Inhibition TcCATL FL ELISA

4.3 Results

4.3.2 Optimisation of inhibition and indirect TcPGP ELISA with Trypanosoma

4.3.3.1 Inhibition TcCATL FL ELISA

Using the optimised TcCATL_FL coating of 0.05 µg/ml in PBS and 0.5 µg/ml affinity purified anti-TcCATLFL IgY from chicken 3, week 6 as described in section 3.3.8, a trial inhibition ELISA was performed with infected and non-infected serum samples. The corrected absorbance values at 405 nm were slightly higher for the non-infected serum samples than for the infected serum samples (Appendix C, Fig. C.5). Upon the calculation of the percentage inhibition values, with the exception of sera 48, 163, 190 and 200, the TcCATLFL inhibition ELISA was able to discriminate between infected and non-infected serum samples (Fig. 4.9).

Figure 4.9: Inhibition TcCATL_FL ELISA using affinity purified anti-TcCATL_FL IgY antibodies with infected and non-infected sera. ELISA plates were coated with TcCATLFL (0.05 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS and incubated with sera (1:10 dilution). Thereafter affinity purified anti-TcCATLFL IgY from chicken 3, week 6 (0.5 μg/ml) was added. 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 triplicate experiments after 60 min development. Each plate had a no serum control in quadruplicate.

The results presented in Fig. 4.9 confirmed that the optimised TcCATL_FL coating and anti-TcCATLFL IgY concentrations were suitable for the inhibition ELISA format and were used to test 82 infected and 29 non-infected serum samples. The corrected absorbance values at 405 nm are found in Appendix C, Fig. C.6. With the exception of the non-infected serum samples 48, 81, 151, 178, 179, 181, 186, 193, 197 and 214 which gave abnormally high percentage inhibition values, the percentage inhibition for the infected sera samples were higher than those for their respective non-infected serum samples (Fig. 4.10).

Figure 4.10: Mass inhibition TcCATL_FL ELISA using affinity purified anti-TcCATL_FL IgY antibodies with infected and non-infected sera. ELISA plates were coated with TcCATLFL (0.05 μg/ml in PBS, pH 7.4), blocked with 0.5% (w/v) BSA-PBS and incubated with sera (1:10 dilution).

Thereafter affinity purified anti-TcCATLFL IgY primary antibodies from chicken 3, week 6 (0.5 μg/ml) were 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 triplicate experiments after 60 min development. Each plate had a no serum control in quadruplicate.

Following the abnormally high percentage inhibition results for some of the non-infected sera seen in Fig. 4.10, non-specific binding might have been the cause.

Optimisation of the blocking buffer was performed by testing the four different buffers against non-infected and infected serum samples from the same test animal , 149 (-7), 149 (+42) and 149 (+49) using: 0.5% (w/v) BSA-PBS (BP); 0.5% (w/v) BSA-PBS, 0.1% (v/v) Tween-20 (BPT); 0.2% (w/v) BSA-PBS, 0.05% (v/v) Tween-20 (BPTT) and 1% (v/v) horse serum-PBS (HSP).

The corrected absorbance values at 405 nm for the non-infected serum samples were lower than those for the infected serum samples, with infected serum 149 (+49) having almost the same values as the no serum control (Appendix C, Fig. C.7). This trend was evident with each blocking buffer. Using the BPT blocking buffer, the highest corrected absorbance values at 405 nm were obtained, followed by BP, HSP and BPTT. Seen in Fig. 4.11, all four blocking buffers resulted in percentage inhibition values which are higher for the non-infected serum samples than for the infected serum samples. The BPT buffer, however, resulted in the smallest difference in percentage inhibition values between the non-infected and the infected serum samples and was the most suitable buffer to use in further optimisation of the TcCATL_FL inhibition ELISA.

Figure 4.11: Inhibition TcCATL_FL ELISA using affinity purified anti-TcCATL_FL IgY antibodies in different blocking buffers with infected and non-infected sera.

ELISA plates were coated with TcCATLFL (0.05 μg/ml in PBS, pH 7.4), 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 and incubated with sera (1:10 dilution). Thereafter affinity purified anti-TcCATLFL IgY from chicken 3, week 6 (0.5 μg/ml) was 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 triplicate experiments after 60 min development. Each plate had a no serum control in quadruplicate.

Along with the new BPT blocking buffer, the ability of the crude and affinity purified anti-TcCATLFL antibodies to discriminate between infected and non-infected serum

samples was compared. The crude anti-TcCATLFL IgY antibodies resulted in higher corrected absorbance values at 405 nm than those for the affinity purified anti-TcCATL_FL IgY but followed the same trend (Appendix C. Fig. C.8). The affinity purified anti-TcCATLFL IgY yielded lower percentage inhibtion values for the non-infected serum samples along with higher values for the infected samples when compared to that obtained with the crude anti-TcCATL_FL IgY (Fig. 4.12). This was expected as the crude anti-TcCATLFL IgY antibodies were able to react with other proteins which may have been present in the serum, resulting in higher absorbance values and subsequently higher percentage inhibition values.

The optimal conditions for the TcCATL_FL inhibition ELISA format for the blinded serum panel was found to be coating with TcCATLFL at 0.05 µg/ml in PBS, blocking with BPT, using affinity purified anti-TcTcCATL_FL IgY from chicken 3, week 8 pool at 0.1 µg/ml and rabbit-chicken IgY HRPO conjugate secondary antibody at a 1:15 000 dilution.

Figure 4.12: Inhibition TcCATLFL ELISA using crude and affinity purified anti-TcCATLFL IgY antibodies with infected and non-infected sera. ELISA plates were coated with TcCATLFL (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 sera (1:10 dilution). Thereafter affinity purified anti-TcCATLFL IgY primary from chicken 3, week 6 (0.1 μg/ml) and crude anti-TcCATLFL IgY from chicken 3, week 8 (10 μg/ml) were 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 triplicate experiments after 45 min development.

Each plate had a no serum control in quadruplicate.