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Quantification of C1 esterase inhibitor in human serum by enzyme-linked immunosorbent assay: Correlation with turbidimetric immunoassay

https://doi.org/10.15789/1563-0625-QOC-2794

Abstract

C1 inhibitor of serine proteases (C1-INH) performs a regulatory function in the complement system and vascular permeability. Deficiency of C1-INH leads to various forms of angioedema, including hereditary angioedema (HAE). The cause of HAE is a genetically determined violation of the synthesis of C1-INH. A decrease in the level of C1-INH to 50% relative to the norm leads to an increase in the production of bradykinin, which is the basis for the diagnosis of HAE. The development of affordable ELISA for the quantitative determination of C1-INH is a popular direction for clinicians. During the development of a new kit for quantitative determination of C1-INH, two mouse monoclonal antibodies (mAb) with different epitope specificities were obtained. On their basis, a sandwich-type ELISA was developed. The specificity of the obtained mAb's was confirmed using the medical device “Berinert”. To prepare calibrators, C1-INH was affinity purified from human blood plasma using a sorbent with immobilized mAbs. The identity of the C1-INH protein was confirmed by PAGE electrophoresis, immunoblotting, and mass spectrometry on MALDI-TOF/TOF UltrafleXtreme mass spectrometer. To assess the quality indicators of developed reagents kit, studies were carried out in accordance with GOST R 51352-2013 and TU 21.20.23-041-01967164-2022. Values of quality indicators: accuracy — 93.53%; measurement linearity interval — 22.00-176.07 ng/mL. Using the developed ELISA test system, we examined 28 blood sera from healthy donors and 7 blood sera from patients with confirmed HAE. In the same samples, the content of C1-INH was determined by turbidimetric method, using the "Diagnostic reagents for in vitro immunochemical studies of specific blood proteins. Model: C1-esterase inhibitor (C1 EsteraseInhibitor)" (Aptec, Belgium). The correlation coefficient was 0.94 (p < 0.05). It was found that the diagnostic sensitivity and specificity of the developed ELISA is 100%. As a result of the study, an original ELISA test system for the quantitative determination of C1-INH was developed "Reagent kit for enzyme-linked immunosorbent assay of human C1-inhibitor (C1-inh PS)".

About the Authors

N. P. Gorbunov
Saint Petersburg Pasteur Institute
Russian Federation

Nikolay P. Gorbunov - Junior Research Associate, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

14 Mira St St. Petersburg 197101

Phone: +7 (921) 786-38-16


Competing Interests:

None



A. V. Zhakhov
Saint Petersburg Pasteur Institute
Russian Federation

Aleksandr V. Zhakhov - Research Associate, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



I. N. Gorbunova
Saint Petersburg Pasteur Institute
Russian Federation

Irina N. Gorbunova - Research Associate, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



A. M. Milichkina
Saint Petersburg Pasteur Institute
Russian Federation

PhD (Medicine), Chief Physician, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



I. V. Drozd
Saint Petersburg Pasteur Institute
Russian Federation

PhD (Biology), Head, Central Clinical Diagnostic Laboratory, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



A. V. Gubanova
Saint Petersburg Pasteur Institute
Russian Federation

Doctor, Clinical Laboratory Diagnostics, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



E. M. Danilova
Saint Petersburg Pasteur Institute
Russian Federation

PhD (Medicine), Head, Dispensary and Polyclinic Department, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



R. N. Kuznecova
Saint Petersburg Pasteur Institute
Russian Federation

PhD (Medicine), Allergist-Immunologist, Dispensary and Polyclinic Department, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



T. V. Savin
Saint Petersburg Pasteur Institute
Russian Federation

Allergist-Immunologist, Dispensary-Polyclinic Department, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



A. G. Burtseva
Saint Petersburg Pasteur Institute
Russian Federation

Anastacia G. Burtseva - Junior Research Associate, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



N. V. Pigareva
Saint Petersburg Pasteur Institute
Russian Federation

Natalya V. Pigareva - PhD (Medicine), Senior Research Associate, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

St. Petersburg


Competing Interests:

None



A. M. Ischenko
Saint Petersburg Pasteur Institute
Russian Federation

Aleksandr M. Ischenko - PhD (Biology), Head, Laboratory of the Hybridoma Technology, Saint Petersburg Pasteur Institute.

St. etersburg


Competing Interests:

None



Areg A. Totolian
Saint Petersburg Pasteur Institute; First St. Petersburg State I. Pavlov Medical University
Russian Federation

Areg A. Totolian - PhD, MD (Medicine), Professor, Full Member, Russian Academy of Sciences, Head, Laboratory of Molecular Immunology, Director, Saint Petersburg Pasteur Institute; Head, Department of Immunology, First St. Petersburg State I. Pavlov Medical University.

St. Petersburg


Competing Interests:

None



References

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5. Tange C.E., Kaur A., Verma N., Hickey A., Grigoriadou S., Scott C., Kiani S., Steven R., Ponsford M., El- Shanawany T., Jolles S., Harding S., Parker A.R. Quantification of human C1 esterase inhibitor protein using an automated turbidimetric immunoassay, J. Clin. Lab. Anal., 2019, Vol. 33, no. 1, e22627. doi: 10.1002/jcla.22627.

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Gorbunov N.P., Zhakhov A.V., Gorbunova I.N., Milichkina A.M., Drozd I.V., Gubanova A.V., Danilova E.M., Kuznecova R.N., Savin T.V., Burtseva A.G., Pigareva N.V., Ischenko A.M., Totolian A.A. Quantification of C1 esterase inhibitor in human serum by enzyme-linked immunosorbent assay: Correlation with turbidimetric immunoassay. Medical Immunology (Russia). 2023;25(5):1197-1204. https://doi.org/10.15789/1563-0625-QOC-2794

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ISSN 1563-0625 (Print)
ISSN 2313-741X (Online)