For a degree of label (DOL) or antibody-drug ratio (DAR) of 2.0
Materials Required but Not Provided：
● Ultrapure water
● DMSO
● BCN/DBCO-toxin
● Centrifuge tubes (1.5 mL /15 mL)
● Ultrafiltration spin columns (0.5 mL, 50 kDa MWCO)
Optimized Protocol for Antibody Azide Labelling
1.1 Add 5 μL of Reagent I to the antibody solution (10 mg/mL, 100 μL, in 1×PBS, pH=7.2) and incubate at 37°C for 2 hours.
1.2 Add 400 μL of 1×PBS (Solution A) to a 50 kDa molecular weight cutoff (MWCO) ultrafiltration tube, then centrifuge at 8,000 × g for 5 minutes at 4°C to wash the ultrafiltration tube.
1.3 Transfer the sample from step 1.1 into the ultrafiltration tube, then add 300 μL of 2×Tris-HCl buffer (Solution B). Centrifuge under the same conditions as in step 1.2. Repeat this step 5 times, adjusting the final volume to approximately 100 μL. Invert the ultrafiltration tube into a new collection tube and centrifuge at 10,000 × g for 2 minutes at 4°C to collect the sample.
1.4 In a new 1.5 mL centrifuge tube, sequentially add 285 μL of ultrapure water, 400 μL of Solution B, 100 μL of Reagent II, 100 μL of Reagent III, 10 μL of Reagent IV, and 5 μL of Reagent V. Mix by inversion, then add 100 μL of the sample from above and mix again. Incubate at 30°C in water bath or with rotation for 16 hours.
(Note: If protein precipitation occurs after the reaction, centrifuge at 10,000 × g for 2 minutes and use the supernatant for subsequent steps.)
1.5 Load 500 μL of Protein A beads into an empty column and equilibrate with 3 mL of Solution B. Apply the reaction mixture from step 1.4 onto the beads and allow the sample to flow through by gravity. Collect the flow-through and repeat the loading 3 times to ensure sufficient antibody binding. Wash the beads with 5 mL of Solution A, then elute the antibody with 300 μL of sodium citrate (Solution C). Collect the eluate and repeat the elution 4 times. Combine all elution fractions and slowly add 1 M Tris (Solution D) to adjust the pH to 7.2.
1.6 Transfer 400 μL of the eluate to a 50 kDa MWCO ultrafiltration tube and centrifuge under the same conditions as in step 1.2. Repeat this step to concentrate all eluates to approximately 100 μL. Add 300 μL of Solution A and centrifuge under the same conditions. Repeat this washing step 5 times. Invert the ultrafiltration tube into a new collection tube and centrifuge at 10,000 × g for 2 minutes at 4°C to collect the sample, yielding the azide-labelled antibody.
1.7 Determine the concentration of the azide-labelled antibody using NanoDrop or the BCA assay.
Recommended Protocol for Toxin Conjugation (Not Included in this Kit)
2.1 Example Using endo-BCN-PEG4-Val-Cit-PAB-MMAE (CAS: 2762519-08-2):
Prepare a 10 mM stock solution of the toxin molecule in DMSO.
Add the toxin molecule to the azide-labelled antibody at a molar ratio of 1:10 (antibody:toxin).
Incubate at 30°C in water bath or with rotation for 16 hours.
2.2 Transfer the reaction product to a 50 kDa molecular weight cutoff ultrafiltration tube.
Centrifuge at 8,000 × g for 5 minutes at 4°C.
Add 300 μL of 1× PBS buffer and repeat centrifugation under the same conditions.
Repeat the washing step a total of 5 times.
Invert the ultrafiltration tube into a new collection tube and centrifuge at 10,000 × g for 2 minutes at 4°C to collect the sample, yielding the MMAE-antibody conjugate.
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