Biotechnology & Immunology


Enhancing immunoassay design through antibody conjugation kits Sharon Sanderson, PhD, Bio-Rad Laboratories, Inc


Antibodies serve as foundational tools across a broad range of research fi elds from immunology to oncology and infectious disease. Advances in reagent development have facilitated the generation of extensive, high-quality toolboxes of specifi c antibodies and compatible labels. Labels, such as fl uorescent dyes, proteins or enzymes, enable target detection when conjugated to antigen-binding antibodies. Using distinct labels supports the simultaneous detection of multiple targets in a single immunoassay experiment, such as western blotting, fl ow cytometry, ELISA, and microscopy.


Target detection can be achieved via either a direct approach using a labelled primary antibody which recognises the target or an indirect approach which requires the use of a labelled secondary antibody specifi c to the primary antibody. The use of direct detection is widely preferred over indirect detection largely due to the requirement for fewer reagents, which reduces costs, simplifi es assay format and design, reduces experimental time, and yields superior data and experiment reliability. It also enables the multiplexed detection of antigens from the same species.


Although many labelled antibodies are commercially available, especially fl uorescent formats, coverage across clones and labels is incomplete. Less popular clones often lack suitable labels, meaning many assays still default to using an indirect format and forgo the benefi ts of direct detection. Consequently, researchers often design experiments based on the commercial availability of labelled antibodies rather than what’s optimal. In this scenario, access to tools that allow researchers to easily conjugate labels to antibodies themselves are greatly advantageous for optimal experiment design.


Conjugation kits address labelled antibody


availability limitations Conjugating antibodies without the use of conjugation kits can take up researchers’ valuable time in the lab and lead to loss of antibody. Antibody conjugation kits offer a fl exible and convenient solution for the direct conjugation of labels to antibodies – with preoptimised reagents and protocols, many kits are easy to use and require minimal hands-on time, often with short protocol durations and incubation periods, or overnight incubation. The use of conjugation kits also doesn’t require the use of specialised equipment, making them very accessible. Many kits are also designed to work with a broad range of purifi ed antibodies, including different isotypes, concentrations and buffers.


By enabling the fast, reliable and reproducible labelling of antibodies, conjugation kits enable researchers to easily expand their toolkit for various research applications. This not only allows researchers to custom label their in-house antibodies but also enables the assembly of optimal panels by pairing the best antibody-label combinations for each assay rather than being limited to whatever pre-labelled clones are commercially available.


While antibody conjugation kits are useful in the context of many research applications, there are many specifi c cases where they might be essential. For instance, in pharma or academic settings, proprietary antibodies often cannot leave the company or institution. Conjugation kits provide a streamlined method for on-site, on-demand, custom-labelling of these antibodies with a wide range of different labels like fl uorescent dyes, enzymes, and biotin/streptavidin.


Beyond these compliance-driven scenarios, conjugation kits also fi ll critical gaps where pre-labelled reagents are scarce – most notably in veterinary immunology. Limited availability of antibodies against veterinary antigens and few conjugated options make experimental design more challenging and constrains multiplexing. Enabling researchers to label available antibodies with suitable tags, conjugation kits expand assay choices and accelerate progress in veterinary immunology.


Real-world applications in veterinary and translational research


The utility of conjugation kits was demonstrated in a recent study investigating avian infl uenza virus (AIV) prevalence in wild ducks, which are considered key natural reservoirs for the virus and therefore play an important role in AIV outbreaks [1]. Immunological studies in ducks have been hindered by the lack of species-specifi c antibody reagent availability. In this study, conjugation kits supported the development of an automated fl ow cytometry-based protocol enabling the monitoring of duck


immune status by detecting changes in white blood cell (WBC) counts. Specifi cally, selected monoclonal antibodies that recognised different types of duck WBCs (e.g., CD4+ and CD8+ T cells, lymphocytes, etc.) were conjugated to unique labels (e.g., FITC, PE, PerCP-Cy5.5, etc.) using a commercially available kit enabling immune system assessment via fl ow cytometry-based WBC quantifi cation. This study demonstrated that age and sex have an effect on WBC counts in ducks – with male ducks naturally infected with low pathogenic AIV showing a reduction of lymphocytes and thrombocytes, which is also commonly observed in humans infected with infl uenza A. The researchers also showed that this protocol can be scaled to analyse samples collected from wild ducks in fi eld studies, allowing broad immune status monitoring of typically understudied avian populations.


Similarly, conjugation kits can also be benefi cial for translational research models, where human disease biology must be mirrored in relevant models. Oral melanoma is a clear example in which human and canine tumours are genetically and biologically similar, making dogs a valuable comparative model. In this model, primary cell cultures present a useful tool for characterising neoplastic cells and investigating cellular pathways that support tumour progression. One study investigated the use of fi ne- needle aspiration (FNA) as a less invasive technique than surgery to sample cells from canine oral melanocytic tumours and nodal metastases for primary cell cultures [2]. In the study, samples were labelled with antibodies conjugated to RPE-Cy7, enabling characterisation of melanocytic cellular phenotypes via fl ow cytometry. This study is the fi rst to demonstrate the establishment of primary tumour cell cultures from FNA samples in dogs for functional studies of oral melanomas, simplifying sample collection for the development of translational in vitro models.


Conjugation kits broaden panel design and multiplex research capabilities


Beyond veterinary and translational research, antibody labelling also enhances the capabilities of fl ow cytometry for other research applications. Conjugation kits can expand the variety of labels conjugated to antigen-specifi c antibodies, resulting in bigger fl ow panels to support multiplexing applications. Conjugation kits also make it easier to combine multiple labelled primary antibodies, which reduces the risk of cross- reactivity that can occur when using secondary antibodies from the same host species in indirect assay formats.


The use of conjugation kits to support multiplexing was demonstrated in a recent study which investigated the role of two transcription factors, Foxn1 and Hif-1α, in the regulation of dermal white adipose tissue (dWAT) during wound healing [3]. Flow cytometry was performed on wounded skin of mice to validate the effi ciency of lentiviral transgene incorporation of Foxn1 and Hif-1α into dermal adipocytes, and to characterise the cell. To enable multiplex labelling, the cells were incubated with multiple antibodies conjugated to varying labels through the use of conjugation kits. This study showed that both transcription factors cooperatively regulate skin dWAT during the proliferative phase of wound healing via the Igf2 signalling pathway. Lentiviral gene delivery of Foxn1 and Hif-1α also facilitated an increased expression of genes involved in lipogenesis and a reduction in macrophage content at the wound site.


By expanding the options of dyes that can be used in fl ow cytometry, conjugation kits make it easier to use a combination of bright fl uorophores that give low spillover and spreading, producing high-quality data to enable clear identifi cation of populations.


Choosing the right kit


Numerous antibody conjugation kits are available to researchers, but the best choice depends on individual applications and experimental set-up. There are a number of factors and questions researchers need to consider to help identify which conjugation kit to use (Figure 1). For example, not all purifi ed antibody storage-buffer components are compatible with every type of conjugation chemistry. Specifi cally, the storage buffer


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