Research paper
Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA

https://doi.org/10.1016/j.jim.2004.11.016Get rights and content

Abstract

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Introduction

The complement system has an essential role in innate immune defence and can be activated by three different pathways (Walport, 2001). The classical pathway is activated by binding of C1q to, e.g., immunoglobulins present on microorganisms or by direct binding to apoptotic cells; the alternative pathway can be directly activated by invading microorganisms, and the lectin pathway is activated by carbohydrate moieties present on the surface of invading microbes. Activation of the complement system generates opsonic components of complement facilitating phagocytosis of microorganisms and other targets (Aderem and Underhill, 1999). Initiation of any of the three pathways of complement is associated with the activation of the terminal complement pathway and formation and deposition of C3 and the terminal C5b-9 complement complex (TCC) also termed the membrane attack complex (MAC).

Deficiencies of complement components of all three pathways are associated with distinct clinical pathology. Deficiencies of the classical pathway (C1, C4, C2) are associated with systemic lupus erythematosus (SLE; Pickering et al., 2000). Deficiency of the central component of all three pathways of complement activation, C3, is associated with SLE, pyogenic infections and glomerulonephritis. Patients with deficiencies of factor D and properdin, components of the alternative pathway, show increased susceptibility to infections with Neisserial species (Sjoholm, 2002). Deficiency of MBL, a major initiator of the lectin pathway of complement, is frequently found in the general population due to point mutations in the coding sequence of the MBL2 gene (Sumiya et al., 1991, Lipscombe et al., 1992, Madsen et al., 1995). MBL deficiency has been shown to be associated with bacterial, fungal and viral infections in both children and adults (Eisen and Minchinton, 2003). Apart from infectious diseases, MBL polymorphisms have been reported to be associated with systemic diseases, such as SLE, rheumatoid arthritis and sepsis (Garred et al., 2000, Garred et al., 2003a, Garred et al., 2003b, Davies et al., 1995). The lectin pathway of complement can also be activated via L-ficolin (Matsushita et al., 2000) and H-ficolin (Matsushita et al., 2002), but deficiencies for these molecules have not been described in the human population. MBL and ficolins use MASP-2 as the C4-activating enzyme of the lectin pathway. Interestingly, a patient with MASP-2 deficiency was recently described (Stengaard-Pedersen et al., 2003). Deficiency of complement components of the common terminal pathway may lead to defective lysis of microorganisms by the C5b-9 complex, particularly of Neisserial species (Jack et al., 2001). Deficiency of regulatory proteins of complement activation is associated with angioedema in the case of C-1 inhibitor and with haemolytic uraemic syndrome, SLE, glomerulonephritis and bacterial infections for factor I and H deficiency (Sjoholm, 2002).

For the assessment of the functional activity of the classical and alternative pathways, haemolysis of erythrocytes by complement activation either via the classical (CH50) or alternative pathway (AP50) is used in most laboratories. Functional ELISA based procedures for the classical and alternative pathways have been developed based on previously reported methodology (Fredrikson et al., 1993, Roos et al., 2003). In view of the clinical relevance of MBL deficiencies, several assays have also been developed to assess MBL functional activity in human serum. These assays are based on the use of mannan as a ligand. Of the two known initiators of the lectin pathway, MBL and ficolins, only MBL binds to mannan. Therefore, these assays specifically detect MBL-dependent activation of the lectin pathway, and we will therefore use the term MBL-pathway to indicate this specificity.

To evaluate the activity of the MBL/MASP complex, Petersen et al. (2001) introduced an ELISA-based procedure with mannan-coated plates. Because of interference from classical pathway activation by antimannan antibodies, sera are incubated in high ionic strength buffers. At this tonicity, however, activation of C4 is also inhibited, and therefore, the activity of the MBL complex is assessed in a second step with exogenously added purified C4. Therefore, with this assay, only the activity of the MBL/MASP complex can be directly assessed.

The functional activity of the whole MBL pathway of complement has also been evaluated by other procedures. Direct haemolysis of erythrocytes coated with mannan and indirect haemolysis of chicken erythrocytes, as innocent bystander cells, have been used (Kuipers et al., 2002, Ikeda et al., 1987). In both assays, exogenous MASP and/or additional complement factors have to be added to the assay system to permit erythrocyte lysis. Furthermore, both types of assay are difficult to perform on a routine basis for clinical use and do not exclude participation of the classical pathway in the assay. In clinical practice, it would be helpful to assess the functional activity of the whole MBL pathway, from MBL through to C9, without the use of additional complement sources. Such ELISA-based procedures have been developed using mannan-coated plates (Roos et al., 2003, Minchinton et al., 2002), and it has been recently demonstrated that the contribution of the classical complement pathway in such an assay can be prevented by addition of an inhibitory antibody directed against C1q (Roos et al., 2003).

A compromised innate immune system resulting from defective activation of complement can be caused by genetically determined deficiencies of any of the complement components. Furthermore, decreased or absent pathway activity may also be caused by acquired complement deficiencies due to consumption. Here, we describe the development of a simple ELISA-based format for the evaluation of all three pathways of complement activation. The assay is now available commercially in kit form. Screening the sera of patients for complement deficiencies or any other functional defect in the complement system can now be performed with one simple assay format for the three pathways analysed in parallel. In the present study, we have standardized and validated these assays for the detection of inherited and acquired complement deficiencies associated with all three activation pathways.

Section snippets

Serum samples

Serum samples were obtained from 120 healthy individuals (registered blood donors), 60 females with a mean age of 44.7 years (20–69 years) and 60 males with a mean age of 45.1 years (20–65 years). For each gender, 12 donors were selected from each decade from age 20 to 70. Serum samples obtained were directly aliquoted and stored at −80 °C. The serum samples were tested in three different laboratories in the novel ELISA-based kit for functional activity of the classical, alternative and MBL

Assessment of complement activity via three pathways in healthy donors

Serum samples from 120 healthy controls were tested for classical pathway activity, alternative pathway activity and MBL pathway activity in three laboratories, as described in the Materials and methods section. The complement activity for each pathway was expressed as a percentage of the activity of a positive standard serum. For both the classical pathway (Fig. 1A) and the alternative pathway (Fig. 1B), complement activity was detectable in all healthy donors. The interindividual variation

Discussion

Haemolytic assays to assess the functional activity of the classical and the alternative pathways of complement activation have been available for some years. However, for the MBL pathway, no comparable assay exists. To assess the functional activity of the MBL pathway, an ELISA has been developed in which the activity of the pathway from MBL through to C9 is assessed in whole serum (Roos et al., 2003). The degree of activity is assessed as the amount of C5b-9 that is generated and bound in

Acknowledgements

This work was supported by grants from the European Union (QLGT-CT2001-01039) and the Dutch Kidney Foundation (PC 95, C98-1763). Research at the Institute of Child Health and the Great Ormond Street Hospital for Children National Health Service Trust benefits from research and development funding received from the National Health Service Executive.

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