Antibody conjugation: the chemistry, the labelling ratio and what to send

Getting a usable conjugate, and where an antibody conjugation kit stops

1. Send the antibody in a compatible buffer. Amine chemistry is consumed by any primary amine in the buffer, so material in a stabiliser containing one cannot be conjugated without a buffer exchange. Carrier protein in the formulation will also be labelled. Establish the buffer requirement before shipping, since this is the commonest avoidable failure.
2. Choose the chemistry from what you can tolerate. Amine chemistry is simple and labels randomly, including within binding regions. Thiol chemistry targets reduced disulphides and is more controlled. Site-specific approaches, whether enzymatic or through engineered residues, give homogeneous products and require more preparation.
3. Set the labelling ratio deliberately. The degree of labelling is a trade between signal and function, and the optimum differs by label and by application. Ask the provider to target a range and to report the measured ratio for the batch rather than the nominal one.
4. Purify away free label. Unreacted label produces background that looks like non-specific binding and is frequently mistaken for a bad antibody. Confirm that removal is part of the service and that residual free label is measured.
5. Verify function after conjugation. Conjugation can reduce affinity, especially with random chemistry at a high ratio. Test the conjugate in the intended application against the unconjugated antibody rather than assuming the label was neutral.

Custom antibody conjugation in house or outsourced

Kits make small-scale conjugation straightforward and are worth it for occasional work and for screening several labels quickly. Outsourcing makes sense at larger scale, for site-specific chemistry and for anything where the conjugate must be characterised properly.

For payload conjugates destined for regulated use, the chemistry, the analytics and the documentation are a different level of work entirely and belong with a provider set up for it.

Characterisation to ask for after HRP antibody conjugation

The measured degree of labelling, the residual free label, aggregate content after conjugation, and concentration by a method valid in the presence of the label. Absorbance-based concentration can be badly wrong when a chromophore has been attached.

Keep an aliquot of the unconjugated antibody. Comparing conjugate and parent in your own assay is the only reliable way to see what the conjugation cost.

An anti cd20 monoclonal antibody and conjugating such a target

An anti cd20 monoclonal antibody binds a B cell surface antigen that does not internalise readily, which is exactly why this target class is used with effector function rather than as a drug carrier. Conjugation chemistry is chosen against internalisation, since a payload that never enters the cell is only a source of toxicity.

antibody synthesis, and what is actually made

antibody synthesis is not chemistry: an antibody is expressed from a gene rather than assembled, so the service is gene synthesis, transient or stable expression and purification, with conjugation afterwards if a label is wanted. Only short fragments and mimetics are genuinely synthesised, which is a different product class.

Common questions

Why did my antibody conjugation fail?

Most often the buffer. Amine chemistry is consumed by primary amines in common stabilisers, and carrier protein in the formulation gets labelled too. Establish the buffer requirement and exchange before shipping.

What degree of labelling should antibody fluorophore conjugation target?

Enough for signal without impairing binding, which varies by label and application. Ask the provider to target a range and to report the measured ratio for your batch rather than a nominal figure.

Random or site-specific conjugation?

Random amine chemistry is simple and can label binding regions. Site-specific approaches give homogeneous conjugates with preserved binding and need more preparation and cost. Function sensitivity decides it.

Should I test the conjugate?

Yes, in the intended application against the unconjugated antibody, which means keeping an aliquot of the parent. Conjugation can reduce affinity and the loss is invisible without that comparison.

How is antibody oligo conjugation different from a dye or enzyme?

The payload is large, charged and made in a different chemistry, so the coupling is usually done in two steps through a linker rather than in one pot, and the product has to be separated from unreacted oligonucleotide by a method that can tell two large molecules apart. Expect a lower yield, a real purification step, and a characterisation that counts oligonucleotides per antibody rather than reading an absorbance ratio.

Browse by service class

• Compare Gene synthesis on advertised prices