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

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.

Full text

Research paper
Functional analysis of the classical, alternative, and MBL pathways
of the complement system: standardization and validation of a
simple ELISA
M.A. Seelen
a
, A. Roos
a
, J. Wieslander
b,1
, T.E. Mollnes
c
, A.G. Sjfholm
d
, R. Wurzner
e
,
M. Loos
f
, F. Tedesco
g
, R.B. Sim
h
, P. Garred
i
, E. Alexopoulos
j
,
M.W. Turner
k
, M.R. Daha
a,
*
a
Department of Nephrology, C3P-29, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
b
Wieslab IDEON Lund, Sweden
c
Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway
d
Institute of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology,Lund University, Lund, Sweden
e
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria
f
Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University Mainz, Germany
g
Department of Physiology and Pathology, University of Trieste, Trieste, Italy
h
Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, UK
i
Tissue Typing Laboratory-7631 of the Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Denmark
j
Department of Nephrology, Hippokration General Hospital, Thessaloniki, Greece
k
Immunobiology Unit, Institute of Child Health, London, UK
Received 24 August 2004; received in revised form 5 November 2004; accepted 15 November 2004
Available online 15 December 2004
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
0022-1759/$ - see front matter D 2004 Published by Elsevier B.V.
doi:10.1016/j.jim.2004.11.016
Abbreviations: AAE, acquired angioedema; AGN, acute poststreptococcal glomerulonephritis; AP, alternative pathway; C1-INH, C1
inhibitor; C3NeF, C3 nephritic factor; CP, classical pathway; HAE, hereditary angioedema; HUVS, hypocomplementaemic urticarial vasculitis
syndrome; MAC, membrane attack complex; MBL, mannose-binding lectin; MBL-P, MBL pathway; SLE, systemic lupus erythematosus; TCC,
terminal complement complex.
* Corresponding author. Tel.: +31 71 526 3964; fax: +31 71 524 8118.
E-mail address: majseelen@lumc.nl (M.R. Daha).
1
This publication represents the results of an EU sponsored collaboration between European academic partners and an industrial partner.
Journal of Immunological Methods 296 (2005) 187 – 198
www.elsevier.com/locate/jim

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.
D 2004 Published by Elsevier B.V.
Keywords: Systemic lupus erythematosus; MBL pathway; ELISA
1. 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 bindi ng to apoptotic cells; the alternative path-
way can be directly activated by invading micro-
organisms, 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). Init iation 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 mem-
brane 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 eryth-
ematosus (SL E; 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, compo-
nents 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). M BL de ficiency 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 poly-
morphisms have been reported to be associated with
systemic diseases, such as SLE, rheumatoid arthritis
and sepsis (Garred et al., 2000, 2003a,b; 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). Defi-
ciency of complement components of the common
termin al 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 activat ion is
associated with angioe dema in th e 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 proce-
dures for the classical and alternative pathways have
been developed based on previously reported method-
ology (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 com-
plex, Petersen et al. (2001) introduced an ELISA-based
procedure with mannan-coated plates. Because of
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198188

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 inhibit ed, 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 innoce nt bystander cells, have been
used (Kuipers et al., 2002; Ikeda et al., 1987). In both
assays, exogenou s MASP and/or additional comple-
ment 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 genetica lly 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 patie nts 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.
2. Materials and methods
2.1. Serum samples
Serum samples were obtai ned 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 8C. The serum
samples were tested in three different laboratories in
the novel ELISA-based kit for functional activity of
the classical, alternative and MBL pathways, sub-
sequently referred to as the complement kit. From six
donors, plasma samples were also collected into
heparin, EDTA or citrate. These samples were tested
in the complement kit, and the results were expressed
as a percentage of pathway activity compared to the
pathway activity assessed in serum samples from the
same donors.
Sixty-four serum samples from patients with differ-
ent well-defined genetically determined complement
deficiencies ( Turner and Hamvas, 2000; Pickering et
al., 2000; Sjoholm, 2002) were collected and tested in
six different laboratories within Europe. Thirt y-eight
samples were taken from patients with low comple-
ment levels caused by complement consumption. From
these patients, five were diagnosed with hypocomple-
mentaemic urticarial vasculitis syndrome (HU VS),
seven with hereditary angioedema (HAE), thirteen
patients with acquired angioedema (AAE), seven
patients with acute poststreptococcal glomeruloneph-
ritis and six sera were from patients positively tested for
the presence of C3 nephritic factor (C3NeF). All
samples taken were directly aliquoted and stored at
80 8C. Forty serum samples were selected from sera
sent to one of the laboratories for diagnostic evaluation
of complement activity. These serum samples were
from patients with SLE, HUVS or recurrent infections.
These samples were tested for total complement
activity in the complement kit.
2.2. Assessment of pathway activity in normal human
serum samples using the complement kit
The complement kit for assessment of classical,
alternative and MBL pathway activity was developed
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198 189

by the EU consortium and prepared centrally at
Wieslab (Sweden). It is now commercially available
(Wielisa COMPL300 Total Complement Functional
Screen kit from Wieslab AB, Lund, Sweden).For the
present studies, the instructions provided in the manua l
were followed. In brief, strips of wells for classical
pathway (CP) evaluation were precoated with IgM,
strips for alternative pathway (AP) determination were
coated with LPS, and MBL pathway (MBL-P) strips
were coated with mannan. Sera were diluted 1/101 for
the CP and MBL-P assay and 1/18 for the AP assay in
specific buffers, which ensured that activation of only
one of the pathways occurred (Roos et al., 2003), and
were incubated for 1 h at 37 8C. After washing the
strips, alkaline phosphatase-conjugated antihuman
C5b-9 was added before incubation at room temper-
ature for 30 min. Additional washing was performed,
substrate was added, and the wells were incubated for
30 min. Finally, absorbance values were read at 405
nm. In each assay, standard posit ive and negative
control sera provided in the kit as lyophilised material
were reconstituted with distilled water. The positive
serum was a pool of five sera from healthy individuals,
and the negative control consisted of sera heat
inactivated at 56 8C for 20 min. Complement activity
was calculated using the following formula: Activi-
ty=100%

(mean A
405
(sample)mean A
405
(negative
control)/(mean A
405
(standard serum)mean A
405
(negative control). Samples as well as standard serum
and negative control serum were tested in duplicate at a
fixed dilution.
2.3. Analysis of intraassay and interassay variation
For the assessment of intraassay variation of the
complement kit, one sample was tested in 40 wells on
one occasion for all three pathways. For calculation of
the interassay variation, three samples were selected
and tested on six different occasions. The mean
values, standard deviation (SD) and the coefficient
of variation (CV=SD/mean

100%) were calculated
for the classical, alternative and MBL pathways.
2.4. Haemolytic assays
For the haemolytic assessment of classical path-
way complement activation, sheep red blood cells
(SRBC) were sensitised using rabbit anti-SRBC Abs
(Ab-coated erythrocytes (EA)). For a classical path-
way test, a total number of 7

10
9
EA diluted in
dextrose gelatin Veronal buffer
2+
(0.5

VBS, 0.05%
gelatin, 167 mM glucose, 0.15 mM CaCl
2
, 0.5 mM
MgCl
2
(DGVB
++
); volume 50 Al) was mixed with
serum (final dilution 1/10 in DGV B
++
, final volume
of 100 Al) for 30 min at 37 8C. For the analysis of
alternative pathway activity, a haemolytic assay for
the alternative pathway was performed. Rabbit
erythrocytes (7

10
9
) suspended in DGVB
++
contain-
ing 10 mM MgEGTA were incubated in a 1:1 ratio
with human serum, final volume of 100 Al, for 30 min
at 37 8C. For both assays, after the addition of 1.5 ml
of PBS and centrifugation, haemolysis was assessed
by measuring absorbance a t 414 nm. The lytic
activity of a sample was expressed in arbitrary units
per ml using the following formula: Lytic activi-
ty=activity of the standard serum (U/ml)

(mean A
414
(sample)mean A
414
(0%))/(mean A
414
(standard
serum)mean A
414
(negative control)). In this for-
mula, the A
414
(0%) represents the incubation of EA
with buffer only, and the A
414
(100%) was assessed
after the addition of H
2
O. Normal haemolytic activity
was defined as higher than 207 U/ml and higher than
52 U/ml for the classical pathway test and the
alternative pathway test, respectively.
2.5. Measurement of MBL serum concentrations
Assessment of MBL concent rations was performed
as described previously (Roos et al., 2001).
2.6. Statistics
The Spearman nonparametric correlation coeffi-
cient was used for statistical analysis. P-values below
0.05 were considered to be statistically significant.
3. Results
3.1. Assessment of complement activity via three
pathways in healthy donors
Serum samples from 120 healthy controls were
tested for classical pathway activity, alternative path-
way activity and MBL pathway activity in three
laboratories, as described in the Materials and methods
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198190

section. The complement activity for each pathway
was expressed as a percentage of the activity of a
positive standard serum. For both the classical path-
way (Fig. 1A) and the alternative pathway (Fig. 1B),
complement activity was detectable in all healthy
donors. The interindividual variation for the alter-
native pathway was somewhat higher than that for the
classical pathway. In contrast, a large interindividual
variation was observed for the activity of the MBL
pathway of complement (Fig. 1C), wi th undetectable
activity in a number of donors. Results for all three
pathways of complement showed a highly significant
correlation between the different laboratories. The
correlation coefficients for the cl assical pathway
activity, the alternative pathway activity and the
MBL pathway activity were above 0.71, above 0.67
and above 0.93 ( Pb0.001), respectively, for all three
pairs of laboratories.
To determine the normal level of activity for the
classical and the alternative pathways of complement
Fig. 1. One hundred and twenty sera from healthy volunteers were tested using the complement kit in three different laboratories for classical
pathway activity (A), alternative pathway activity (B) and MBL pathway activity (C). The solid lines indicate the mean values of the samples tested.
The mean absorbance values of the serum samples tested in the three laboratories for classical pathway activity (D), alternative pathway (E) and
MBL pathway (F) were 2.079, 1.558 and 1.009, respectively. The MBL pathway activity is plotted against the MBL concentrations assessed in the
same serum samples (F). The solid lines indicate the mean values of the samples tested, and the dotted lines indicate the cut off values for normal
pathway activity. The distribution of pathway activity for CP, AP and MBL-P activity is shown in panels G, H and I, respectively.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198 191

activation, the mean percentage of activity of the results
assessed in the three laboratories was calculated (Fig.
1D,E). The lower cutoff value of normal pathway
activity for these pathways was defined as the mean
percentage of activity minus two times the standard
deviation. For the classical pathway activity, the mean
level of activity was 98%, and the lower cutoff value of
normal pathway activity was 74% (Fig 1D). For the
alternative pathway, the mean level o f activity was
74%, and the lower cutoff value was 39% (Fig 1E). In
contrast to the classical and alternative pathways of
complement activation, the MBL pathway activity of
the 120 samples showed a large variation. This
variation was stro ngly dependent on the seru m
concentration of MBL (Fig. 1F). The threshold level
of normal MBL pathway activity was arbitrarily set at
10%, resultin g in 28% of the healthy donor sera falling
below this threshold (Fig. 1F). More than 90% of these
sera had serum MBL concentrations below 300 ng/ml.
The distributions of pathw ay activity for the 120 sera
tested are show n in Fig. 1G,H and I for CP activity, AP
activity and MBL-P activity, respectively.
As shown in Table 1, the intraassay variation for the
three pathways was below 7%, whereas the interassay
variation was below or equal to 20% (Table 2).
The results obtained by assessment of plasma
samples either collected in heparin, EDTA or citrate
showed reduced pathway activity when compared to
the activity assessed in serum samples from the same
patients (Fig 2).
3.2. Correlation with hemolytic assessment of comple-
ment activity
Currently in most diagnostic laboratories, the
functional activity of the classical and the alternative
pathways of complement is assessed by a haemolytic
assay, such as the CH50 for the classical pathway and
the AP 50 for the alternative pathway. We selected 40
serum samples showing differing levels of classical
pathway and alternative pathway haemolytic activity
and measured these samples in parallel for classical
and alternati ve pathway activity using both hemolytic
assays and the complement kit. As indicated in Fig. 3 ,
the results obtained in the complement kit showed a
good correlation with the results obtained in the
quantitative haemolytic assays both for the classical
pathway (R=0.89, Pb0.001) and for the alternative
Table 1
Intraassay variation for the three pathways of complement activation
evaluated with the complement kit
Mean
activity (%)
S.D. CV
(%)
Classical pathway 85 2.9 3
Alternative pathway 83 5.7 7
MBL pathway 74 3.9 5
One serum sample was tested for classical, alternative and MBL
pathway activity in 40 wells on one occasion.
Fig. 2. Serum samples, heparin plasma, EDTA plasma and citrate
plasma samples were obtained from six donors for assessment of
classical, alternative and MBL pathway activity. The pathway
activity detected in serum samples was set at 100% for all three
pathways. The mean pathway activity and standard deviations for
each condition are shown.
Table 2
Interassay variation for the three pathways of complement activation
evaluated with the complement kit
Mean
activity (%)
S.D. CV
(%)
Classical pathway S1 98 4.3 4
S2 92 3.9 4.2
S3 21 1.7 8
Alternative pathway S1 48 5.1 11
S2 89 8.0 9
S3 16 3.1 20
MBL pathway S1 91 3.3 4
S2 37 4.0 11
S3 16 2.3 15
Three different serum samples (S1, S2, S3) were tested in triplicate
on six occasions for complement activity in the classical, alternative
and MBL pathways.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198192

pathway (R=0.84, Pb0.001). For the alternative path-
way, the complement kit showed undetectable func-
tional activity for all serum samples w ith an
alternative pathway haemolytic activity below the
normal value.
3.3. Detection of complement deficiencies with the
complement kit
A total of 64 sera with different defined comple-
ment deficienci es were assessed for classical pathway,
alternative pathway and MBL pathway activity in the
complement kit (Fig 4A–C).
C1q deficient serum samples showed undetectable
classical pathway activity, whereas alternative pathway
activity was within the normal range, and MBL
pathway activity showed a distribution similar to that
seen in healthy donors. Serum samples from well-
characterized MBL varia nt genotypes showed no
detectable MBL pathway activity, whereas classical
and alternative pathway activities were normal. The
MBL pat hw ay was also deficien t in a MA S P-2-
deficient serum sample. Serum samples deficient in
C4 or C2 showed normal alternative pathway activity
and undetectable classical and MBL pathway activity.
Alternative pathway activity was decreased in all
properdin-deficient sera, whereas classical pathway
activity was normal, and MBL pathway activity was
low in some but not all of the sera. As expected,
because of the central position of C3 in all three
pathways of complement act ivation, n o detectable
activity was found in any of the pathways when C3-
deficient sera were analyzed. Similarly, sera deficient
in complement components of the final common
pathway (C5b-9) showed, as anticipated, no activity
in any of the three pathways. Taken together, in wel l-
defined complement-deficient sera, the activity of the
involved pathway(s), when assessed in the complement
kit, were below 10% compared to a standard serum.
Among the samples tested, one C9-deficient serum
showed slightly more than 10% classical pathway
activity, while one serum with incomplete properdin
deficiency (properdin deficiency type 2) showed
slightly more than 10% alternative pathway activity.
With this possible exception, the three known pheno-
typic variants of properdin deficiency (Sjoholm, 2002)
were all correctly identified with the present ELISA.
MBL deficiency is frequently found in apparently
healthy individuals. Therefore, a combined deficiency
of properdin and MBL or C1q and M BL was
suspected in sera showing a decreased activity in
more than one of the pathways. By adding purified
MBL to these sera the MBL pathway activity was
restored, demonstrating a combined properdin and
MBL deficiency, and combined C1q and MBL
deficiency, respectively, in these sera (Fig 5A and B).
Impaired complement activation can be found in
sera from patients with a genetically determined
Fig. 3. Functional activity of the classical pathway of 40 serum samples from patients tested in a quantitative haemolytic assay plotted against
the functional activity assessed with the complement kit (A). For the activity of the alternative pathway, the results of a quantitative haemolytic
assay are plotted against results obtained in the complement kit (B). The dotted lines indicate the cut off values for healthy controls.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198 193

complement deficiency, as shown above, but comple-
ment consumption can also be an explanation for
diminished pathway activity. Therefore, sera were
collected from patients with disease o r having
deficiencies of regulatory proteins that might cause
complement consumption. For this purpose, sera
from patients with HUVS, HAE, AAE, acute post-
AGN and sera from patients with C3NeF were tested
in the complement kit. In addition, serum samples
with a genetic factor I or factor H deficiency were
tested.
Sustained in vivo complement activation as found
in the diseases mentioned above leads to complement
deficiencies with decreased activity of one or more of
the pathways of complement activation, and this was
confirmed using the present kit (Fig. 6). In addition,
deficiencies of regulatory proteins of the alternative
pathway of complement causes consumption of
Fig. 4. Serum samples with well-defined complement deficiencies were tested in the complement kit for complement pathway activity in the
classical pathway (A), the alternative pathway (B) and the MBL pathway (C). The group indicated as C5–C9 includes sera deficient in C5
(N=3), C6 (N=4), C7 (N=5), C8 (N=4) and C9 (N=2). The group indicated as MBL 0/0 includes sera from donors known to be homozygous (C/
C, D/D) or compound homozygous (B/D) for MBL variant alleles. The group indicated as properdin (P) deficiency included complete
properdin deficiency (N=5), incomplete properdin deficiency (N=2) and properdin dysfunction (N=2). The solid lines indicate the mean value of
activity in the different assays for healthy control samples. The dotted lines indicate the lower cut off values for normal activity for the different
pathways.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198194

complement components including C3 and therefore
decreased pathway activity in all three pathways of
activation.
4. 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 ELISA wells
coated with mannan. At the same time, it is possible to
measure classical and alternative pathway activati on
using specific reagents and coatings. These three
assays have now been combined in one kit, called the
complement kit, and make it possible to identify
defects in any of the three initiating pathways and the
terminal sequence of complement activation in any
given serum sample. The standardisation and valida-
tion of this assay revealed it to be a sensitive, specific
and simple assay for the detect ion of complement
deficiencies.
The results obtained in three different laboratories
using 120 serum samples from healthy donors tested
for the three pathways correlated well. Because of the
limited variation of complement activity betw een
different sera, particularly in the classical pathway
but also in the alternative pathway, a cutoff value for
normal pathway activity could be defined as the mean
value of activity minus two times the standard
deviation. This approach resulted in 2.5% of the
healthy population falling below the cutoff value for
normal complement activity. The mean value of
activity in the alternative pathway for the 120 sera
was 74% of the standard serum alternative pathway
activity. Because of the variation in alternative path-
way activity observed in sera from healthy individu-
als, we conclude that the pool of sera used in the
standard had relative ly high alternative pathway
activity compared to the donor samples tested.
For MBL pathway activity, a different method was
required to define a cutoff value. Because of the
variation in MBL concentration in the normal healthy
population, which is mainly genetically determined
(Garred et al., 2003a,b), there is also a large variation in
MBL pathway activity, with a distribution skewed to
the left, as confirmed in the present study. Here, an
arbitrary minimum level for normal MBL pathway
activity was set at 10% of the standard, which
corresponded to MBL concentrations below 300 ng/
ml. Using this threshold level, 98% of the serum
samples with reduced MBL pa thway activity had
serum MBL concentrations below 300 ng/ml. Studies
on the association of low MBL serum concentrations
and susceptibility to disease such as infections have
shown that patients with serum MBL levels below
approximately 300 ng/ml are at risk (Sumiya et al.,
1991; Peterslund et al., 2001). Therefore, a cutoff value
of 10% for MBL-P activity is expected to be useful for
the detection of MBL deficiency in the complement kit.
Fig. 5. Three serum samples with known properdin deficiency (A) and two samples with C1q deficiency (B) had decreased MBL pathway
activity. After reconstitution with purified MBL, MBL pathway activity was restored. The solid lines indicate the mean activity for MBL
pathway activity as assessed in healthy controls.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198 195

An important practical observation was that plasma
samples showed decreased pathway activity compared
to serum samples from the same patients. This was
especially true for heparinised plasma used for MBL
pathway estimations. Reduced pathway activity in
plasma samples could be explained by dissociation of
C1 complexes and MBL–MASP complexes in a
calcium-free environment. It takes time to reassociate
these complexes, which makes the complement acti-
vation less efficient. Furthermore, anticoagulants may
bind to complement factors, thereby influencin g their
activity. This binding is not reversed when diluting the
sample. Therefore, only serum samples should be used
to quantify pathway activity in the complement kit.
Complement activity in sera deficient in individual
complement components was below 10% in either
Fig. 6. Serum samples with acquired complement deficiencies and deficiencies of complement regulatory proteins were tested for complement
activity via the classical pathway (A), the alternative pathway (B) and the MBL pathway (C) in the complement kit. The solid lines indicate the
mean activity for the different pathways as assessed in healthy controls. The dotted lines are the cut off values for normal activity in the different
pathways.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198196

one or more pathways. Combined complement
deficiencies in the MBL pathway and the alternative
pathway were demonstrated, as well as combined
complement deficiencies in the MBL pathway and
classical pathway of complement activation. MBL
pathway activity was reconstituted when purified
MBL was added to these sera. The results obtained
demonstrate the value of the complement kit in the
detection of combined deficiencies in one assay.
Sera from patients diagnosed with diseases known
to cause complement consumption were also tested
with the complement kit. Decreased activity in all
three pathways was demonstrated in most of these
sera. Consumption of components of the classical
pathway in patients with hereditary and acquired
angioedema is caused by deficient or nonfunctional
C1-INH (Carugati et al., 2001). C4 and C2 are
consumed depending on the degree of disease activity,
and in some patients with AAE, C3 levels are also
low. The present findings were consistent with this. In
patients with HUVS, C1q in serum is depleted in the
presence of anti-C1q autoantibodies, and the classical
pathway is activated (Wisnieski, 2000). In patients
with acute poststreptococcal glomerulonephritis, the
alternative pathway is predominantly activated, and
the classical pathway of complement can also be
activated via immune complexes (Sjoholm, 1979).
The autoantibody C3NeF stabilises the C3 convertase
causing enhanced C3 activation (Daha and van Es,
1979). Accordingly, all these conditions are associated
with strongly enhanced complement consumption,
which was reflected in our data showing decreased
complement activity in all three pathways. Comple-
ment deficiency is also found in patients with a
deficiency in regulatory complement components of
the alternative pathway. Patie nts with factor H and I
deficiency showed decreased activity in all p athways
because of secondary C3 deficiency (Sjoholm, 2002).
Collectively, the results demonstrate the value of the
complement kit in the detection of acquired comple-
ment deficiencies, as well as genetic defects.
Assays measuring haemolysis of eryth rocytes by
complement activation either via the classical or
alternative pathway are used on a routine basis to
assess the functional activity of these pathways. To
compare the results of complement activation by the
classical and alternative pathways assessed by
haemolytic assays or the complement kit, serum
samples were tested in parallel using both methods.
A strong correlation was found between the results
obtained by the haemolytic assay compared with
those in the c omp lement kit. Compar ed to the
original definition of AP50 and CH50, different type
of calculation is used to obtain the results in the
present complement assays. Results from the latter
assays were originally defined by titration. Therefore,
the results of the complement kit, expressed as
percent of activity of a standard serum, cannot be
used as a direct quantitative description of the
complement defe ct. Howeve r, in view of the
reproducibility and simplicity of the complement
kit, this method should be regarded as preferable to
haemolytic assays for the screening of classical and
alternative pathway activity in clinical pract ice. In
patients suspected of having a deficiency of the
humoral immune system, the complement system
should be screened for deficiencies, and immuno-
globulin quantity should be assessed. Complement
deficiencies in any of the three pathways of comple-
ment activation can easily be detected in the new
combined assays. We have shown the results of
assaying 64 sera deficient in one or more of 11
different components of the classical, alternative and
MBL pathways. When, after c onfirmation in a
second independent sample, the screening assays
suggest a complement deficiency, samples should be
further analysed in specialist laboratories for precise
identification of the deficient component.
In conclusion, a simple assay with a uniform
design has been developed by which it is possible to
evaluate functional activity of the three pathways of
complement activation in parallel. The assay results
are reproducible between different laboratories, and
standardization of the assay will permit its use for
patient diagnostics. In this respect, we have shown
that the assay is able to detect genetic complement
deficiencies at all levels of the complement cascade,
as well as acquired complement deficiencies associ-
ated with in vivo complement consumption.
Acknowledgements
This work was s upported by grants from the
European Union (QLGT-CT2001-01039) and the
Dutch Kidney Fo undation (PC 95, C 98-1763).
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198 197

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.
References
Aderem, A., Underhill, D.M., 1999. Mechanisms of phagocytosis in
macrophages. Annu. Rev. Immunol. 17, 593.
Carugati, A., Pappalardo, E., Zingale, L.C., Cicardi, M., 2001. C1-
inhibitor deficiency and angioedema. Mol. Immunol. 38, 161.
Daha, M.R., van Es, L.A., 1979. Activation of the classical pathway
of complement by the C3NeF-stabilized cell-bound amplifica-
tion convertase. J. Immunol. 122, 801.
Davies, E.J., Snowden, N., Hillarby, M.C., Carthy, D., Grennan,
D.M., Thomson, W., Ollier, W.E., 1995. Mannose-binding
protein gene polymorphism in systemic lupus erythematosus.
Arthritis Rheum. 38, 110.
Eisen, D.P., Minchinton, R.M., 2003. Impact of mannose-binding
lectin on susceptibility to infectious diseases. Clin. Infect. Dis.
37, 1496.
Fredrikson, G.N., Truedsson, L., Sjoholm, A.G., 1993. New
procedure for the detection of complement deficiency by
ELISA. Analysis of activation pathways and circumvention of
rheumatoid factor influence. J. Immunol. Methods 166, 263.
Garred, P., Madsen, H.O., Marquart, H., Hansen, T.M., Sorensen,
S.F., Petersen, J., Volck, B., Svejgaard, A., Graudal, N.A., Rudd,
P.M., Dwek, R.A., Sim, R.B., Andersen, V., 2000. Two edged
role of mannose binding lectin in rheumatoid arthritis: a cross
sectional study. J. Rheumatol. 27, 26.
Garred, P., Larsen, F., Madsen, H.O., Koch, C., 2003a. Mannose-
binding lectin deficiency-revisited. Mol. Immunol. 40, 73.
Garred, P., Strom, J., Quist, L., Taaning, E., Madsen, H.O., 2003b.
Association of mannose-binding lectin polymorphisms with
sepsis and fatal outcome, in patients with systemic inflammatory
response syndrome. J. Infect. Dis. 188, 1394.
Ikeda, K., Sannoh, T., Kawasaki, N., Kawasaki, T., Yamashina, I.,
1987. Serum lectin with known structure activates complement
through the classical pathway. J. Biol. Chem. 262, 7451.
Jack, D.L., Klein, N.J., Turner, M.W., 2001. Mannose-binding
lectin: targeting the microbial world for complement attack and
opsonophagocytosis. Immunol. Rev. 180, 86.
Kuipers, S., Aerts, P.C., Sjoholm, A.G., Harmsen, T., van Dijk, H.,
2002. A hemolytic assay for the estimation of functional
mannose-binding lectin levels in human serum. J. Immunol.
Methods 268, 149.
Lipscombe, R.J., Sumiya, M., Hill, A.V., Lau, Y.L., Levinsky, R.J.,
Summerfield, J.A., Turner, M.W., 1992. High frequencies in
African and non-African populations of independent mutations
in the mannose binding protein gene. Hum. Mol. Genet. 1,
709– 715.
Madsen, H.O., Garred, P., Thiel, S., Kurtzhals, J.A., Lamm, L.U.,
Ryder, L.P., Svejgaard, A., 1995. Interplay between promoter
and structural gene varian ts control basal serum level of
mannan-binding protein. J. Immunol. 155, 3013.
Matsushita, M., Endo, Y., Fujita, T., 2000. Cutting edge: comple-
ment-activating complex of ficolin and mannose-binding lectin-
associated serine protease. J. Immunol. 164, 2281.
Matsushita, M., Kuraya, M., Hamasaki, N., Tsujimura, M., Shiraki,
H., Fujita, T., 2002. Activation of the lectin complement
pathway by H-ficolin (Hakata antigen). J. Immunol. 168, 3502.
Minchinton, R.M., Dean, M.M., Clark, T.R., Heatley, S., Mullighan,
C.G., 2002. Analysis of the relationship between mannose-
binding lectin (MBL) genotype, MBL levels and function in an
Australian blood donor population. Scand. J. Immunol. 56, 630.
Petersen, S.V., Thiel, S., Jensen, L., Steffensen, R., Jensenius, J.C.,
2001. An assay for the mannan-binding lectin pathway of
complement activation. J. Immunol. Methods 257, 107.
Peterslund, N.A., Koch, C., Jensenius, J.C., Thiel, S., 2001.
Association between deficiency of mannose-binding lectin and
severe infections after chemotherapy. Lancet 358, 637.
Pickering, M.C., Botto, M., Taylor, P.R., Lachmann, P.J., Walport,
M.J., 2000. Systemic lupus erythematosus, complement defi-
ciency, and apoptosis. Adv. Immunol. 76, 227.
Roos, A., Bouwman, L.H., Gijlswijk-Janssen, D.J., Faber-Krol,
M.C., Stahl, G.L., Daha, M.R., 2001. Human IgA activates
the complement system via the mannan-binding lectin path-
way. J. Immunol. 167, 2861.
Roos, A., Bouwman, L.H., Munoz, J., Zuiverloon, T., Faber-Krol,
M.C., Fallaux-van den Houten, F.C., Klar-Mohamad, N., Hack,
C.E., Tilanus, M.G., Daha, M.R., 2003. Functional character-
ization of the lectin pathway of complement in human serum.
Mol. Immunol. 39, 655.
Sjoholm, A.G., 1979. Complement components and complement
activation in acute poststreptococcal glomerulonephritis. Int.
Arch. Allergy Appl. Immunol. 58, 274.
Sjoholm, A.G., 2002. Deficiencies of mannose-binding lectin,
the alternative pathway, and the late complement compo-
nents. In: Rose, N.R., Hamilton, R.G., Detrick, B. (Eds.),
Manual of Clinical Laboratory Immunology. ASM Press,
Washington, p. 847.
Stengaard-Pedersen, K., Thiel, S., Gadjeva, M., Moller-Kristensen,
M., Sorensen, R., Jensen, L.T., Sjoholm, A.G., Fugger, L.,
Jensenius, J.C., 2003. Inherited deficiency of mannan-binding
lectin-associated serine protease 2. N. Engl. J. Med. 349, 554.
Sumiya, M., Super, M., Tabona, P., Levinsky, R.J., Arai, T., Turner,
M.W., Summerfield, J.A., 1991. Molecular basis of opsonic
defect in immunodeficient children. Lancet 337, 1569.
Turner, M.W., Hamvas, R.M., 2000. Mannose-binding lectin:
structure, functi on, genetics and disease associations. Rev.
Immunogenet. 2, 305.
Walport, M.J., 2001. Complement. First of two parts. N. Engl. J.
Med. 344, 1058.
Wisnieski, J.J., 2000. Urticarial vasculitis. Curr. Opin. Rheumatol.
12, 24.
M.A. Seelen et al. / Journal of Immunological Methods 296 (2005) 187–198198