Novel assays to assess the functional capacity of the classical, the alternative and the lectin pathways of the complement system

Abstract

Deficiencies in many of the complement proteins and their regulatory molecules have been described and a variety of diseases, such as recurrent infections, systemic lupus erythematosus (SLE) and renal diseases, may be linked to deficiency in the complement system. Screening for complement defects is therefore of great importance. In this study, we present novel improved enzyme-linked immunosorbent assays for the functional assessment of the three individual pathways of the complement system. The method is applicable at high serum concentrations and we demonstrate that it minimizes both false negative as well as false positive results. In particular, for the functional mannose-binding lectin activity it represents an improvement on the existing assays. In this respect, the present assays represent novel improved diagnostic protocols for patients with suspected immunodeficiencies related to the complement system.

Fig. 1

(a–d) An example of a regression analysis on logistically transformed optical density (OD) values used to determine the complement activity as percentage of the activity of a positive standard serum. (a) Duplicate raw data of the lectin pathway (LP) C3 deposition of two serum samples: a calibrator serum (solid symbols) and a donor serum (open symbols), with OD as a function of the power of the repeated twofold dilutions from each dilution series. (b) Due to the maximum limit of the OD determination, only the last value of OD = 3·5 is maintained in each dilution series, while the prior maximum determinations are omitted. (c) OD values are divided by 3·6 to transform the OD data to values between 0 and 1, which are then logistically transformed, y' = ln[y/(1–y)]. (d) A background level of OD = 0·15 is observed, and values below the corresponding logistically transformed value of −3·135 are omitted. A linear regression is then fitted to the remaining data points and dilution factors are compared at 50% of the maximum OD of 3·5, i.e. at OD = 1·75, equal to a transformed value of −0·056.

Fig. 2

(a) Normal activity of complement activity of the alternative, classical and mannose-binding lectin (MBL) pathway measured in 150 healthy blood donors. The quantile box-plot shows the median (line) and the quartiles (25th and 75th percentiles) defining the interquartile range. The ends of the whiskers are the outermost data points from their respective quartiles that fall within the distance computed as 1·5 × the interquartile range. (b) The distribution of the normal complement activity for the alternative, classical and MBL pathway measured in 150 healthy blood donors. The quantile box-plot shows the median (line) and the quartiles (25th and 75th percentiles) defining the interquartile range. The ends of the whiskers are the outermost data points from their respective quartiles that fall within the distance computed as 1·5 × the interquartile range. The mean diamond identifies the mean and the 95% confidence interval about the mean.

Fig. 3

Relation between serum concentration of oligomerized mannose-binding lectin (MBL) (ng/ml) and MBL pathway activity (%) in healthy blood donors; linear regression: r² = 0·70, P < 0·0001; n = 150.

Fig. 4

(a–b) Comparison of mannose-binding lectin (MBL) pathway assays. (a) % MBL pathway activity measured using the Wielisa MBL pathway kit (WIESLAB® Complement System Screen COMPL 300; Euro-Diagnostica). Seven homozygous MBL-deficient sera (O/O) diluted 1:10 (a, left panel) and 10 serum samples with reduced, but measurable MBL activity diluted 1:101 as instructed according to the accompanying Wielisa protocol (a, right panel) were analysed for MBL pathway activity using the Wielisa kit. Readout: C5b-9. (b) % MBL pathway activity measured using the MBL pathway assay described in this study. The same seven homozygous MBL-deficient sera (O/O) (b, left panel) and 10 serum samples with reduced MBL activity (a, right panel) as analysed with the Wielisa kit. Readout: C5b-9.

Fig. 5

(a–c) Serum samples with well-defined defects in complement components were analysed in our assay for alternative (a), classical (b) and mannose-binding lectin (MBL) (c) pathway activity. The dotted lines indicate the lower cut-off values for normal activity for the different pathways.