. The sensitivity of LFIA strips was evaluated by testing a series of different concentration of the purified BBrMV protein solution 0, 5, 10, 20, 40, 80, 160, 320 and 640 ng per milli litre. Each dilution was then added to the LFIA test strip, and the sensitivity was determined by finding the endpoint dilution. A series of crude extract dilutions from BBrMV-infected leaves were tested using the LFIA, crude extract from BBrMV-uninfected leaves was used as the negative control.
The LFIA was validated using 114 samples of commercial banana cultivars suspected to have BBrMV infection and collected from different orchards located in six states of India and also tissue culture samples received under the NCS-TCP being operative in India. In addition, the same samples were also subjected to ELISA assay to provide a comparison. Based on the results obtained, the samples were classified as true positive (TP), true negative (TN), false positive (FP), or false negative (FN). The diagnostic sensitivity and specificity of the LFIA were calculated using the Cohen’s kappa index 6.

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