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Sunday, October 1, 2023

How to detect a protein by antibodies?

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As the scientific world advances, a variety of techniques to detect proteins continue to evolve as essential tools that are widely used for both diagnostic and research purposes. Among these techniques is detecting proteins by antibodies through immunofluorescence and immunohistochemistry. These two methods use antibodies in detecting and localizing proteins and other antigens within biological samples including cells or tissues, which are important in identifying binding regions of proteins and specific antigens (Quintero-Ronderos et al., 2013).

Immunofluorescence and immunohistochemistry have the same fundamental procedure as enzyme-linked immunosorbent assays (ELISA) and Western blot. In addition, in a study in 2001, multiple proteins were detected in an antibody-based protein microarray system that combined the specificity of ELISA, sensitivity of enhanced chemiluminescence (ECL), and high-throughput microspot (Huang, 2001). Furthermore, the use of antibodies to detect proteins have extended into the use of monoclonal antibodies (mAbs) as tools that are vital in biochemistry, molecular biology, and medicine research (Zhang et al., 2017).

The first approach in using mAbs was described by Kohler and Milstein in 1975 through the mouse hybridoma method in which the spleen cells from an immunized mouse was fused with a nonsecretory murine myeloma cell line, which was widely useful in diagnostic and therapeutic settings (Rossi et al., 2005). This technique was followed by many for the past few decades to produce mAbs including phage display, yeast surface display, ribosome display, and mRNA display technologies.

One method was recently developed, the single B cell-based method, which mainly allows a more thorough interrogation of the B-cell population by direct sampling of the immune repertoire from a single B-cell and retaining the natural heavy and light chain pairing, unlike the inefficient fusion step in hybridoma (Zhang et al., 2017). The single B cell-based method utilizes the affinity, specificity, and stability profiles of mAbs. Fluorescence-activated cell sorting (FACS) and manual micromanipulation are currently used for single-cell isolation, and the antibody genes are transferred to mammalian cells for mAb production and subsequent characterization. The immune system of rabbits is different from those of mice and other rodents due to its ability to generate high titers of high-affinity antibodies (Rossi et al., 2005).

Rabbit monoclonal antibodies

Rabbit monoclonal antibodies have become an outstanding tool in laboratory research especially in diagnostic and therapeutic applications. This is because of the promising ontogeny of rabbit B cells that has high diversity, affinity, and specificity of antibody repertoire (Weber et al., 2017). In addition, when compared to conventional mouse mAbs, rabbit mAbs show higher affinity and specificity for antigens, as well as broader epitope recognition, and significantly enhanced reactivity to small-size epitopes and mouse antigens (Zhang et al., 2017). As an important model in immunology for many years, rabbits have been used for producing monoclonal and polyclonal antibodies. Monoclonal antibodies have specific antigen-binding sites or paratopes that bind to only one epitope, which also provide a molecularly defined and reproducible product from a pharmaceutical perspective (Weber et al., 2017). More than 9,500 rabbit mAbs were produced as a result of these beneficial characteristics, and they were identified for significant signaling pathways such as apoptosis, cell cycle, epidermal growth factor receptor signaling, and transforming growth factor-β signaling (Zhang et al., 2017). Moreover, 11 rabbit mAbs for in vitro diagnostic tools in the clinic have been approved by FDA, Ten of these FDA-approved rabbit mAbs are used to identify expression of tumor-associated antigens such as HER2, estrogen receptors, progesterone receptors and PD-L1. Out of the 11 FDA-approved rabbit mAbs, one rabbit monoclonal antibody is used in detection of Helicobacter pylori infections (Weber et al., 2017).

Neuronal nuclear antigen (NeUN)

Another tool has been extensively utilized in immunohistochemical studies of neuronal differentiation to determine the functional condition of norm and pathological neurons for more than 20 years (Gusel’nikova & Korzhevskiy, 2015). This tool is monoclonal antibody (mAb) A60, specifically neuronal nuclear antigen (NeuN), first described by Mullen et al. (1992). The DNA-binding, neuron-specific protein NeuN is localized in the nuclei and perinuclear cytoplasm of the majority of the neurons of the mammalian central nervous system (Soylemezoglu et al., 2003). NeuN antibodies have recently been used in the differential morphological diagnosis of cancer (Gusel’nikova & Korzhevskiy, 2015). In addition, the reliability of NeuN as a neuronal marker in the differential diagnosis of clear cell neoplasms of the central nervous system was found in 23 cases of biopsy series (Gusel’nikova & Korzhevskiy, 2015).

Plasma cell discovery

Different services are offered by biological companies to help clients with their goals either for diagnostic, therapeutic, or research purposes. One of these services is plasma-cell discovery (PCD) technology, which involves an initial screening through FACS and a secondary testing of individual plasma cells. FACS is used to isolate antigen-specific plasma cells from a number of splenocytes whereas the secondary testing is for the recombinantly expressed antibodies in the supernatant. PCD technique improves the number of antibody clones that can be tested 500-fold by isolating antigen-specific plasma cells on FACS. Therefore, this technology screens individual cells treated with proprietary chemistry using flow cytometry, which will keep B cells from secreting antibodies, thus retaining on cell membranes. PCD platform also allows screening of the entire rabbit spleen through the incubation of splenocytes with fluorochrome conjugated antigens and plasma cells. The brightest clones have the highest affinity and are isolated for further screening and processing, which allows for screening as much and as early as possible according to the custom antibody project’s objectives. With the PCD platform, there is a high chance of finding rare clones with highest affinity for the selected biomarker.

Custom rabbit monoclonal antibodies are currently offered by biotechnology companies such as Boster Bio with minimal upfront fees and fast delivery. The plasma cell discovery (PCD) platform offered by Boster Bio is best suited for diagnostics and therapeutics targets due to its ability to generate clones with the highest affinity and specificity amongst all antibody discovery technologies. Learn more about the technology of the PCD platform here.

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