A microplate assay to measure classical and alternative complement activity
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Bénédicte Puissant-Lubrano
Abstract
Background:
We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum.
Methods:
The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily.
Results:
The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs.
Conclusions:
This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.
Acknowledgments
The authors thank the residents David Metsu and Patricia Dubot as well as Gauthier Benat (Master’s student) for their excellent technical assistance directed by B.P.L. during their stay in the laboratory. The authors are grateful to Drs. Catherine Viguie and Stéphane Bertagnoli for providing blood samples of sheep and rabbit.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Reagents for the liposome immunoassay were provided by The Binding Site.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References
1. Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:1058–66.10.1056/NEJM200104053441406Search in Google Scholar
2. Walport MJ. Complement. Second of two parts. N Engl J Med 2001;344:1140–4.10.1056/NEJM200104123441506Search in Google Scholar
3. Ahmed AE, Peter JB. Clinical utility of complement assessment. Clin Diagn Lab Immunol 1995;2:509–17.10.1128/cdli.2.5.509-517.1995Search in Google Scholar
4. Kirschfink M, Mollnes TE. Modern complement analysis. Clin Diagn Lab Immunol 2003;10:982–9.10.1128/CDLI.10.6.982-989.2003Search in Google Scholar
5. Ricklin D, Lambris JD. Complement in immune and inflammatory disorders: pathophysiological mechanisms. J Immunol 2013;190:3831–8.10.4049/jimmunol.1203487Search in Google Scholar
6. Sahu A, Lambris JD. Complement inhibitors: a resurgent concept in anti-inflammatory therapeutics. Immunopharmacology 2000;49:133–48.10.1016/S0162-3109(00)80299-4Search in Google Scholar
7. Wagner E, Frank MM. Therapeutic potential of complement modulation. Nat Rev Drug Discov 2010;9:43–56.10.1038/nrd3011Search in Google Scholar PubMed
8. Mayer MM. In: Experimental immunochemistry. EA Kabat and MM Mayer, editors. IL: Thomas Springfield, 1961:Ch4.Search in Google Scholar
9. Abbal M, Tkaczuk J, Praud C, Msayeh F, Ohayon E. Computer-assisted kinetic assay for quantification of total complement activity. Complement Inflamm 1991;8:92–103.10.1159/000463185Search in Google Scholar PubMed
10. Kolde HJ, Deubel R. Development of a rapid kinetic assay for the function of the classical pathway of the complement system and for C2 and C4. J Clin Lab Immunol 1986;21:201–7.Search in Google Scholar
11. Akots G, Braman JC, Broeze RJ, Bowden DW. Rapid, homogeneous phase, liposome-based assays for total complement activity. Complement 1984;1:125–33.10.1159/000467828Search in Google Scholar PubMed
12. Jaskowski TD, Martins TB, Litwin CM, Hill HR. Comparison of three different methods for measuring classical pathway complement activity. Clin Diagn Lab Immunol 1999;6:137–9.10.1128/CDLI.6.1.137-139.1999Search in Google Scholar PubMed PubMed Central
13. Glick MR, Ryder KW, Jackson SA. Graphical comparisons of interferences in clinical chemistry instrumentation. Clin Chem 1986;32:470–5.10.1093/clinchem/32.3.470Search in Google Scholar
14. Liu CC, Young JD. A semi-automated microassay for complement activity. J Immunol Methods 1988;114:33–9.10.1016/0022-1759(88)90150-0Search in Google Scholar
15. Muzykantov VR, Samokhin GP, Smirnov MD, Domogatsky SP. Hemolytic complement activity assay in microtitration plates. J Appl Biochem 1985;7:223–7.Search in Google Scholar
16. Narchi G, Milani S, Tedesco F. A computerized microassay for complement hemolytic activity using an automated spectrophotometer. Ric Clin Lab 1987;17:27–33.10.1007/BF02909385Search in Google Scholar
17. Nilsson UR, Nilsson B. Simplified assays of hemolytic activity of the classical and alternative complement pathways. J Immunol Methods 1984;72:49–59.10.1016/0022-1759(84)90432-0Search in Google Scholar
18. Yamamoto S, Kubotsu K, Kida M, Kondo K, Matsuura S, Uchiyama S, et al. Automated homogeneous liposome-based assay system for total complement activity. Clin Chem 1995;41:586–90.10.1093/clinchem/41.4.586Search in Google Scholar
Supplemental Material:
The online version of this article (DOI: 10.1515/cclm-2016-0553) offers supplementary material, available to authorized users.
©2017 Walter de Gruyter GmbH, Berlin/Boston
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