Validating the Quality of Antibody Kits

The past 20 years have seen vast growth in the use of kits for a wide variety of applications in the biological sciences. The protocols these kits replace were often time-consuming and sometimes dangerous. Kit use has also democratized science, making many applications simple and reliable. One application where a kit is frequently used is the fluorescent labeling of antibodies. Sherri Fraser spoke with Judie Berlier, technical applications specialist in the life sciences solutions group at Thermo Fisher Scientific, about choosing antibody kits, what quality control to expect from your supplier, and what validation measures you may need to do after using a kit. Note: these responses have been edited for clarity and style.

Q: Why would someone want to use an antibody kit?

A: In many antibody-based applications, such as western blots, a primary antibody is used that binds directly to a protein target. To visualize the target, a secondary antibody labeled with fluorescent dye is used to bind the primary antibody, creating a sandwich with the primary antibody between the target protein and the secondary antibody.

For applications such as flow cytometry, researchers don’t use a secondary antibody. In such cases, customers have a unique antibody that is not commercialized nor available labeled with the dye of choice. Kits give them the option to easily make fluorescently labeled antibodies following simple directions. Furthermore, with increasing emphasis on multiplexing in experiments, users are working with panels of multiple antibodies, all with different visual tags. Using a kit allows for ultimate customization when planning such experiments. 

Q: What quality control measures should clients look for from manufacturers when purchasing an antibody kit?

A: A kit is only as good as the user guide. There should be parameters well defined for each kit. These parameters should specify the amount of antibody that it is optimized to label, and what size ranges are suitable for the purification step.

Look at the literature from the vendor and consider the conditions, such as pH, that the antibody will be subjected to by using the kit. Use knowledge of these conditions to determine how that may impact your experiment or antibody sensitivity and choose a kit best suited for your antibody. Another thing to look for would be citations showing that the kit has been used successfully by others—a new kit may not have those, but if there is no data for an older product, it’s a red flag. 

Q: What are the advantages of using an antibody kit? 

A: The kits are easy and straightforward, and faster than sending the antibody out to have it labeled by a company. Kits are like using a box-meal recipe. They tell you how much of and when to add each component, all of which are provided in the box.

A kit is only as good as the user guide.

Using a kit helps scientists move on with the real work instead of spending time developing methods and creating components. It’s efficient, it’s easy, it’s going to work probably 95 percent of the time. When it doesn’t, that’s when you start looking at options, and everybody has options right now in the industry.

Q: What should a lab manager consider when buying antibody kits?

A: Choose the right tool for the job. Many problems can be avoided with some pre-planning. Some things to consider include:

•    How much antibody do you have?

There are kits designed for different antibody amounts. You need a different kit based on the mass that you need to label. Don’t use the wrong kit or it won’t work. 

•    What is your antibody currently stored in?

Purified antibodies can be easily labeled using amine reactive chemistry, while antibodies with carrier proteins or in media or ascites need to be addressed differently. For best results, you should purify your antibody before labeling it, if possible.

•    What color do you need?

As researchers switch to multiplex panels, with many different targets to be visualized in a single sample, the color of each label must be considered in terms of the entire experiment. 

• What expression level is your target?

The level of expression of your target will influence the fluorophore you use to label your antibody. You may want to use the brightest fluorophore on the lowest expressing target. Plan your experiments considering how many channels you will need and the expected expression levels for each target. Browse your supplier’s catalog to determine which dyes fit the desired wavelengths. 

Q: Once you have a kit, how do you take care of it?

A: Pay close attention to the storage conditions of the kit components. Some kits have components that should be stored in a refrigerator, but another part should be stored in a freezer. Improper storage can ruin components.

Q: What validation steps should be taken by the user after antibody labeling?

A: When buying off-the-shelf antibodies, most of the validation work has been done by the manufacturer. They should also tell you which applications the antibody has been tested for. However, when you label your own, you need to do some of that legwork yourself.

Kit use has also democratized science, making many applications simple and reliable.

Normally the labeled antibody would be characterized to determine the degree of labeling (DOL) and the concentration of the labeled antibody using the correction factor provided by the vendor. After that, it would need to be tested in the user platform to make sure that it still binds specifically to the antigen. In rare cases, antibodies can lose binding avidity.

If labeling is too high, you will probably see non-specific staining, resulting in high background. If labeling is too low, unlabeled antibody will outcompete the labeled antibody, and your signal will be dim. Sorting these issues may be difficult but contact your vendor if you need advice.

 Q: What are the risks or disadvantages of using an antibody kit?

A: In some circumstances, some or all avidity of the antibody can be lost post-labeling. Usually this occurs when the fluorophore is in the binding site. If this happens with your antibody, there are kits that don’t rely on random labeling.

If you do a pilot experiment, it may not be easy to scale up to a larger conjugation. Your supplier should have experts available who would be happy to help you figure out how to scale up using their kits.

Q: Are there any exciting prospects on the horizon in the development of antibody kits?

A: The Nobel Prize for Chemistry in 2022 was awarded for the discovery of “click chemistry.”  It was discovered that complex molecules can be easily made using principles similar to LEGO or IKEA—modules can be linked together with simple “click” reactions, creating large complex structures from smaller subunits. 

Click chemistry is being enthusiastically embraced to make designer molecules quickly and easily, including antibodies. It is possible to design where a reactive group should be and, basically at will, have it placed there with click chemistry. Much of the forthcoming work will be on what kinds of reactive partners can be used in antibody design. We will continue to see more sophisticated, faster kits to help do all the antibody-based work you can imagine.

Judie Berlier, technical applications scientist in the life sciences solutions group at Thermo Fisher Scientific, has spent over 20 years in R&D testing new fluorescent dyes and developing hundreds of new products, including antibody labeling kits. She started with Molecular Probes, which became part of Thermo Fisher Scientific. She now works as an applications scientist helping customers use Thermo Fisher’s cell analysis products.