Quantitative Immunohistochemistry Using an Antibody-Fused Bioluminescent Protein

Wang, Ke-Yong; Wu, Chun; Shimajiri, Shohei; Enomoto, Toshiteru; Kubota, Hidehiro; Akiyama, Hidefumi; Ohmiya, Yoshihiro

doi:10.2144/btn-2020-0006

Cited by 4 publications

(3 citation statements)

References 13 publications

(17 reference statements)

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“…Figure 5a shows the procedure of IHC visualization, that is, immunohistochemical staining of serial paraffin sections using antibody-fused luciferase (B), in comparison with the peroxidase labeling procedure (A) [71]. The optical signal of BL-IHC can be absolutely counted by the imaging system, whereas the signal using the indirect secondary labeling system visualizes antigen distribution.…”

Section: Application Of Bioluminescent Immunohistochemistrymentioning

confidence: 99%

Quantitative Analysis of Bioluminescence Optical Signal

et al. 2023

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Bioluminescence is light emission based on the luciferin–luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document.

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Section: Application Of Bioluminescent Immunohistochemistrymentioning

confidence: 99%

Quantitative Analysis of Bioluminescence Optical Signal

et al. 2023

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“…Immunohistochemistry (IHC) has long been the gold standard for detection the expression of a target protein in tissue samples. Developed in 1941, IHC involves detecting a target protein via the detection of antigen‐specific antibodies, which are often conjugated to fluorescent probes, bioluminescent probes, or colorogenic substrates [25]. Whilst immunohistochemistry has become a routine process, there are several advantages and disadvantages associated to it.…”

Section: Elucidating Biomarkers Within the Immune System: The Role Of...mentioning

confidence: 99%

The fusion of quantitative molecular proteomics and immune‐oncology: a step towards precision medicine in cancer therapeutics

2022

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Innate and adaptive immune systems are built-in homeostatic functions of many multicellular organisms and protect the host against foreign pathogens and infections. Dysregulation of the molecular mechanisms of the immune system can result in autoimmune diseases. The immune system can also be harnessed and manipulated to provide targeted cancer therapies, some of them relying on the blockade of immune-checkpoint receptors. Two prominent immune checkpoints, PD-1/PD-L1 and CTLA-4/CD80, comprise receptor-ligand pairs that prevent the host immune cells from attacking host tissues. However, cancer cells upregulate the respective PD-L1 and CD80 ligands for PD-1 and CTLA-4 and thereby evade the host-immune response. Therapeutic drugs that block PD-1/PD-L1 and CTLA-4/CD80 interactions re-enable the immune system to attack cancer cells, but their prognostic biomarker remains challenging. In this review, we discuss how the use of quantitative molecular imaging can be exploited to predict the response to anti-PD-1/PD-L1 therapies and to identify cancer patients who would benefit from them.

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“…A quantitative bioluminescence immunoassay for immunohistochemistry based on Cypridina luciferase conjugated secondary antibody and its luciferin, was also developed and successfully used to detect the tumor marker carcinoembryonic antigen ( Wang et al, 2020 ). Bioluminescent sensors to detect multiple antibodies based on microfluidics and BRET were also proposed (Kosuke et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

Viviani

Silva

2021

Front. Bioeng. Biotechnol.

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Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

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Quantitative Immunohistochemistry Using an Antibody-Fused Bioluminescent Protein

Cited by 4 publications

References 13 publications

Quantitative Analysis of Bioluminescence Optical Signal

Quantitative Analysis of Bioluminescence Optical Signal

The fusion of quantitative molecular proteomics and immune‐oncology: a step towards precision medicine in cancer therapeutics

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

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