The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs

Hennig, Christian; Rink, Lothar; Fagin, Ursula; Jabs, Wolfram J.; Kirchner, Holger

doi:10.1016/s0022-1759(99)00206-9

Cited by 105 publications

(62 citation statements)

References 18 publications

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“…The peptide standard refers to the 13 separated by LC retention time as well. In contrast, autoantibodies and heterophilic anti-reagent antibodies are known to produce false positive and false negative results in sandwich immunoassays (33)(34)(35). These types of interferences are not observed in immuno-MRM-MS because the auto-and heterophilic antibodies are inactivated by reduction, alkylation, and enzymatic digestion during sample preparation for SISCAPA (36 -38).…”

Section: Discussionmentioning

confidence: 99%

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Kuhn

Whiteaker

Mani

et al. 2012

Molecular & Cellular Proteomics

170

158

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The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 l of plasma. Assay reproducibility was acceptable for verification studies, with median intra-and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-

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Section: Discussionmentioning

confidence: 99%

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Kuhn

Whiteaker

Mani

et al. 2012

Molecular & Cellular Proteomics

170

158

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“…Since this multiplex assay has been set up as a sandwich immunoassay, it will enhance specificity and reduce the risk of cross-recognition with other proteins (7). Hence, specificity could be increased when additional antibodies are added to the multiplex assay.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

Jager

Velthuis

Prakken

et al. 2003

Clin Vaccine Immunol

494

365

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Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.Cytokines are soluble proteins that are secreted by cells of the immune system. These proteins can alter the behavior and properties of different cell types. Although cytokine functions are complex, cytokine profiles are highly relevant parameters of an immune response. Different cytokines possess biological overlapping functions, and they have the ability to regulate production of other cytokines. Therefore, analysis of the function of the complete set of cytokines expressed within microenvironments (e.g., a site of inflammation) is often of more value than analysis of a single isolated cytokine (13).Cytokines can be quantitated at various levels. mRNA can be detected by real-time PCR; intracellular proteins can be measured by fluorescence-activated cell sorter staining of permeabilized cells, and secreted cytokines can be quantified with bioassays, enzyme-linked immunosorbent assays (ELISAs), radioactive immunosorbent assays, and microarrays. Multiplex assays for detection of cytokines at the mRNA (6) and cellular levels (16, 18) are commonly used. However, these assays have one or more limitations, like the need for a large sample volume or detection of precursor proteins rather than native secreted proteins. In addition, these techniques are time-consuming and laborious.Recent advances concerning applications for the simultaneous detection of proteins have resulted in different particlebased flow cytometric assays. These assays have proven to be very useful in the simultaneous detection of cytokines in body fluids. Unfortunately, at present, either the number of different microspheres or the availability of predefined kits limits these assays (1, 3). The Bio-Plex system employing the Luminex multianalyte profiling technology (Lab-MAP) allows individual and multiplex analysis of up to 100 different analytes in a single microtiter well (20).Our laboratory focuses on immunoregulation and immunotherapy of children with autoimmune diseases-in particular, juvenile idiopathic arthritis (JIA). Sample volumes are relatively small due to our patient population. For a number of cytokines, ready-to-use beads are available, but not for the full spectrum. To overcome these limitations, we chose to develop and validate our own multiplex assays with the Bio-Plex system. With this assay, we were able to detect human cytokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) culture supernatants from both autoimmune and healthy individuals. We showed that it is a reliable, fast, and reproducible technique with a sensitivity that is comparable to that of conventional ELISAs. MATERIALS AND METHODSCell isolation and cultures. Heparinized blood ...

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“…In our experience before the surge in therapeutic use of murine or humanized murine monoclonal antibodies, we found little evidence of interfering immunoglobulins from the immunoglobulin classes that normally exhibit higher affinities, i.e., IgA or IgG (personal observation). Low-affinity human antibodies often bind to both animal and human immunoglobulins, making heterophilic antibodies (human anti-animal) and rheumatoid factors (human anti-human) overlapping and sometimes confusing entities (4 ). Such antibodies do not form complexes in vivo, although they bind the stacked capture antibodies on the solid phase in immunometric assays.…”

Section: The War On Heterophilic Antibody Interferencementioning

confidence: 99%

The War on Heterophilic Antibody Interference

Bjerner¹,

Børmer

Nustad

2005

Clinical Chemistry

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The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs

Cited by 105 publications

References 18 publications

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

The War on Heterophilic Antibody Interference

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