3.3 Results
3.3.5 Antibody preparation and ELISA optimisation for TvOPB
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.
3.3.5 Antibody preparation and ELISA optimisation for
Figure 3.17: ELISA of anti-TvOPB IgY antibodies isolated from the egg yolks of immunised chickens. ELISA plates were coated with TvOPB (1 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TvOPB IgY from chickens 1 to 3, weeks 1 to 11 (100 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 45 min development.
Due to the poor results for the antibody production of anti-TvOPB IgY seen in Fig. 3.17, the ELISA was repeated using a higher TvOPB coating concentration of 5 µg/ml in carbonate coating buffer, pH 9.6 (CCB), instead of PBS. This change in coating conditions resulted in a marked increase in the absorbance values at 405 nm especially for the antibodies produced by chicken 2 and in a shorter development time (Fig. 3.18). It appeared that chicken 1 produced less anti-TvOPB IgY antibodies when compared to chickens 2 and 3, with the absorbance values at 405 nm being less than those obtained from the pre-immune IgY antibodies, which were still abnormally high.
Figure 3.18: ELISA of anti-TvOPB IgY antibodies isolated from the egg yolks of immunised chickens using a different coating diluent. ELISA plates were coated with TvOPB (5 μg/ml in CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TvOPB IgY from chickens 1 to 3, weeks 1 to 12 (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.
Further optimisation of the TvOPB coating conditions needed to be performed. Using anti-TvOPB IgY antibodies from chicken 3, weeks 6 and 10, which gave reasonable absorbance values and were mid range between those from chicken 3 and chicken 2, as seen in Fig. 3.18, an optimisation ELISA was performed. From Fig. 3.19, coating at 5 μg/ml TvOPB in CCB, pH 9.6, gave the highest absorbance values at 405 nm after 45 min of development for the anti-TvOPB IgY weeks 6 and 10, but also for its pre-immune IgY. This highlighted the need to obtain a new pre-immune IgY.
Figure 3.19: Optimisation of ELISA of TvOPB coating concentrations with anti-TvOPB IgY antibodies. ELISA plates were coated with TvOPB (5 and 1 μg/ml in PBS, pH 7.4 and CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TvOPB IgY from chicken 3, pre-immune and weeks 6 and 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 45 min development.
Using the TvOPB coating conditions determined from Fig. 3.19, the concentration of the secondary detection antibody, rabbit anti-chicken IgY HRPO conjugate, was optimised. Anti-TvOPB IgY was isolated from each of the weeks from all three chickens and along with 1:15 000 and 1:20 000 dilutions of the secondary antibody, were used to confirm the trend in antibody production as seen in Fig. 3.17.
With a 1:20 000 dilution of the secondary rabbit anti-chicken IgY HRPO conjugate (Fig. 3.20, panel A), lower absorbance values at 405 nm were obtained when compared to those resulting from the 1:15 000 dilution of the secondary antibody (Fig. 3.20, panel B). Furthermore, the 1:15 000 secondary antibody dilution diminished the intensity of the fluctuations between each week as compared to those seen when a 1:20 000 dilution was used. In addition, when comparing the trend of antibody production for each chicken shown in Fig. 3.20 to those seen in Fig. 3.18, chicken 3 now seemed to have produced the least amount of anti-TvOPB IgY antibodies instead of chicken 1.
Figure 3.20: Optimisation of secondary antibody in the ELISA of TvOPB against anti-TvOPB IgY antibodies. ELISA plates were coated with TvOPB (5 μg/ml in CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TvOPB IgY from chickens 1 to 3, weeks 4 to 14 (100 μg/ml). Rabbit anti-chicken IgY HRPO secondary antibody (1:20 000 and 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 45 min development. Panel A depicts the 1:15 000 dilution whilst panel B depicts the 1:20 000 dilution.
From the previous optimisation ELISAs, the optimal TvOPB coating concentration, diluent and secondary detection antibody concentration had been determined. Using the anti-TvOPB IgY from chicken 2, week 8, which gave the highest absorbance value at 405 nm with a 1:15 000 secondary antibody dilution, a checkerboard ELISA was performed for further optimisation. A no coat control was included for each of the samples to account for any background interference which may have occurred. The corrected absorbance values at 405 nm were below 0.1 after 45 min of development;
however the highest corrected absorbance values at 405 nm and thus optimal conditions for future inhibition and indirect ELISA formats were found at a coating concentration of 1 μg/ml TvOPB and between 1 and 5 µg/ml anti-TvOPB IgY antibodies (Fig. 3.21).
Figure 3.21: Checkerboard ELISA of TvOPB coating and anti-TvOPB IgY antibody concentrations. ELISA plates were coated with TvOPB (1, 0.5 and 0.1 μg/ml in CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS and incubated with anti-TvOPB IgY from chicken 2, week 8 (50, 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 45 min development.
Panel A1 is the expanded view of the 0.1 to 1 μg/ml anti-TvOPB IgY concentrations.
Investigation into the optimisation of the blocking step was performed using four different blocking agents: (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. A no coat control was included for each of the samples to account for any background interference which may have occurred. From Fig. 3.22, no significant difference was evident in panels A to C, with panel D showing the same trend but at lower corrected absorbance values at 405 nm. In order to standardise the blocking reagent amongst the different ELISA antigens, the 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 was chosen as the optimum blocking buffer as was the case for the TcOPB antigen.
Figure 3.22: Checkerboard ELISA of TvOPB and anti-TvOPB IgY antibody titrations using different blocking agents. ELISA plates were coated with TvOPB (2.5 and 1 μg/ml in CCB, pH 9.6) 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-TvOPB IgY from chicken 2, week 8 (50, 10 and 1 μg/ml) were subsequently added. 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.
A new pre-immune IgY antibody was isolated from an egg from a non-immunised chicken and used in a checkerboard ELISA together with the previously optimised TvOPB ELISA conditions. A no coat control was included for each of the samples to account for any background interference which may have occurred. Even with an increased coating concentration of 6 µg/ml TvOPB, Fig. 3.23, panel A, showed low to negative corrected absorbance values at 405 nm. Low absorbance values at 405 nm,
which were not corrected, were still evident after 60 min of development (Fig. 3.23, panel B).
Figure 3.23: Checkerboard ELISA of TvOPB and different anti-TvOPB IgY antibody concentrations. ELISA plates were coated with TvOPB (6 μg/ml in CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 and incubated with anti-TvOPB IgY from chicken 1, week 7 and chicken 2, weeks 8 and 9 (20, 10, 5, 2.5, 1.25 and 0.625 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 A shows where the no coat control was subtracted from the absorbance readings and panel B shows were the no coat control was ignored.
From the poor ELISA results obtained even after extensive optimisation steps, it was thought that the amount of antigen used for immunisation, 50 µg/immunisation, was not sufficient to elicit an immune response in the chicken. A new immunisation schedule was implemented using 50 and 100 μg/immunisation antigen per chicken.
Anti-TvOPB IgY antibodies were isolated from a single egg from each chicken at each week during the new immunisation schedule and used in an ELISA shown in Fig. 3.24.
A no coat control was included for each of the samples to account for any background interference which may have occurred. In Fig. 3.24 the production of anti-TvOPB IgY production in chickens 1 and 2 is depicted, of which both were immunised with 50 μg/immunisation TvOPB. The corrected (Panel A) and non corrected (Panel B) absorbance values at 405 nm of the anti-TvOPB IgY from each of the weeks were lower than that of the new pre-immune IgY antibody, only chicken 2 had a single corrected absorbance value at 405 nm at week 9 which was above the pre-immune IgY antibody (Panel B). Panels C and D from Fig. 3.24, depict the anti-TvOPB IgY production in chickens 3 and 4, which were both immunised with 100 μg/immunisation TvOPB. These chickens seem to have elicited a higher response than that seen in chickens 1 and 2, with chicken 3 producing the highest amount of anti-TvOPB IgY from weeks 4 to 10, even after the absorbance values at 405 nm had been corrected (Panel C). In addition, the antibodies from chicken 3, weeks 3 to 10 were the only
weeks which the corrected absorbance values at 405 nm were higher than those of the pre-immune IgY antibody.
Figure 3.24: ELISA of anti-TvOPB IgY antibodies isolated from the egg yolks of chickens immunised with 50 and 100 μg/immunisation of TvOPB. ELISA plates were coated with TvOPB (2.5 μg/ml in CCB, pH 9.6), blocked with 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 and incubated with anti-TvOPB IgY from (A and B) chickens 1 and 2 immunised with 50 µg/immunisation and (C and D) chickens 3 and 4 immunised with 100 µg/immunisation, weeks 1 to 15. Rabbit anti-chicken IgY HRPO secondary antibody (1:30 000) 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. Panels A and C show where the no coat control was subtracted from the absorbance readings. Panels B and D show were the no coat control was ignored.