Fig 1.
Composition of the rSP03B sero-strip.
The rSP03B sero-strip consists of a backing card to which a lower absorbent and an upper absorbent pad are attached. Both of these pads overlap an NC membrane located in the middle of the test. On the NC membrane 3 lines are coated: the sample line, a test line and a control line. The lower absorbent pad is impregnated with a colloidal gold-conjugate (conjugate pad), consisting of a mixture of the conjugates for the test and the control line. NC = nitrocellulose.
Fig 2.
Stepwise description of the rSP03B sero-strip principle.
To start the test a buffer is deposited on the sample line (NC membrane) (1). Immediately after applying the buffer, the sample is spotted on the same position (2). Both the buffer and the sample will migrate to the upper part of the strip. The anti-rSP03B Abs present in the sample get captured by the rSP03B Ag coated on the test line (3). The strip is then immediately dipped into the migration buffer solution (4). After which the colloidal gold conjugate mixture gets hydrated and starts migrating upwards together with the moving liquid (5). The anti-dog IgG Ab-gold conjugate binds to the dog IgG Abs on the test line. The colloidal gold control conjugate is captured by the GAC Abs present at the control line (6). NC, Nitrocellulose; Abs, Antibodies; Ag, Antigen; GAC, Goat anti-chicken.
Fig 3.
Intensity of test line relates to amount of target Ab deposited.
Dilution series of four highly positive sera samples indicate that the intensity of the purple color at the test line decreases when a lower amount of target Ab is deposited on the strip. The sample appears negative when a dilution of 1/50 is used. A: undiluted sample; B: 1/10 sample dilution; C: 1/20 sample dilution; D: 1/30 sample dilution; E: 1/40 sample dilution; F: 1/50 sample dilution.
Fig 4.
Interpretation of the results.
The test is only valid if the migration control line is present (A and B). A positive test result is observed when two lines (test and control) are visible on the NC membrane (A). The test is considered negative when only the control line is present (B). When the migration control line is not present, the test is considered invalid and should be redone (C and D). NC, Nitrocellulose.
Fig 5.
Distribution of results from ELISA and the rSP03B sero-strip.
SOD (%) distribution of positive sera samples (experimentally exposed dogs) and negative sera samples (non-bitten dogs) tested by SGH-ELISA (A) and rSP03B-ELISA (B) is shown. The cut-off for each ELISA was calculated by the mean of non-bitten negative control sera +3SD. Results from the rSP03B sero-strip were classified according to the intensity of the observed band, starting from (0) a negative test result; (1) a very faint signal; (2) a low positive signal; (3) a positive but less intense signal than the control band and (4) a strong positive signal, same intensity as the control band. All samples classified in categories (1), (2), (3) and (4) were considered positive. Distribution of positive sera samples and negative sera samples is shown in (C). Correlation between SOD (%) values of SGH-ELISA and rSP03B-ELISA was performed using Pearson’s r correlation and is shown in (D). SD, Standard Deviation; SOD, Standardized Optical Density; SGH, Salivary Gland Homogenate; r, Correlation index; CI, Confidence Interval.
Table 1.
Sensitivity and specificity of each serological method (SGH-ELISA, rSP03B-ELISA and the rSP03B sero-strip).
Table 2.
Comparison of rSP03B-ELISA and rSP03B sero-strip with standard SGH-ELISA.