3.3 Results
3.3.4 Antibody preparation and ELISA optimisation for TcOPB
Figure 3.12: Purification of recombinant TcCATL using a HiPrep™ 16/16 Sephacryl™ S200 HR molecular exclusion resin. (A) Elution profile of TcCATL on a HiPrep™ 16/16 Sephacryl™ S200 HR resin (16 x 600 mm, flow rate 0.5 min/min, 0.08 MPa), previously equilibrated with MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and eluted using MEC buffer. Fractions were monitored by recording the absorbance at 280 nm. (B) Samples of the eluted fractions were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were visualised using silver staining.
3.3.4 Antibody preparation and ELISA optimisation for
Figure 3.13: ELISA of anti-TcOPB IgY antibodies isolated from the egg yolks of immunised chickens. ELISA plates were coated with TcOPB (1 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TcOPB IgY from chickens 1 to 3, weeks 1 to 10 (100 μg/ml). Rabbit anti-chicken IgY HRPO secondary antibody (1:20 000) and ABTS∙H2O2 were used as the detection system. The absorbance readings at 405 nm represent the average of duplicate experiments after 15 min development.
A checkerboard ELISA was employed for the optimisation of the TcOPB coating and the anti-TcOPB IgY concentrations as described by Crowther (2000). A no coat control was included for each of the samples to account for any background interference which may have occurred. The anti-TcOPB IgY antibody from chicken 1 at week 7 gave the best mid range absorbance at 405 nm and was used in the checkerboard ELISA. As shown in Fig. 3.14, coating with 0.1 and 0.05 μg/ml TcOPB and using 10 µg/ml anti-TcOPB IgY as the primary antibody, gave the highest corrected absorbance readings at 405 nm. Using these concentrations, the effect of different blocking buffers was investigated.
Figure 3.14: Checkerboard ELISA of TcOPB coating and anti-TcOPB IgY antibody concentrations. ELISA plates were coated with TcOPB (0.1, 0.05 and 0.025 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TcOPB IgY from chicken 1, week 7 (50, 10, 5, 2.5, 1, 0.5, 0.25 and 0.1 μg/). Rabbit anti-chicken IgY HRPO secondary antibody (1:20 000) and ABTS∙H2O2 were used as the detection system. The absorbance readings at 405 nm represent the average of duplicate experiments after 60 min development.
Panel A1 is the expanded view of the 0.1 to 1 μg/ml anti-TcOPB IgY concentrations.
A variety of blocking buffers have been reported for ELISA diagnostics, 1% (w/v) casein-PBS (Tran et al., 2009), 0.5% (v/v) mouse serum-PBS (Eisler et al., 1998), 5% (w/v) skim milk powder-PBS, 0.1% (v/v) Tween-20 (OIE, 2013) to name but a few.
Thus four different blocking buffers were tested: (A) 0.5% (w/v) BSA-PBS, (B) 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20, (C) 0.2% (w/v) BSA-PBS, 0.05% (v/v) Tween-20 and (D) 1% (v/v) horse serum-PBS. The blocking step is essential to prevent any non-specific binding of the subsequent reagents to the unoccupied hydrophobic sites in the plate well. Detergents and proteins may be used in combination for this purpose as both have their unique advantages and disadvantages (KPL, 2013). A no coat control was included for each of the samples to account for any background interference which may have occurred. From Fig. 3.15, the trends in the corrected absorbance values were similar for panels B and C with low values being obtained in panel D. Using 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 as the blocking buffer (Fig. 3.15, panel B), higher corrected absorbance values at 405 nm were obtained at 1 µg/ml anti-TcOPB IgY antibody than that obtained when 0.5% (w/v) BSA-PBS was used (Fig 3.15, panel A). Since low concentrations of primary antibody are used for ELISAs, it follows that blocking with 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 is optimal and was chosen as the standardised blocking reagent amongst the different ELISA antigens.
Figure 3.15: Checkerboard ELISA of TcOPB and anti-TcOPB IgY antibody concentrations using different blocking agents. ELISA plates were coated with TcOPB (0.1 and 0.05 μg/ml in PBS, pH 7.4) and blocked with (A) 0.5% (w/v) BSA-PBS, (B) 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20, (C) 0.2% (w/v) BSA-PBS, 0.05% (v/v) Tween-20 and (D) 1% (v/v) horse serum-PBS. Anti-TcOPB IgY from chicken 1, week 7 (50, 10 and 1 μg/ml) were subsequently added. Rabbit anti-chicken IgY HRPO secondary antibody (1:10 000) and ABTS∙H2O2 were used as the detection system. The absorbance readings at 405 nm represent the average of duplicate experiments after 60 min development.
A checkerboard ELISA was repeated using higher TcOPB coating concentrations along with the new blocking buffer, 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20. A no coat control was included for each of the samples to account for any background interference which may have occurred. The results were compared to those shown in Fig. 3.14, at 60 min of development, in which 0.5% (w/v) BSA-PBS was used as the blocking buffer. From Fig. 3.16 below, it was evident that the new blocking buffer resulted in higher corrected absorbance values at 405 nm, when comparing the 0.1 μg/ml TcOPB coating, at an anti-TcOPB IgY antibody concentration of 1 μg/ml, from an absorbance value of 0.07 to > 0.1. It was thus determined that coating with TcOPB at 1 µg/ml in PBS, blocking with 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 and anti-TcOPB IgY at 1 µg/ml were the optimal conditions for future inhibition and indirect ELISA formats.
Figure 3.16: Checkerboard ELISA of a range of TcOPB coating and anti-TcOPB IgY antibody concentrations. ELISA plates were coated with TcOPB (2, 1.5, 1, 0.5, 0.1 and 0.05 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 and incubated with anti-TcOPB IgY from chicken 1, week 7 (10, 5, 2.5, 1, 0.5, 0.25 and 0.1 μg/ml).
Rabbit anti-chicken IgY HRPO secondary antibody (1:15 000) and ABTS∙H2O2 were used as the detection system. The absorbance readings at 405 nm represent the average of duplicate experiments after 60 min development. Panel A1 is the expanded view of the 0.1 to 1 μg/ml anti-TcOPB IgY concentrations.