Team:Queens Canada/Demonstrate

Overview

We successfully produced a fluorescently tagged recombinant antibody fragment that was able to bind to tetrahydrocannbinol (THC). To test the antibodies' ability to bind THC we developed a novel one-step immunoassay (Protocol: Novel Immunoassay for Lipophilic Antigens). The protocol uses a lipophilic membrane to isolate THC, and relies on the antibody for the specificity (Fig. 1). To determine the validity of the assay, we first tested it with commercially available anti-THC antibodies, with a fluorescent secondary antibody (Fig. 2).

Figure 1. Explanation of the theory behind the novel immunoassay for THC detection. Note that this concept could be adapted to other lipophilic antigens.

Figure 2. THC membrane assay using commercial goat anti-THC and varying concentrations of THC. The signal was detected via an anti-goat fluorescent secondary antibody.

THC Binding Limit

We examined the detection limit of the anti-THC ScFv-mNG via THC membrane assay. The antibody signal was distinguishable from the background down to 0.1 mg/mL of THC (Fig. 3.) At 0.1 mg/mL, the fluorescent intensity was ~3x as bright as the CTRL, which did not contain any THC (Fig. 4).

Figure 3. THC membrane assay using ScFv-mNG and varying concentrations of THC. A) The signal in 0.1 mg/mL is distinguishable from the control (CTRL), indicating that the sensitivity is below 0.1 mg/mL. B) The signal in 0.05 mg/mL is not distinguishable from the control (CTRL). Therefore, the detection limit is between 0.1 and 0.05 mg/mL.

Figure 4. Fluorescent intensity of the membrane assay using ScFv-mNG and varying concentrations of THC. A) The signal in 0.1 mg/mL is distinguishable from the control (CTRL), indicating that the sensitivity is below 0.1 mg/mL. B) The signal in 0.05 mg/mL is not distinguishable from the control (CTRL). Therefore, the detection limit is between 0.1 and 0.05 mg/mL.

Hardware integration

The designed circuit was tested in darkroom conditions with different concentrations of GFP samples, the results of which can be seen below.

Taking the average of the data, it can be shown that the concentration data and the level of illuminance had a somewhat linear relationship.

Future Directions

The recombinant antibody was able to detect THC on a lipophilic membrane, within 30 minutes, down to a concentration of 0.1 mg/mL of THC. However, it seems that this assay is limited by the lipophilic membranes. Our team used PIG® Oil-Only Absorbent Mats, which are designed for cleaning up oil spills and hence are not manufactured uniformly. Future studies may aim at testing a large library of membranes for this test in order to absorb THC uniformly, and ultimately increase the sensitivity of this assay. The sample data collected using the prototype circuit could have been affected by numerous factors such as light emitted by the nearby laptop, inconsistent distance to the sensor, and differing quantities of the GFP sample itself. Future testing would be done more rigorously, and this example was just to demonstrate the functionality of the circuit.