Antibody Glycan Site-specific Conjugation Kit II

An antibody site-specific conjugation kit developed based on antibody glycan editing technology uses the conserved N-297 glycan in the CH2 region of the antibody IgG heavy chain to mediate site-specific conjugation, employing glycosidases and glycosyltransferases for precise modification.

It enables rapid site-specific conjugation of monoclonal antibodies, overcoming the drawbacks of conventional conjugation methods, such as random conjugation sites, heterogeneous products, or the need for complex antibody engineering. This kit provides a complete set of reagents for azide modification of antibodies and can be completed within two days.

As shown in the figure below, first, the glycosidase EndoS is used to expose the conserved N-glycan on the constant region of the monoclonal antibody, revealing the N-acetylglucosamine (blue square). Then, a mutant bovine galactosyltransferase (b.GalTY298L) is used to attach an acetylgalactosamine with an azide functional group (yellow square-N3) is attached to acetylglucosamine. Subsequently, biotin, fluorescein, or toxin molecules, among others, can be linked to the monoclonal antibody using a copper-free click chemistry reaction (such as SPAAC).

Product Features

No antibody modification required; antibody structure remains intact.

Homogeneous product with high and stable conjugation efficiency.

Applicable to antibodies with glycosylation modifications, compatible with a variety of linkers and drugs.

*After thawing, store at 2-8 ℃ for up to 1 week; do not freeze-thaw repeatedly.

For a drug-to-antibody ratio (DAR) of 4.0

I. Reagents and Instruments to Bring Yourself:
1. Ultrapure water
2. DMSO
3. BCN/DBCO-toxin molecules
4. Centrifuge tubes (1.5 mL / 15 mL)
5. Ultrafiltration tubes (0.5 mL, 50 kDa MWCO)
6. Low-temperature refrigerated centrifuge (compatible with 1.5 mL / 2 mL centrifuge tubes)
7. Water bath or constant temperature and humidity incubator
8. Vortex mixer

II. Operating Procedures:
1. Azide modification of antibodies (1 mg antibody)
(1) Add 10 μL of Reagent I to the antibody solution (10 mg/mL, 100 μL, in 1×PBS, pH=7.2), and react at 37°C for 2 hours;
(2) Add 400 μL of 1× PBS (Solution A) to an ultrafiltration tube with a molecular weight cut-off of 50 kDa, and centrifuge at 8,000 × g for 5 minutes at 4°C to wash the ultrafiltration tube.
(3) Transfer the sample from Step (1) into the ultrafiltration tube, add 300 μL of 2× Tris-HCl buffer (Solution B), and centrifuge using the same method as in Step (2). Repeat this step five times, adjusting the final volume to approximately 100 μL. Invert the inner membrane of the ultrafiltration tube into a new outer sleeve, centrifuge at 10,000 × g for 2 minutes at 4°C, and collect the sample.
(4) In a new 1.5 mL centrifuge tube, sequentially add 177.5 μL of ultrapure water, 400 μL of Solution B, 150 μL of Reagent II, 150 μL of Reagent III, 15 μL of Reagent IV, and 7.5 μL of Reagent V. Invert to mix thoroughly, then add 100 μL of the above sample, invert again to mix, and incubate at 30°C in a water bath or on a rotator for 16 hours (Note: If a small amount of protein precipitates after the reaction, centrifuge at 10,000×g for 2 minutes and use the supernatant for subsequent experiments).
(5) Transfer 500 μL of Protein A beads to an empty column, equilibrate the beads with 3 mL of Solution B, and load the reaction solution from Step (4) onto the beads. Allow the sample to pass through the beads under gravity, collect the filtrate, and repeat the loading three times to ensure complete binding of the antibody to the beads. Wash the beads with 5 mL of Solution A, then elute the antibody using 300 μL of sodium citrate (Solution C), collecting the eluate. Repeat this elution four times, combine the elution fractions, and slowly add 1 M Tris (Solution D) to the eluate while stirring to adjust the pH to 7.2.
(6) Transfer 400 μL of elution buffer into an ultrafiltration tube with a molecular weight cut-off of 50 kDa, centrifuge under the same conditions as in step (2), and repeat this step to concentrate all the eluate to approximately 100 μL. Add 300 μL of solution A, centrifuge under the same conditions, and repeat five times. Invert the inner tube of the ultrafiltration device into a new outer tube, centrifuge at 10,000×g for 2 minutes at 4°C, collect the sample to obtain the azide-modified antibody.
(7) Determine the concentration of the azide-modified antibody using NanoDrop or the BCA method.

2. Azide Modification of Antibodies (500 μg of antibody)
(1) Add 5 μL of Reagent I to the antibody (5 mg/mL, 100 μL, in 1×PBS, pH = 7.2), and react at 37°C for 2 hours.
(2) Add 400 μL of 1× PBS (Solution A) to an ultrafiltration tube with a molecular weight cut-off of 50 kDa, and centrifuge at 8,000 × g for 5 minutes at 4°C to wash the ultrafiltration tube.
(3) Transfer the sample from Step (1) into the ultrafiltration tube, add 300 μL of 2× Tris-HCl buffer (Solution B), and centrifuge using the same method as in Step (2). Repeat this step five times, adjusting the final volume to approximately 100 μL. Invert the inner membrane of the ultrafiltration tube into a new outer sleeve, centrifuge at 10,000 × g for 2 minutes at 4°C, and collect the sample.
(4) In a new 1.5 mL centrifuge tube, sequentially add 38.5 μL of ultrapure water, 200 μL of Solution B, 75 μL of Reagent II, 75 μL of Reagent III, 7.5 μL of Reagent IV, and 4 μL of Reagent V. Invert to mix thoroughly, then add 100 μL of the above sample, invert again to mix, and incubate at 30°C in a water bath or on a rotator for 16 hours. Note: After the reaction is complete, if a small amount of protein precipitates, centrifuge at 10,000×g for 2 minutes and use the supernatant for subsequent experiments.
(5) Transfer 250 μL of Protein A microbeads into an empty column, equilibrate the microbeads with 2 mL of Solution B, and load the reaction solution from Step (4) onto the microbeads. Allow the sample to pass through the microbeads under gravity, collect the filtrate, and repeat the loading three times to ensure complete binding of the antibody to the microbeads. Wash the microbeads with 5 mL of Solution A, then elute the antibody using 150 μL of sodium citrate (Solution C), collecting the eluate. Repeat this elution four times, combine the eluate fractions, and slowly add 1 M Tris (Solution D) to the eluate while stirring to adjust the pH to 7.2.
(6) Transfer 400 μL of elution buffer into an ultrafiltration tube with a molecular weight cut-off of 50 kDa, centrifuge under the same conditions as in step (2), and repeat this step to concentrate all the eluate to approximately 100 μL. Add 300 μL of solution A, centrifuge under the same conditions, and repeat five times. Invert the inner tube of the ultrafiltration device into a new outer tube, centrifuge at 10,000 × g for 2 minutes at 4°C, collect the sample to obtain the azide-modified antibody.
(7) Determine the concentration of the azide-modified antibody using NanoDrop or the BCA method.

3. Recommended steps for toxin molecule conjugation (not included in this kit)
(1) Use endo-BCN-PEG4-Val-Cit-PAB-MMAE (CAS: 2762519-08-2, recommended product:abs823512orabs823511) as an example, use DMSO to prepare a stock solution of the toxin molecule at a concentration of 10 mM. Add the toxin molecule to the azide-modified antibody at a molar ratio of 1:10 (antibody:toxin molecule), and incubate in a 30°C water bath or rotate for 16 hours.
(2) Transfer the product into an ultrafiltration tube with a molecular weight cut-off of 50 kDa. Centrifuge at 4°C at 8,000×g for 5 minutes, add 300 μL of 1× PBS buffer, and centrifuge under the same conditions. Repeat this process five times. Invert the inner membrane of the ultrafiltration tube into a new outer sleeve, centrifuge at 4°C at 10,000×g for 2 minutes, and collect the sample to obtain the MMAE-antibody conjugate.

1. This kit is intended for research use only and shall not be used in clinical or human experiments.
2. Please read this instruction manual and the precautions before performing any experimental procedures.
3. For your safety and health, please wear gloves and a mask while handling the materials.
4. Use only the reagents provided with this kit; do not mix them with components from other kits.