A conjugated or labeled antibody is a polyclonal or monoclonal antibody with an attached reporter molecule that generates a signal through color, fluorescence, radioactivity, or enzymatic activity, which can be detected and measured.

If you intend to conjugate your antibody, the quality and purity of the starting antibody are critical. Ensure that it has been stored at an appropriate concentration to maintain activity. According to Novus Biologicals, the storage buffer should be amine-free (e.g., PBS, HEPES, or MOPS) and free of components such as BSA, sodium azide, glycine, or tris, as these can quench certain labeling reactions.

Attaching the conjugate

In a Cold Spring Harbor Protocol1, Berg and Fishman explain that most antibody-labeling strategies use one of four modes of attaching a small molecule to an antibody. For labeling or for crosslinking biomolecules, the primary sites used are the charged amino acids found on the outer surfaces of antibodies.

Primary amines are frequently the targets for the coupling chemistry because they are abundant, widely distributed, and easily modified due to their reactivity. But they are often involved in protein–protein interactions that are necessary for the antibody’s function. Therefore, their chemical modification may interfere and significantly decrease the antigen-binding activity of the antibody.

A second target is the carbohydrate moieties found on the often highly glycosylated tail of the antibody called the crystallizable or Fc fragment of the antibody, which are distant from the antigen recognition sites. Therefore, this label is less likely to interfere with antigen binding. Other targets include the sulfhydryl moiety contained in cysteine residues, and tyrosine residues that can be specifically iodinated (e.g., with 125I) to radiolabel the antibody.

Many antibody conjugating methods use chemical cross-linkers to facilitate the addition of a desired modification. Important factors to consider include the chemical specificity of the cross-linker for a particular functional group along with specific side reactions that could occur, whether the required cross-linker should be hetero- or homo-bifunctional, the spacer arm length that represents the distance between the two cross-linking moieties, and the solubility of the cross-linker in water or an appropriate organic solvent.Homo-bifunctional cross-linkers can result in self-conjugation and polymerization whereas hetero-bifunctional agents allow controlled sequential reactions that minimize unintended intramolecular crosslinking.

Labels

In the simplest approach, enzymes such as horseradish peroxidase or alkaline phosphatase can be covalently attached to antibodies and used in immunoassays, western blots, immunohistochemistry assays, and other applications. Labeling antibodies with biotin is also a useful and simple technique. The affinity of avidin for biotin enables detection of biotin-conjugated antibodies. In addition, fluorescently-labeled bioactive reagents are suitable for use in immunofluorescence, flow cytometry, and other biological applications.1

Purification

After conjugation, the antibody must be purified. Different types of chromatography—size-exclusion, ion-exchange, reverse-phase, or affinity—or dialysis can be used. All clean-up approaches have method-specific advantages and disadvantages.

Storage

Depending on the amount of labeled antibody and experimental needs, short- or long-term storage could be required. Typically, the recommendation is to store antibody conjugates at 4°C. Sterile filtration can help extend long-term stability, by inhibiting microbial contamination. If an antimicrobial agent, like sodium azide, is to be added to the antibody conjugate, ensure it will not affect performance in intended assays. In general, storage of an antibody or antibody conjugates at a protein concentration >1 mg/mL will retain higher activity.1

New labeling methods

Affinity-based methods have been developed to overcome the limitations of nonspecific antibody conjugation using traditional random conjugation methods, to direct the conjugation to a specific location on the antibody, to avoid interference with the antibody’s target binding properties, and to gain control of the number of reporter molecules attached per antibody. The possibility of using these affinity-based methods to label antibodies even when present in complex mixtures makes them broadly applicable to many different samples.2

Transferring the readout signal from the protein to the DNA level with an oligonucleotide-conjugated antibody increases the sensitivity of protein assays by orders of magnitude and enables new multiplexing strategies. Wiener et al., addressed bottlenecks in the generation of larger oligonucleotide-conjugated antibody panels by combining a nonsite-directed antibody conjugation technique using copper-free click chemistry with ion-exchange chromatography to obtain purified single and double oligonucleotide-conjugated antibodies. They achieved conjugation yields of 30% with a starting quantity as low as tens of nanograms of antibody.3

Despite innovations in labeling methods and reported molecules, it is important to always test and characterize the newly generated antibody-conjugate before application. You can check for proper labeling by, for example, determining the dye-to-protein ratio and comparing the labeled antibodies to unlabeled ones in applications like western blots or immunofluorescence to ensure that their specificity and staining patterns remain the same.

 

References

  1. Berg EA and Fishman JB. Labeling Antibodies, Cold Spring Harb Protoc; doi:10.1101/pdb.top099242
  2. von Witting E, Hober S, and Kanje S. Affinity-Based Methods for Site-Specific Conjugation of Antibodies. Bioconjugate Chem. 2021, 32, 1515−1524
  3. Wiener, J., Kokotek, D., Rosowski, S. et al.Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics. Sci Rep 10, 1457 (2020). org/10.1038/s41598-020-58238-6