The efficiency of conjugation reactions
August 8th, 2017
There are several reasons why you would want to measure conjugation efficiency, with both qualitative and more labour-intensive quantitative methods available. In many cases a qualitative answer may be sufficient, for example to provide reassurance before embarking on a lengthy set of experiments with a new conjugate; but where absolute consistency from batch to batch must be demonstrated, a quantitative test may be required. This article focuses primarily on methods of assessing conjugation efficiency which require little or no specialist equipment, with some reference to more sophisticated tests for those wishing to carry out extensive characterisation of conjugates prior to use.
In many cases, testing in the intended downstream application may be too time-consuming to routinely be used to confirm the success of a conjugation reaction, but some relatively simple analytical tests are available. Methods of conjugate detection include:
- Direct assessment of a measurable property of the label (e.g. absorbance, fluorescence, enzyme activity) following physical separation of excess label from the conjugate
- Evaluation of a new binding property of the conjugate
- Observation of a significant change in the molecular weight of an antibody following conjugation (this is a useful test if the label is a large molecule and can be separated easily from the free antibody)
Organic dye labels
When labeling an antibody with an organic dye, the most appropriate detection method depends on the conjugation approach that has been taken. NHS ester-type reactions are popular, but necessitate a dye separation step to avoid high background staining. During the separation step any free dye is removed, albeit with the associated loss of some conjugate, and it is then relatively easy to determine whether the antibody has any dye attached e.g. by measurement of absorbance or fluorescence, or potentially even by eye.
The dye separation step can be avoided using Lightning-Link®, an innovative technology that enables direct conjugation of antibodies to a wide variety of labels, including organic dyes. The Lightning-Link® kits are incredibly quick and easy to use and, since the conjugation process involves no separation steps, no antibody conjugate is lost. The kits are fully scalable, allowing the conjugation of just 10µg antibody, up to 1g or more; during the initial stages of conjugate generation, we recommend preparing and testing microscale amounts of conjugate to optimize the conjugation conditions. Microscale conjugations are also useful if there is some uncertainty over the antibody buffer composition, and thus the presence of potentially interfering substances in the sample.
To quickly and easily determine the success of antibody conjugation to a colored label such as an organic dye, fluorescent protein, gold nanoparticle or latex bead we have developed Conjugate Check&Go!, a dipstick lateral flow assay in which the appearance of a colored line on a nitrocellulose membrane signifies that a conjugate has been formed. The kit is compatible with IgG antibodies from multiple species, provided that they have affinity for either Protein A or G, which are immobilized at the Test line on the membrane. The kit is not specific to a particular type of conjugation technology, and requires only small volumes of antibody conjugate, thereby saving precious material.
Since most protein labels are of high purity, they give distinct bands on SDS-gels. These bands will become weaker or disappear when the protein is attached to an antibody chain, due to a band shift to a higher molecular weight. After attachment of a protein label to an antibody, the greatest band shift will be seen for the light chains on a reducing gel, although it is rare to see a high percentage of light chains labeled since the probability of labeling the larger heavy chains is greater. Although SDS gels are very useful for showing a change in profile following conjugation, it is essential to run the unconjugated antibody and the label alongside the conjugate for accurate interpretation of results. SDS-PAGE profiles can be quite complex for several reasons:
- Each individual antibody molecule will have a different number of labels attached
- The protein label itself may have multiple sub-units
- The most common type of SDS-PAGE analysis (reducing conditions) separates the four antibody chains, which further complicates the profiles and can have the effect of making the labeling appear inefficient – for example, if all antibodies have one label attached, three quarters of the antibody chains on a reducing gel would exhibit no band shift. A solution to this problem is to run non-reducing gels, in which case every antibody with a label attached will exhibit a band shift since the four chains migrate as one. One should use gradient gels for this type of analysis as the 160kDa IgG antibody and larger conjugates otherwise do not migrate very far into the gel.
Whether a reducing or a non-reducing gel is run, gel scanning software can be used to quantify the intensity of the bands, allowing batch-to-batch consistency to be checked. While SDS gel analysis can become extremely complex, it is still useful if demonstration of a shift in the antibody bands is all that is required.
Our Conjugate Check&Go! Kit is superior to SDS-PAGE or gel filtration as a method of proving successful conjugation of an antibody to a multi-subunit fluorescent protein. If SDS-gels are not routinely being run in the laboratory, or a multi-track gel to test a single conjugate cannot be justified, Conjugate Check&Go! is a rapid and cost-effective solution. We have used Conjugate Check&Go! to test antibody conjugates produced using our Lightning-Link® antibody labeling kits.
It is usually very difficult, short of testing in the actual assay application, to verify that conjugation to gold nanoparticles has taken place successfully. In the case of passive antibody adsorption to gold, where the antibody binds non-covalently, the gold nanoparticles may be stabilized by antibody binding and therefore more resistant in a salt stress test. Although this is a qualitative test, it is widely used to show that conjugation is likely to have taken place. Another method of assessing antibody conjugation to gold is to look for a detectable change in the absorbance profile, which occurs because the ‘coating’ of antibody subtly changes the plasmon band. Again, this sort of result is reassuring but qualitative and indirect.
To overcome the difficulties associated with conjugating antibodies to gold nanoparticles, we have developed InnovaCoat® GOLD. The nanoparticles within the InnovaCoat® GOLD Conjugation Kits have a proprietary surface coating which prevents metal-protein interactions and covalently binds the antibody to form highly stable conjugates. The kits are incredibly easy to use and require significantly less antibody than passive conjugation methods. Furthermore, with different nanoparticle sizes and different chemistries available, InnovaCoat® GOLD provides excellent flexibility.
Our Conjugate Check&Go! Kit is by far the easiest and most convincing test available for evaluating whether antibody conjugation to gold nanoparticles, whether by passive or covalent means, has been successful.
Successful conjugation of an antibody to latex beads is very difficult to assess. Unlike antibody conjugation to gold, latex conjugation cannot be evaluated in a salt stress test; furthermore, latex does not have the potentially diagnostic plasmon shift.
Traditionally, the only way to evaluate the success of an antibody conjugation to latex has been to test the conjugate in the intended downstream application. Traditional methods of conjugating antibodies to latex beads are technically complex and can waste a lot of precious material. Latex Conjugation Kits from Expedeon have been designed to greatly simplify the process. The kits are quick and easy to use, and the latex beads are specially treated, making them resistant to aggregation and meaning that extensive pH optimization is not required. With several different colors available, our Latex Conjugation Kits also provide the opportunity for multiplexing.
Conjugate Check&Go! is the quickest and easiest method of proving successful antibody conjugation to latex beads.
Streptavidin is a protein label and, as discussed, the success of an antibody conjugation to streptavidin may be readily assessed by SDS-PAGE. Since streptavidin is not colored, antibody conjugation to streptavidin cannot be evaluated using Conjugate Check&Go!
Biotinylation is far harder to quantify. It is most commonly evaluated using the HABA assay, a test which is not straightforward and requires very careful calibration. The HABA assay involves addition of the biotinylated antibody to a solution containing 4-Hydroxyazobenzene-2-carboxylic acid (HABA) and avidin. Since it has a higher affinity for avidin, biotin displaces the HABA, resulting in a measurable decrease in absorbance at 500nm.
Our Biotin Check&Go! Kit is a quick and easy to use alternative to the HABA assay, and exploits the ability of biotinylated antibodies to bind to streptavidin gold, thus providing a simple readout with little effort at all. Reference materials provided within the kit allow semi-quantitative assessment of biotinylation efficiency.
Expedeon offers a wide range of bioconjugation kits, including Lightning-Link® for direct labeling of antibodies with fluorescent dyes, fluorescent proteins, enzymes, biotin or streptavidin, InnovaCoat® GOLD for conjugation of antibodies to high quality gold nanoparticles, and Latex Conjugation Kits for one-step covalent conjugation of antibodies to specially-treated latex beads.
For more information about any of our products please contact us.