Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor

Hoshina, Motoki; Ojima, Shiomi; Kawasaki, Atsushi; Doi, Kosuke; Ohta, Satoshi; Inoue, Asuka; Murayama, Hiroshi

doi:10.1016/j.jim.2024.113655

Cited by 1 publication

(3 citation statements)

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“…While RIA requires the extraction of AVP from the plasma, our assay is homogeneous and does not require time-consuming procedures 7 , 8 . The assay developed in this study requires time for cell seeding and transfection, but we believe that this problem can be solved using cryopreserved cells, as reported previously 9 , 10 . These findings demonstrate the potential of this simplified assay for the quantification of plasma AVP.…”

Section: Discussionmentioning

confidence: 74%

“…We hypothesized that a biosensor-based cAMP accumulation assay could be an alternative method for measuring blood AVP levels. A recent study reported the successful application of a similar biosensor-based assay to measure levels of thyroid-stimulating autoantibodies (TSAbs) using cells co-expressing the thyroid-stimulating hormone receptor (TSHR) and a cAMP biosensor 9 , 10 . In this assay, the cAMP biosensor measured the increase in intracellular cAMP levels triggered by TSHR activation promoted by blood TSAbs.…”

Section: Introductionmentioning

confidence: 99%

“…Notably, the assay is both simple and rapid, requiring only a brief handling period. This biosensor-based technology has been approved by the regulatory agency in Japan for in vitro diagnosis of Graves’ disease and is already routinely used in clinical laboratories 10 . Considering that V2R elevates intracellular cAMP concentrations via the Gs protein, this biosensor-based approach can be adapted for our purposes.…”

Section: Introductionmentioning

confidence: 99%

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A cAMP-biosensor-based assay for measuring plasma arginine–vasopressin levels

Doi,

Kawakami,

Ikuta

et al. 2024

Sci Rep

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Arginine–vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

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