Bioconjugation is the establishment of covalent bonds between a biomolecule, eg. a protein, a carbohydrate, a lipid, or a nucleic acid, and another molecule or material. This method is used in manufacturing of ELISA, ELISpot, WB, IHC, IF, or other antibody-based methods, such as Antibody-drug conjugates (ADCs). Bioconjugation modifies the structures of the biomolecules, giving them new functions that they cannot exhibit by themselves.

Bioconjugation must-haves:

• high concentration, high purity and quality of starting compounds are necessary
• high level of reaction, minimum energy input, minimum byproduct, maximum yield
• needs to be site-specific
• functional groups must be accessible
• appropriate linkers must be used
• in many cases, bioconjugation must occur with cellular processes intact

Basics of bioconjugation techniques – commonly used chemical reactions

• Amide bond is formed between amino acids to form peptides or proteins when an amine group reacts with carboxylic acid. This bond is stable under wide pH and temperature range, and is used in pharma and nanoparticle-mediated drug delivery.
• Thiol-maleimide reaction is commonly used to modify the thiol group of cysteine residue of proteins to produce functional therapeutics i.e. ADCs. Because available cysteine residues are few, reaction is selective and uniform.
• Click chemistry, eg. CuAAC copper-catalyzed azide-alkyne cycloaddition occurs where an alkyne and an azide form a ring in a process of cycloaddition in which copper is needed to bring both compounds closer together. This method is used, for example, in polymer synthesis, and protein-small drug conjugates. The reaction is highly selective, but cannot be used in living cells or tissues or organisms because of the toxicity of copper.
• Copper free click chemistry uses the functional group DBCO (dibenzocyclooctyne) for in vivo studies.

Applications of bioconjugation

In Therapeutics: Bioconjugation is used to attach a therapeutic agent (a cytotoxin) to an antibody that can specifically target tumor-associated antigens on cancer cells. These ADCs, are selectively taken up by tumor cells, and cytotoxin exerts its effects in the tumor cells. Glycans (such as mucin) are good targets because they differ chemically between tumor cells and  healthy cells. The antibodies can be radiolabeled in order to track and ensure delivery to target. Therapeutic ADCs are being developed for example against breast cancer, lymphoma, lung cancer, prostate cancer, and gastric tumors.

In Diagnostics: Enzyme-linked immunosorbent assays (ELISAs) are used to study the antigen-antibody interactions often by using a secondary antibody for detection and quantification. Then a colorimetric detection (eg. HRP, fluorophore) is used to quantify and visualize between healthy and diseased cells.

In Protein Research: Biotin is the most widely used detection label for protein detection and isolation studies. Conjugation of biotin to antibodies and cell surface receptors can be used to highly enhance the detection signal since streptavidin has a high affinity towards biotinand can be used instead of a secondary detection antibody. In protein research, bioconjugation is used to create fusion proteins that allow the study of protein-protein interactions. These protein-protein units are created by genetic fusion, enzyme-mediated conjugation, or chemical ligation (eg. click chemistry). Fusion proteins can function as a single unit.

In Nanotechnology: This field of research aims to make use of nanoparticles to improve e.g. therapeutic antibodies and imaging agents (such that are used in MRI).

Advantages of bioconjugation

Specific tissues and cells can be targeted by using conjugations of proteins and peptides to nanoparticles, drugs, and imaging agents. This often helps to enhance the signal, and minimize off-target effects.

Bioconjugation can improve drug efficacy. For example, drug molecules can be coupled with biomolecules to keep them inactive until they are taken up by the target host cells. Drugs are thus protected from degradation, which in turn extends their half-life and reduces dosages needed. By attaching hydrophilic biomolecules, the solubility of a drug can be increased to enhance the circulation and absorption of injectable or orally administered drugs. 

Bionconjugation offers biocompatibility. By using biomolecules in functional enhancement ensures that the targeted therapeutic or diagnostic method is not cytotoxic to live, healthy cells. Therefore, bioconjugation provides an opportunity to visualize and track molecular interactions in live cells in real-time.

Challenges and considerations of bioconjugation

The components of a bioconjugate are linked together through a group that involves the reactive functional groups of these molecules. Lysine and cysteine are ideal reactive sites for conjugation in proteins. However, their high relative abundance in proteins can result in variability in the number and location of conjugation sites, potentially disrupting the natural function of the protein and reducing its effectiveness. Site-specific bioconjugation methods provide control over structure and function of protein conjugates. By ensuring the covalent attachment occurs only at a specific site, unexpected changes that could negatively impact the function of the biomolecule can be prevented.

Future prospects

Bioconjugation technologies continue to evolve thanks to new innovations in chemical, enzymatic, genomic, and computational methods. These methods include click chemistry, directed site-specific labeling, high-throughput screening (eg. mass spectrometry), photo-click chemistry, precision microfluidic devices, and stem cell research (eg. 3D models).

 Our Bionconjugation Suppliers

VectorLabs

BioNordika supplies bioconjugation reagents and solutions from Vector Laboratories. Their bioconjugation portfolio includes:

1. Functionalized dyes (dyes, IR Fluors, quenchers)
2. Functionalized beads (agarose, magnetic)
3. Linkers and reagents (Click Chemistry, SoluLINK, ChromaLINK)
4. Linker kits (Click Chemistry, SoluLINK)
5. Classic fluorescent dyes (IR dyes, cyanine)

Functionalized dyes are customized conjugates that are modified chemically by adding reactive groups and linkers to dyes.

Dye Brands:

• AzDyes are a direct replacement for Alexa Fluor Dyes.
• Cal Fluor Dyes are only fluorescent when bound to target (for in vivo studies)
• AQuora Dyes from Quanta BioDesign enable higher degree of labelling
• MB Dyes are a mix of Alexa and ATTO dyes that are less susceptible for self-quenching. These are used e.g. in histology, microbiology and cytology etc.

How to create a functionalized dye?

1. select brand (e.g. AZDyes)
2. choose reactive group (e.g. azide)
3. select laser (e.g. 488 nm).

Vector Laboratories´ dye selection pages can be found here

Cell Signaling Technology

BioNordika offers custom conjugation services from Cell Signaling Technology (CST). These services are available for antibodies produced by CST.

• Custom conjugation services are available for the following: fluorophores, haptens (e.g. biotin), enzymes (e.g. HRP), beads (e.g. agarose, magnetic) and nucleotides (e.g. 10x and Akoya oligo-antibody conjugation services
• CST produces high purity, high yield conjugates that are free from unlabeled material
• Tiered service offerings range from basic antibody conjugation to full validation and stability testing
• Conjugates are tested in key applications using biologically relevant cell systems and controls
• Identification of optimal degree of labeling (DOL) is used to achieve the best signal-to-noise ratio
• Technical support from CST conjugation team