Improved Catalyzed Reporter Deposition, iCARD

Lohse, Jesper; Petersen, Kenneth H.; Woller, Nina Claire; Pedersen, Hans Christian; Skladtchikova, Galina; Jørgensen, Rikke Malene

doi:10.1021/bc400311g

Cited by 12 publications

(13 citation statements)

References 12 publications

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“…The qIHC method addresses the need for a robust and quantitative assay, which can be combined with the morphological information gained from immunohistochemistry. The assay is based on the iCARD chemistry, which has been described in Lohse et al 54 It converts antibody/antigen complexes into red dots, which can subsequently be counted and quantified.…”

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confidence: 99%

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2

Jensen

Krusenstjerna-Hafstrøm

Lohse

et al. 2017

Modern Pathology

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In clinical routine pathology today, detection of protein in intact formalin-fixed, paraffin-embedded tissue is limited to immunohistochemistry, which is semi-quantitative. This study presents a new and reliable quantitative immunohistochemistry method, qIHC, based on a novel amplification system that enables quantification of protein directly in formalin-fixed, paraffin-embedded tissue by counting of dots. The qIHC technology can be combined with standard immunohistochemistry, and assessed using standard bright-field microscopy or image analysis. The objective was to study analytical performance of the qIHC method. qIHC was tested under requirements for an analytical quantitative test, and compared with ELISA and flow cytometry for quantitative protein measurements. Human epidermal growth factor receptor 2 (HER2) protein expression was measured in five different cell lines with HER2 expression from undetectable with immunohistochemistry to strong positive staining (IHC 3+). Repeatability, reproducibility, robustness, linearity, dynamic range, sensitivity, and quantification limits were evaluated. Reproducibility and robustness were assessed in a setup to resemble daily work in a laboratory using a commercial immunohistochemistry platform. In addition, qIHC was correlated to standard HER2 immunohistochemistry in 44 breast cancer specimens. For all evaluated parameters, qIHC performance was either comparable or better than the reference methods. Furthermore, qIHC has a lower limit of detection than both immunohistochemistry and the ELISA reference method, and demonstrated ability to measure HER2 accurately and precise within a large dynamic range. In conclusion, the results show that qIHC provides a sensitive, quantitative, accurate, and robust assay for measurement of protein expression in formalin-fixed, paraffin-embedded cell lines, and tissue.

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mentioning

confidence: 99%

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2

Jensen

Krusenstjerna-Hafstrøm

Lohse

et al. 2017

Modern Pathology

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“…#019–19741) (1:8000) antibodies, which identify endothelial cells, leukocytes, and microglia was used as primary antibodies for standard IHC/3'3-diaminobenzidine tetra-hydrochloride (DAB) EnVision Flex staining (20 min). qIHC staining was performed as described previously [22]. Conceptually, a polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) was used as secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

“…Further, the level of APNG was examined using both a quantitative immunofluorescence (IF) method used previously in our lab [19, 20], and the new method of dot technique in quantitative immunohistochemistry (qIHC) [21]. qIHC is a novel methodology developed by DAKO, which combined with image analysis, allows for quantitation of the expression of proteins in paraffin embedded tissue samples using bright field assessments [22]. The current IF method has previously been used successfully in other biomarker studies on the same cohort [19, 20].…”

Section: Introductionmentioning

confidence: 99%

APNG as a prognostic marker in patients with glioblastoma

et al. 2017

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AimExpression of the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG) has been correlated to temozolomide resistance. Our aim was to evaluate the prognostic value of APNG in a population-based cohort with 242 gliomas including 185 glioblastomas (GBMs). Cellular heterogeneity of GBMs was taken into account by excluding APNG expression in non-tumor cells from the analysis.MethodsAPNG expression was evaluated using automated image analysis and a novel quantitative immunohistochemical (IHC) assay (qIHC), where APNG protein expression was evaluated through countable dots. Non-tumor cells were excluded using an IHC/qIHC double-staining. For verification, APNG was measured by a quantitative double-immunofluorescence (IF) assay. As validation APNG mRNA expression was evaluated using independent TCGA data.ResultsUsing qIHC, high levels of APNG were associated with better overall survival (OS) in univariate (HR = 0.50; P < 0.001) and multivariate analysis (HR = 0.53; P = 0.001). Patients with methylated MGMT promoters and high APNG expression demonstrated better OS, than patients with methylated MGMT promoters and low APNG expression (HR = 0.59; P = 0.08). Retesting the cohort using IF showed similar results in both univariate (HR = 0.61; P = 0.002) and multivariate analysis (HR = 0.81; P = 0.2). The results were supported by data from the TCGA database.ConclusionsUsing two different assays combined with quantitative image analysis excluding non-tumour cells, APNG was an independent prognostic factor among patients with a methylated MGMT promoter. We expect that APNG qIHC can potentially identify GBM patients who will not benefit from treatment with temozolomide.

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“…In the latest improvement of the biotin-free CSA method, fluorescein is conserved in the substrate, while the tyramine is substituted with ferulic acid, which is a much better peroxidase substrate and increases signal-to-noise ratio. In this system, the incubation time in each step can be significantly reduced, making it possible to stain a tissue in less than 1 h [112]. Detection Systems in Immunohistochemistry DOI: http://dx.doi.org/10.5772/intechopen.82072…”

Section: Biotin-free Tsa/csamentioning

confidence: 99%

Detection Systems in Immunohistochemistry

Shojaeian¹,

Lay²,

Zarnani³

2020

Immunohistochemistry - The Ageless Biotechnology

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Immunohistochemistry (IHC) is a process of selectively imaging antigens in cells or tissue sections by exploiting antibody specificity. This technique is widely used in diagnostic pathology and research experiments for tracking specific molecular markers characteristic of a particular cell type or cellular events such as cancerous cell development, cell proliferation, or apoptosis. Visualizing the target antigen following an antibody-antigen interaction is accomplished by different detection systems. In the simplest instance, primary antibody directly conjugated to an enzyme is responsible for both specifically binding to the antigen and catalyzing a color-producing reaction. Alternatively, complex detection systems could be designed to profoundly improve minimal detection level of the antigen. During the past years, there has been a considerable improvement in designing and introduction of new and highly sensitive detection systems. The choice of an IHC detection system is a compromise of a variety of variables including desired sensitivity, cost, and the time needed for an IHC staining to be performed. This chapter covers the immunohistochemistry detection systems with emphasis on their principle, history, advantages, and limitations and delineates factors needed to be considered for choosing an appropriate detection system for IHC applications.

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Improved Catalyzed Reporter Deposition, iCARD

Cited by 12 publications

References 12 publications

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2

APNG as a prognostic marker in patients with glioblastoma

Detection Systems in Immunohistochemistry

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