We utilized synthetic biology techniques to express a newly discovered dual target nanobody in E. coli EcN expression using nanomaterials to effectively and thoroughly block the TNF signaling pathway in inflammatory intestinal inflammation, achieving efficient, safe, convenient, and low-cost treatment of intestinal inflammation. Throughout the process, we encountered two experiments that needed to be quantified:
1. ELISA method for detecting the blocking ability of TNFa binding to TNFR (There are currently no reagent kits available on the market)
2. Blocking ability of TNFa induced U937 apoptosis
Measurement Background
TNFa naturally binds to TNFR on the cell membrane, triggering the TNF signaling pathway. When nanobodies are co incubated with human TNFa protein, anti TNFa nanobody of Ozoralizumab (A) and anti TNFa nanobody of V565 (B) can specifically bind to human TNFa, interfering with the natural binding between TNFa and TNFR on the cell membrane (Figure 1).
Figure 1: TNFα signaling pathway [1]
Measurement Principle
TNFa and TNFR naturally bind to each other, producing a pro-inflammatory TNF signaling pathway. When TNFR is fixed at the bottom of the tablet, adding human TNFa binds to TNFR at the bottom of the tablet.
Therefore, TNFa is captured at the bottom of the plate, and HRP enzyme conjugated anti TNFa antibodies are added after elution. At this time, a human TNFa antibody HRP enzyme conjugated TNFa antibody complex is formed at the bottom of the plate. The addition of HRP enzyme substrate can cause color development, and the strength of the color development indicates the amount of the complex, that is, the amount of TNFa bound to TNFR (Figure 2).
We used ELISA to detect the ability of anti TNFa nanobody of Ozoralizumab (A) and anti TNFa nanobody of V565 (B) to block the binding of TNFa to TNFR. The principle is that A/B can specifically bind to human TNFa protein and block its natural binding ability to TNFR.
Figure 2. Principle of ELISA for detecting the blocking ability of TNFa binding to TNFR
Measurement Protocols
Control：
Blank control: PBS
Positive control:Posi+antibody
Materials:
Protein samples,THF-a, HRP, Bradford reagent, Clear, flat-bottom 96-well microplate
Microplate reader, Pipettes and tips, Distilled water
Procedures:
Step 1: Prepare 1x wash solution by diluting 20x wash solution with distilled water at a ratio of 2:38.
Step 2: Prepare 1x assay buffer by diluting 10x buffer with distilled water at a ratio of 1:10.
Step 3: Prepare the detection antibody working solution by diluting 1x antibody working solution with distilled water at a ratio of 1:10.
Step 4: Dilute the sample with 200 µl of 1x assay buffer.
Step 5: Centrifuge at 10,000g for 1 minute and let it stand for 10 minutes.
Step 6: Take 230 µl of reconstituted standard, add 230 µl of standard diluent, and perform a 1:1 dilution using the high concentration standard.
Step 7: Take 230 µl of reconstituted standard, add 230 µl of cell culture medium, and perform a 1:1 dilution using the high concentration standard.
Step 8: Detach any unnecessary strips from the plate and reseal them to avoid contact with the inner surface of the microwells.
Step 9: Add 300 µl of 1x wash solution to each well on the ELISA plate and let it soak for 30 seconds.
Step 10: Discard the wash solution and blot the microwell plate dry on absorbent paper. After washing, do not let the microwell plate dry out.
Step 11: Add 100 µl of 2-fold serially diluted standard to the standard wells.
Step 12: Add 50 µl of 1x assay buffer and 50 µl of the sample to the sample wells.
Step 13: Add 100 µl of cell culture supernatant to the sample wells.
Step 14: Add detection antibody: Add 50 µl of detection antibody working solution to each well to be tested.
Step 15: Incubate at 100-300 rpm at room temperature (25°C ± 3°C) for 2 hours.
Step 16: Discard the liquid, add 300 µl of wash solution to each well, and wash the plate 6 times. Blot the plate dry on absorbent paper, ensuring all residual liquid is removed.
Step 17: Add enzyme: Add 100 µl of streptavidin working solution to each well.
Step 18: For non-acidic liquid waste, add 1.0% sodium hypochlorite and soak for 30 minutes. For acidic liquid waste, neutralize it first, then add sodium hypochlorite.
Step 19: Incubate: Seal the plate with a new adhesive cover. Incubate at 100-300 rpm at room temperature (25°C ± 3°C).
Step 20: Repeat Step 16.
Step 21: Add 100 µl of the substrate solution to each well, incubate in the dark at room temperature (25°C ± 3°C) for 5-30 minutes.
Step 22: Add 100 µl of stop solution to each well; the color will change from blue to yellow.
Measurement Discussions
Figure 3: The ELISA verified the blocking effect of A/B proteins on the interaction of TNFα-TNFR.The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.
We use the above method to quantify the blocking ability of TNF. Initially, we coated the ELISA plate with TNFR, followed by the addition of various antibody sera mixed with TNFα at four distinct concentrations. Finally, we quantified the resultant light signal by detecting antibodies of anti-TNFα-HRP. We find that blank control group need to higher than positive control group in the Figure 3. So, we found that only in the concentration of 1ng/ml and 0.5ng/ml TNFα, the nano-antibody A shows block effect on the binding of TNFα-TNFR.
Measurement Part 2: Cell viability testing
TNFa naturally binds to TNFR on the cell membrane, triggering the TNF signaling pathway and inducing cell apoptosis upon binding.
U937 cells are a human lymphoma cell line that expresses TNFR on the cell membrane. Adding human TNFa to U937 cell culture medium resulted in rapid apoptosis of the cells.
When nano antibodies are co incubated with human TNFa protein, A/B in the supernatant can bind specifically and with high affinity to human TNFa, which interferes with the natural binding between TNFa and TNFR on the cell membrane. Therefore, after adding a mixture of human TNFa and bacterial culture supernatant to U937 cell culture medium, the TNF signaling pathway was not activated and U937 cells did not undergo apoptosis.
Measurement Principle
ATP, as the most important energy molecule, it plays a crucial role in various physiological and pathological processes of cells. ATP is an important indicator of cellular metabolism and a significant marker molecule for metabolically active cells, a good linear relationship with the number of live cells.
The principle of detecting cell viability through ATP is shown in Figure 4. By utilizing ATP dependent luciferase to catalyze the luminescent reaction of luciferin.ATP can be quantified by measuring chemiluminescence. Due to the fact that ATP content can well reflect the number of live cells, and ATP content is directly proportional to luminescence intensity, cell viability can be easily calculated through chemiluminescence intensity[2].
Figure 4. CellTiter Lumi ™ Schematic diagram of ATP detection using luminescent cell viability assay kit.
Measurement Protocols
Control：
Blank control: PBS
Materials
Well plate, U937 and mouse primary CD8-positive inguinal lymph node T cells, TNFα+PBS, Protein samples, microplate reader
Procedures
Step 1. Add 100μl of cells (30,000 cells) (U937 and mouse primary CD8-positive inguinal lymph node T cells) to a 96-well plate and incubate it overnight in a cell incubator.
Step 2. Create a control group by adding TNFα+PBS. Create an experimental group by adding TNFα and 5 different concentration antibodies, each with three concentration gradients, with the concentration halved sequentially. Then incubate the 96-well plate at 37°C for 6 hours.
Step 3. Prepare CellTiter-Lumi solution and bring it to room temperature.
Step 4. Take out the 96-well plate and balance it to room temperature.
Step 5. Add 100μl of CellTiter-Lumi solution to each well, cover the plate, oscillate for 2 minutes, and incubate for 20 minutes.
Step 6. Measure chemiluminescence using a microplate reader.
Measurement Discussions
After TNF-α binds to the TNF receptor (TNFR) on the cell surface, it triggers apoptosis and necrosis pathways, ultimately leading to cell death. However, when TNF-α binds to a nanoantibody, it is prevented from binding to TNFR, thereby inhibiting the cell death process. To further explore the effect of nanoantibodies in inhibiting inflammatory factors, we conducted cell experiments. it.
TNF-α was mixed with a series of nanoantibodies at different concentrations (five nanoantibodies obtained from culture, each diluted stepwise by half), and TNF-α mixed with PBS was used as a negative control. This mixture was incubated with U937 cells for 6 hours. Cell viability in each group was then assessed using the CellTiter-Lumi k
The results (Figure 5) demonstrated that, compared to the control group, the nanoantibodies produced by the five culture methods exhibited strong inhibition of cell death. Furthermore, this inhibitory effect was positively correlated with the concentration of the nanoantibody—meaning that as the concentration of the nanoantibody increased, the ability to prevent cell death also improved.
Figure 5: The luminescence-based cell viability assay for antibody A and B under different temperatures and growth conditions.
Other contributions
1.As the ELISA method, we used to detect the blocking ability of TNFa and TNFR binding. It is currently not sold in the market, so the future of the iGEM team contribution is very large.The future team can use this method to detect other nano-antibody protein blocking the effect of TNFR binding, and can be quantitative protein expression.The method will play an important role in drug development.
2.The CellTiter Lumi TM Schematic diagram of ATP detection, using luminescent cell viability assay kit, we used can also be used to detect the effects of other drugs or antibodies on cells, and can be widely used in pharmaceutical development.
1.Webster, J. D., & Vucic, D. (2020). The Balance of TNF Mediated Pathways Regulates Inflammatory Cell Death Signaling in Healthy and Diseased Tissues. Frontiers in cell and developmental biology, 8, 365. https://doi.org/10.3389/fcell.2020.00365
2.Haoyuan L ,Wenjun Z ,Weiyan M , et al.High-Glucose Concentration Aggravates TNF-alpha-Induced Cell Viability Reduction in Human CD146-Positive Periodontal Ligament Cells via TNFR-1 Gene Demethylation.[J].Cell biology international,2020,44(12):2383-2394.