Antibody-Oligonucleotide Conjugation Chemistries

A Comparison of Amine Conjugation and Light-Activated Site-Specific Conjugation Techniques

Antibody-oligonucleotide conjugates are gaining popularity for their ability to precisely detect very small quantities of proteins, therefore significantly increasing assay sensitivity and specificity, whilst also enabling new multiplexing strategies. However, it is well established that the antibody-oligonucleotide conjugation process is expensive, time consuming and requires high levels of expertise. 

Fortunately, the development of commercially available kits that simplify labeling of antibodies to oligos has removed much of this complexity. There are many oligonucleotide conjugation products now available that are helping researchers (even those with limited antibody labeling experience) perform their own antibody-oligonucleotide conjugations in the lab, without having to outsource or hire an expert.

There are several chemical approaches that can be used to conjugate an antibody to an oligo including amine conjugation, labeling of carbohydrate residues, or via reducing disulfide bridges on the antibody to expose their reactive group. In this blog we compare the most common conjugation approach adopted by commercially available technologies, amine conjugation, to AlphaThera’s innovative Light-Activated Site-Specific Conjugation approach.

Amine Conjugation Technologies

Primary amines (-NH2) are common targets on antibodies for linking oligonucleotides (as well as other labels). They present throughout every antibody, mainly on the lysine residues but also on the N-terminus of each polypeptide chain. The abundance and distribution of amine groups across the antibody, alongside their ability to be easily modified because of their location and reactivity, establishes amine directed conjugation as the most popular technique adopted by commercially available antibody-oligonucleotide labeling technologies.

Products using an amine conjugation approach have played a significant role in the progression of research using oligo conjugates, however there are some drawbacks to these technologies which should be addressed:

• Potential blocking of antibody binding sites: Primary amines are distributed across the entire antibody, meaning there is the potential for the oligo to attach at the antibody binding site. This then would render the conjugate useless, wasting expensive antibody and oligo, as well the time required for the conjugation procedure and the application itself.

• Long protocols with many steps: Although the conjugation procedure is made simpler with the development of these conjugation technologies, there are still lengthy protocols associated with the labeling process.  For example purification of the antibody and oligo is required prior to conjugation, as well as oligo and antibody activation, and desalting steps.

• Large quantities of material required: These technologies require sizable quantities of antibody (usually over 100ug), as well as large quantities and a molar excess of oligo to ensure successful conjugation, resulting in additional cost.

• Pre-conjugation purification of antibody required:  In addition to the already lengthy labeling procedure, any antibody used with amine directed conjugation technologies will need to be purified in advance of conjugation, as proteins in the antibody buffer such as BSA, or even some salts such as Tris and sodium azide would otherwise interfere with the conjugation reaction.

   

Light-activated site-specific conjugation (oYo-Link®)

Controlled, reliable and rapid antibody-oligo conjugation in just 2 steps.

The researcher simply has to submit their oligonucleotide sequence. We then source from our partner provider (IDT) before combining with our oYo-Link® technology. We then send the oYo-Link Oligo reagent back to the researcher ready for conjugation to the antibody of their choice. All that is needed is an LED Crosslinking Device.

2 steps – mix & illuminate 

One of the main advantages of this conjugation approach is the significantly reduced hands-on time. Unlike other conjugation technologies which demand antibody purification, as well as  activation of the oligo and antibody prior to conjugation,  oYo-Link® Oligo requires only 2 steps – mix and illuminate. Furthermore, after just 2 hours the conjugates are ready to use!

Site-Specific – controlled reactions & no loss of antibody functionality

oYo-Link® Oligo offers several more benefits over other commercially available technologies and kits. In particular, the ability to site-specifically label the antibody on the Fc region only ensures controlled reactions with a consistent number of oligo attached, and confidence that there will be no attachment of the Oligo in the antigen binding site.

oYo-Link Oligo also enables conjugation of oligonucleotides with very small quantities of antibody in diluted concentrations – as little as 1ug in concentrations as low as than 0.05mg/ml!

Below we compare the main features of oYo-Link® and LASIC approaches to conjugation with other commercially available technologies using amine directed oligonucleotide conjugation: