Primary antibodies for flow cytometry: conjugated or not?

Designing a successful flow cytometry experiment requires effort on multiple fronts, including selecting the right antibodies for the experiment. The antibody selection process, often referred to as “building an antibody panel,” is the pre-experimental step researchers spend the most time on. Here we provide pointers for choosing between conjugated and unconjugated antibodies for flow cytometry experiments.

Before you start:

1. Define clear goals for your flow cytometry experiment.
2. Make sure you know which fluorochromes your instrument can detect.
3. Design your experimental controls with care, especially if fluorescence compensation is needed.
4. Outline your gating strategy, such as excluding debris, doublets or clumps, or isolating specific cell types in a complex population.
5. Select your antibodies

When it comes to selecting antibodies, researchers can choose between conjugated antibodies and unconjugated antibodies depending on their needs. Below, we discuss when it makes the most sense to choose one against the other.

Conjugated versus Unconjugated Flow Cytometry Antibodies

Conjugated flow cytometry antibodies are primary antibodies that have a fluorochrome, like fluorescein (FITC), phycoerythrin (PE), or allophycocyanin (APC), directly coupled to the antibody. Unconjugated antibodies on the other hand do not have a linked fluorochrome.

Unconjugated antibodies

If you choose to use unconjugated antibodies in your flow cytometry experiment, you will have to perform a secondary staining with an antibody that recognizes the primary and carries the fluorochrome, which increases the number of steps in your protocol compared to conjugated antibodies. However, unconjugated antibodies can be used across multiple types of experiments more easily than a conjugated antibody, adding flexibility.
Additionally, a scientist who wants to conjugate their antibodies with specific fluorochromes that are not available commercially can do so with unconjugated antibodies, allowing for more customization. Secondary staining is also known to amplify signals and can help with sensitivity if the target is expressed at low levels. On the other hand, they may also produce higher background signals.

Advantages of unconjugated antibodies for flow cytometry

• Higher signal sensitivity due to signal amplification.
• Increased flexibility, since the same secondary can be used for different primary antibodies in various experiments.
• Commercial availability, as conjugated secondaries are more common than conjugated primary antibodies.

Conjugated antibodies

Conjugated antibodies enable simple one-step staining of the cells you’re studying. This means using conjugated antibodies offers a simpler, faster workflow compared to unconjugated antibodies. They also lead to lower background signals compared to primary/secondary antibody combinations. They may cost less than having to purchase primary plus secondary antibodies. Conjugated antibodies are also useful for complex, multi-color panels allowing researchers to perform their experiments without cross-reactivity between secondary antibodies. In addition, if the sample you’re analyzing is precious because perhaps the cell population is difficult to obtain, using conjugated antibodies in a shorter staining workflow helps you avoid multiple wash steps, and preserves the sample.

In summary, conjugated antibodies are preferred when the goal is rapid, reproducible, multiparameter flow cytometry with limited or valuable samples, whereas unconjugated antibodies are preferred when maximum sensitivity through signal amplification or greater experimental flexibility is required.

Advantages of conjugated antibodies for flow cytometry:

• Faster, simpler, and possibly cheaper workflow without secondary antibody, reducing hands-on time.
• Reduced non-specific binding due to secondary antibody.
• Ideal for multicolor flow cytometry panels, eliminating cross-reactivity between secondary antibodies.
• Fewer wash steps, minimizing cell loss and preserving precious or limited samples.
• Reduced experimental variability, since the staining procedure involves fewer reagents and manipulations.

FluoSite™ Antibodies

BPS Bioscience offers recombinant, conjugated antibodies for your flow cytometry studies. Their FluoSite antibodies are tagged with fluorochromes using a method that is highly site-specific. The process ensures that the labeling site is far from the antigen-binding domain to preserve antibody functionality. The precise labeling strategy ensures the antibodies are useful for sensitive flow cytometry experiments. It also ensures homogeneity of the antibody preparation, lot-to-lot consistency, and experimental reproducibility.

BPS Biosciences FluoSite antibodies:

1. Are recombinant and monoclonal
2. Bind to human targets
3. Have been validated for flow cytometry
4. Are purified from a human HEK293 expression system

We currently offer BPS Bioscience’s PE, FITC, and APC-labeled antibodies that bind to popular targets such as PSMA, KLK2, STEAP1, GPRC5D, CD19, CD20, CD4, CD8, EGFR, Claudin-18.2, and many more, as well as isotype controls.

Building your flow cytometry panel?

Choosing the right antibodies can take time, especially when you are working with complex panels or valuable samples. Our colleagues are happy to think along with you and help you find the BPS Bioscience FluoSite™ antibodies that fit your flow cytometry experiment. Contact our team for personal advice or product availability.