Hook effect in immunometric assays for prostate-specific antigen

Charrié, Anne; Charrière, G; Guerrier, A

doi:10.1093/clinchem/41.3.480

Cited by 11 publications

(3 citation statements)

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“…This so-called “hook effect” has been observed in other immunoassays and can result from monovalent antibody binding driven by Fab arms on the same IgG molecule competing for limiting binding sites on the target antigen. 43 Consistent with this observation, MEDI9447 Fab did not inhibit sCD73 activity ( Fig. 5A ).…”

Section: Resultssupporting

confidence: 83%

Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action

Geoghegan¹,

Diedrich²,

Lu³

et al. 2016

mAbs

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M. Damschroder (2016) Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action, mAbs, 8:3, 454-467, DOI: 10.1080/19420862.2016 ABSTRACT CD73 (ecto-5 0 -nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.

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Section: Resultssupporting

confidence: 83%

Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action

Geoghegan¹,

Diedrich²,

Lu³

et al. 2016

mAbs

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“…Our THBS1 and CTSD ELISAs are two-site immunoassays in which capture and detection antibodies are simultaneously exposed to the analyte. High-dose hook effect has been shown for several analytes (PSA, growth hormone, ferritin) [33][34][35] and is dependent on the sample to antibody ratio, where in the presence of high concentrations of analyte, binding of the labelled antibody-bound analyte may be decreased rather than increased (due to competitive binding of free analyte), resulting in erroneous results. When sera were tested at lower…”

Section: Discussionmentioning

confidence: 99%

Analytical performance of thrombospondin-1 and cathepsin D immunoassays part of a novel CE-IVD marked test as an aid in the diagnosis of prostate cancer

Macagno¹,

Athanasiou²,

Wittig³

et al. 2020

PLoS ONE

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The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4-6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total withinlaboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80-120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used.

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“…The 'hook effect' is a well-documented cause of falsely low results in sera with high analyte concentrations in immunometric (sandwich) assays. [126][127][128][129][130] The reduced assay signal is caused by excessively high concentrations of the analyte simultaneously binding both capture and detecting antibodies. This prevents the formation of the required complexes with capture antibody, analyte and detecting antibodies.…”

Section: Hook Effectmentioning

confidence: 99%