Inter-assay Comparison Research Articles

Design and implementation of a TaqMan® real-time PCR method for detection and quantification of bovine leukemia virus.

The bovine leukemia virus (BLV) is an important infectious agent transmitted from cattle to humans. It is considered one of the oncogenic viruses in breast cancer, so an accurate detection of this virus is important. The study aimed to design a specific and sensitive method based on TaqMan® real-time polymerase chain reaction (RT-PCR) for BLV detection. Probes and primers were designed using bioinformatics software for a 108 pairs region of the BLV tax gene. Criteria employed for determining analytical sensitivity were prepared using in-vitro RNA transcriptions. The National Center for Biotechnology Information (NCBI), basic local alignment search tool (BLAST) databases various viral panels and genomic samples from healthy individuals (Qom Province, Iran in 2023) were used to verify analytical specificity and clinical specificity, respectively. This method can measure a minimum of 10 copies of DNA and RNA mL-1. Moreover, the assay is linear in the range of 100 - 109 copies mL-1. By testing negative specimens, the method specificity was 100%. The reproducibility results of the reaction were examined at the intra- and inter-assay comparison. In fact, 10 technical replicates of each concentration of the control sample were analyzed in each working reaction. Due to the locally made kit, exact sensitivity and specificity, rapid analysis, and relatively low cost, as compared to commercial kits of other countries, the method introduced in the present study could be suitable for accurate detection of the BLV. Also, the TaqMan® real-time PCR method could be detected in cattle and human and before malignant changes of breast cancer which could reduce infection and breast cancer.

Patient-Centric Quantitative Microsampling for Accurate Determination of Urine Albumin to Creatinine Ratio (UACR) in a Clinical Setting.

Developing and implementing new patient-centric strategies for drug trials lowers the barrier to participation for some patients by reducing the need to travel to research sites. In early chronic kidney disease (CKD) trials, albuminuria is the key measure for determining treatment effect prior to pivotal kidney outcome trials. To facilitate albuminuria sample collection outside of a clinical research site, we developed 2 quantitative microsampling methods to determine the urinary albumin to creatinine ratio (UACR). Readout was performed by LC-MS/MS. For the Mitra device the within-batch precision (CV%) was 2.8% to 4.6% and the between-batch precision was 5.3% to 6.1%. Corresponding data for the Capitainer device were 4.0% to 8.6% and 6.7% to 9.0%, respectively. The storage stability at room temperature for 3 weeks was 98% to 103% for both devices. The recovery for the Mitra and Capitainer devices was 104% (SD 7.0%) and 95 (SD 7.4%), respectively. The inter-assay comparison of UACR assessment generated results that were indistinguishable regardless of microsampling technique. The accuracy based on LC-MS/MS vs analysis of neat urine using a clinical chemistry analyzer was assessed in a clinical setting, resulting in 102 ± 8.0% for the Mitra device and 95 ± 10.0% for the Capitainer device. Both UACR microsampling measurements exhibit excellent accuracy and precision compared to a clinical chemistry analyzer using neat urine. We applied our patient-centric sampling strategy to subjects with heart failure in a clinical setting. Precise UACR measurements using quantitative microsampling at home would be beneficial in clinical drug development for kidney therapies.

Comparison of Circulating Tumor DNA Assays for Molecular Residual Disease Detection in Early-Stage Triple-Negative Breast Cancer.

Detection of circulating tumor DNA (ctDNA) in patients who have completed treatment for early-stage breast cancer is associated with a high risk of relapse, yet the optimal assay for ctDNA detection is unknown. The cTRAK-TN clinical trial prospectively used tumor-informed digital PCR (dPCR) assays for ctDNA molecular residual disease (MRD) detection in early-stage triple-negative breast cancer. We compared tumor-informed dPCR assays with tumor-informed personalized multimutation sequencing assays in 141 patients from cTRAK-TN. MRD was first detected by personalized sequencing in 47.9% of patients, 0% first detected by dPCR, and 52.1% with both assays simultaneously (P < 0.001; Fisher exact test). The median lead time from ctDNA detection to relapse was 6.1 months with personalized sequencing and 3.9 months with dPCR (P = 0.004, mixed-effects Cox model). Detection of MRD at the first time point was associated with a shorter time to relapse compared with detection at subsequent time points (median lead time 4.2 vs. 7.1 months; P = 0.02). Personalized multimutation sequencing assays have potential clinically important improvements in clinical outcome in the early detection of MRD.

Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray

Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.

Midpregnancy testing for soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF): An inter-assay comparison of three automated immunoassay platforms

We performed an inter-assay comparison among three immunoassay platforms for midpregnancy testing of sFlt-1, PlGF and the sFlt-1/PlGF ratio, which are established markers for pre-eclampsia. Maternal blood was collected 19–22 weeks’ gestation. Raw data values were converted to multiples of the median (MoM). PlGF and sFlt-1 values among platforms were highly correlated (p < 0.001). There was significant variation in raw data values for PlGF and sFlt-1 among platforms, eliminated following conversion to MoM. When directly comparing raw data values among platforms, platform-specific reference ranges values should be used. MoM values were equivalent among platforms, allowing direct inter-assay result comparison.

Development of monoclonal antibody-based enzyme-linked immunosorbent assay for quantitative quality control of Derris scandens (Roxb.) Benth

ABSTRACTDerris scandens (Roxb.) Benth. is a medicinal plant used for treatment of musculoskeletal pain in Thai traditional medicines. Its stem contains active compound genistein-7-O-[α-rhamnopyranosyl-(1 to 6)-β-glucopyranoside] (GTG) which is used as a biomarker for standardization of D. scandens extracts. As an alternative for rapid quantitation of GTG, a monoclonal antibody against GTG was prepared and applied for an indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine GTG in plants and herbal products. The established method provided a quantification range of 0.31–10 µg/mL with a limit of detection of 0.29 µg/mL. The assay was validated for precision and accuracy by intra- and interassay variation analyses, recovery test, and comparison analysis between the amounts of GTG determined by ELISA and HPLC. The results exhibited that the developed ELISA is sensitive and effective for determination of GTG in D. scandens plant materials and herbal products.

A Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Determination of Homoharringtonine.

Homoharringtonine (HHT), also known as omacetaxine, is a natural compound found in the genus Cephalotaxus and is a promising pharmaceutical drug used for the treatment of chronic or accelerated phase chronic myeloid leukemia. As a tool for the quantitative determination of HHT, a specific monoclonal antibody against HHT (MAb 6A1) was generated by conjugates prepared via sodium periodate-mediated oxidation. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) using MAb 6A1 was found to be highly specific and sensitive with a limit of detection for HHT of 48.8 ng/mL. Validation assays to evaluate precision and accuracy of the method were conducted by the use of intra- and inter-assay analysis, recovery test, and comparison analysis between the amounts of HHT determined by ELISA and high-performance liquid chromatography. These results revealed that the established icELISA using MAb 6A1 is specific, sensitive, and reliable enough to be applied to the qualitative analysis for HHT. Furthermore, the results of this study support the usefulness of sodium periodate as a reagent for the conjugation between Cephalotaxus alkaloids and proteins for producing specific antibodies.

Abstract LB-B11: Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of ColoScape™, a highly sensitive test powered by XNA technology to detect novel multigene mutation biomarkers in CRC by real-time PCR. The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated in tissue biopsy (FFPE) and plasma samples.Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers with natural nucleoside bases (A, T, C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes cannot be extended by DNA polymerase thus suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. For each of the selected mutation sites, primers and FAM-labeled TaqMan probes were designed and tested together with the selected XNA oligomers. An internal PCR control selected in the Human -Actin (ACTB) gene was employed utilizing an HEX-labeled TaqMan probe.Results: The ColoScape™ kit was demonstrated to have robust analytical performance and clinical accuracy for FFPE, stool and plasma samples. The rapid, precise and sensitive molecular assay for mutation detection in colon cancer has key benefits listed below. The assays showed a sensitivity of as low as 0.1% mutation in 5-10 ng of WT DNA/well. No cross-reactivity was observed with wild-type up to 320ng purified gDNA and up to 20ng FFPE DNA per reaction demonstrating high specificity of the ColoScape™ assay. Intra-assay, inter-assay, lot-to-lot and operator variation comparison showed CV% between 3% and 8%. Excluding pre-cancer samples, the assay clinical specificity and sensitivity were 95% and 100%, respectively. Pre-cancer detection sensitivity was 60% and 62.5% for stool samples. For tested FFPE clinical samples, the assay specificity and sensitivity were 95% and 91% respectively while the assay clinical specificity and sensitivity were both 100% for plasma samples.Conclusion: The ColoScape™ Colorectal Cancer Mutation Detection qPCR assay is shown to be a sensitive tool intended to aide in the early detection of colorectal cancer, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Qing Sun, Larry Pastor, Aiguo Zhang, Kamila Koprowska, Walter Bodmer. Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™ [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B11.

In vitro observations and in silico predictions of xenoestrogen mixture effects in T47D-based receptor transactivation and proliferation assays

Within endocrine disruptor research, evaluation and interpretation of mixture effects and the predictive value for downstream responses still warrant more in-depth investigations.We used an estrogen receptor (ER)-mediated reporter gene assay (ER-CALUX®) and a cell proliferation assay (WST-1 assay), both based on the T47D breast cancer cell line, to test mixtures of heterogeneous xenoestrogens. Observed concentration-response curves were compared to those predicted by the concepts of concentration addition (CA), generalized concentration addition (GCA), and a novel full logistic model (FLM).CA performed better regarding mixture potency (EC50 values), whereas GCA was superior in predicting mixture efficacy (maximal response). In comparison, FLM proved to be highly suitable for in silico mixture effect prediction, combining advantages of both CA and GCA. The inter-assay comparison revealed that ER activation is not necessarily predictive for induction of cell proliferation.The results support the use of models like CA, GCA, or FLM in mixture effect evaluation. However, we conclude that reliable estimations regarding the disruptive potential of mixtures of endocrine active substances require an integrative approach considering more than one assay/endpoint to avoid misinterpretations. The formazan-based WST-1 proliferation assay might be a possible alternative to commonly used proliferation assays in endocrine disrupter research.

Interassay Comparison of the Tumor Markers CA125, CA15.3, and CA27.29.

Cancer antigens 125, 27.29, and 15-3 (CA125, CA27.29, and CA15-3) are markers of ovarian and breast cancer. Comparing tumor marker results across methods is challenging because of the lack of harmonization. Documenting comparability of results is important. Siemens Advia Centaur CA125 and CA27.29 assays were compared to their corresponding Beckman Coulter DxI CA125 and CA15-3 assays. The interassay bias was determined and the manufacturer-recommended reference intervals were evaluated. The DxI CA125 assay demonstrated an overall positive 29% bias relative to the Centaur CA125 assay. The DxI CA15-3 assay demonstrated an overall negative 65% bias relative to the Centaur CA27.29 assay. For patients with multiple comparisons during the study period, the trend of results over time was similar across both sets of assays. Implementing the manufacturer-recommended reference interval for the DxI CA125 assay increased the abnormal flagging rate by 4.5%. In contrast, implementing the manufacturer-recommended reference interval for the DxI CA15-3 assay decreased the abnormal flagging rate by 13.0%. The overall trends for the majority of patients were similar. Therefore, despite the overall biases, transitioning tumor marker assays should not affect clinical interpretation of results.

Infliximab quantitation in human plasma by liquid chromatography-tandem mass spectrometry: towards a standardization of the methods?

Infliximab (IFX) is a chimeric monoclonal antibody targeting tumor necrosis factor-alpha. It is currently approved for the treatment of certain rheumatic diseases or inflammatory bowel diseases. Clinical studies have suggested that monitoring IFX concentrations could improve treatment response. However, in most studies, IFX was quantified using ELISA assays, the resulting discrepancies of which raised concerns about their reliability. Here, we describe the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for IFX quantification in human plasma. Full-length stable-isotope-labeled antibody (SIL-IFX) was added to plasma samples as internal standard. Samples were then prepared using Mass Spectrometry Immuno Assay (MSIA™) followed by trypsin digestion and submitted to multiple reaction monitoring (MRM) for quantification of IFX. The chromatographic run lasted 13min. The range of quantification was 1 to 26mg/L. For two internal quality controls spiked with 6 and 12mg/L of IFX, the method was reproducible (coefficients of variation (CV%): 12.7 and 2.1), repeatable (intra-day CV%: 5.5 and 5.0), and accurate (inter-day and intra-day deviations from nominal values: +6.4 to +3.7% and 5.5 to 9.2%, respectively). There was no cross - contamination effect. Samples from 45 patients treated with IFX were retrospectively analyzed by LC-MS/MS and results were compared to those obtained with an in-house ELISA assay and the commercial Lisa Tracker® method. Good agreement was found between LC-MS/MS and in-house ELISA (mean underestimation of 13% for in-house ELISA), but a significant bias was found with commercial ELISA (mean underestimation of 136% for commercial ELISA). This method will make it possible to standardize IFX quantification between laboratories. Graphical Abstract Interassay comparison of the three methods: LC-MS/MS vs inhouse ELISA assay or vs Lisa Tracker® ELISA assays, Passing & Bablok (a) and Bland & Altman (b) for the comparison of LC-MS/MS vs in-house ELISA assay; Passing & Bablok

Accurate Determination of Soluble Axl by Enzyme-Linked Immunosorbent Assay.

Levels of soluble Axl (sAxl) are routinely assessed in human sera by sandwich enzyme-linked immunosorbent assay (ELISA). Although sAxl values are suggested to diagnose different types of disorders, no uniform ELISA method is available, allowing the reliable interassay comparison between results. Furthermore, little is known about the stability of sAxl under storage conditions, which is a relevant parameter for biomedical trials. The evaluation of sAxl stability under various stress conditions and the determination of proper conditions to use the sAxl ELISA for routine clinical applications are of great interest. In this study, serum samples were subjected to freeze-thaw cycles and incubation at different temperatures to analyze the stability of sAxl by ELISA. Dilution and spike-in experiments were carried out to examine the impact of serum and diluent components on the ELISA performance. Various diluents and media were employed to resolve masking effects of the serum. The assay components were further optimized for long-term usability by treatment with stabilizers and validation under temperature stress. Indeed, sAxl showed long-term stability in serum during freeze-thaw cycles and incubation under temperature stress conditions. The dilution experiments revealed that unknown components in the serum caused masking effects that can be reduced by proper dilutions. The assay performance was further increased by using a standardized buffer system to dilute serum samples. Stabilization of coated plates and of streptavidin-horseradish peroxidase allowed long-term storage for up to 6 months. In sum, our data demonstrate proper ELISA conditions, allowing the accurate analysis of sAxl levels in human serum.

Simultaneous analysis of highly polar pharmaceutical adulterants in slimming products by hydrophilic interaction liquid chromatography

ABSTRACTA chromatographic method utilizing hydrophilic interaction liquid chromatography was developed for the estimation of highly polar pharmaceutical adulterants in commercial slimming products. Five adulterants, phenylpropanolamine, salbutamol, phenformin, buformin, and metformin, were successfully separated on an XBridge™ amide column (3.5 µm, 4.6 × 250 mm) using a mobile phase composed of acetonitrile/26.5 mM ammonium formate containing 0.1% formic acid (85:15) flowing at the rate of 0.8 mL/min. Wavelengths for monitoring the elution of these compounds were determined based on the wavelength of their absorption maxima (λmax = 208, 226, and 236 nm). The developed method was validated for specificity, linearity, accuracy, precision, and robustness. Interassay comparison showed that the performance of the hydrophilic interaction liquid chromatography-based method was superior to that of the traditional reversed-phase liquid chromatography-based method. Therefore, the proposed method is useful to analyze highly polar pharmaceutical adulterants in slimming products.

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples

Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry

Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.

Specific IgE measurement using AdvanSure® system: Comparison of detection performance with ImmunoCAP® system in Korean allergy patients

BackgroundAdvanSure enzyme immunoassay (EIA) system is a recently developed multiple allergen screen test with specific immunoglobulin E (sIgE) detection assay, while ImmunoCAP fluorescent EIA (FEIA) has been widely used for sIgE detection. There has been no inter-assay comparison data for these two systems. We determined the detection performance of AdvanSure system compared to that of ImmunoCAP. MethodsWe performed an inter-method comparison using sera from 199 Korean allergy patients, including asthma (39.7%), allergic rhinitis (54.8%), atopic dermatitis (36.2%) and food allergies (21.6%). We compared the sIgE detection performance for nine major inhalant and four food allergens. Results950 paired assay results were analyzed. Most allergen sIgE results showed above 0.5 intraclass correlation coefficient except Blattella germanica, alternaria and mugwort allergen. Intermethod comparison results showed multiple differences in a few allergens. The inter-method concordance was moderate to substantial for most allergens (κ=0.528–0.778, p<0.001), except for cat dander. ConclusionAdvanSure system showed a good detection performance compared with ImmunoCAP in correlation and agreement in Korean allergy patients. However, in terms of differences in the methodologies used by these two systems, careful clinical correlation is needed for interpretation of AdvanSure EIA results.

Standardized LC–MS/MS based steroid hormone profile-analysis

In order to overcome many limitations of immunoassays, high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC–MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01–32ng/mL, ULOQ 5–8000ng/mL), linear correlation coefficient of calibration (R2>0.9966), intra- and inter-day precision (intra-day 1.1–8.8%, inter-day 5.2–14.8% and 8.2–18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3–115.5% and 109.3–128.2% for 11-deoxycorticosterone, inter-day 91.4–117.2% and 102.3–137.1% for 11-deoxycorticosterone), analytical total error (3.6–17.8%), proficiency test accuracy (85.4–113.4%), recovery (68–99%), and metabolite stability (freeze/thaw stability 95.5–108.1%, short term stability 86.9–107.2%). Inter-assay comparison with a routine reference HPLC–MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC–MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC–MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.

Validation of in vitro screening models for progestagenic activities: Inter-assay comparison and correlation with in vivo activity in rabbits

The PR CALUX® cell line is a stably transfected human U2-OS cell line expressing the human PR and a luciferase reporter construct containing three progesterone-responsive elements coupled to a minimal promoter. The validity of this assay has been studied as an alternative to the McPhail assay in rabbits, an in vivo assay to detect progestins. The PR CALUX assay was characterized by its stable expression of PR protein which leads to induction of endogenous PR target genes by progestins. It was found to have a highly selective response to low levels of different progestins, as well as an insignificant response to other nuclear hormone receptor ligands. As an important step in their validation, the PR CALUX bioassay was compared with another earlier described in vitro bioassay, a Chinese Hamster Ovary (CHO) cell-based PR-CHO reporter gene assay as well as with an in vitro PR-binding (PR-BIN) assay, and the in vivo McPhail assay. This was done using 35 (with the most accurate potency determinations in all tests) and 50 (with less reliable potency determinations in some tests) compounds tested in all assays. The correlation scores between PR CALUX and PR-CHO were r2=0.77, and 0.93, respectively; between PR CALUX and PR-BIN r2=0.69 and 0.80. Comparison between either the PR CALUX or the PR-CHO transactivation assay and the in vivo McPhail assay revealed very good correlations of r2=0.68 (n=35), and 0.85 (n=50). The transactivation assays can discriminate very potent, from potent, weak and inactive compounds rather easily. Besides testing the biological activity of pure chemicals and pharmaceuticals in vitro, the PR CALUX and PR-CHO transactivation assays proved to be relatively good predictors of in vivo progestagenic activity, allowing the use of these assays as prescreening methods or in vitro alternatives.

Immunosuppressant drug monitoring – a routine undertaking?1

Abstract The quantitative assessment of immunosuppressant drug levels is still one of the most challenging therapeutic drug monitoring procedures in clinical routine. During the past years, several technical developments matured to usable methods. In addition to immunoassays, liquid chromatography-tandem mass spectrometry has become a key method in immunosuppressant therapeutic drug monitoring. This overview should aid in understanding the advantages and disadvantages of the various assays and methods. UK-NEQAS proficiency testing results are used for an inter-assay comparison approach.

Immunsuppressiva-Medikamentenspiegelmessung – reine Routine? / Immunosuppressant drug monitoring: a routine undertaking?

Zusammenfassung Die Erfassung von Immunsuppressivaspiegel ist eine der anspruchsvollsten Medikamentenspiegelmessungen in der klinischen Routine. Die vergangenen Jahre haben eine Reihe von technologischen Fortschritten gebracht. Neben neuen immunologischen Analysesystemen hat sich die LC-MS/MS zu einem fixen Bestandteil des Geschehens entwickelt. Der vorliegende Beitrag soll helfen, Vor- und Nachteile der unterschiedlichen Assayformate zu erkennen. UKNEQAS-Ringversuchsergebnisse werden zur vergleichenden Assay-beurteilung herangezogen.