**2.4 Standardization of TcISP2 enzyme-linked immunosorbent assay (ELISA)**

To perform the indirect ELISA test for antibodies against TcISP2r, the binding efficiency of the recombinant protein was initially verified in 96-well plates with low, medium, and high binding capacity (**Figure 2**). Each of the plaque types was sensitized with 1.0, 2.0, and 5 μg of TcISP2r protein diluted in 0.2 M


## **Figure 2.**

*Indirect ELISA to standardize the ligation of TcISP2r in 96 wells plates with different binding capabilities using anti-histidine antibody. TcISP2r histidine tailed protein, purified by histidine affinity chromatography, were ligated to plates of high, medium, and low binding capabilities as indicated in the figure. Columns 1 to 4 contain 1 ug of protein per well; 5 to 8, 2 ug; and 9 to 12, 5 ug. Columns 1, 5, and 9 started with 1 ug of anti-his antibody; columns 2, 6 e 10, started with 0,1 ug of anti-his antibody; colunms 3, 7, and 11, started with 0,05 ug of anti-his antibody; in columns 4, 8, and 12 no anti-his antibody were used. From a to H, anti-his antibodies were serially diluted in order of 2 (1:2; 1:4; 1:6; 1:8; 1:10; 1:12: 1:14; 1:16).*

carbonate-bicarbonate buffer, pH 9.2. The plates were filled with 100 μL of buffer containing 1, 2, or 5 μg of protein and kept in the refrigerator for 16 hours. Subsequently, the plates were washed with 20 mM of phosphate buffer pH 7.4 and 0.05% of tween 20 and subsequently incubated with blocking solution (5% skim milk, 20 mM of phosphate buffer pH 7.4 and 0, 05% tween 20) for 30 minutes. Then, the blocking solution was removed and different dilutions of the anti-histidine monoclonal antibody (1 ug, 0.1 ug, and 0.05 ug/mL) produced in mice (Invitrogen) in 50 uL of blocking solution were added per well. Serial dilutions in the order of 2 of the anti-histidine antibodies were also made in columns A to H, followed by incubation for 2 hours at room temperature. After washing three times with 20 mM of phosphate buffer pH 7.4 and 0.05% tween 20, 0.1 ug/mL of the anti-mouse IgG second antibody, bound with alkaline phosphatase, was added to 50 uL of blocking solution and incubated for 1 hour at room temperature. The plate was then washed 3 times with 20 mM phosphate buffer pH 7.4 and 0.05% tween 20 and 2 times with 20 mM phosphate buffer pH 7.4 without tween 20. Reactions were developed with PNPP substrate for alkaline phosphatase (Invitrogen), according to the manufacturer's instructions and the plates were incubated for 30 minutes at room temperature and read in a spectrophotometer at a wavelength of 405 nm.

ELISA tests were performed in 96-well plates with medium binding capacity and three concentrations of TcISP2r protein (2, 5, and 10 μg). Recombinant protein binding was carried out for 16 hours in 100 uL of 0.1 M carbonate-bicarbonate buffer pH 9.2, in the refrigerator. Subsequently, the plates were washed with 20 mM of

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phosphate buffer pH 7.4 and 0.05% of tween 20 and subsequently incubated with blocking solution (5% skim milk, 20 mM of phosphate buffer pH 7.4 and 0, 05% tween 20) for 30 minutes. Then, the blocking solution was removed and 50 uL of serial dilutions of serum from 3 mice collected at 7-, 15-, and 60-days post-infection were added to the wells and the plate incubated for 2 hours at room temperature. After three washes with 20 mM of phosphate buffer pH 7.4 and 0.05% tween 20, 0.1 ug/mL of the anti-mouse IgG second antibody, bound with alkaline phosphatase, was added to 50 uL of blocking solution and incubated for 1 hour at room temperature. The plate was then washed 3 times with 20 mM phosphate buffer pH 7.4 and 0.05% tween 20 and 2 times with 20 mM phosphate buffer pH 7.4 without tween 20. Reactions were developed with PNPP substrate for alkaline phosphatase (Invitrogen), according to the manufacturer's instructions and the plates were incubated for 30 minutes at room temperature and read in a spectrophotometer at a wavelength of 405 nm.
