Multiplex immunofluorescence staining of three or more proteins in fixed cells or tissues can be challenging due to the lack of diversity in species used to produce commercial antibodies. Most antibodies are raised in either mouse or rabbit, so researchers are limited to probing only two proteins or cellular targets simultaneously.
Enter chicken antibodies.
Avian-derived antibodies are an excellent choice for complex multiplex immunofluorescence imaging, immunohistochemistry, or immunocytochemistry experiments with more protein targets as they show exceptionally high sensitivity and specificity, with no cross-reactivity to mammalian-derived antibodies.
In this article, we explore the limitations of multiplex immunofluorescence while showcasing what polyclonal chicken antibodies are and how they can help you overcome these limitations with numerous advantages for your next experiment. We also highlight their efficient, non-invasive method of production and some exciting examples of how custom chicken antibodies and current products are advancing our understanding of health and disease.
The Problem with Multiplex Immunofluorescence
Continual advances in immunofluorescence techniques, fluorophore-conjugated antibody design, and complex microscopes now give researchers more tools than ever to pinpoint how the location of one protein in a cell or tissue relates to multiple other proteins simultaneously.
Our guide to immunofluorescence provides a broad overview of direct or indirect approaches, combined with some crucial considerations when planning multiplex immunofluorescence experiments. For instance, studies probing multiple proteins simultaneously could use direct immunofluorescence, where primary antibodies to specific protein targets are conjugated to fluorophores with non-overlapping emission wavelengths and then imaged as different colors.
While many fluorophore-conjugated antibodies are available from Antibodies Inc., in some cases, the appropriate primary antibody-fluorophore conjugate combinations aren’t commercially available. This limits researchers and their experimental options, ultimately hindering scientific discoveries.
A more flexible approach to this is to perform indirect immunofluorescence, where unconjugated primary antibodies raised in different species are combined with fluorophore-coupled secondary antibodies recognizing that particular species. But, researchers are limited by the species used to produce the primary antibody. Usually, primary antibodies are raised in mouse or rabbit, limiting studies to two proteins.
The addition of antibodies raised in a third species, such as chicken, allows researchers to perform robust three-color multiplex immunofluorescence experiments to unravel the complexities of multi-protein localization, potential interactions, and novel protein functions in health and disease.
What Are Chicken Polyclonal Antibodies?
In contrast to mammalian serum, where the main immunoglobulin is IgG with multiple different isotypes, chicken egg yolk contains only one class of immunoglobulin called IgY.
The absence of different immunoglobulin isotypes and the sheer abundance of IgY in eggs allows efficient antibody purification with simple precipitation techniques, reducing cost and boosting efficiency.
IgY accumulates in egg yolks in much greater quantities than IgG in mouse or rabbit serum. For instance, the amount of purified egg IgY produced in one month is 18 times higher than that of IgG produced in rabbit. Similarly, egg yolk contains 15-25 mg/ml of IgY, a much larger quantity than the 10-12 mg/ml of IgG in human serum (Akita and Nakai, 1993).
Both IgY and its mammalian counterpart IgG mount rapid responses to infectious agents but have subtle structural differences, meaning there is no cross-reactivity between antibodies from chickens and mammals. This makes them an excellent addition to multiplex immunofluorescence experiments.
Both contain two heavy and two light chains consisting of a variable domain and four constant domains. IgY has a shorter hinge region than IgG, making it less flexible, and IgY has a higher molecular weight due to an extra heavy chain constant domain.
Unlike IgG, IgY lacks the Fc domains in its heavy chain that binds complement and protein G or protein A, reducing background in immunofluorescence experiments. But if protein G or A are routinely used for pull-downs in your immunoprecipitation experiments, we recommend our PrecipHen® affinity-purified goat anti-chicken IgY coupled to agarose to ensure effective pull-downs with chicken antibodies.
The fact that IgY and IgG are so similar in their structure and biological function yet differ enough to avoid cross-reactivity provides researchers with several advantages when performing multiplex immunohistochemistry, immunocytochemistry, immunofluorescence, or even western blot and ELISA experiments.
Advantages of Chicken Polyclonal Antibodies
- Higher Avidity: Birds and mammals diverged around 300 million years ago, so they are extremely evolutionarily distinct. This means chickens mount more potent immune responses to most antigens, even proteins highly conserved in mammals and thus weakly immunogenic in mouse or rabbit hosts. This allows highly specific antibodies to recognize diverse epitopes even where there are only minor differences in the amino acid sequences of the host homolog and the target protein.
- Abundant and Convenient: One chicken can produce vast quantities of IgY in egg yolk against even low amounts of antigen. Compared to rabbit serum, chickens produce up to 20 times more antibodies, making them highly efficient, convenient, and straightforward to produce in a matter of weeks. Storage is also simple as chicken antibodies are stable for months at room temperature thanks to chickens' relatively high core body temperature at 41°C. However, we recommend storage at 4°C for consistently robust results.
- Humane and Non-Invasive: IgY antibodies are harvested only from eggs, so laying hens don’t undergo invasive blood harvesting procedures, reducing stress and increasing animal welfare, all with no compromise on the final antibody product. It is a win-win for both animals and researchers.
- No Cross-Reactivity: Avian IgY is distinct from mammalian IgG, so there is no cross-reactivity with mammalian-derived antibodies.
- Less Background: Chicken IgY antibodies lack an Fc domain, so they don't recognize other aspects of the mammalian immune system, such as rheumatoid factors, complement, Fc-receptors, or proteins A or G. This means less background signal and fewer false positives in mammalian in vivo studies than with IgG-based antibodies. Using our highly cited BlokHen® blocking reagent can reduce background staining even further in applications such as immunocytochemistry, immunohistochemistry, ELISA, and western blot.
- Compatible with IgG-based Assays: Both IgG and IgY are functionally equivalent in most biochemical or cellular applications, so IgY can largely be used as a direct alternative to IgG in immunofluorescence, immunohistochemistry, immunocytochemistry, ELISA, western blot, and many other approaches (Lee et al.,2017). They can also be conjugated to different fluorophores or other molecules, such as Aves Labs' popular FITC-conjugated chicken anti-rabbit or anti-mouse IgG secondary antibodies.
How Are Chicken Polyclonal Antibodies Produced?
At Aves Labs, we produce our high-quality chicken polyclonal antibodies by combining rigorous computational analyses, to ensure the most immunogenic peptide is used to elicit a strong immune response, with humane, non-invasive protocols for antibody purification.
For custom antibody requests, we begin by providing the amino acid sequence of your protein of interest to our proprietary Immunogenicity Algorithm®, which looks for a combination of hydrophilicity, chain flexibility, and specific residues. Once appropriate peptides are identified and synthesized, we follow our standard injection protocol, where the peptide is injected into the breast muscles of laying hens multiple times over four weeks. After the final injection, we collect eggs over a period of weeks and use our proprietary methods to purify IgY. From this IgY preparation, we can then further affinity-purify the antibody if required.
As the hens are unharmed during this entire process and antibodies are purified from eggs instead of invasive blood collection procedures, as with mouse or rabbit-derived antibodies, we can perform additional injections and purifications to suit the customer's needs. We also provide storage of eggs or boosting of hens where the antibody is required in high volume or at later dates.
We have used this humane, non-invasive process to develop custom antibodies for various proteins and immunohistochemical studies. These studies often wish to combine their custom antibody with other commercial antibody markers made using rabbit and mouse hosts, so chicken antibodies were the best option to perform multiplex immunofluorescence experiments while avoiding cross-reactivity.
Study Success With Custom Chicken Antibodies
The custom antibodies we have developed have been used in many studies informing on mammalian health and disease. For instance, we produced a custom polyclonal chicken antibody to the non-structural viral protein NS5 of Zika virus, which encodes an RNA-dependent RNA polymerase, to investigate the neurotropic properties of Zika virus and the cell types that it infects in the brain (Winkler et al, 2017).
Researchers successfully used this antibody to probe Rag1-/- adult mice lacking a critical B-cell maturation factor, making them susceptible to Zika virus infection. In Rag1-/- adult mouse brains, researchers found widespread ZIKV NS5 immunoreactivity specific to neurons but absent from other cells like astrocytes and microglia.
To show this, they performed multiplex immunohistochemistry with the ZIKV NS5-specific antibody in combination with the neuronal marker neuron-specific nuclear antigen (NeuN), the astrocyte marker GFAP, or the microglia marker Iba1/AIF1 in different regions of the mouse hippocampus, the dentate gyrus and cerebral cortex. As the antibodies for these marker genes were made using rabbit or mouse hosts, an NS5 antibody raised in chicken was the optimal choice for multiplex immunofluorescence, limiting cross-reactivity and boosting sensitivity.
We now also provide the microglia marker protein Iba1/AIF1 as a chicken IgY polyclonal antibody. This protein also has essential roles in inflammation and also marks macrophages, T-cells, synoviocytes, and adipocytes (Watano et al., 2001).
Similarly, other researchers wanted to use a chicken antibody to differentiate between different Pannexin proteins that specifically recognized Panx1 but not Panx2 and Panx3. Panx1 is the most widely expressed on the surface of glial cells and sensory neurons in the peripheral nervous system.
Pannexin proteins allow for the release of intracellular signaling molecules such as ATP, and the scientists hypothesized that these proteins could underlie some aspects of pain perception and might be novel analgesic therapeutic targets (Hanstein et al., 2016).
We used our Immunogenicity Algorithm® to identify appropriate peptide sequences within Panx1 and deployed our development pipeline to generate a highly specific polyclonal chicken antibody that the researchers used to show Panx1 expression in sensory glia, but not sensory neurons and that Panx1 is involved in pain hypersensitivity (Hanstein et al., 2016).
Hot Products: From Embryogenesis to Brain Development and Disease
Our portfolio of chicken polyclonal antibodies is constantly expanding, with over 50 primary antibodies available to date. Here are some of our recent additions and popular products to drive insights into mammalian development and beyond.
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SOX2 - a core regulator of embryogenesis, cell differentiation, and disease
We recently developed our new SOX2 affinity-purified polyclonal IgY chicken antibody suitable for immunocytochemistry, immunohistochemistry, and western blot with reactivity to humans, mice, and rats. SOX2 is a crucial transcription factor in maintaining the pluripotency of embryonic stem cells and the formation of the central nervous system and neural progenitor cells. But the importance of SOX2 also extends to adult tissues, with core roles in neural plasticity and maintaining round tissue homeostasis. When these processes go wrong, SOX2 is also strongly linked to diseases like glioma. As SOX2 is present in a vast number of cell types, each with different marker genes, our anti-SOX2 polyclonal chicken antibody serves as another string in the bow of researchers performing multiplex immunofluorescence or other experiments previously limited by the species antibodies were raised in. -
Beta-Catenin - Many faces in health and disease
Beta-catenin is involved in countless biological processes and is the main component of many crucial cellular pathways, such as the Wnt pathway. The protein is essential for correct tissue homeostasis, and when beta-catenin dysregulation occurs, excessive cellular proliferation and invasion leads to tumor development. With so many roles in different tissues and processes, the ability to perform more extensive multiplex immunofluorescence experiments is paramount to improving our understanding of its role in both known and unknown biological processes and pathways. Check out our affinity-purified chicken anti-Beta-catenin polyclonal antibody to streamline your next multiplex experiment. -
S100B - An astrocyte and disease marker promoting cell growth and survival
S100B is primarily an astrocyte marker associated with calcium binding, but recent discoveries suggest widespread roles in neurodegenerative and neuropsychiatric disorders, even extending to novel functions in different cancers like benign and malignant melanoma, and cardiovascular diseases. Elevated levels of S100B in the blood are crucial biomarkers of neurological conditions and brain injury with potential as therapeutic targets in various diseases. With its extensive roles in human health and disease, our new affinity-purified chicken polyclonal anti-S100B antibody detects human, mouse, and rat S100B and is suitable for use in western blot, immunocytochemistry, and immunohistochemistry.
Overall, chicken polyclonal antibodies offer exquisite sensitivity with minimal cross-reactivity with mammalian-derived antibodies to drive successful multiplex immunofluorescence experiments informing on countless biological processes, diseases, and cellular functions. Please contact us to see how polyclonal chicken antibodies can help in your next study.
References
Akita, E.M. and Nakai, S., 1993. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. Journal of immunological methods, 160(2), pp.207-214.
Hanstein, R., Hanani, M., Scemes, E. and Spray, D.C., 2016. Glial pannexin1 contributes to tactile hypersensitivity in a mouse model of orofacial pain. Scientific reports, 6(1), p.38266.
Lee, W., Atif, A.S., Tan, S.C. and Leow, C.H., 2017. Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies. Journal of immunological methods, 447, pp.71-85.
Watano, K., Iwabuchi, K., Fujii, S., Ishimori, N., Mitsuhashi, S., Ato, M., Kitabatake, A. and Onoé, K., 2001. Allograft inflammatory factor‐1 augments production of interleukin‐6,‐10and‐12 by a mouse macrophage line. Immunology, 104(3), pp.307-316.
Winkler, C.W., Myers, L.M., Woods, T.A., Messer, R.J., Carmody, A.B., McNally, K.L., Scott, D.P., Hasenkrug, K.J., Best, S.M. and Peterson, K.E., 2017.
Adaptive immune responses to Zika virus are important for controlling virus infection and preventing infection in brain and testes. The Journal of Immunology, 198(9), pp.3526-3535.
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