The overall goal of this procedure is to measure the relative potency of anti-TNF-alpha molecules. In this example, we compare two anti-TNF-alpha monoclonal antibodies in order to determine the neutralization efficiency of one reference mAb versus a sample mAb under investigation. This tutorial shows detailed steps for the determination of the TNF-alpha neutralization potency, which are not included in the literature.
Cells can be stressed by different environmental factors, for instance, starvation. Each cell has different proteins on its surface called receptors. Some of them are specific for the cytokine TNF-alpha.
When this molecule interacts with its receptor and a specific signal is developed, this signaling combined with an environmental stress factor undergo a programmed cell death called apoptosis. However, in the presence of an anti-TNF-alpha molecule which can be an monoclonal antibody, the cytokine can be titrated by the mAb, undergoing a neutralization process helping the cells to survive. Thus under standardized methodology, the neutralization strength of a molecule can be measured.
This protocol is currently performed during five days. On the first day, solutions for cell culturing must be prepared. Additionally, WEHI 164 cells will be thawed and subcultured.
On the second and the third day, confluency of cells must be verified. After this step, cells must be detached, counted, cell density adjusted, and finally, cells should be cultured again. All these steps must be performed each day.
At the fourth day, mAb concentration must be determined by UV absorption and antibody dilutions should be performed. Apoptosis induction solutions will be prepared. And finally, cell density of the WEHI 164 cells should be adjusted to a half million cells per mL.
At the fifth day, substrate solution for the detection of apoptotic cells will be prepared, and the spectrophotometric analysis performed. Finally, in silico analysis of the results will be discussed. All the solutions employed in this protocol are depicted in this slide.
At the first day, this solution must be prepared previously to the protocol execution with the exception of apoptosis induction and substrate solutions which must be used immediately after reconstitution. At the beginning of this protocol, frozen cells must be removed from the liquid nitrogen container. This vial with frozen WEHI cells must be kept on ice until use.
Keep your workplace clean and sanitized previously to execute any activity in order to avoid cross-contamination. Transfer the isotherm rack to the laminar flow hood and dispense nine mL of prewarmed culture medium into a 15-milliliter sterile tube. With one mL of prewarmed culture medium, wash WEHI 164 cells for gentle up-down pipetting until frozen cells detach from the microtube.
Mix by inversion five times until the frozen cells completely thaw. Then centrifuge cells at 125 g's for three minutes. Discard the supernatant and disaggregate the cell pellet.
Add five mL of the prewarmed culture medium to the tube and mix the suspension until the cells were completely resuspended. Recover cells into a T-culture flask. And incubate overnight at 37 Celsius and carbon dioxide at 5%Each day previously to detach, count cells of culturing, it is mandatory to verify culture health under microscope.
The researcher must verify cell health, viability and cell density for subculturing. This verification must be performed also for the biological assay. First, it must be verified, the absence of biological contamination, for example, bacteria.
Secondly, cell health can be verified by confluency. A confluency above 90%is enough to achieve good results. However, if cells have not reached the desired cell density, the flask must be returned to the incubator and wait until the cells have reached the desired cell density and confluency.
Aged culture medium must be removed from the T flask. Wash attached cells on the T flask walls with five mL of the cell wash solution. Take care of not removing attached cells.
Mix manually to eliminate culture medium residues and cell debris. This step must be executed twice. Remove residual wash solutions using vacuum and add three mL of the cell detachment solution.
Agitate and incubate during three minutes. Once incubation is finished, add prewarmed culture medium to the T flask in order to inactive the enzyme. And recover cells.
Transfer the cell suspension to a 15-mL tube and centrifuge. Identify the cell pellet and discard the supernatant. Removing aged culture medium is critical for the cultivation of healthy cells.
Disaggregate the cell pellet and reconstitute cells with five mL of the prewarmed culture medium. Transfer 50 microliters of the cell suspension to a microtube. Count cells using the trypan blue exclusion method.
Count cells and detemine cell viability. Once having the number of viable cells, adjust 15 mL of the culture medium to a cell density of a half million cells per mL using the following formula. Transfer 15 mL of the adjusted cell suspension in a T-culture flask and incubate overnight.
This overall culture process must be repeated at least one more time for a total of three cell passages before beginning the neutralization assay. At the fourth day, measure optical density of reference substance, analytical sample and control sample by UV absorption at 280 nanometers. Perform an initial dilution to two milligrams per mL with assay culture medium for each samples by independent triplicates.
Execute a second dilution down to two micrograms per mL for each mAb sample. Dispense 75 microliters of the assay culture medium to the 60 wells present in the middle of a polystyrene microplate. Add 230 microliters of reference substance in the wells 2B and 2C.
Analytical samples in the wells, 2D and 2E. And finally, the control sample in the wells, 2F and 2G. A control sample is a molecule that has a known relative potency.
Then make serial dilutions into the microplate. An example is depicted in following slides. Remember to mix solutions previously to dispense into the wells.
This operation is critical for the success of this protocol. Adjust previously to a half million cells per mL to a dispenser. Add 50 microliters of cell suspension to the internal 60 wells of the assay plate starting from the column two to the 11th.
Mix cell suspension previously to add liquid to the microplate. Immediately transfer 50 microliters of the mAb dilutions to the assay plate. For paired lanes, dispense the dilutions by turning around the micropipette.
The mAb dilutions can be dispensed into the wells in the following order, controls dispensed in the surrounding wells. Dilute TNF-alpha with 500 microliters of ultrapure water. Mix by vortex mixer at 2200 rpms.
Transfer to a 15-mL tube and finally, dilute to 40 nanograms per mL with assay culture medium. Dispense 50 microliters of the apoptosis induction solution into the 60 internal assay wells. Dispense 50 microliters of the apoptosis induction solution into the the 60 internal assay wells.
Finally, incubate for 16 hours. The fifth day, reconstitute substrate with this buffer solution as directed in the manufacturer's instructions. Mix by inverting five times.
Dispense substrate solution. And add 50 microliters of the substrate solution to each sample well in the assay plate. Also add substrate to controls.
Agitate the assay plate in a vortex mixer at 400 rpms for three minutes. Take out the assay plate from the mixer and incubate at room temperature. Turn on a plate reader-spectrophotometer.
Open the SoftMax Pro software or any equivalent program. Load the microplate into the spectrophotometer and adjust the following parameters. Activate the luminescence function and the Read Type into Endpoint function.
Adjust the Read Area, excluding columns one and 12. Select the Plate Type to 96 Well Standard clear bottom microplate. Alter the Integration Time up to 1250 milliseconds.
Adjust the shake time for the microplate to 10 seconds. Select the well in which Reference Substance, Analytic Substance and Control Sample will be placed. Begin to read.
The results in this assay are those response curves expressed in luminescence compared to the log scales of the mAb concentration. Inflection points of these curves are the effective concentrations, EC50 for each mAb. C or EC50 is expressed in nanograms per mL.
However this value has no meaning by itself, if it's not compared to the reference sample. Thus, potency of an analytical sample is expressed in percentage and called relative potency. The relative potency is the average of three independent microplate results which comes accordingly from the EC50 value from each plate.
Comparison between control samples and reference samples helps to determine any deviation or bias in the current assay. Comparison of reference sample with an analytical sample helps to determine the relative potency. This table shows the relative potency of the mAbs and its confidence interval at 95%Also it displays the relative standard deviation from each sample related to potency of the reference substance.
Relative standard deviation above 15%is considered not acceptable. This characterization helps to determine apriori, the biotic behavior of a molecule that is under development before conducting expensive and time-consuming clinical trials. Also it useful for batch-to-batch release of an approved product.
It is our determination that these assays are helpful to determine if a molecule has an adequate biological effect regarding to its mechanism of action. As a part of biophysical methods, this methodology is able to determine by biological means the potency and the efficacy of a drug's quality and attributes thus showing a complete and full significance of its effector functions.
