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Biotinylated Antibodies: A Closer Look at the Chemistry and Optimization

Biotinylated antibodies are antibodies that have been chemically modified to include biotin, a small molecule that strongly binds to proteins like streptavidin or avidin. Primarily, you’d use biotinylated antibodies in assays that need signal amplification and detection, like immunohistochemistry (IHC), enzyme-linked immunosorbent assays (ELISAs), and flow cytometry. 

Let’s look a little deeper at the chemistry behind the biotinylation process and try to understand why this sticky reaction has become such a central part of so many immunoassays.

Biotin and Streptavidin Chemistry

The part that makes biotinylated antibodies what they are is the interaction between biotin, which is actually a small molecule vitamin (B7), and streptavidin, a tetrameric protein with a high affinity for biotin. This interaction is incredibly strong – with a dissociation constant in the order of 10-15 M – that rivals some strongest known protein-ligand interactions. The reason behind this high-affinity binding is primarily due to the super complementary fit between biotin and the streptavidin binding site, which also work alongside multiple hydrogen bonds and hydrophobic interactions to lock the biotin in place. 

You can use this strong interaction to your advantage if you’re running assays that require high degrees of sensitivity and specificity, knowing that the biotin-streptavidin complex will remain intact even under stringent conditions.

Biotinylation: The Attachment Process

Attaching biotin to antibodies (biotinylation) involves the formation of a covalent bond between biotin and specific amino acids on the antibody, typically lysine residues or the N-terminus. We can do this with reagents such as N-hydroxysuccinimide (NHS) esters of biotin, which react with the primary amine groups of lysines to form a stable amide linkage. At Alomone labs, we offer the medium-chain (spacer length 22.4 Å), NHS-ester activated biotinylation reagent that labels primary amines (lysines)/

Your choice of biotinylation reagent and the conditions you choose for biotinylation are critical as they impact the efficiency of biotin attachment and the preservation of antibody affinity and specificity.

  • Carefully controlling the pH and temperature during the biotinylation process will help you not only maximize efficiency but also preserve the antibody’s affinity. Typically, using a pH around 7.2–7.5 and at room temperature is optimal for most antibodies.

Optimizing Your Experiments

Your success with biotinylated antibodies hinges on how much you choose to optimize various factors, from the degree of biotinylation to the choice of streptavidin conjugate. Over-biotinylation can lead to aggregation or reduced affinity for the antigen, while under-biotinylation may result in weak signals. Furthermore, the choice of streptavidin conjugate – whether it’s labeled with fluorophores for imaging or enzymes for ELISA – can greatly impact the sensitivity and specificity of your assays.

  • Try adjusting the molar ratio of biotin-to-antibody to reach optimal biotinylation without compromising the antibody’s function. A ratio of 20:1 (biotin:antibody) is a good starting point, but this may need adjustment based on the specific antibody and application.
  • Select an appropriate streptavidin conjugate (fluorophore-labeled for imaging, enzyme-labeled for ELISAs) based on your assay’s detection method. Consider using streptavidin with a high purity and low biotin-binding capacity to minimize background noise.

Overcoming Challenges

Despite their utility, biotinylated antibodies can come with several challenges, such as background noise from endogenous biotin or non-specific binding. It’s therefore essential that you carry out blocking steps, careful washing, and optimizing assay conditions to mitigate these issues, if you want data that are both reliable and reproducible.

  • Use blocking agents that do not contain biotin or streptavidin to prevent non-specific binding. A common choice here is a solution containing a non-biotinylated protein, like BSA, to sit in nonspecific binding sites.
  • You can play around with washing steps, incubation times, and temperatures to minimize background noise and enhance signal-to-noise ratio; this will likely mean experimenting with different buffer compositions and washing solutions.
  • Always include controls without biotin or with free biotin to help determine the specificity and efficiency of the biotinylation and detection processes.

Let Us Help

You can absolutely undertake the biotinylation process yourself. However, if you’d rather save yourself the time and expense of that, we have an easy service available in which all you need to do is tell us a few simple details and we’ll then send you the biotinylated antibody (which we also validate by western blot). We’ll also fully establish the extent of labeling, i.e. the amount of biotin molecules per antibody molecule, so you have all the relevant details before you start your work. Together, this frees you up to focus on the research.

To wrap it up, biotinylated antibodies give you a blend of specificity, sensitivity, and versatility, underpinned by robust biochemical interactions. By understanding the chemistry behind biotinylation and strategically applying this knowledge to your experimental design, you can use these tools to explore biological questions. Whether you’re mapping protein distributions, quantifying interactions, or developing novel diagnostics, biotinylated antibodies are your allies in the quest for scientific discovery.

Have us biotinylate your antibodies