Detection of Trypanosoma congolense antigen by Nanobody sandwich ELISA

2022-11-04

Detection of Trypanosoma congolense antigen by Nanobody sandwich ELISA 

The ELISA plate's wells were coated with a nanobody specific to T. congolense antigen (Nb474-His) overnight.

The next day, wells are washed and blocked with a milk solution to prevent non-specific binding. Another round of washing follows to ensure cleanliness.

A 2-fold serial dilution of the positive control (T. congolense aldolase) and serum samples from infected animals were prepared.

Positive control samples were added to specific rows, and test samples from potentially infected patients were added to designated rows. Negative control (non-infected serum) goes into separate rows.

Incubation and Detection

After incubation, the plate is washed, and a second nanobody (Nb474-HA) was added to bind the antigen.

Following further washing, a mouse anti-HA antibody was applied, which will detect the HA-tagged nanobody.

Signal Amplification

After incubation with the mouse antibody, goat anti-mouse IgG HRP was added to enable colorimetric detection.

TMB substrate was then added, resulting in a color change that indicates the presence of the antigen. The reaction was stopped with an acid solution.

Reading Results

The optical density (OD) was measured to quantify the antigen level, with higher absorbance indicating a positive reaction.

This assay provides a sensitive and specific method to detect T. congolense antigen, allowing the differentiation between positive, negative, and test samples based on the colorimetric signal.

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