Improving Immunoassay Performance by Antibody Engineering

Stenman, Ulf‐Hǎkan

doi:10.1373/clinchem.2005.049221

Cited by 8 publications

(5 citation statements)

References 16 publications

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“…The revolution in molecular biology towards the end of the 20 th century, particularly the availability of recombinant DNA technology, gave new and promising alternatives to antibodies in immunoassay design (75,76). Use of non-immunoglobulin proteins or engineered antibodies as capture or detection proteins, such as affibodies/aptamers, fusion proteins or single-chain fragments (scFv), effectively limit interference from heterophilic antibodies (77)(78)(79)(80)(81)(82).…”

Section: Recombinant Proteins Non-mammalian and Non-immunoglobulin Amentioning

confidence: 99%

Heterophilic antibody interference in immunometric assays

Bolstad

Warren

Nustad

2013

Best Practice & Research Clinical Endocrinology & Metab

183

133

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Immunometric assays are inherently vulnerable to interference from heterophilic antibodies, endogenous antibodies that bind assay antibodies. The consequences of such interference can be devastating. In this review, we discuss strategies that reduce the damage caused by heterophilic antibodies. Clinicians should only order blood tests that are indicated for the patient and clinical setting at hand, and have the confidence to question laboratory results discordant with the clinical picture. Laboratorians should familiarize themselves with the vulnerability of the assays they offer, and be able to perform and interpret adequate confirmatory measures correctly. When designing immunoassays, the immunoassay industry should invest the necessary resources in specific protective measures against heterophilic antibody interference. Examples include using antibody fragments and the addition of effective blockers to assay reagents. The increasing use of modified monoclonal mouse antibodies both in therapy and diagnostics could present a particular challenge in the future.

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Section: Recombinant Proteins Non-mammalian and Non-immunoglobulin Amentioning

confidence: 99%

Heterophilic antibody interference in immunometric assays

Bolstad

Warren

Nustad

2013

Best Practice & Research Clinical Endocrinology & Metab

183

133

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“…Indeed, site-specific biotinylation can be easily performed in vivo during Escherichia coli expression to produce milligram quantities of uniformly biotinylated scFv, facilitating optimal orientation of the binder. 39 In addition, upon removal of the immunoglobulin constant domains, the scFv format seems to present a resistance to heterophilic antibody interference in serum immunoassays. 38 Molecular models of engineering effort Figures 2-4 illustrate the molecular models used to understand the likely effects of changes within the engineered 5D3D11 CDRs loops compared to the wild-type antibody.…”

Section: Structure-guided Mutagenesis Strategymentioning

confidence: 99%

In Vitro Affinity Maturation of an Anti-PSA Antibody for Prostate Cancer Diagnostic Assay

Müller¹,

Savatier²,

L’Hostis³

et al. 2011

Journal of Molecular Biology

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“…These methods are however not well suited to the high-throughput assays used in clinical laboratories. Indeed, optimal reductions in assay interference can most probably only be achieved by focusing on this problem during the assay design phase (16).…”

Section: Introductionmentioning

confidence: 99%

Heterophilic antibody interference in commercial immunoassays; a screening study using paired native and pre-blocked sera

Bolstad

Warren

Bjerner³

et al. 2011

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: Heterophilic antibodies are still an important source of interference in immunoassays. We have conducted a screening study for interference in a panel of commercially available assays using two sera known to contain high titer Fc-reactive heterophilic antibodies. Methods: The sera were distributed to laboratories participating in the Nordic External Quality Assessment cooperation (EQANord). Duplicate samples pre-blocked with aggregated murine monoclonal MAK33 were also supplied. Discrepancies ()50%) between the results for native and blocked samples were used to classify the tested assays as susceptible to interference. A total of 170 different assay kits covering 91 analytes were tested. Results: We found that 21 assays, covering 19 different analytes, were susceptible to interference from the heterophilic antibodies in the two sera. Many of these are clinically and commercially important assays. Some of the false results were grossly elevated and could have been detrimental to patient care in a clinical setting. Conclusions: Heterophilic antibodies with Fc-reactivity remain a threat. A more widespread use of antibody fragments and aggregated immunoglobulin could potentially improve the heterophilic antibody resistance of assays intended for clinical use.

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Improving Immunoassay Performance by Antibody Engineering

Cited by 8 publications

References 16 publications

Heterophilic antibody interference in immunometric assays

Heterophilic antibody interference in immunometric assays

In Vitro Affinity Maturation of an Anti-PSA Antibody for Prostate Cancer Diagnostic Assay

Heterophilic antibody interference in commercial immunoassays; a screening study using paired native and pre-blocked sera

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