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.




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.

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

LifeTein Leads the Way in Revolutionary Peptide Conjugation Methods

In the realm of peptide synthesis and bioconjugation, LifeTein stands at the forefront, offering innovative solutions for linking peptides to other biomolecules. Typically, peptides have three biological functional groups available for conjugation: amino (–NH2), carboxyl (–COOH), and thiol (–SH). Among these, the thiol group, particularly from cysteine residues, is often the most effective for bioconjugation. The reaction between maleimides and thiols is a widely recognized method for the bioconjugation and labeling of biomolecules, and LifeTein has mastered this technique to offer superior results.

Click Chemistry: A Revolution in Peptide Conjugation by LifeTein

LifeTein has embraced Click Chemistry, an efficient method for conjugating peptides with various biomolecules. This technique involves modifying the peptide with azide groups (–N3). A standout feature in LifeTein’s arsenal is the novel Copper-free Click Chemistry, which is based on the reaction of a diaryl cyclooctene moiety (DBCO) with an azide-modified peptide. This reaction is not only rapid at room temperature but also avoids the use of cytotoxic Cu(I) catalysts, leading to almost quantitative yields of stable triazoles.

The DBCO component allows copper-free click chemistry to be safely employed with live cells, whole organisms, and non-living samples, which is a significant advantage in various biological applications. Importantly, within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are abundantly present in many biomolecules. The reaction of the DBCO group with the azide group is notably faster than with the sulfhydryl group (–SH, thiol), making it a preferred choice for many of LifeTein’s clients.

Practical Applications: Peptide Drug Conjugations

A prime example of the application of these techniques is in the creation of antibody-biomolecule conjugates. LifeTein’s protocol for Click chemistry of antibody-DNA conjugation is straightforward and efficient:

  1. Pre-conjugation Preparations: Remove all additives from antibody solutions using methods like dialysis or desalting. It’s crucial to eliminate BSA and gelatin from these solutions and concentrate the antibody post-purification.

  2. Activation with DBCO-NHS Ester: The antibody is mixed with a 20-30 fold molar excess of DBCO-NHS ester dissolved in DMSO and incubated at room temperature or on ice.

  3. Quenching the Activation Reaction: This step involves adding Tris-HCl (50-100mM, pH 8) to the reaction mixture, followed by incubation at room temperature or on ice to stabilize the reaction.
  1. Equilibration and Removal of Non-reactive DBCO-NHS Ester: This is achieved using a Zeba column, following the manufacturer’s instructions to ensure precision and effectiveness.

  2. Copper-Free Click Reaction: The DBCO-NHS ester labeled antibody is then mixed with a 2-4 times molar excess of azide-modified oligos. This mixture is incubated overnight at 4°C or for a few hours at room temperature, facilitating the conjugation process.

  3. Validation and Purification: The final step involves validating the conjugation and purifying the product using HPLC, ensuring the high quality and efficacy of the conjugate.

LifeTein’s expertise in peptide synthesis and conjugation is further exemplified by their application of Click Chemistry and thiol-maleimide bioconjugation techniques. These methods are not only efficient but also versatile, opening up new possibilities in the field of peptide-based therapeutics and research.

Selected References:

  • Simon et al. (2012). Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
  • 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.
  • Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.

Through these advanced techniques, LifeTein continues to be a leader in the field of peptide synthesis and bioconjugation, contributing significantly to the advancement of biomedical research and therapeutic development.

One example of peptide drug conjugations is the antibody-biomoleule 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