Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

We describe a simple method for preparing antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates and discuss its applications in drug delivery and new drug design. Conjugation is based on alkyne-azide cycloaddition. This Cu-free click reaction starts from the dibenzocyclooctyne (DBCO) moiety-activated antibodies and subsequently linked covalently with an azide-modified peptide, oligonucleotide or compounds. The reaction is performed under physiological conditions and has no adverse effects on antibodies or proteins. This can also be used as the click chemistry fluorescence labeling and the click chemistry in peptide-based drug design.

However, the copper-catalyzed alkyne-azide cycloaddition (CuAAC) is not suitable for applications involving functional biomolecules because copper ions can cause protein denaturation.

Measuring the protein levels directly is challenging. However, the signals can be amplified by immuno-PCR using oligonucleotide-attached antibodies to detect protein indirectly.

Antibody-Conjugate

Antibody-Conjugate

 

Preparing Antibody-Peptide, Antibody Oligonucleotide or Antibody-Compound Conjugates

1. Conjugation of DBCO to the Antibody. The DBCO-PEG5-NHS was used to react with the NH2 groups on the antibody. The inclusion of a PEG5 linker improves the water solubility of the hydrophobic DBCO, introduces a spacer and flexibility between the antibody molecule and the peptide/oligonucleotide or compounds. This will alleviate the steric effect of the antibody on the enzymatic reactions.

2. Prepare the azido-Peptide or azido-oligonucleotide. LifeTein provides click chemistry modified peptide synthesis: N-terminal azide-peptide/oligo or C-terminal peptide/oligo-azide.

3. Covalent attachment of the peptide/oligonucleotide to the antibody. The reaction between DBCO and azide is slow compared to CuAAC reaction. The reaction time of 16–18 h in PBS at 4 °C is ideal to increase the final product yield. The DBCO-antibody in the intermediate reaction is stable.

https://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.5b00613

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Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

Copper-Free Click Chemistry Antibody-DNA Conjugation

Typically, there are three biological functional groups on the peptide for the further conjugation: amino ( –NH2 ), carboxyl ( –COOH), and thiol ( –SH). The most effective way is to utilize the free thiol groups from cysteine. The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules.

Click Chemistry to Conjugate Peptides With Other Biomolecules

The click chemistry is another efficient method to conjugate the peptide with other biomolecules. The peptide can be modified with azide groups (–N3). The novel Copper-free Click Chemistry is based on the reaction of a diarylcyclooctyne moiety (DBCO) with an azide-peptide reaction partner. This click reaction is very fast at room temperature and does not require a cytotoxic Cu(I) catalyst, resulting in almost quantitative yields of stable triazoles. The DBCO allows Copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. The reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (–SH, thiol). One example of the peptide drug conjugations is the antibody-biomolecule conjugate.
click chemistry: DBCO-azide

click chemistry: DBCO-azide

A simple protocol: Click chemistry of antibody-DNA conjugation Pre-conjugation considerations
  • Remove all additives from antibody solutions using dialysis or desalting.
  • Remove BSA and gelatin from antibody solutions.
  • Concentrate the antibody after dialysis or purification.
Activation of antibodies with DBCO-NHS ester
  • Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
  • Incubates at room temperature for 30 min or 2 hours on ice.
Quenching activation reaction
  • Add Tis-Hcl (50-100mM, pH 8) to the reaction.
  • Incubate at RT for 5 min or 15 minutes on ice.
Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction) Copper-Free click reaction
  • Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
  • Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.
Validation of conjugation and purification by HPLC Selected References:
  1. Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
  2. Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst-free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
  3. Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.

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Our Services: COVID-19 Services & Products Custom Antibody Services Rush Peptide Synthesis Peptide Nucleic Acids (PNAs) Custom Peptide Synthesis Services Gene Synthesis Service Custom Chemical Synthesis Other Posts: Personalized treatment using synthetic peptides Long peptide synthesis by click chemistry Post-translational modifications: Methylated peptides Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates