The path to VICTORy – a beginner's guide to success using commercial research antibodies

S, Goodman

doi:10.1242/jcs.216416

Cited by 11 publications

(6 citation statements)

References 76 publications

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“…S3). A growing literature surrounds the use of antibodies that are not carefully validated for IHC analysis, which result in inconsistent, and often misleading, correlations with patient outcome 27,28 . While the antibodies against BCL-2 and BCL-X L exhibited specificity in both Western blots (characterized by a specific band at the predicted molecular weight) and IHC (characterized by specific mitochondrial staining, which decreased following siRNA transfections), problems were evident with the different MCL-1 antibodies used (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Carter¹,

Milani²,

Butterworth³

et al. 2019

Cell Death Dis

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Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.

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Section: Resultsmentioning

confidence: 99%

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Carter¹,

Milani²,

Butterworth³

et al. 2019

Cell Death Dis

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“…Our phenotypic multifaceted characterization using antibodies and pharmacological tools confirms and expands previous published results showing the presence of AT2R at sites where its activation could lead to the regulation of nociception (Anand et al ; Chakrabarty et al ; Benitez et al ). Note that the use of commercially available antibodies has some limitations (Goodman ). The specificity of the antibodies against AT2R and AT1R has been questioned (Hafko et al ).…”

Section: Discussionmentioning

confidence: 99%

Cutaneous inflammation differentially regulates the expression and function of Angiotensin‐II types 1 and 2 receptors in rat primary sensory neurons

Benitez

Seltzer

Messina

et al. 2019

Journal of Neurochemistry

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Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type‐2 receptor for Angiotensin‐II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type‐1 receptor for Angiotensin‐II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT‐PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation‐induced neuritogenesis. We demonstrated that AT2R‐neurons express C‐ or A‐neuron markers, primarily IB4, trkA, and substance‐P. AT1R expression was highest in small neurons and co‐localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co‐operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non‐peptidergic C‐neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous‐induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: .

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“…One might infer that this problem is poorly documented in the scientific literature. However, this is distinctly not the case as there are a number of published articles that directly deal with problems of antibody specificity for many types of assays, including those that focus on problems with IHC [8] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] . In addition, a number of organizations, including the United State National Institutes of Health (NIH) have been focusing on the problem of antibody validation and the NIH now requires a section in grant applications that describes efforts to authenticate antibodies [12] .…”

Section: Introductionmentioning

confidence: 99%

“…When flawed IHC studies are published, this leads to biased results that can confound or “pollute” the literature [8] , [13] , [24] . This results in potential wasted time and research dollars for those performing these studies, as well as for future researchers, and can even have implications for pharmaceutical research and patient care (see below section on cytomegalovirus [CMV] and gliomas).…”

Section: Introductionmentioning

confidence: 99%

If this is true, what does it imply? How end-user antibody validation facilitates insights into biology and disease

Sfanos

Yegnasubramanian

Nelson

et al. 2019

Asian Journal of Urology

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Antibodies are employed ubiquitously in biomedical sciences, including for diagnostics and therapeutics. One of the most important uses is for immunohistochemical (IHC) staining, a process that has been improving and evolving over decades. IHC is useful when properly employed, yet misuse of the method is widespread and contributes to the “reproducibility crisis” in science. We report some of the common problems encountered with IHC assays, and direct readers to a wealth of literature documenting and providing some solutions to this problem. We also describe a series of vignettes that include our approach to analytical validation of antibodies and IHC assays that have facilitated a number of biological insights into prostate cancer and the refutation of a controversial association of a viral etiology in gliomas. We postulate that a great deal of the problem with lack of accuracy in IHC assays stems from the lack of awareness by researchers for the critical necessity for end-users to validate IHC antibodies and assays in their laboratories, regardless of manufacturer claims or past publications. We suggest that one reason for the pervasive lack of end-user validation for research antibodies is that researchers fail to realize that there are two general classes of antibodies employed in IHC. First, there are antibodies that are “clinical grade” reagents used by pathologists to help render diagnoses that influence patient treatment. Such diagnostic antibodies, which tend to be highly validated prior to clinical implementation, are in the vast minority (e.g. < 500). The other main class of antibodies are “research grade” antibodies (now numbering >3 800 000), which are often not extensively validated prior to commercialization. Given increased awareness of the problem, both the United States, National Institutes of Health and some journals are requiring investigators to provide evidence of specificity of their antibody-based assays.

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The path to VICTORy – a beginner's guide to success using commercial research antibodies

Cited by 11 publications

References 76 publications

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Cutaneous inflammation differentially regulates the expression and function of Angiotensin‐II types 1 and 2 receptors in rat primary sensory neurons

If this is true, what does it imply? How end-user antibody validation facilitates insights into biology and disease

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