A: anti-TNFa nanobody antibody—A (TNFa binding fragment of Ozoralizumab)
B: anti-TNFa nanobody antibody—B (TNFa binding fragment of V565)
- Gene synthesis of the nanoAb-A/B sequence by GeneScript.
- Preparation of culture medium of the bacterial, including liquid and solid form of LB and SB medium.
- PCR amplification of target gene A，B and linearization of vector backbone
Gene synthesis:
We download and verify the sequence of gene A and B then submitted to the gene synthesis company GeneScript.
PCR amplification of A, B and vector backbone
-1ul of DNA template
-1ul of primers for P1 and P2 respectively
-10ul of Polymerase
-7ul of double distilled water
PCR steps: 40× cycles1. Adjust the numbers with a pipette first, and then mix the materials
2. Inactivate at a high temperature of 98 °C Celsius, causing DNA to change from double stranded to single stranded
3. Deactivate 56 °C primer binding to DNA
4. 72 °C extended DNA pairing for 1 minute
5. Repeat about 40 cycles to obtain a large amount of DNA
-add tryptone(1%), yeast extract(0.5%), NaCl(1%) into double distilled water and make it dissolved in it.
-sterilize the mixture at the temperature of 121 Celsius degree for 20 minutes.
- Add chloromycetin into the sterile medium and poured the solid LB medium into plates and allowed it to solidify
- Gel Electrophoresis and extraction to purify the target PCR fragment
- Homologous Recombination
Prepare the TAE buffer:
-Add TAE buffer powder (sufficient for 1L).
-Add 1L of water and shake until fully dissolved.
-Weigh the agarose (1%) and mixed with TAE buffer then boiled in microwave.
-Add about 1:10000 amount of Gel Red to the melted agarose in the flask and continue to mix gently by swirling until the Gel Red is completely mixed with the melted agarose.
-Pour into a mold and allow it to solidify.
Load the Gel:
Once solidified, remove the gel from the mold and place it in the electrophoresis apparatus.
-Add marker to the first well.
Mix 4 μL of 6× loading buffer with 20 μL of sample, and load into alternating wells.
-Run the gel for 30 minutes.
Remove the gel and place it under UV light to document the results.
1. cut out the needed parts of the gel that contain DNA
2. add buffer B2 (3-6 times of the amount of the cut gel)
3. place the substances at 50℃ for 5-10 minutes
4. move the solution into a collection tube with a spin column
5. centrifuge 9000g 30sec
6. remove the flowed liquid, add 500ul washing agent and centrifuge 9000g 30sec
7. repeat step 6
8. centrifuge the spin column 9000g 1min
9. transfer the spin column into a clean 1.5 mL tube and add 15~40 uL Elution Buffer. place in room temperature for 1 minute then centrifuge for one minute.
10. Discard the spin column and preserve the flow-through DNA solution.
Mix 0.5 μL each of A/B+ backbone of vector.
Add 5 μL enzyme mix.
Incubate at 50°C for 5 minutes.
Transfer the reaction mixture to ice.
Transformation:
1. Prepare Cells:
2. Mix and Incubate:
3. Smear the transformed bacterial cell to culture plate
First eletrophoresis: DNA marker is degraded and the result is faild
Second electrophoresis: choose a new DNA marker and redo the PCR reaction
Step 1: We took out the Escherichia coil we cultured and put them into the PCR machine to verify not being mistaken in our experiment.
The culture disk on the left side is the bacterium that added DH5A with A-antibody, while the one on the right of this picture is added with B-antibody, CHR and DH5A.
Step 2: Use PCR technique and gel electrophoresis to verify the bacterium we cultured.
Take eight tubes of monoclonal bacterial strain by using the pipette tips. After that, we put 10 ul of sterile water and used the system of PCR including 2 ul of bacterial solution, 1 ul of primer number 1, 1 ul of primer number 2, 10 ul of mixture and 6 ul of double distilled water.
load the product into gelOnce solidified, remove the gel from the mould and place it in the electrophoresis apparatus.
Add marker to the first well.
Mix 4 μL of 6× loading buffer with 20 μL of the PCR sample, and load into alternating wells.
Run the gel for 30 minutes.
Remove the gel and place it under UV light to document the results.
The expected bands will be visible if successful, indicating successful plasmid construction.
After verifying the bacterium we cultured, we kept culturing the bacterium by adding 3 ml LB culture medium and CHR (1:1000) for each tube of bacterial solution.
Inoculate each tube with a small amount of the confirmed bacterial suspension.
Incubate overnight with shaking at 37℃ and 220rpm
A1, A2, A3, A4
B1, B2, B3, B4
Preparation:
Add 500 μL of Buffer S to a spin column.
Centrifuge at 12000g for 1 minute.
Discard the flow-through from the collection tube.
Bacterial Cell Harvesting:
Add 2 mL of the overnight bacterial culture to an Eppendorf tube.
Centrifuge at 8000g for 1 minute.
Discard the supernatant.
Cell Lysis and Neutralization:
Add 250 μL of Buffer SP1 to the cell pellet and mix thoroughly.
Add 250 μL of Buffer SP2 and invert the tube 4 times to mix.
Add 350 μL of Buffer SP3 and centrifuge at 12000g for 5 minutes.
Plasmid Binding:
Transfer the supernatant to the prepared spin column.
Centrifuge at 8000g for 30 seconds.
Discard the flow-through and reapply the liquid to the spin column.
Centrifuge again at 8000g for 30 seconds.
Wash Steps:
Add 500 μL of DW1 to the spin column.
Centrifuge at 9000g for 30 seconds.
Add 500 μL of Wash Solution.
Centrifuge at 9000g for 30 seconds.
Repeat the wash step with 500 μL of Wash Solution and centrifuge at 9000g for 30 seconds.
Drying:
Centrifuge the empty spin column at 9000g for 1 minute to remove any residual wash solution.
Elution:
Transfer the spin column to a new 1.5 mL Eppendorf tube.
Add 50 μL of Elution Buffer to the center of the spin column membrane.
Let it stand for 1 minute.
Centrifuge at 9000g for 1 minute.
Reapply the eluate to the spin column and centrifuge at 9000g for another minute.
Remove the spin column and retain the Eppendorf tube containing the purified plasmid DNA.
Culture of successful engineered bacteria overnight
Sequence result of Nanobody antibody A
Sequence of Nanobody antibody B
Prepare Cells:
Use 100 μL of competent ECN cells.
- Add 100 uL of competent cell (EcN1917) to the 1 uL constructed plasmid A or B seprately.
- Place on the ice for 30 minutes.
- After the ice incubation, heat shock the tubes at 42 degree for 90 seconds.
- Return to the ice for 2-3 minutes.
- Add 900 uL of LB broth to each tube.
- Vortex each tube and incubate them at 37°C for 60 minutes while shaking.
- When the bacterial recovery is complete, centrifuge the tubes for 5 minutes at full speed.
- Discard but leave 100 uL of supernatant left. Resuspend the cell pellet. Make sure the entire pellet is resuspended before plating.
- Aliquot the entire 100 uL to an LB agar plate.
- Incubate the plate at 37 degree for 24 hours.
- Add 10 uL of LB broth in each tube and gently mix.
- Obtain the plates and gently remove 6 bacteria colonies from the surface of the plate to sterile tubes.
- Obtain a 0.2 mL tube strips and place on the bench.
- Add 2 uL of each mixed solution, 1 uL of P1, 1 uL of P2, 6 uL of ddH2O, and 10 uL of PCR reaction mix in each 0.2 mL tube strips. The total amount of mixture in each tube of the 0.2 mL tube strips should be 20 uL.
- Do a PCR of the tube in the PCR apparatus
- After PCR, to check the plasmid is right or wrong.
Make SB medium
Weigh and solve the various regent by ddH2O
Trypton 3.2%
Yeast extract 2%
NaCl 0.5%
Culture of EcN1917- A/B at LB/SB at 16/37 Celsius and collect the supernatant of medium
The preceding night, the bacteria were inoculated into LB liquid culture medium and incubated overnight. Chloramphenicol was subsequently added to achieve a final concentration of 25 micrograms per milliliter, specifically for Nissle1917 expressing antibodies A and B.
On the following evening, measure the optical density (OD) and select the strain with the highest OD value. Inoculate this strain at a concentration of 1% of its volume into SB medium containing chloramphenicol and adjust the volumes of the remaining strains to match this maximum OD value. Based on the added volume, proportionally increase the inoculation volume in LB medium while incubating at 16°C and shaking at 220 rpm for 12 hours; prior to incubation at 37°C, measure the OD to ensure that all inoculated liquid starts from an equivalent initial volume.
Centrifuge at 3000 g for 5 minutes at 4°C, collect the supernatant, filter it through a 0.22-micron filter, and store the resulting solution at -80°C.
37 Celsius
16 Celsius
Materials Required:
Protein lysates (containing your samples)
SDS-PAGE gel system
Transfer apparatus (e.g., semi-dry transfer cell or tank transfer system)
Nitrocellulose membrane or PVDF membrane
Blocking buffer (such as 5% non-fat milk in TBS-T)
Primary antibody (anti-His tag antibody)
Secondary antibody conjugated with an enzyme (such as HRP-conjugated anti-mouse IgG if the primary is mouse-derived)
ECL substrate for chemiluminescence detection
Imaging system (e.g., ChemiDoc MP Imaging System)
Procedure:
Step 1: Sample Preparation
Prepare protein lysates: Ensure that your lysates contain a reducing agent like DTT or β-mercaptoethanol.
Boil samples: Boil the lysates at 95°C for 5 minutes to denature the proteins and allow them to bind to the SDS.
Step 2: Electrophoresis (SDS-PAGE)
Load samples: Load equal amounts of protein lysates into the wells of the pre-cast polyacrylamide gel.
Run the gel: Apply a constant voltage (e.g., 80V for stacking gel, then increase to 120V for resolving gel) until the tracking dye reaches the bottom of the gel.
Step 3: Transfer (Blotting)
Assemble transfer sandwich: Place the nitrocellulose or PVDF membrane on top of the gel, ensuring there are no air bubbles.
Perform transfer: Use either a semi-dry transfer apparatus or a wet transfer tank. Apply a constant current (e.g., 300mA for 90 minutes) to transfer the proteins from the gel to the membrane.
Step 4: Blocking
Block non-specific binding sites: Incubate the membrane in blocking buffer for 1 hour at room temperature or overnight at 4°C to prevent non-specific antibody binding.
Step 5: Primary Antibody Incubation
Prepare primary antibody: Dilute the anti-His tag antibody in blocking buffer according to the manufacturer's instructions.
Incubate: Incubate the membrane with the diluted primary antibody for 1-2 hours at room temperature or overnight at 4°C.
Step 6: Wash
Wash the membrane: Wash the membrane several times with TBS-T (Tris-buffered saline with Tween-20) to remove unbound primary antibody.
Step 7: Secondary Antibody Incubation
Prepare secondary antibody: Dilute the HRP-conjugated secondary antibody in blocking buffer.
Incubate: Incubate the membrane with the diluted secondary antibody for 1 hour at room temperature.
Step 8: Final Wash
Wash the membrane: Wash the membrane several times with TBS-T followed by a final wash with TBS alone.
Step 9: Detection
Apply ECL substrate: Apply the chemiluminescent substrate to the membrane.
Develop the signal: Place the membrane in a cassette and expose it to film or use a digital imaging system to visualize the bands.
Step 10: Analysis
Evaluate results: Analyze the bands using software such as Image Lab to confirm the presence and size of the His-tagged protein.
ELISA assay：TNFa coated plate
A final concentration of 100 ng/ml TNF-α is diluted by 1x DBPS and 100 µl of this solution is added to each well of the ELISA plate, followed by incubation at 4°C overnight.
After overnight, perform three washes with PBST, utilizing 300 microliters for each wash
Position the board inverted on the paper to ensure complete absorption of any residual moisture
Block: PBS containing 1% BSA and 0.1% Tween 20 (no Tween 20 was added today), incubate at room temperature for one hour, gently mixing with a decolorizing shaker.
Wash with PBST for one time
Add sample and primary antibody: incubate at room temperature for 2 hours
Positive group：100ul/well, 2.5ug of TNFRSF1A-His protein
Negative group: 100ul/well PBS
Experimental group：
16°C Antibody Supernatant (A-sb, B-sb, A-LB, B-LB), diluted in 5 gradients (0, 2×, 4×, 8×, 16×)
37°C Antibody Supernatant (A-sb, B-sb, A-LB, B-LB), diluted in 5 gradients (0, 2×, 4×, 8×, 16×)
Perform three washes with PBST, utilizing 300 microliters for each wash
Add detection antibody：Anti-His-HRP antibody diluted at a ratio of 1:2000, 10 mL of blocking solution (PBS + 1% BSA + 0.1% Tween 20) diluted, incubated at 4°C overnight
Perform three washes with PBST, utilizing 300 microliters for each wash
Add 100ul of TMB substrate to each well.
Add the stop solution to each well at a volume of 100ul.
Measure the absorbance at OD450 utilizing Thermofisher MultiScan microplate reader
Standard curve of the ELISA kit to detect TNFa
Prepare the ELISA TNFR coated plate
ELISA test to verify the effect of nanobody antibody A/B supernatant to block TNFa binding to TNFR
Standard curve of TNFa ELISA kit
Experimental steps:
1. Prepare 1x wash solution by diluting 20x wash solution with distilled water at a ratio of 2:38.
2. Prepare 1x assay buffer by diluting 10x buffer with distilled water at a ratio of 1:10.
3. Prepare the detection antibody working solution by diluting 1x antibody working solution with distilled water at a ratio of 1:10.
4. Dilute the sample with 200 µl of 1x assay buffer.
5. Centrifuge at 10,000g for 1 minute and let it stand for 10 minutes.
6. Take 230 µl of reconstituted standard, add 230 µl of standard diluent, and perform a 1:1 dilution using the high concentration standard.
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.
8. Detach any unnecessary strips from the plate and reseal them to avoid contact with the inner surface of the microwells.
9. Add 300 µl of 1x wash solution to each well on the ELISA plate and let it soak for 30 seconds.
10. Discard the wash solution and blot the microwell plate dry on absorbent paper. After washing, do not let the microwell plate dry out.
11. Add 100 µl of 2-fold serially diluted standard to the standard wells.
12. Add 50 µl of 1x assay buffer and 50 µl of the sample to the sample wells.
13. Add 100 µl of cell culture supernatant to the sample wells.
14. Add detection antibody: Add 50 µl of detection antibody working solution to each well to be tested.
15. Incubate at 100-300 rpm at room temperature (25°C ± 3°C) for 2 hours.
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.
17. Add enzyme: Add 100 µl of streptavidin working solution to each well.
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.
19. Incubate: Seal the plate with a new adhesive cover. Incubate at 100-300 rpm at room temperature (25°C ± 3°C).
20. Repeat Step 16.
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.
22. Add 100 µl of stop solution to each well; the color will change from blue to yellow.
A final concentration of 100 ng/ml (10ng/well) TNFR is diluted by 1x DBPS and 100 µl of this solution is added to each well of the ELISA plate, followed by incubation at 4°C overnight.
After overnight, perform three washes with PBST, utilizing 300 microliters for each wash
Position the board inverted on the paper to ensure complete absorption of any residual moisture
Block: PBS containing 1% BSA and 0.1% Tween 20 (no Tween 20 was added today), incubate at room temperature for one hour, gently mixing with a decolorizing shaker.
Wash with PBST for two times
Pre-treatment：Add nanoAb supernatant, Positive Ab and PBS with different concentration of TNFa at room temperature for one hour
TNFa concentration: 0.25ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, and 100ul/well
Positive group：Positive-Ab(1:10000) + TNFa
Negative group: PBS+TNFa
Experimental group：
16°C Antibody Supernatant (A-sb, B-sb, A-LB, B-LB) + TNFa
37°C Antibody Supernatant (A-sb, B-sb, A-LB, B-LB) + TNFa
Add the mixture of step 18 to the coated plate and incubate at room temperature for 2 hours
Perform three washes with PBST, utilizing 300 microliters for each wash
Add detection antibody：Anti-TNFa-biotin antibody diluted at a ratio of 1:1000, 10 mL of blocking solution (PBS + 1% BSA + 0.1% Tween 20) diluted, incubated at room temperature for 1 hour
Add SA-HRP (1:250) and the final concentration is 2ug/ml, incubate at room temperature for 1 hour
Perform three washes with PBST, utilizing 300 microliters for each wash
Add 100ul of TMB substrate to each well.
Add the stop solution to each well at a volume of 100ul.
Measure the absorbance at OD450 utilizing Thermofisher MultiScan microplate reader
Cell isolation and culture
TNFa stimulate the cell and verify the protect effect of nanobody antibody A/B by cell viability assay
Experimental steps:
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.
2. Create a control group by adding TNFα+PBS. Create an experimental group by adding TNFα and 5 different antibodies, each with three concentration gradients, with the concentration halved sequentially. Then incubate the 96-well plate at 37°C for 6 hours.
3. Prepare CellTiter-Lumi solution and bring it to room temperature.
4. Take out the 96-well plate and balance it to room temperature.
5. Add 100μl of CellTiter-Lumi solution to each well, cover the plate, oscillate for 2 minutes, and incubate for 20 minutes.
6. Measure chemiluminescence using a microplate reader.
