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Hormone Receptors and Endocrine Therapy in Breast Cancer
Published in Sherry X. Yang, Janet E. Dancey, Handbook of Therapeutic Biomarkers in Cancer, 2021
Sherry X. Yang, Nancy E. Davidson
Since 1970s, assays with different biochemical principles, analytic sensitivity, and analytic precision have been used for evaluation of ER/PgR expression. The initial assays developed were ligand-binding assay (LBA), which had significant limitations due to the technical complexity, requirement for fresh-frozen tissues, high variability, and non-specific to tumor cells [122]. Nonetheless, earlier randomized trials that used LBAs led to our understanding of the relationship between the presence of ER and response to endocrine therapy. In the late 1980s, immunoassays using polyclonal and monoclonal antibodies to detect ER emerged. The enzyme immunoassay (EIA) was developed for quantification of ER from the cytosol extracts of fresh tumor tissues. The amount of steroid hormone receptors detected by EIA was highly correlated with that measured through LBAs, and EIA was less labor-intensive [123]. Immunohistochemistry (IHC) was subsequently developed to allow for detection of ER in formalin-fixed and paraffin-embedded tissues [124, 125]. It is the use of IHC methodology to detect the hormone receptors in the malignant nuclei that has resulted in a practical reality for routine and convenient evaluation of ER/PgR [124].
Analytical Method and Assay
Published in Emmanuel Lesaffre, Gianluca Baio, Bruno Boulanger, Bayesian Methods in Pharmaceutical Research, 2020
Ligand-binding assays, in particular ELISA, are widely used to quantify proteins. The mechanism of detection is based on biology by opposition as for chromatographic methods relying on physico-chemical properties. Quantification of the amount of analyte existing in samples is usually performed by relating the signal of the assay (optical density) to concentration of a standard sample, namely a calibration curve. Typical models used to fit the standard curve are non-linear, such as 4 or 5-parameter logistic functions (see Figure 20.6). Numerous assay parameters can be tuned to optimize such assays such as the nature of enzymes, volumes of products, different incubation times, etc. (Yarovoi et al., 2013; Verch, 2014). Each combination of these parameters will influence the shape of the calibration curve and hence alter the predicted concentration of analyte. The final aim of such assays is to be able to provide accurate analytical results. This is generally translated in an operational way by requiring having results with less than λ% total error, e.g. 30%. To this end, the path to optimization is to select the ranges of the assay parameters that will reach high probability to provide accurate results.
Identification Of Receptors In Vitro
Published in William C. Eckelman, Lelio G. Colombetti, Receptor-Binding Radiotracers, 2019
One of the most promising methods for the study of receptors and the drugs that bind to them is the in vitro ligand-binding assay. It allows both the characterization of the receptors and a more detailed analysis of the mechanism of drug action than has been available previously. Furthermore, binding studies do not require that the receptor of interest be purified — simple membrane preparations or even crude homogenates can be used in most cases.
Targeting the TGF-β signaling pathway for fibrosis therapy: a patent review (2015–2020)
Published in Expert Opinion on Therapeutic Patents, 2021
Xuanyi Li, Ziang Ding, Zixuan Wu, Yinqiu Xu, Hequan Yao, Kejiang Lin
Owing to the large difference in the IC50-values of integrin-specific ligands developed in the past decades, there is a lack of accurate numerical value for research purposes. Kessler et al. evaluated the affinity of various ligands with integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and αIIbβ3 that bind to the RGD motif through homogenous ELISA-like solid-phase binding assay [46]. Next, Hatley et al. supplemented these findings by performing the radio ligand binding assay. Based on the pharmacological and physicochemical characteristics, such as solubility, permeability, protein binding, and lipophilicity, an av-RGD integrin inhibitor toolbox containing small molecules and antibodies was constructed [47]. The summary of av-RGD integrin inhibitor in clinical trials and as a valuable tool, demonstrates that the toolbox provides a complete set of av-RGD integrin affinity comparison for drugs, and it is expected to guide the design of inhibitors and improve the reproducibility of results. Under the premise of ideal efficacy and acceptable safety, inhibitors targeting at multiple integrin affinities can be accepted to achieve resistance to adverse reactions [48], such as cilengitide, or change the key regions to achieve high specificity for specific integrin, such as an RGD Aza-glycine lead [49].
N-terminal selective conjugation method widens the therapeutic window of antibody–drug conjugates by improving tolerability and stability
Published in mAbs, 2021
Min Ji Ko, Daehae Song, Juhee Kim, Jae Yong Kim, Jaehyun Eom, Byungje Sung, Yong-Gyu Son, Young Min Kim, Sang Hoon Lee, Weon-Kyoo You, Jinwon Jung
The test samples (trastuzumab, T-N-F, T-C-F, or T-K-F) were intravenously administered at 2.5 mpk into a female SD rat (6-week-old, Orient Bio). Blood samples were collected at 0.5, 1, 2, 4, 6, 24, 72, 144, 336, 400, and 504 h after the administration. The concentrations of each sample were measured using two methods. Total antibody concentration was measured using an ELISA method that captured the sample with an ErbB2 ectodomain (1129-ER, R&D biosystems) and the captured samples were then detected using a goat anti-human kappa light chain-HRP conjugate antibody. The concentrations of drug-conjugated antibodies were measured using a sandwich ELISA method that captures the sample with a polyclonal rabbit anti-MMAF antibody and the captured samples were then detected using biotinylated ErbB2 (HE2-H8225, ACROBiosystems) and streptavidin-HRP. Both the samples and the standards were diluted with PBS containing 1% BSA and 10% (v/v) rat serum. The other ELISA method is described in the “ligand binding assay” subsection. PK parameters were analyzed using WinNonlin software (ver. 6.1, Certara) and statistical analysis was performed using Minitab software (version 16, Minitab LLC).
Hematocrit effect on dried blood spots in adults: a computational study and theoretical considerations
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2019
Chrysa Daousani, Vangelis Karalis, Anđelija Malenović, Yannis Dotsikas
The potential influence of Ht on DBS assay accuracy and precision are well described in the literature [13]. The Ht may also influence matrix effect and DBS homogeneity [14]. Finally, Ht can affect analyte recovery [15]. Based on the above, it is acknowledged that, if the Ht values of the unknown blood samples vary significantly from Ht value of the calibration standards, this definitely influences assay bias; positive error for higher Ht values and negative for lower Ht values, when a punch of fixed diameter from DBS is utilized. Consequently, the increased variability in the assay performance, may sometimes, stand beyond the current applicable acceptance criteria for the total amount of bias for validated assays in regulated bioanalysis; in general, these acceptance criteria for accuracy of chromatographic assays are set at ±15% (% bias) and at ±20% for the lower limit of quantitation (LLOQ) [16]; for other methods, such as ligand-binding assays, broader acceptance criteria are applied [16]. Regarding adults, the largest impact is expected for DBS samples obtained from subjects where the clinical condition largely influences Ht value (e.g. oncology or anemic patients) [13].