Reduction of Dilution Error in Elisas Using an Internal Standard

Verch, Thorsten; Roselle, Christopher; Shank-Retzlaff, Mary

doi:10.4155/bio-2016-0053

Cited by 6 publications

(4 citation statements)

References 33 publications

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“…However, assays that support potency or those that do not have a standard curve may benefit from an IS to control the assay performance. A case study was discussed where the IS concept was applied to monitor sample dilution bias in a relative potency ELISA under GMP [42]. A fluorescent dye was spiked into test articles and reference standards at the first assay step and carried downstream through all subsequent sample dilution steps.…”

Section: Pk Assays Use Of Is In Lba To Improve Precision and Accuracy: mentioning

confidence: 99%

2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3 – Lba: Immunogenicity, Biomarkers and PK Assays)

et al. 2017

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The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3–7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC–MS, hybrid ligand-binding assay (LBA)/LC–MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC–MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

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Section: Pk Assays Use Of Is In Lba To Improve Precision and Accuracy: mentioning

confidence: 99%

2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3 – Lba: Immunogenicity, Biomarkers and PK Assays)

et al. 2017

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“…The imprecision can largely be attributed to inconsistencies in the upper asymptote. Although the precision is higher in this assay format, we suspect this could be improved dramatically through further assay optimization, replication, plate layout randomization, and/or automation. , …”

Section: Resultsmentioning

confidence: 99%

“…Although the precision is higher in this assay format, we suspect this could be improved dramatically through further assay optimization, replication, plate layout randomization, and/or automation. 39,40 ■ CONCLUSION In this study, we explored the feasibility of using aptamers as affinity reagents in an HPV potency assay. We generated an aptamer, HPV-07, that has many of the attributes of a high quality reagent such as (a) high affinity, (b) high specificity, (c) stability indication, (d) competition with a neutralizing reagent, (e) precision, (f) accuracy, (g) and a large linear range in standard assay conditions.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Development and Characterization of an HPV Type-16 Specific Modified DNA Aptamer for the Improvement of Potency Assays

2017

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Measuring vaccine potency is critical for vaccine release and is often accomplished using antibody-based ELISAs. Antibodies can be associated with significant drawbacks that are often overlooked including lot-to-lot variability, problems with cell-line maintenance, limited stability, high cost, and long discovery lead times. Here, we address many of these issues through the development of an aptamer, known as a slow off-rate modified DNA aptamer (SOMAmer), which targets a vaccine antigen in the human papillomavirus (HPV) vaccine Gardasil. The aptamer, termed HPV-07, was selected to bind the Type 16 virus-like-particle (VLP) formed by the self-assembling capsid protein L1. It is capable of binding with high sensitivity (EC of 0.1 to 0.4 μg/mL depending on assay format) while strongly discriminating against other VLP types. The aptamer competes for binding with the neutralizing antibody H16.V5, indicating at least partial recognition of a neutralizing and clinically relevant epitope. This makes it a useful reagent for measuring both potency and stability. When used in an ELISA format, the aptamer displays both high precision (intermediate precision of 6.3%) and a large linear range spanning from 25% to 200% of a typical formulation. To further exploit the advantages of aptamers, a simplified mix and read assay was also developed. This assay format offers significant time and resource reductions compared to a traditional ELISA. These results show aptamers are suitable reagents for biological potency assays, and we expect that their implementation could improve upon current assay formats.

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“…For example, infliximab, somatropin and nivolumab quantification with signature peptides containing a methionine were previously reported [47,66,68]. Also, endogenous insulin-like growth factor 1 was successfully quantified in serum with a signature peptide containing two cysteines that were protected from oxidation by attaching a carboxymethyl group on the thiol moiety through iodoacetamide alkylation and acid hydrolysis following disulfide bond reduction [88]. Nevertheless, care must be taken when these peptides are used since in vivo degradation of the therapeutic mAb via the above mentioned pathways can lead to an underestimation of circulating mAb as was demonstrated by Bults and colleagues in their research into deamidation of trastuzumab in human plasma [67].…”

Section: Signature Peptide Selectionmentioning

confidence: 99%

Innovative drug monitoring of factor VIII and emicizumab in haemophilia A

Donners¹

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People with haemophilia A (PwHA) have a deficiency of coagulation factor VIII and experience recurrent bleeding. Currently, PwHA are treated effectively with drugs such as FVIII products or emicizumab. Measuring and monitoring these drugs is important to determine the dose, which needs to be effective but not unnecessarily high due to costs (i.e., ~€100.000‒€400.000 per person per year). Mass-spectrometry (MS) is a beneficial technique to measure drug concentrations in blood and is used in clinical laboratories worldwide. Prior to this thesis, MS analysis was not used in haemophilia A care. This thesis aimed to optimize drug monitoring of FVIII and emicizumab in PwHA in three parts. Firstly, the development of MS methods measuring drugs similar to FVIII and emicizumab was summarized in a simple six-step workflow. Subsequently, two MS methods measuring FVIII and emicizumab were set-up and validated. Secondly, the studies on the concentration–biomarker relationship of FVIII yielded more questions than answers, while studies on literature and in our clinic indicated that emicizumab concentrations on standard dosing were often two-times above the minimal effective concentration. Thirdly, this thesis formed the groundwork for the design of a study protocol where the emicizumab dose is adjusted individually by targeting the minimum effective concentration. Finally, future perspectives were shared by giving four new applications of MS analysis quantifying FVIII and a framework for cost-efficient dosing strategies for other drugs similar to emicizumab.

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Reduction of Dilution Error in Elisas Using an Internal Standard

Abstract: Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.

Cited by 6 publications

References 33 publications

2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3 – Lba: Immunogenicity, Biomarkers and PK Assays)

2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3 – Lba: Immunogenicity, Biomarkers and PK Assays)

Development and Characterization of an HPV Type-16 Specific Modified DNA Aptamer for the Improvement of Potency Assays

Innovative drug monitoring of factor VIII and emicizumab in haemophilia A

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