The results suggest that 3 μg of TSWV-IgG is sufficient for 1 ml of AuNPs and the optimal conju¬gation between TSWV-IgG and AuNP is done at pH8.0.
In general, the analytical performance of the RIGS strip is af¬fected by many parameters, such as the type and pore size of the membrane, blocking buffer and immune-reagent amount. In this work, blocking buffers for the conjugate and sample pads were evaluated to study its effect on polyclonal IgG spe¬cific to TSWV and analytes. In addition, the blocking buffers with the BSA or ovalbumin were evaluated to get the optimal block¬ing buffer. The results show that 10 mM PBS (pH 7.4) containing 0.25% BSA, 1% sucrose and 0.03% NaN3 was chosen as the opti¬mal blocking buffer for the conjugate pads.
The competitive RIGS strip for TSWV detection was defined as the lowest TSWV concentration producing the color on the test line significantly weaker than that of the negative control strip.
A serial end-point dilution of crude saps from TSWV-infected tomato and tobacco leaves was used for the sensitivity of the RIGS. The sample pad of RIGS strip was soaked in aliquot of crude saps, showing that the color intensity was readily shown after 5 min (Fig. 4).
The intensity of the test lines decreased with the dilution of TSWV virion concentration. It is clearly shown that TSWV in plant tissues can be successful detected as amount as 7.83 × 10-4 g/ml of tomato tissue and 1.57 × 10-3 g/ml of tobacco tissue using the developed RIGS kit (Fig. 4).