This is in agreement with previous observations, that 5–10% of the Caucasian population is MBL-deficient [7]. It is well known that mannan besides activating the MBL pathway also has the potential to trigger activation of the CP and the AP [22]. In assays that are not able to block the influence of the AP when measuring the MBL activity, it
is necessary to dilute the serum up to a level where the contribution from the AP is minimal. This may result in false negative MBL measurements in samples where the MBL activity is only reduced. The results obtained by Seelen et al. [21] showed that 28% of the 120 sera from healthy donors had functional MBL activities below a normal threshold set at 10%, which is an unrealistically
high proportion selleck chemicals llc of MBL-deficient individuals in a normal population. This may be due to the fact selleck compound that the serum samples were diluted 1:101 prior to analysis, and thus samples with low MBL activity will read out as negative. This present ELISA set-up using SPS for assessment of the MBL activity completely blocks the interference from the AP and the CP, allowing valid analysis of samples in high serum concentration. By analysing serum samples in twofold serial dilutions starting at a high serum concentration (10%), a more precise determination of MBL activity is obtained, which removes the risk of generating false negative measurements. Data were analysed using regression analysis on logistically transformed values taking the dilution factor into account. To illustrate the influence of the AP when measuring MBL pathway activity on a mannan-coated surface, seven samples with no MBL pathway activity Adenosine triphosphate (all either homozygous
or compound heterozygous for the structural MBL-2 gene mutations) in our MBL activity assay were analysed using the commercial kit Wielisa for assessment of MBL pathway. Each sample was analysed using 1:10 dilutions. All samples, which should have no functional MBL pathway activity, showed measureable false positive MBL pathway activities. This clearly indicates interference from the AP and illustrates why it is necessary to dilute samples in order to minimize the influence of the AP using this commercial kit. The concerns regarding diluting samples at 1:101 prior to analysing MBL pathway activity, which may give false negative results, were also tested using the Wielisa kit. The results obtained from the kit showed that six of 10 samples (two XA/XA, four XA/O and four YA/O), which had measurable MBL activity in our assay, showed no MBL pathway activity using the Wielisa kit. Taken together, the data indicate a risk of both false negative and false positive results using MBL pathway assays that do not block the AP. Although the terminal complement complex (C5b-9) is used as readout in the above-mentioned commercial assay, we recommend the use of the central complement factor C3 as readout in the assays presented in this study.