Multilayer peroxidase‐labeled antibody method: Comparison with labeled streptavidin‐biotin method, avidin‐biotin‐peroxidase complex method, and peroxidase‐antiperoxidase method

Shin, Masashi; Izumi, Shinichi; Nakane, Paul K.

doi:10.1002/jcla.1860090615

Cited by 16 publications

(14 citation statements)

References 20 publications

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“…The indirect HRP-labeled antibody method was employed. 29 Briefly, sections were placed on positively charged glass slides and deparaffinized in xylene. After rehydration in a series of graded alcohols, heat-induced antigen retrieval in a solution of either citrate buffer (pH 6.0) or Tris-EDTA (pH 8.0) was carried out, before to incubate the samples with 3% hydrogen peroxide to inactivate endogenous peroxidases.…”

Section: Methodsmentioning

confidence: 99%

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

et al. 2010

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Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxic effects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumour growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumour regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.

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Section: Methodsmentioning

confidence: 99%

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

et al. 2010

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“…For quantification, images of electron microscopic photographs (both sample and control specimens) taken under constant conditions were digitized into a computer through a video camera head (ITC-370M; Olympus-Ikegami, Tokyo, Japan) according to the method of Shin et al (1995). The optical density of the regions of the neural rough endoplasmic reticulum, nucleus, and nucleolus in the photographs was measured by a computer-assisted image analyzer (Olympus-SP500; Tokyo, Japan) under constant conditions.…”

Section: Electron Microscopymentioning

confidence: 99%

Immunoelectron Microscopic Study for Polyamines

Fujiwara

Bai²,

Kitagawa³

et al. 1998

J Histochem Cytochem.

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SUMMARYThe polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization. In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata. The nuclei, axons, and nerve endings were devoid of PA. This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis.

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“…In the ABC (avidin-biotin complex) method and related procedures, the detection reagent is assembled during the course of the assay by sequential addition of biotin-labeled antibody, avidin, and biotin-labeled enzyme (7,8 ). Multiple layers of enzyme can also be built up by stepwise addition of enzyme conjugated to antibody directed against the host species of the antibody in the previous layer (9 ). These methods can give highly sensitive measurements, but at the cost of increasing the number of steps in the assay.…”

Section: Discussionmentioning

confidence: 99%

Defined Protein Conjugates as Signaling Agents in Immunoassays

et al. 2004

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Background: Conventional methods for conjugation of macromolecules, such as antibodies and reporter groups, typically yield a mixture ranging from unconjugated starting materials to large aggregates. We explored the use of a solid-phase process to allow improved control in conjugation of macromolecules for use in immunodiagnostic reagents. Methods: Activated components were sequentially delivered to an immobilized core protein, linking in concentric layers. For immunodiagnostic reagents, proteins with the desired signaling properties were added as interior layers and binding proteins were placed in the final surface layer. After assembly, the conjugates were released into solution by cleaving the linker holding the core protein to the support. Conjugates were prepared with use of three different reporter agents: R-phycoerythrin for microsphere fluorescence flow immunoassay, alkaline phosphatase for enzyme immunoassay, and acridinium for magnetic chemiluminescence immunoassay. For each reporter, six conjugates were prepared with various concentrations of both the reporter and an antibody directed against the ␣-subunit of thyroidstimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. Results: Products ranged in mass from ϳ1 to ϳ20 MDa. HPLC analysis of the conjugates on a gel-permeation column showed sizes and chromophore contents highly consistent with the intended structures. In appropriate assay formats, the signal generated by a conjugate increased with incubation time, then plateaued at an intensity approximately proportional to the reporter content but relatively independent of the antibody con-

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Multilayer peroxidase‐labeled antibody method: Comparison with labeled streptavidin‐biotin method, avidin‐biotin‐peroxidase complex method, and peroxidase‐antiperoxidase method

Cited by 16 publications

References 20 publications

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Immunoelectron Microscopic Study for Polyamines

Defined Protein Conjugates as Signaling Agents in Immunoassays

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