Complement: Measurement

Abstract

Complement comprises more than 30 proteins present in plasma, other biological fluids and on cell surfaces that collaborate in the host defence against infection. The components of the pathway interact in a sequential manner to efficiently label pathogens; self‐cells are protected by plasma and membrane regulators, and cells bind and respond to products of activation through specific membrane receptors. Measurement of complement components, activation products and activity can be clinically useful in several situations: first, where there is a suspicion of a complement deficiency; second, where complement activation is suspected in a disease and third, to monitor effect of a therapeutic intervention on complement activation or activity. This article describes methods for measuring levels of complement proteins, activation products and complement activity in biological fluids and discusses recent developments in the field. In particular, the interindividual variability in complement activity is described and the relevance of this inherited ‘complotype’ on disease susceptibility discussed. The advent of effective anticomplement drugs, already in clinical use in several disease areas, opens a new area in which effective measurement of complement will be an essential requirement.

Key Concepts

  • Correct sample collection and storage is essential for effective measurement of complement.
  • Functional assays of complement haemolytic activity provide the best screen for suspected deficiencies.
  • Immunochemical measurement of circulating complement components and activation fragments can be performed by commercial assays, nephelometric, turbidimetric or ELISA.
  • Pathway‐specific and individual component‐specific functional assays of complement activity are required to detect deficiencies that do not affect immunochemical levels.
  • Functional complement assays are highly dependent on the choice of target cell and buffer composition, particularly the presence of calcium and magnesium ions.
  • Modern structural analyses and biosensor assays allow an in‐depth analysis of the affinity and kinetics of complement component interactions that reveals the impact of mutations and polymorphisms.
  • National and international quality control of complement assays has been lacking in the past and is required for meaningful interlaboratory comparisons.

Keywords: complement; CH50; EA; ELISA; haemolytic assays

Figure 1. Schematic diagram of the complement system. The pathways, regulation and active products are highlighted. Abbreviations are as in text. Regulators are in black boxes.
Figure 2. Complement deficiencies cause clinical problems that are dictated by the part of the system affected.
Figure 3. Route to diagnosis in a patient suspected of harbouring a complement deficiency. Note that activation must always be considered and activation markers such as TCC used as appropriate before embarking on a hunt for deficiency.
Figure 4. A simple haemolysis assay and calculation of lytic activity. The percentage haemolysis (%) obtained in the haemolysis assay at each serum dilution for control standard (•) and test (▪) samples are plotted against the dilution. A line is drawn from the 50% haemolysis point on the Y‐axis to intersect the plotted line for each sample; a line is then plotted vertically down from this point to intersect the X‐axis at the dilution value that gives 50% haemolysis. In the example shown, 50% lysis was achieved in control serum at a dilution of 0.13 and in test serum at a dilution of 0.22. By assigning the standard control serum a value of 100 and applying these figures in the equation: CH50(Test) = 100 × (50% lysis dilution Standard)/(50% lysis dilution Test), the test serum has a calculated CH50 of 59.1 – low haemolytic activity compared to the standard.
Figure 5. ELISA used in complement diagnostics. (a) For measuring complement proteins, an antibody (mAb preferred) is immobilised on the plastic, sample incubated to capture protein of interest, and then a second, noncompeting antibody (mAb preferred) labelled with HRP (*) added to detect bound protein of interest. The assay is developed by incubating with HRP substrate (OPD) and results read spectrophotometrically. (b) For measuring complement activation products, the capture antibody (always a mAb) is against a neoantigen in the fragment absent in the native protein and selectively captures the activation fragment, ignoring native protein. The assay is developed as in (a). (c) For measuring autoantibodies against complement proteins, the protein of interest is immobilised directly on the plastic, sample incubated to capture autoantibody from the test serum, and then an anti‐human IgG (or IgM) antibody labelled with HRP added. The assay is developed as in (a).
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Further Reading

De Cordoba SR, Tortajada A, Harris CL and Morgan BP (2012) Complement dysregulation and disease: from genes and proteins to diagnostics and drugs. Immunobiology 217: 1034–1046.

Liu CC, Manzi S, Danchenko N and Ahearn JM (2004) New advances in measurement of complement activation: lessons of systemic lupus erythematosus. Current Rheumatology Reports 6: 375–381.

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Morgan BP (ed) (2000) Complement Methods and Protocols (Methods in Molecular Biology). Totowa: Humana Press.

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Morgan, Bryan Paul(Sep 2015) Complement: Measurement. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001279.pub4]