Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective

Findlay, J. W. A.; Smith, Wendell C.; Lee, J.W.; Nordblom, Gerald D.; Das, Ira; DeSilva, Binodh; Khan, Masood N.; Bowsher, Ronald R.

doi:10.1016/s0731-7085(99)00244-7

Cited by 519 publications

(323 citation statements)

References 48 publications

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“…The complete assay was fitted using a standard sigmoidal logistics fit (see below, left hand side) and the low concentrations of human IgG were fitted using an allometric power function (see below, right hand side) [38]. …”

Section: Fluorescence Linked Immunosorbent Assay (Flisa)mentioning

confidence: 99%

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance

Gubala

Guével

Nooney

et al. 2010

Talanta

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When designing devices for biomedical diagnostics, increasing the signal to noise ratio is often critical for achieving clinically relevant sensitivity and limits of detection (LOD). In antibodybased assays, the measured signal can be amplified through the replacement of molecular fluorophores with doped nanoparticles (NP). However, the benefits of using NPs can only be realized if the NPs are coated efficiently with detection antibody, have good colloidal stability and the ratio of specific to non-specific binding (NSB) is high enough. The main focus of this paper is on the optimization of the bioconjugation protocol for antibody labeling of NPs leading to improved assay performance. Two types of linkers were used: monovalent linkers (glutaraldehyde; sulfo-SMCC; and sulfo-SIAB), and three generations of dendrimers endowed with multivalent carboxylic functionality. Overall, the NP-IgG conjugates prepared using multivalent linkers showed a significantly lower LOD and higher sensitivity than their homo-or hetero-functional counterparts. The multivalent dendrimers also improved NP stability and reduced aggregation. Moreover, the dendrimers showed a higher reactivity with biological material, a feature that could significantly reduce the cost of high-throughput biodiagnostics tests. Introduction:

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Section: Fluorescence Linked Immunosorbent Assay (Flisa)mentioning

confidence: 99%

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance

Gubala

Guével

Nooney

et al. 2010

Talanta

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“…Recent methodological advances, particularly enzyme-linked immunospot (ELISPOT) assays and cytokine-based flow cytometry (CFC) assays coupled with overlapping pooled peptide technology, give the opportunity for detailed and precise analyses of specific cellular immune responses (1,3,11,19,22,29). As cellular immune response assays proceed from being used primarily as research tools to be being used as tools for clinical evaluation and assessment of end points in different phases of vaccine development, the required standards of quality assurance (QA)/quality control (QC) for these assays rise dramatically (4,10,16,17,21,27,28). While assay techniques and equipment can be standardized by employing standard operating procedures and assay reagents and can be standardized by use of manufacturer-certified assay kits, a critical component of the assay that is not typically subject to standardized QA/QC is the synthetic peptides used for stimulating the T cells.…”

mentioning

confidence: 99%

Peptide Impurities in Commercial Synthetic Peptides and Their Implications for Vaccine Trial Assessment

Currier

Galley

Wenschuh

et al. 2008

Clin Vaccine Immunol

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The advent of T-cell assay methodologies that are amenable to high throughput coupled with the availability of large libraries of overlapping peptides have revolutionized the fields of vaccine efficacy testing and cellular immune response assessment. Since T-cell assay performance is critically dependent upon the quality and specificity of the stimulating peptides, assurance of high-quality and reliable input peptides is an important aspect of assay validation. Herein, we demonstrate that individual peptides from large human immunodeficiency virus (HIV)-based peptide library sets obtained directly from two independent custom peptide suppliers contained contaminating peptides capable of giving false-positive results, which were consistent with nominal antigen-specific CD8 ؉ T-cell responses. In-depth investigation of the cellular response in terms of responding CD8 ؉ T-cell frequency and human leukocyte antigen (HLA) restriction led to the conclusion that one set of HIV type 1 (HIV-1)-derived peptides was contaminated with a peptide from human cytomegalovirus (HCMV), which is commonly used in cellular immunology research applications. Analytical characterization of the original stock of the suspect HIV-1 peptide confirmed the presence of ϳ1% by weight of the HCMV peptide. These observations have critical implications for quality assurance (QA) and quality control (QC) of peptides used in clinical trials where cellular immune-based assays are important end-point determinants. We propose a simple schema of biological QA/QC protocols to augment the standard biochemical QA/QC analyses as a means to circumvent this and other problems that can affect cellular immune-based assay outcome and interpretation.The identification and characterization of immunogenic Tcell epitope-containing regions of the proteomes of infectious agents and candidate cancer-and tumor-specific proteins are an essential phase in the rational development of prophylactic vaccines and immunotherapeutic strategies. Recent methodological advances, particularly enzyme-linked immunospot (ELISPOT) assays and cytokine-based flow cytometry (CFC) assays coupled with overlapping pooled peptide technology, give the opportunity for detailed and precise analyses of specific cellular immune responses (1,3,11,19,22,29). As cellular immune response assays proceed from being used primarily as research tools to be being used as tools for clinical evaluation and assessment of end points in different phases of vaccine development, the required standards of quality assurance (QA)/quality control (QC) for these assays rise dramatically (4,10,16,17,21,27,28). While assay techniques and equipment can be standardized by employing standard operating procedures and assay reagents and can be standardized by use of manufacturer-certified assay kits, a critical component of the assay that is not typically subject to standardized QA/QC is the synthetic peptides used for stimulating the T cells. Synthetic peptides are usually specific to the particular application of the assay, d...

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“…It is recommended that a minimum of six non-zero concentrations be used when fitting a nonlinear (sigmoidal) concentration-response curve (8,13). We used the bulk stock reference material to prepare a ninepoint standard curve, including two anchor points, sufficient to define the four-parameter logistic function.…”

Section: Methods Modificationmentioning

confidence: 99%

“…The results of accuracy and precision experiments were used to define the sensitivity, assay range, and set acceptance criteria for QCs. For drug development support, the assay range is defined by the LLOQ to ULOQ with well-characterized parameters meeting a priori criteria and for which interpolated results are generated (13). For a diagnostic kit assay, the assay sensitivity is often defined using limit of detection (LOD).…”

Section: Methods Modificationmentioning

confidence: 99%

“…When it is difficult to find "blank" control matrix for standards or QC preparation for a biomarker assay, standards and QCs can be prepared in assay buffer if no matrix effect is demonstrated for the method (13). In addition, SCs prepared from pooling human sera were used to represent the endogenous analyte in serum samples.…”

Section: Use Of Serum Sample Controlmentioning

confidence: 99%

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“Fit-for-Purpose” Method Validation and Application of a Biomarker (C-terminal Telopeptides of Type 1 Collagen) in Denosumab Clinical Studies

Wang

Lee

Burns

et al. 2009

AAPS J

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Abstract. Biomarkers are used to study drug effects, exposure-response relationships, and facilitate early decision making during development. Denosumab, a fully human monoclonal antibody against receptor activator of nuclear factor-κB ligand, profoundly inhibits bone resorption. C-terminal telopeptides of type I collagen (CTx), a bone resorption biomarker, provides early indications of denosumab effectiveness and informs protracted clinical outcomes (e.g., bone mineral density). Because of the dynamic relationship between denosumab and CTx, a precise and robust assay was desired. Thus, we adopted a fit-for-purpose approach to modify and validate a commercial CTx diagnostic kit to meet the intended applications of a quantitative pharmacodynamic biomarker for denosumab development. Seven standards were prepared to replace five calibrators provided in the kit. Three quality controls (QC) and two sample controls were used to characterize and monitor assay performance. Robotic workstations were used for standard and QC preparation and assay execution. Method validation experiments were conducted with rigor and procedures similar to those used for drug bioanalysis. The method demonstrated a linear range of 0.0490-2.34 ng/mL with four-parameter logistic regression. Inter-assay total error of validation samples in serum was ≤26.7%. Extensive tests were conducted on selectivity in sera from target populations, specificity, stability, parallelism, and dilutional linearity. Applications to samples from numerous clinical studies confirmed that the CTx method was reliable, robust, and fit for use as an early indicator of denosumab effectiveness. Refinement supported the confidence for use in pharmacokinetic/pharmacodynamic modeling, dose selections, correlation to clinical effects, and formulation bioequivalence work.KEY WORDS: bone resorption marker; commercial immunoassay kit; method validation; pharmacodynamic (PD) biomarker; serum C-terminal telopeptides of type 1 (CTx).

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Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective

Cited by 519 publications

References 48 publications

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance

Peptide Impurities in Commercial Synthetic Peptides and Their Implications for Vaccine Trial Assessment

“Fit-for-Purpose” Method Validation and Application of a Biomarker (C-terminal Telopeptides of Type 1 Collagen) in Denosumab Clinical Studies

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