Antibody Visualization

We are only part way finished with our immunoassay detection system story. We now have an antibody to bind to our Bt protein in solution with other proteins (BT protein is illustrated by green hexagons in Fig 9), but we still are not able to “see” our Bt protein. The final step is the visualization process, which gives us the pink bands on the lateral flow strip (Figure 5) or the blue color in the tube and microwell formats (Figs 3, 4). 

Figure 9 Merely binding an antibody to a target protein is not enough for protein detection. It somehow must also be visualized.

Antibodies are ‘labeled’ with detectable substances to provide a means for visualizing and quantifying the presence of the target protein. They can be labeled with colored particles (e.g., colloidal gold, latex), enzymes, fluorescent molecules or chemiluminescent molecules (Fig 10). In the lateral flow strips formats described here (Fig.5), antibodies have been labeled with gold particles or enzymes which can catalyze reactions in the solution to give the color for the ELISA tests (Figs. 3,and 4). 

Figure 10 Examples of detectable substances used in antibodies.

To assist in separating the target protein from the other proteins in the sample, antibodies are purified and attached to a ‘solid phase’. Examples of solid phases are plastic wells, tubes, capillaries, membranes, and magnetic particles. For the lateral flow formats, the solid phases are the filters on the test strip. In the coated tube format, it would be the test tube. The lateral flow tests utilize a double antibody sandwich model (Fig 11). Using our ongoing case study of detecting the Bt protein, the 'target analyte' in Figure 11 would be our Bt protein. The 'capture antibody' would be the one which is attached to the filter on the test, the solid phase. The 'detector antibody' binds to a different site on the Bt protein than the capture antibody, and its role is to provide visualization.

Figure 11 Detection system used in lateral flow strips. The 'target analyte' is our Bt protein.