A microplate assay to measure classical and alternative complement activity

Bénédicte Puissant-Lubrano 1 , 2 , Françoise Fortenfant 1 , Peter Winterton 3 ,  and Antoine Blancher 2 , 4
  • 1 Laboratoire d’Immunologie, CHU de Toulouse, Hôpital Rangueil, TSA 50032, Toulouse, France
  • 2 Laboratoire d’Immunogénétique Moléculaire, Université Paul Sabatier, Faculté de médecine Toulouse-Rangueil, Toulouse, France
  • 3 Département de Langues Vivantes et Gestion, Université Paul Sabatier, Toulouse, France
  • 4 Laboratoire d’Immunologie du CHU de Toulouse, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France
Bénédicte Puissant-Lubrano, Françoise Fortenfant, Peter Winterton and Antoine Blancher

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.

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Clinical Chemistry and Laboratory Medicine ( CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor of over three. CCLM is the official journal of nine national clinical societies and associated with EFLM.

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