Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach

2020 ◽  
pp. 030098582096013
Author(s):  
Joshua D. Webster ◽  
Margaret Solon ◽  
Katherine N. Gibson-Corley
Keyword(s):  
Protein Expression ◽  
Experimental Studies ◽  
Negative Control ◽  
Weight Of Evidence ◽  
Molecular Technique ◽  
Western Blots ◽  
Cumulative Evidence ◽  
Antibody Validation ◽  
Careful Design

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay’s specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay’s specificity and build confidence in a study’s conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study’s results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.

34 Multi-step antibody validation for the geomx® digital spatial profiler

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A35-A35
Author(s):  
Alyssa Rosenbloom ◽  
Jenny Cronin ◽  
Shilah Bonnett
Keyword(s):  
Protein Expression ◽  
Limit Of Detection ◽  
False Positive Rate ◽  
Expression Patterns ◽  
Negative Control ◽  
False Positives ◽  
List Type ◽  
Validation Process ◽  
Tissue Slices ◽  
Antibody Validation

BackgroundUnderstanding protein expression patterns within tissue compartments is imperative to investigating a range of biological questions. Historically, low plex immunohistochemical (IHC) approaches have been employed to assess the spatial heterogeneity of protein expression in tissue slices, but these techniques are of limited utility due to the challenge of measuring multiple targets in parallel. Compounding this limitation is the necessity of validating all antibodies which is resource intensive. Antibodies with poor quality have led to wasted time and resources, including false positives and non-reproducible results.1 2 Here we review the antibody validation process for the GeoMx® Digital Spatial Profiler (DSP) which enables investigation of high-plex, validated, spatially resolved protein targets from a single slide mounted formalin-fixed paraffin-embedded (FFPE) or fresh frozen sample. The robust validation process is in line with recent suggestions for antibody validation from SITC.3MethodsUnconjugated and oligo-conjugated antibodies are screened by IHC to assess staining sensitivity, patterns, and more importantly ensure that the oligo-conjugation has not adversely affected antibody performance. Upon approval by a pathologist, the antibodies are incorporated into a core or module and further validated using the GeoMx DSP. Using FFPE cell pellet arrays (CPAs) containing positive and negative control pellets, we assess the specificity as defined as a lack of signal in negative control pellets and a robust signal in positive control pellets. Antibodies with robust signals are then screened on tissue microarrays (TMAs) composed of healthy and diseased tissues to ensure that they will perform as expected in real samples and yield sufficient signal over background. Finally, after antibodies pass functional validation, we assess the performance of antibodies within panels of antibodies that will be commercialized.ResultsIn total, approximately 60% of off-the-shelf antibodies tested for use in GeoMx assays pass the entire validation process and are put into commercial assays. Passing requirements include exhibiting a maximum positive signal divided by the limit of detection, plus two standard deviations (SD) that is greater than or equal to 5 in both CPAs and TMAs for individual antibodies; such a threshold gives a false positive rate of less than 10%.ConclusionsUnvalidated or poorly validated antibodies can result in false positives and non-reproducible results. Following the robust validation process outlined here, approximately 40% of off-the-shelf antibodies are removed from panels, underscoring the importance of antibody validation prior to incorporating new antibodies into experiments.ReferencesTaussig MJ, Fonseca C, and Trimmer JS. Antibody validation: a view from the mountains. N Biotechnol. 2018; 45:1–8.Bordeaux J, Welsh AW, Agarwal S, Killiam E, Baquero MT, Hanna JA, Anagnostou VK, Rimm DL. Antibody validation. Biotechniques 2010;48(3):197–209.Taube, et al. The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother Cancer 2020;8(1):e000155.

scholarly journals ABC Transporter Function and Regulation at the Blood–Spinal Cord Barrier

2012 ◽  
Vol 32 (8) ◽  
pp. 1559-1566 ◽  
Author(s):  
Christopher R Campos ◽  
Christian Schröter ◽  
Xueqian Wang ◽  
David S Miller
Keyword(s):  
Spinal Cord ◽  
Protein Expression ◽  
Abc Transporter ◽  
Transport Function ◽  
Western Blots ◽  
Aryl Hydrocarbon ◽  
P Glycoprotein ◽  
Blood Spinal Cord Barrier ◽  
Brain And Spinal Cord

We present here an initial characterization of ATP binding cassette (ABC) transporter function and regulation at the blood–spinal cord barrier. We isolated capillaries from rat spinal cords and studied transport function using a confocal microscopy-based assay and protein expression using western blots. These capillaries exhibited transport function and protein expression of P-glycoprotein (Abcb1), multidrug resistance protein 2 (Mrp2, Abcc2), and breast cancer-related protein (Bcrp, Abcg2). Exposing isolated capillaries to dioxin (activates aryl hydrocarbon receptor) increased transport mediated by all three transporters. Brain and spinal cord capillaries from dioxin-dosed rats exhibited increased P-glycoprotein-mediated transport and increased protein expression for all three ABC transporters. These findings indicate similar ABC transporter expression, function, and regulation at the blood–spinal cord and blood–brain barriers.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Synthesis and characterization of L-threonine ammonium bromide: grown on single crystal with experimental studies on NLO

2021 ◽  
Vol 44 (3) ◽  
Author(s):  
T KALAIARASI ◽  
M SENTHILKUMAR ◽  
S SHANMUGAN ◽  
T JARIN ◽  
V CHITHAMBARAM ◽  
...  
Keyword(s):  
Single Crystal ◽  
Experimental Studies ◽  
Synthesis And Characterization ◽  
Ammonium Bromide

scholarly journals Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Systems ◽  
Free Protein

scholarly journals Antiangiogenic potential of Jatropha curcas latex in the chick chorioallantoic membrane model

2019 ◽  
Vol 29 (1) ◽  
pp. 32157
Author(s):  
Luciane Madureira Almeida ◽  
Elisa Flávia Luiz Cardoso Bailão ◽  
Illana Reis Pereira ◽  
Fabrício Alves Ferreira ◽  
Patrícia Lima D'Abadia ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Jatropha Curcas ◽  
Physicochemical Characterization ◽  
Chorioallantoic Membrane ◽  
Angiogenesis Inhibitor ◽  
Negative Control ◽  
New Drugs ◽  
Membrane Model ◽  
Chick Chorioallantoic Membrane

AIMS: To perform a physicochemical and phytochemical characterization of Jatropha curcas latex and to investigate its antiangiogenic potential. METHODS: We performed an initial physicochemical characterization of J. curcas latex using thermal gravimetric analyses and Fourier Transform Infrared spectroscopy. After that, phenols, tannins and flavonoids were quantified. Finally, the potential of J. curcas latex to inhibit angiogenesis was evaluated using the chick chorioallantoic membrane model. Five groups of 20 fertilized chicken eggs each had the chorioallantoic membrane exposed to the following solutions: (1) water, negative control; (2) dexamethasone, angiogenesis inhibitor; (3) Regederm®, positive control; (4) 25% J. curcas latex diluted in water; (5) 50% J. curcas latex diluted in water; and (6) J. curcas crude latex. Analysis of the newly-formed vascular net was made through captured images and quantification of the number of pixels. Histological analyses were performed to evaluate the inflammation, neovascularization, and hyperemia parameters. The results were statically analyzed with a significance level set at p ˂0.05.RESULTS: Physicochemical characterization showed that J. curcas latex presented a low amount of cis-1.4-polyisoprene, which reduced its elasticity and thermal stability. Phytochemical analyses of J. curcas latex identified a substantial amount of phenols, tannins, and flavonoids (51.9%, 11.8%, and 0.07% respectively). Using a chick chorioallantoic membrane assay, we demonstrated the antiangiogenic potential of J. curcas latex. The latex induced a decrease in the vascularization of the membranes when compared with neutral and positive controls (water and Regederm®). However, when compared with the negative control (dexamethasone), higher J. curcas latex concentrations showed no significant differences.CONCLUSIONS: J. curcas latex showed low thermal stability, and consisted of phenols, tannins, and flavonoids, but little or no rubber. Moreover, this latex demonstrated a significant antiangiogenic activity on a chick chorioallantoic membrane model. The combination of antimutagenic, cytotoxic, antioxidant and antiangiogenic properties makes J. curcas latex a potential target for the development of new drugs.

scholarly journals Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells

2014 ◽  
Vol 306 (2) ◽  
pp. G123-G131 ◽  
Author(s):  
Arivarasu N. Anbazhagan ◽  
Shubha Priyamvada ◽  
Anoop Kumar ◽  
Daniel B. Maher ◽  
Alip Borthakur ◽  
...  
Keyword(s):  
Protein Expression ◽  
Therapeutic Strategy ◽  
Luciferase Activity ◽  
Negative Control ◽  
Intestinal Epithelial ◽  
Diarrheal Diseases ◽  
Regulate Protein ◽  
Putative Binding Site ◽  
Mammalian Intestine ◽  
Relative Luciferase Activity

SLC26A3 [downregulated in adenoma (DRA)] is a Cl−/HCO3− exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3′-untranslated region (3′-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3′-UTR (pmirGLO-3′-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3′-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3′-UTR of DRA abrogated the effect of miR-494 on 3′-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.

Characterization of JEOL 3000f TEM Equipped for Electron Holography

2000 ◽  
Vol 6 (S2) ◽  
pp. 228-229
Author(s):  
M. A. Schofield ◽  
Y. Zhu
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Design Of Experiment ◽  
Electron Holography ◽  
Fringe Spacing ◽  
Interference Fringes ◽  
Transmission Electron ◽  
Careful Design

Quantitative off-axis electron holography in a transmission electron microscope (TEM) requires careful design of experiment specific to instrumental characteristics. For example, the spatial resolution desired for a particular holography experiment imposes requirements on the spacing of the interference fringes to be recorded. This fringe spacing depends upon the geometric configuration of the TEM/electron biprism system, which is experimentally fixed, but also upon the voltage applied to the biprism wire of the holography unit, which is experimentally adjustable. Hence, knowledge of the holographic interference fringe spacing as a function of applied voltage to the electron biprism is essential to the design of a specific holography experiment. Furthermore, additional instrumental parameters, such as the coherence and virtual size of the electron source, for example, affect the quality of recorded holograms through their effect on the contrast of the holographic fringes.

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