The presented protocol is used for high-throughput screening and identification of inhibitory and non-inhibitory nanobodies targeting electrogenic transporters reconstituted in proteoliposomes or present in membrane vesicles. Designing transfer assays can be difficult in multiple cases, especially if labeled substrates are not available. If the same electrophysiology allows studying the effect of nanobodies on transport for virtually any electrogenic transporter, since SSM electrophysiology doesn't require labeled substrates.
Nanobodies have been investigated for their usage in medical applications. This technique can help screen potential inhibitors targeting specific electrogenic transporters of humans or human pathogens. To begin, take 10 clean tubes and transfer 10 milliliters of the nonactivating solid-supported membrane, or SSM, buffer into each tube.
To prepare the activating SSM buffers, add the substrate into the tubes using a series of concentrations around the expected half maximum concentration. Start the SSM software and let the machine initialize automatically. Set the saving path for data.
Confirm by hitting the OK button. Select the standard initial cleaning protocol in the workflow options and click run. Next, mount the proteoliposome-coated chip on the socket.
Move the arm to lock the chip. Enclose the mounted chip with the cap. Select the program CapCom in the workflow, and let it run to determine the conductivity and capacitance.
Confirm that the conductivity is below five nanosiemens and the capacitance is between 15 and 35 nanofarad before using it for the measurement. Transfer the activating solutions into vials and position the buffers in the probe sampler. Transfer the nonactivating buffer into a reservoir and place it next to the chip holder at the reservoir position on the right.
Create a protocol for the workflow using a sequence of nonactivating, activating, and nonactivating solution sequence, or a BAB sequence, in a loop that performs three measurements and moves to the next activating buffer for all 10 buffers. Use the default flow rate of 200 microliters per second with one second, one second, one second flow times for the BAB sequence and click play to start the measurement. Save the protocol and let the workflow run by clicking on the play button.
Then perform the same experiment using protein-free liposomes. Use any preferred software for data analysis to plot the measured current versus time and use the function for peak height estimation in the range of the addition of the activating buffer. Plot the peak current against the substrate concentration to determine the half maximal effect of concentration, or EC50, of the substrate via nonlinear regression.
Transfer 50 milliliters of the nonactivating SSM buffer into a clean tube. Add the substrate choline to a final concentration of five millimolar and use this for a positive control measurement. Transfer 10 milliliters of the non activating SSM buffer into a clean tube.
Add substrate choline to five millimolar and nanobody to 500 nanomolar final concentration. Repeat the step for each nanobody to prepare the activating solutions as demonstrated. Start the SSM machine and measure the capacitance and conductivity of the proteoliposome-coated chip, as demonstrated earlier.
Transfer the activating solution without a nanobody into a vial and place the buffer in the probe sampler. Transfer the nonactivating buffer without a nanobody into a reservoir and position it in the probe sampler. Then repeat this process for all solutions containing nanobodies with activating and nonactivating solution.
Create a protocol for the workflow Using a BAB sequence. Create a loop that performs three measurements of the BAB sequence using buffers without nanobodies, two measurements of the BAB sequence with buffers containing a nanobody, 120 second delay time incubation with the nanobody, and three measurements of the BAB sequence with buffers containing the nanobody. Save the workflow and let it run by clicking the play button.
Next, create a new protocol for the workflow using a BAB sequence and a loop of five measurements to wash out the reversibly bound nano body. Save the workflow and let it run by clicking the play button. Compare the last peak current of the measurements with the initial substrate-only measurement.
If the peak current reaches the initial value, the nanobody has been successfully washed out and the initial conditions have been reestablished. Otherwise, repeat the workflow or change to a new chip. Repeat this process using individual chips for each nanobody screen, or repeat with multiple nanobodies using the same chip.
Use any preferred software for data analysis to plot the measured current versus time. Then the software automatically selects the function for peak height estimation in the range of the addition of the activation buffer. Normalize the peak current and the presence of the nanobody based on the proceeding substrate-only measurement.
Plot the peak currents in the histogram and compare the peak currents of the substrate-only measurements to the peak currents measured in the presence of nanobodies to identify inhibitory nanobodies. Transfer 50 milliliters of the nonactivating SSM buffer into a clean tube and add the substrate choline to a final concentration of five millimolar, and use this as the activating solution for positive control. Add five milliliters of the nonactivating solution to eight clean tubes, then add substrate choline and inhibitory nanobody to the tubes at concentrations in the expected IC50 range.
Add 10 milliliters of the nonactivating solution to the eight tubes and add inhibitory nanobody to each tube corresponding to the nonactivation buffer. Start the SSM setup and measure the capacitance and conductivity of the proteoliposome-coated chip. Transfer the activating solution without nanobody into a vial and place it in the probe sampler.
Then transfer the nonactivating buffer without nanobody into a reservoir and position it at the reservoir position next to the chip holder on the right. Transfer the activating and nonactivating solutions containing nanobodies into vials and position the buffers in the probe sampler. Create a protocol for the workflow using a BAB sequence.
Include a loop to measure each concentration twice, incubate for 120 seconds and measure three more times. Use any preferred software for data analysis to plot the measured current versus time and select the function for the peak height estimation in the range of the addition of the activation buffer. Plot the peak currents against the nanobody concentration to determine the IC50 via nonlinear regression.
For determining the substrate concentration to be used during a screening of nanobodies, electrogenic transport was measured under different substrate concentrations to determine EC50. A substrate concentration corresponding to saturating conditions, five millimolar, was selected and kept constant in all activating buffers. The effect of inhibitory nanobodies on electrogenic transport was visualized from the decrease in peak currents'amplitudes.
After running the washing protocol to allow nanobody unbinding, a recovery of 80 to 95%of the initial peak current amplitude was observed. When changing from nonactivating to activating conditions, no significant artifact currents were introduced by nanobodies present in these buffers. After selecting nanobodies with inhibitory properties, the IC50 values for individual nanobodies were determined.
It is crucial to give enough time for the calibration of the proteoliposomes on the chip with the buffer containing the nanobody. Since the binding is reversible, the nanobody needs to be present in both activating and nonactivating buffers.
