A robust multiplexed assay to quantify C1-inhibitor, C1q, and C4 proteins for in vitro diagnosis of hereditary angioedema from dried blood spot.

Abstract:

:Hereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Plasma C1-INH activity and concentrations of C1-INH and complement components 1q and 4 (C1q, C4) are critical to the HAE diagnosis. We describe a novel multiplexed assay to simultaneously measure C1-INH, C1q, and C4 levels in dried blood spot (DBS) of HAE patients. The blood proteins were extracted from 3 mm punches of DBS samples and were subsequently digested by trypsin. The signature peptide derived from each protein was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyte-depleted blood was generated as a surrogate matrix for the preparation of calibration curves to overcome the interference of endogenous proteins, and the assay reproducibility was further monitored by assessing the signal of plasma transferrin as a house-keeping protein. The assay was fully validated following regulatory guideline, with a quantification range of 12.5-800 μg/mL for C1-INH and C4 and 3.13-200 μg/mL for C1q. The precision and accuracy ranged from 3.3%-9.8% and -8.2%-12.6%, respectively. All the patient samples exhibited C1-INH levels lower than normal range except the Type II patient and the C4 and C1q concentrations were as expected. Results from the DBS-based LC-MS assay were highly correlated with the ELISA data measured in plasma of the same subjects. The method described here offers unique advantages such as less invasive sampling, minimal blood processing, and easy transportation and sample storage, allowing, for the first time, C1-INH, C4, and C1q levels to be simultaneously determined in a drop of dried blood.

journal_name

J Pharm Biomed Anal

authors

Lai Y,Zhang G,Inhaber N,Bernstein JA,Cwik M,Zhou Z,Chockalingam PS,Wu J

doi

10.1016/j.jpba.2020.113844

subject

Has Abstract

pub_date

2021-02-20 00:00:00

pages

113844

eissn

0731-7085

issn

1873-264X

pii

S0731-7085(20)31731-3

journal_volume

195

pub_type

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