Monoclonal Antibodies against Complement 3 Neoantigens for Detection
of Immune Complexes and Complement Activation
Relationship between Immune Complex Levels, State of C3, and Numbers
M. Teresa Aguado, John D. Lambris, George C. Tsokos, Reinhard Burger, Dieter Bitter-Suermann, John D. Tamerius,
Frank J. Dixon, and Argyrios N. Theofilopoulos
Department ofImmunology, Research Institute ofScripps Clinic, La Jolla, California 92037; National Institutes ofHealth, National
Institute ofArthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland 20205; and Institutfur Immunologie und Serologie,
Im Neuenheimer Feld 305, 6900 Heidelberg, Federal Republic ofGermany
C3-bearing immune complexes and C3 activation products were
detected by using two monoclonal antibodies, one specific for a
neoantigenic determinant on C3c and the other for C3d. To
quantitate immune complexes, the anti-C3c or anti-C3d anti-
bodies were fixed to microtiter plates and reacted with test
plasma. The binding of C3-bearing immune complexes in this
plasma was then measured with radioisotope- or enzyme-labeled
anti-human IgG. To test for C3 breakdown products, solid-phase
monoclonal antibody to the C3d neoantigen was reacted with
EDTA-plasma samples, and fixed iC3b or C3d was measured
with a polyclonal anti-C3 antibody. Patients with autoimmune
diseases, such as systemic lupus erythematosus, rheumatoid ar-
thritis, and Sjogren's syndrome, and paracoccidioidomycosis were
found to contain immune complexes bearing C3b/iC3b or C3d.
In most conditions, there were more C3d-containing immune
complexes than C3b/iC3b. Although CR1 (C3b receptors) rap-
idly converted immune complex-bound iC3b to C3dg/C3d and
lupus patients had reduced CR1, no correlation between the state
of C3 on circulating immune complexes or levels of immune
complexes and CR1 numbers was seen. However, levels of C3-
fixing ICs correlated with levels of C3 activation products.
This assay system with monoclonal antibodies to neoantigens
expressed on activated, but not native, C3 provides sensitive and
specific means for detecting and classifying C3-fixing immune
complexes and for assessing C3 activation.
Many assay systems have been devised for immune complex
(IC)' detection (1, 2). Since most pathogenic ICs activate the
This is publication No. 3725-IMM from the Immunology Department,
Research Institute of Scripps Clinic. Dr. Aguado is a recipient of an
Arthritis Foundation Postdoctoral Fellowship.
Receivedfor publication 17 December 1984 and in revisedform 15
1. Abbreviations used in this paper: AHG, aggregated human gamma-
globulin; ARG, aggregated rabbit gamma-globulin; BBS, borate-buffered
saline; C, complement; CR1, complement receptorforC3b; CR2,com-
plement receptor for C3d; CR3, complement receptor for iC3b; HSA,
human serum albumin; IC, immune complex; mAb, monoclonal anti-
body; NHS, normal human serum; PEG, polyethylene glycol; PCM,
paracoccidioidomycosis; RA, rheumatoid arthritis; RF,rheumatoid fac-
tor; SLE, systemic lupus erythematosus; SS, Sjogren's Syndrome.
complement (C) system, most of these assays (Clq , conglu-
tinin , and Raji cell ) are based on interactions of certain
substances with IC-bound C components. Although these tech-
niques have provided useful information regarding immuno-
pathology, prognosis, and follow-up ofimmunologic disorders,
several drawbacks, particularly lack of specificity, have limited
To improve the specificity ofIC assays, we recently described
a method based on the interaction of solid phase-bound poly-
clonal F(ab')2 anti-human C3 and test samples and the subse-
quent detection of bound C-fixing ICs by labeled anti-IgG or
protein A (6). This assay was specific, but competition between
IC-bound and free C3 limited its sensitivity. Moreover, since
the polyclonal anti-C3 antibody did not discriminate between
C3b, iC3b, C3c, or C3dg/C3d, C-fixing ICs could not be sub-
categorized according to the C3 fragment they bore. Such dif-
ferentiation may be of value, since cell types that handle or are
affected by ICs differ in their expression ofthe main C3 receptors
(CR1, C3b; CR2, C3d; CR3, iC3b) (7, 8). The implication is
that the effects of ICs in health and disease may be modified by
the C3 fragment they bear. Thus, an estimation of relative
amounts of ICs according to their C3 fragment may enhance
the prediction of their consequences.
We report here a modification of the solid-phase anti-C3
assay whereby monoclonal antibodies (mAb) recognizing
neoantigenic determinants on C3c or C3d, instead ofpolyclonal
anti-C3 antibodies, have been used as substrates. This modifi-
cation achieves three goals: (a) the assay shows exquisite speci-
ficity and sensitivity; (b) C3-fixng ICs in patient plasmas can be
characterized with regard to the C3 fragment they express; and
(c) simultaneous with IC detection, levels ofC3 activation prod-
ucts can be ascertained by replacing the final anti-Ig reagent
used for IC estimation with a polyclonal anti-C3 antibody.
Proteins. C fragments C3b, iC3b, C3c, and C3d were either supplied to
us by Dr. M. K. Pangburn (Scripps Clinic and Research Foundation)
(9) or prepared as described (10). Human serum albumin (HSA) was
purchased from Miles Laboratories (Elkhart, IN), and bovine serum al-
bumin (BSA) from Calbiochem-Behring Corp. (La Jolla, CA). Human
IgG was purchased from Sigma Chemical Co. (St. Louis, MO), and further
purified by ion exchange chromatography in a DE-52 column. Mouse,
rat, and rabbit IgG were obtained from Miles Laboratories. Cohn Fraction
II, inulin, and zymosan were purchased from Sigma Chemical Co.
Antibodies. Six mAb and one polyclonal anti-C3 antibody were used.
Three of the available monoclonals, mAb-105 (1 1), mAb-4 (12), mAb-
BRL (purchased from Bethesda Research Laboratories, Gaithersburg,
MD) were against antigenic determinants on the C3c fragment; the other
three, mAb- 1 30 (13), mAb-3 (12), and mAb-ORTHO (Ortho Diagnostics,
Aguado et al.
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
Volume 76, October 1985, 1418-1426
Raritan, NJ) were against C3d. Two antibodies, mAb-3 and mAb-4,
originated from rat, and the remainder from mouse. Animals were im-
munized with human C3 or fragments thereof, except for mAb-105,
which was raised against guinea pig C3 but was shown to cross-react
with human C3 (1 1). mAb-105 and mAb-BRL were used directly in the
form ofascites fluids, whereas all others were used as IgG fractions after
ammonium sulfate precipitation of ascites fluids and subsequent IgG
purification on DE-52 columns or protein A-Sepharose columns. The
IgG and its F(ab')2 fraction of the polyclonal IgG goat anti-human C3
antibody were prepared as described previously (6).
Other antibodies were the following: (a) rabbit anti-human IgG pre-
pared by affinity purification on human IgG-Sepharose columns; (b)
peroxidase-conjugated sheep anti-human IgG, obtained from Cappel
Laboratories (Cochranville, PA); (c) goat anti-rabbit IgG purchased from
Miles Laboratories; (d) rabbit anti-BSA isolated by affinity chromatog-
raphy on a BSA-Sepharose column and renderedaggregate-freeby passage
through a Sephadex G-200 column; (e) polyclonal anti-C3b receptor
(anti-CR 1) antibody from rabbits immunized with human CR1 as de-
scribed (14); and (I) peroxidase-conjugated goat anti-mouse IgG obtained
from Cappel Laboratories. The above antisera were used as whole IgG
or as F(ab')2 fragments prepared by pepsin digestion (15).
In vitro models ofICs. Aggregated human gamma-globulin (AHG)
was prepared by heating ofCohn Fraction II in phosphate-buffered saline
(PBS), pH 7.5, at 630C for 30 min. Large aggregates were removed by
centrifugation at 3,000 rpm for 15 min, and aliquots were stored at
-700C. Aggregated rabbit IgG (ARG) was similarly made, with heating
at 72°C for 20 min. Soluble BSA-anti-BSA complexes were prepared at
five times antigen excess ( 16).
Radioiodination. 1251I antibody labeling was performed by the chlo-
ramine T method (17), whereas C3 fragments were labeled with the
iodogen method (18).
Test samples. Venous blood was collected in EDTA (0.01 M final
concentration). Plasma samples from normal individuals (n = 18) and
patients with systemic lupus erythematosus (SLE, n = 120), rheumatoid
arthritis (RA, n = 92), Sjogren's syndrome (SS, n = 28), and paracoc-
cidioidomycosis (PCM, n = 31) at various stages ofdisease activity were
analyzed. Plasma aliquots were stored at -70°C and thawed once. DNA
binding capacity and CH50 levels in serial studies with SLE patients
were provided by Dr. R. G. Lahita, The Rockefeller University, New
Binding ofC3fragments to anti-C3 antibodies. The test was carried
out in two systems: (a) Microtiter wells were coated with C3 fragments,
free sites blocked with 1% HSA-borate-buffered saline (BBS), and serial
dilutions of the mAbs were allowed to react for 30 min at room tem-
perature. Plates were washed three timesand peroxidase-conjugated goat
anti-mouse IgG was added. After 30-min incubation at 37°C and color
development, plates were read at 412 nm in a Dynatech MR600. (b)
Microtiter wells were coated with mAbs, nonreacting sites were blocked
with 1% HSA-BBS, pH 7.5, and serial dilutions ofradiolabeled C3 frag-
ments were added. After 2-h incubation at 37°C, plates were washed
five times, and wells cut and counted.
IC assay. The assay is a modification ofthat described by Pereira et
al. (6). Flexible microtiter plates with U-shaped wells (Dynatech Labo-
ratories, Inc., Alexandria, VA) were coated with 50Alofan appropriate
dilution ofanti-C3 antibodies (see Results) in BBS, pH 7.5, and incubated
at 4°C overnight. The material was then aspirated, and 150IMl of 1%
HSA-BBS was added to each well to cover unreacted sites. After 1 h at
room temperature, wells were washed once with BBS. Then, 50Alof
test plasma diluted 1:21 in BBS that contained 1% HSA, 0.2% Tween
20, 0.01 M EDTA, and 0.1% aggregated mouse or rat IgG (depending
on the mAb origin) was added in duplicate wells. Excess heterologous
IgGs were added to avoid false positive results caused by rheumatoid
factors (RFs). Simultaneously, as a standard, various amounts ofAHG
(3.12 to 800 Mg/ml) were incubated for 15 min at 37°C with normal
human serum (NHS) to fix C, diluted as above, and added to antibody-
coated wells. After 2 h at 37°C, the wells were washed three times with
0.2% Tween 20-BBS, and 50 ul (200 ng) radioiodinated F(ab')2 rabbit
anti-human IgG was added. After further incubation for 2 h at 370C,
the plateswere washed three times and cut out wells counted. The uptake
of radioiodinated anti-human IgG bound perwell was calculated from
the specific activity ofthe labeled antibody.
Data were analyzed by the leastsquaremethod from standard curves
using the nanograms ofanti-human IgG bound and the micrograms of
added AHG (log2 concentration). Linear, logarithmic, exponential,and
polynomical curves were tested to identify the best fit. A logarithmic
transformation of the nanograms of anti-IgG bound proved optimal,
based on an analysis of variances and the F distribution. The IC level
results were expressed as microgram equivalent AHG/ml. When enzyme
immunoassay was used, peroxidase-conjugated anti-human IgG was
employed as final reagent. For comparison, the Raji cell assay was also
performed in some samples, as describedpreviously (5).
Detection of C3 activation products. Microtiter wells were coated
with mAb- 130 as described above. This mAb reacts onlywithactivated,
not native C3, and bindsiC3b and C3d fragmentseither fixed to substrates
or free in solution (see Results). Plasmas to be tested for C3 activation
were diluted 1:50 in BBS that contained 1% HSA, 0.2% Tween 20,0.01
M EDTA, and 50 Ml was added to the wells. After45 min at4VC, samples
were aspirated and wells were washed three times with 0.2% Tween 20-
BBS. Thereafter, 50 Ml (1 Mg) of 251I-labeled polyclonal F(ab')2anti-human
C3 was added, incubation was carried out for 1 h at 370C, and, after
three washes, wells were cut out and counted. Controls included normal
plasmasand NHS treated (370C, 30 min)with variousCsystemactivators
(inulin, 20 mg/ml; zymosan, 5 mg/ml; AHG, 1,600 Mg/ml serum). Before
testing, inulin and zymosan were removed by centrifugation (10 min at
3,000 rpm). AHG were removed in some instancesby precipitationwith
polyethylene glycol (PEG [3%finalconcentration]).
Measurement ofCRI receptors on erythrocytes. Erythrocytes from
venous blood of normal individuals (n = 12) and IC-positive SLE
(n=32) patients that had been anti-coagulated in 0.01 M EDTA and
held at 4°C were washed three times with PBS containing 0.1% rabbit
IgG and counted in a Coulter counter. Erythrocytes (3.3 X 106 in 0.1%
rabbit IgG-PBS) were incubated for45 min at 200C with 1 Mg '251-labeled
F(ab')2 anti-CR1 or, as control, radiolabeled F(ab')2 anti-BSA. This
amount of anti-CRI antibody was found to exceed that required for
CR1 receptorsaturation in several normalpersonsandpatients. Duplicate
cell samples in 0.1 ml were removed from each mixture, layered on 0.3
ml of 20% sucrose in PBS in polyethylene microfuge tubes (VWR Sci-
entific, Inc., Gibbstown, NJ), and pelleted by centrifugation at 8,000 g
for 5 min. Tube tips were cut and the pellets counted. The counts per
minute obtained with anti-BSA were subtracted, and the results expressed
as specific counts of anti-CR1 antibody bound to cells. The data were
10 20 40 80 160
mAb Added (ng)
FragmentsAdded (Molecules x10-)
Figure 1. Specificity ofmAb-105 for C3 fragments. (A) Microtiter
wells were coated with C3c or C3d, reacted with mAb- 105, and finally
incubated with peroxidase-conjugated anti-mouse IgG. (B) Alterna-
tively, wells were coated with mAb-105 and reacted with radiolabeled
C3b, iC3b, C3c, and C3d.
Antibodies to C3 Neoantigens
10 2040 80160
4080 160 320
Fragments Added (Molecules x 1 0"1
Figure 2. Specificity of mAb-1 30 for C3 fragments. (A) Microtiter
wells were coated with C3c or C3d, reacted with mAb-130, and finally
incubated with peroxidase-conjugated anti-mouse IgG. (B) Alterna-
tively, wells were coated with mAb-1 30 and reacted with radiolabeled
C3b, iC3b, C3c, and C3d.
then transformed to numbers ofCR1 antigenic sites per cell as described
by Wilson et al. (19), using a standard curve derived from bindings of
incremental amounts of '25I-dimeric C3b and "25I F(ab')2 anti-CR
tibody on similar numbers of normal erythrocytes.
Statistical methods. The Spearman rank correlation coefficient was
used in all statistical analyses.
Specificities of mAbs to C3. The C3 fragment specificities of
some ofthe mAbs in this study have been defined, primarily by
agglutination or binding to antibody and/or C-sensitized particles
(1 1-13). Verification ofthese specificities in our solid-phase assay
systems was sought. As depicted in Fig.
bound in microtiter wells, mAb- 105 reacted with C3c, but not
C3d. Conversely, when mAb-105 was added to microtiter wells
before assessment of radiolabeled soluble C3b, iC3b, C3c, or
C3d uptake, weak binding with the first three fragments, but no
binding with C3d, followed (Fig.
interacted with C3d, but not C3c; binding ofC3d to mAb-130
was independent ofwhether this fragment was offered on a solid
matrix or in fluid phase (Fig. 2, A and B). Binding offluid phase
iC3b to mAb-130 was almost as strong as that ofC3d, whereas
binding of fluid phase C3b was weak (Fig. 2 B). When mAbs
1 A, when C3 fragments
B). In contrast, mAb-130
were first bound in the wells before incubation with radiolabeled
fragments, mAb-4 and mAb-BRL reacted with C3b; iC3b, and
C3c, but not C3d, whereas mAb-3 and mAb-ORTHO reacted
with C3b, iC3b, and C3d, but not C3c (data not shown).
mAbs reactive with C3 neoantigenic determinants. By using
sensitized erythrocytes, Tamerius et al. (13) observed that mAb-
130 recognized determinants expressed on activated, but not
native, C3. We devised a solid-phase assay system to determine
which of the mAbs could discriminate between activated and
native C3. Microtiter wells were coated with each ofthe mAbs
and reacted with EDTA-plasma or serum to which C activators
(AHG, inulin, and zymosan) had been added; bound C3 was
then detected with a radiolabeled polyclonal anti-C3 antibody.
As shown in Table I, among the anti-C3c mAbs, only mAb-105
clearly discriminated activated from nonactivated C3, i.e., 53
ng of anti-C3 bound to mAb-105 coated wells incubated with
serum + AHG, but only 7 ng in EDTA-plasma. Similarly, among
the anti-C3d mAbs, only mAb-130 substantially differentiated
activated from native C3 (72 vs. 11 ng anti-C3 uptake, respec-
tively). Of considerable interest, the amount ofC3 (as assessed
by uptake ofpolyclonal anti-C3) bound to mAb-105 was mark-
edly reduced if the activator AHG had been removed by 3%
PEG before testing. Furthermore, C3 binding to mAb-105 was
low when particulate activators such as inulin and zymosan had
been removed by centrifugation before assaying. In contrast,
mAb-130 bound almost equal amounts of C3 whether the ac-
tivating principle (AHG, zymosan, or inulin) was present in the
incubation or had been removed. Therefore, mAb-105 prefer-
entially recognizes an activator- or surface-bound neoantigenic
determinant on C3c, whereas mAb-130 reacts equally well with
activator- or surface-bound and free in fluid phase neoantigenic
determinant on C3d.
Modifiedanti-C3 assayfor IC detection. We next modified
the solid-phase anti-C3 IC assay (6) by substituting the polyclonal
anti-C3 antibody for one ofthe two mAbs reactive with neoan-
tigenic determinants on C3c or C3d. Bound IgG was detected
with radiolabeled or enzyme-conjugated anti-IgG. Fig. 3 shows
sensitivity curves in detecting model ICs(AHG) in human serum
with mAb-105 or mAb-l 30, and a polyclonal antibody for com-
parison. Both mAbs detected as little as 6.25,ggAHG/ml serum.
The polyclonal IgG anti-C3 antibody showed much lower sen-
sitivity, with uptake higher than that in serum alone only when
AHG exceeded 50 ug/ml.
Absence ofnon-IC-derivedlow molecular weight C3-bearing
IgG in serum samples with added ICs. It has been reported that
generation ofnascent C3b by ICs in serum results in a bystander
Table I. Ability ofMonoclonal Anti-C3 Antibodies to Discriminate between Native andActivated C3
Monoclonal Anti-C3c Antibodies
Monoclonal Anti-C3d Antibodies
Serum + EDTA
Serum + AHG
Serum + AHG + PEGt
Serum + inulin§
Serum + zymosan§
*Serum was activated with AHG (1,600 yg/ml), inulin (20 mg/ml), or zymosan (5 mg/ml).
§ Inulin and zymosan were removed by centrifugation.
Results are expressed as nanograms of polyclonal anti-C3 bound per well.
t AHG was removed with 3% PEG beforetesting.
1Numbers represent mean values ofthree experiments with three different normal sera.
Aguado et al.
3.12 6.25 12.5
100 200 400 800
Figure 3. Detection ofC-fixing ICs by a solid-phase assay using anti-
C3 antibodies. Microtiter wells were coated with monoclonal or poly-
clonal anti-C3 antibodies, reacted with serum that contained increas-
ing amounts ofAHG as model of ICs, and finally incubated with ra-
diolabeled anti-human IgG. Nanograms ofbound anti-IgG were mea-
binding or metastasis of IC-fixed C3b to monomeric IgG (20).
If so, the anti-C3 assay may give false positive results and/or
inaccurate estimation of true serum IC levels. To address this
issue, we coated microtiter wells with mAb-105 (anti-C3c) or
mAb-130 (anti-C3d) and reacted them with a mixture containing
NHS as a C source and ICs made with rabbit antibodies (BSA-
rabbit anti-BSA or ARG). Bound rabbit IgG in the form ofcom-
plex or human IgG present in monomeric form in the C source
were detected with radiolabeled anti-rabbit IgG and anti-human
IgG, respectively. As shown in Table II, mAb- 105 or mAb-130
coated wells incubated with human serum to which ICs con-
taining rabbit antibody had been added reacted only with anti-
rabbit, but not anti-human, IgG. Wells incubated with human
serum containing AHG registered binding with anti-human IgG,
as expected. Therefore, under the conditions ofour experiment,
we did not observe significant metastatic or bystander binding
of IC-generated C3 fragments to monomeric IgG.
Kinetics ofC3fragments' retention by ICs in mixtures with
serum or serum + CRI. AHG in serum have been reported to
Table I. Absence ofC3 on Monomeric IgG
after C3 Activation by ICs
Materials added to human
serum (C source)*
*Serum was incubated (60 min) with BSA-rabbit-anti-BSA, ARG, or
AHG at a final concentration of 1,600Ag/ml.Increasing the incuba-
tion period to 24 h did not modify the results.
f Numbers represent mean values ofthree experiments with three dif-
ferent normal sera.
retain C3 fragments and reactivity with anti-C3 polyclonal an-
tibodies for only -60 min (20). Recently, Medoffand co-workers
(21, 22) and Ross et al. (23) demonstrated that cell surface CR1
(the bulk ofwhich is represented on the large numbers oferyth-
rocytes) participate as co-factors with factor I, and perhaps factor
H, ofthe C system in rapidly degrading IC-bound C3b to iC3b
and finally to C3dg/C3d. We tested whether model ICs (AHG
and BSA-anti-BSA) incubated for various times with human
serum alone or with erythrocytes (a more physiologic combi-
nation) (a) retained mAb reactivity, (b) registered different values
with anti-C3c or anti-C3d mAbs, and (c) had values affected by
the numbers ofCR1 in the mixture? A representative experiment
of this type with erythrocytes from two individuals, of which
one had high CRI phenotype (736 CRl/erythrocyte) and the
other low CRI (140 CRI/erythrocyte) is shown in Fig. 4. Similar
results were found in four other individuals, two with low CR1
numbers and two with high CR1 numbers. As illustrated in Fig.
4 A, the amount ofanti-human IgG (reflecting the quantity of
C-fixing AHG bound) reacting with mAb-105 (anti-C3c) coated
wells decreased substantially when the aggregates were left for
long periods in serum (50% reduction by 24 h). Reductions were
similar when erythrocytes with low CR1 receptors were added
to the AHG and serum mixture. However, iferythrocytes with
high numbers ofCR1 receptors were included, the AHG level
reduction registered with the anti-C3c mAb-105 was greatly ex-
pedited (Fig. 4 A, 50% reduction by 3-4 h). In contrast, the
levels ofAHG detected with the anti-C3d mAb-l 30 decreased
5' 10' 15' 30' lh 2h 3h 4h 5h 6h 16h 24h
.-. serum+ IC+ CR1(high)
ot-oserum + IC+CRlffew)
5' 10' 15' 30' lh 2h 3h 4h 5h 6h 16h 24h
Figure 4. Effect ofmembrane-associated CR1 (erythrocytes) on the
state ofIC-bound C3 fragment. AHG (400Ag/ml)was incubated with
fresh NHS for 15 min at 370C before testing. Subsequently, sera con-
taining AHG or AHG and CR1 (4.5 X 109 cells/ml; high number, 736
CRl/erythrocyte; low number, 140 CR1/erythrocyte) were incubated
for different periods at 370C. C3b/iC3b-containing complexes were
measured with mAb-105 (anti-C3c, A), whereas iC3b/C3d-containing
complexes were measured with mAb- 30 (anti-C3d, B).
Antibodies to C3 Neoantigens
Figure 5. Levels ofC3b/iC3b- (0) and iC3b/C3d- (0) containing ICs in pathologic plasmas.
slightly with time (Fig. 4 B, -20-30% over a 24-h period), and,
as expected, were not influenced by the presence and/or numbers
ofCR 1, since mAb130 reacts with iC3b and C3d. This slight to
moderate reduction might be caused by release of some C3d
from the complex, as described by Law et al. (24). Results were
similar when BSA-rabbit-anti-BSA ICs were used instead of
AHG, i.e., a 50% reduction occurred after 2 h with the anti-C3c
antibody, but only a 20-30% after 24 h with the anti-C3d an-
tibody (data not shown). These results indicate that mAb-130
reacting with iC3b as well as the end product ofC3 degradation
(C3dg/C3d) detects most of the C3-bearing ICs independently
of the duration of exposure to C3b/iC3b-converting factors.
Detection ofICs in human plasma. 18 plasmas from healthy
volunteers were analyzed using mAb- 130 (anti-C3d) and mAb-
105 (anti-C3c), giving a mean value±2 SD of 2.36±4.08 ggof
AHG eq/ml and 2.01±4.78 ,g ofAHG eq/ml. High levels (>2
SD of normal) of IC were detected with both monoclonal an-
tibodies in several patients with autoimmune syndromes or a
parasitic disease (Fig. 5). A good correlation was found between
values obtained with anti-C3d and anti-C3c monoclonal anti-
bodies in each group of patients with the following correlation
coefficients: SLE (r = 0.66), RA (r = 0.73), SS (r = 0.54), and
PCM (r = 0.75), with P < 0.01 in all cases.
To determine whether the IC expressed predominantly C3dg/
C3d or C3b/iC3b, the IC-positive plasmas were compared. A
large proportion of patients with autoimmune diseases (SLE,
RA, and SS) registered higher values in the sample with anti-
C3d than anti-C3c mAb as substrate, whereas most ofthe PCM
patients registered equivalent values with both antibodies (Fig.
6). Serial studies were performed on five SLE patients. In Fig.
7, the results ofthe different parameters studied on one ofthem
are shown. Exacerbation ofthe individual's disease, correspond-
ing to increased anti-DNA antibodies and decreased complement
levels, correlated, in general, with increases in the levels of IC,
Finally, the results obtained with mAb- 130 in RA and SLE pa-
tients were compared with IC levels registered by the Raji cell
assay. In RA patients, a good correlation between these two
assays was noted (r = 0.61, P < 0.01). However, in SLE patients,
Log yg eq AHGWiN ImAb-105)
Figure 6. Relationship between C3b/iC3b- andiC3b/C3d-containing
ICs in patients' plasmas. The former were detected by using mAb-105
(anti-C3c) and the latter by using mAb-1 30 (anti-C3d). Nanograms of
immunoglobulin bound were transformed into ggofeq. AHG/ml
Aguado et al.
1 8.2 93.5
Figure 7. Serial study ofan SLE patient, showing the relationship of
circulating ICs as detected with mAb-130 with levels ofanti-DNA an-
tibodies and total hemolytic complement. The shaded area represents
the normal IC range.
* .- *
frequent divergence of results were observed with 42% of the
samples being positive in one, but not the other assay; 40% of
the samples, however, were positive in both systems.
Correlation between CR1 and IC levels and state ofC3 on
circulating ICs. The number ofCR1 in SLE patients' peripheral
erythrocytes is considered lower than normal (19, 25-28). Al-
though the number ofCR1 is genetically determined (19), it is
not known whether this abnormality is genetic in patients with
systemic autoimmunity (19, 25, 26) or secondary to IC-mediated
occupation of the CRI (26, 27). The mean± 1 SD of CR1 for
our SLE patients was 387±200 vs. 625±246 for normals. Com-
parison ofCR1 values to levels ofC3b/iC3b orC3d ICs revealed
no correlation, as assessed with mAb-105 (r = 0.22) and mAb-
130 (r = 0.12), respectively (Fig. 8).
Correlation ofIC levels with C3-activation products. Since
mAb-130 binds to a C3 neoantigen, a system was devised and
applied to detect C3 activation in plasmas, and these results
were correlated with IC levels. For detection of C3 activation
products, mAb-130 was bound to microtiter wells and reacted
with EDTA-plasmas. Fixed activated C3 was then measured with
a radiolabeled (or enzyme-conjugated) polyclonal anti-C3 an-
tibody. As shown in Table III, little C3 bound to wells incubated
with normal plasmas or SLE and RA plasmas without ICs.
However, significant levels ofC3 activation products correlated
with high levels ofICs (r=0.71; P < 0.01) in SLE orRA patients.
This report demonstrates the usefulness ofmAbs that recognize
activated C3 neoantigenic determinants to detect and classify
ICs, and identify C3 activation products in human sera.
Activation of the C system by immunoglobulin aggregates
and other substances results in cleavage of native C3 into C3a
(an anaphylatoxin) and nascent C3b. This enables the nascent
C3b to attach covalently to a variety ofactivation principles via
an ester bond formed between a carboxyl group ofC3b and the
activators' surface hydroxyl group. Subsequently, bound C3b
interacts with factor B that, under the influence ofa serine pro-
tease factor D, is cleaved to Bb and forms a C3bBb convertase
that cleaves C3 and C5 molecules (reviewed in reference 29).
Under the influence of CR1 and factor H acting as co-factors
for factor I, particle-bound C3b is inactivated to iC3b, and finally
Log CR1 numberlerythrocyte
Figure 8. Relationship between the number ofCRl/erythrocyte and
C3b/iC3b or iC3b/C3d-containing ICs in SLE patients. CR1 sites were
assessed with a polyclonal anti-CR1 antibody. C3b/iC3b and iC3b/
C3d-containing ICs were determined with mAb-105 (anti-C3c) and
mAb-130 (anti-C3d), respectively. Regression analyses showed no sig-
nificant correlation between these parameters.
degraded to C3dg and C3c, with the latter being eluted from the
particle (30). In vitro, various enzymes finally split C3dg to C3d
and C3g, but it is debatable whether such breakdown of C3dg
occurs in vivo (9, 23, 31). Antigenic analysis with polyclonal
antisera to C3 identified distinct antigenic determinants in C3,
C3a, C3c, and C3d (32-34). Further topographic mapping of
the C3 molcule and its fragments with respect to immunochem-
Table III. Relationship between IC Levels
and Complement Activation as Detected with mAb-130
*10 plasma samples tested in each group.
f C activation was measured in microtiter wells coated with mAb-130
(anti-C3d neoantigen), subsequently incubated with plasma samples,
and finally with radiolabeled polyclonal anti-C3 antibody. The levels
ofC activation products in plasmas of IC-positive SLE and RA
showed a statistically significant increase when compared by the t test
to those in normal plasmas (P < 0.02).
Antibodies to C3 Neoantigens
ical domains has been achieved by mAbs developed in several
laboratories ( I1-13). Thus, antigenic determinants on the g por-
tion ofthe molecule (9, 31, 35) and neoantigenic determinants
expressed on activated, but not native, C3 (11-13) were revealed.
Among the anti-C3 mAbs available to us, two (mAb-105
and mAb-130) specifically detected C-fixing ICs, subcategorized
ICs according to the C3 fragment they express, and detected C3
activation products. mAb-105, developed by Burger et al. (1 1),
reacted primarily with a neoantigenic determinant on C3c, pres-
ent on particle-bound C3b and iC3b. mAb-130, developed by
Tamerius et al. ( 13), recognized a neoantigenic determinant on
C3d, present also on iC3b, and bound to its respective epitope
whether these fragments were free in fluid phase or on substrates.
Our data indicate that the epitope recognized by mAb-105 is
inside the folded activated C3 molcule when free in solution,
and is thus inaccessible to the antibody. Conceivably, the binding
of activated C3 to substrates induces a conformational change
which results in the generation or exposure of the mAb-105-
directed epitope. Examples of similar divergence in reactivities
ofantibodies with the relevant antigens in fluid phase or bound
to substrates have also been described by others with certain
antibodies against Factor D (36), interleukin 2 (37), and Esch-
erichia coli tryptophan synthase (38).
On the basis ofbinding characteristics ofmAb-105 and mAb-
130, a modified anti-C3 assay was developed to detect C-fixing
ICs in biologic fluids. With either mAb, a sensitivity of as little
as 6.25;gAHG/ml serum was achieved, which is comparable
or superior to the most commonly used Raji cell (5) and Clq (3)
assays. The reagents used in this highly sensitive assay are avail-
able in almost unlimited quantities and, therefore, provide a
superior and reproducible substrate for IC detection and appro-
priate standardization. Since the assay is based on the interaction
ofC3 fragments with specific mAbs, it can detect ICs that activate
C via the classical or alternative pathways. This procedure is
therefore superior to fluid (3) or solid-phase Clq (39) assays in
which only classical C pathway activating ICs can be detected,
and to the Raji cell assay in which anti-cellular antibodies, com-
monly found in autoimmune diseases, may give false positive
In the initial description ofthe anti-C3 assay, we used F(ab')2
fragments of the polyclonal anti-C3 antibody (6) to avoid false
positive results caused by RFs in the test sample. However, a
recent report indicated that false positive results can sometimes
still be obtained due to the presence ofantibodies against F(ab')2
(pepsin agglutinators), but not whole IgGs from differentspecies
(41). To circumvent this problem, we used whole IgG fractions
ofthe mAbs instead ofF(ab')2, and to avoid false results caused
by RFs incorporated in the test sample excess aggregated rat or
mouse IgG similar to the species from which the mAbs were
Our previous studies have shown that in some IC-positive
plasma samples in the polyclonal antibody assay, the anti-C3
bound IgG was sedimenting at the 7-8S position in sucrose gra-
dients (6). Such low molecular weight IgG associated with C3
fragments was primarily found in arthritic and lupus patients
whose disease was not associated with systemic IC-mediated
manifestations. In contrast, patients with systemic organ in-
volvement (vasculitis and/or glomerulonephritis) had anti-C3
bound IgG at an I 1- I9S position. This suggested to us IgG dis-
sociation from antigen, but retention ofsmall covalently bound
C3 fragments in patients with efficient C-mediated solubilizing
activity. In patients with poor C-mediated solubilizing activity,
however, most ofthe IgG remains complexed with antigen and
C fragments. This concept was later challenged by others (20),
who remarked that the anti-C3 assay may give false positive
results due to C activation by ICs or other substances and sub-
sequent bystander binding or transfer of activated C3 from IC
to monomeric IgG. Although such a phenomenon might occur,
our experiments with a given antigen-antibody system did not
reveal significant bystander C3 binding to monomeric IgG.
Therefore, the suggestion that low molecular weight IgG detected
by anti-C3 might be derived from solubilized C-fixing IC remains
a likely possibility.
Ross et al. (23) demonstrated that treatment with isotonic
serum supplemented with purified CR1 for I h at 370C cleaved
50% of activator-bound C3b into bound C3dg and fluid-phase
C3c. Our experiments largely confirm this finding, since levels
of model ICs incubated with serum plus CR1 expressing eryth-
rocytes did not register efficiently (50% reduction by 3-4 h) when
the mAb-105 was the detecting reagent. However, when mAb-
130 was used, very little effect in accurately registering the actual
IC levels was observed over a 24-h period. Regarding the kinetics
ofthese experiments, it should be noted that some degradation
ofC3b to iC3b may have occurred during the preincubation of
ICs with serum to allow C fixation before CR1 addition. Fur-
thermore, the amount of ICs used in the experiment can influ-
ence the speed of bound C3b to C3dg conversion. However,
detection of the ICs using anti-C3d mAb-130 should remain
With the mAbs to neoantigens on C3c and C3d, increased
levels of C-fixing ICs were detected in plasmas of patients with
autoimmune diseases (SLE, RA, and SS) and PCM. ICs in sera
from similar patients have also been detected by othertechniques
(2). The nature ofthe antigen involved in such ICs has not been
investigated. Although not extensively studied, a relationship of
the IC levels with clinical activity in a patient with SLE is shown
in this report. Additional studies correlating IC levels and clinical
activity are in progress. It is of interest that, in some patients
with all ofthe above conditions, ICs were detectable by the anti-
C3c mAb, which could mean that a relatively large part of the
ICs were newly formed and so contained C3b or iC3b. Nev-
ertheless, in most ofthese conditions the levels recorded by the
mAb-130, which recognizes iC3b and C3d, were higher than
those with the mAb-105, which recognizes C3b, iC3b, and C3c,
suggesting by this subtractive approach that a larger proportion
ofthe ICs carried the end product (dg) ofC3 degradation. The
exception was plasmas of patients with PCM, in whom levels
were usually equivalent for both mAbs. This finding suggests
either a rapid turnover and continuous formation ofnew ICs in
which the bound C3b/iC3b is not yet degraded to C3dg, or a
considerable defect in the C3b/iC3bdegrading factors, i.e., CR 1,
factor H and factor I, in these patients.
Medofet al. (21) and others (23, 42, 43) clearly documented
the importance of the CRI in the conversion of C3b to iC3b
and finally to C3dg. Recent studies indicated a diminished num-
ber of CR1 receptors on erythrocytes of SLE and RA patients
vs. normals (19, 25-27). Some investigators believe this defect
is genetic (19, 25), whereas others indicate that it is acquired
and secondary to IC-mediated receptor occupation (27). Our
studies on a limited number of SLE patients confirmed lower
mean numbers oferythrocyte CR1 receptors, but failed to discern
a significant correlation between IC levels and CR1 numbers,
in agreement with others (44), although opposite results have
also been published (45). We also found no correlation between
Aguado et al.
amounts ofICs detected by the anti-C3c or anti-C3d mAbs and
numbers of CR1. One might expect that IC-positive patients
with low CR1 numbers, in which breakdown of C3b will be
slow, would register equal values with the anti-C3c mAb-105
and anti-C3d mAb-130, since both can recognize their respective
epitopes on C3b and/or iC3b. However, since the rate of IC
production, C fixation, and C degradation are dynamic and
asynchronous events, such correlations in a given blood sample
may be precluded. Additionally, even so-called "low numbers"
of CR1 may be sufficient to convert C3b to iC3b and C3dg.
Future analyses oflarger numbers ofsamples and ofserial bleed-
ings over time may permit more definitive conclusions.
The availability ofmAbs that recognize C3 neoantigenic de-
terminants has allowed us to develop a simple solid-phase assay
system for detecting C3 activation products. The present system
appears to be a considerable improvement over existing pro-
cedures, which rely on differential precipitation and mobility
characteristics ofthe C3 breakdown products vs. native molecule
(46, 47). This assay identified significant C3 activation in plasmas
ofpatients with a variety ofdiseases, and correlated C3 activation
products with IC levels, as previously observed by others (47).
To conclude, mAbs to neoantigenic determinants on acti-
vated C3 and its fragments are useful substrates in simple, ac-
curate, and specific assays for IC and C3 activation product es-
timation in biologic fluids.
The authors wish to thank Drs. Robert 1. Fox, Maura M. Bacchi, Richard
S. Smith, and Robert G. Lahita, for providing some ofthe patient samples
used in this study, and Drs. Hans J. Muller-Eberhard and Peter J. Lach-
mann for providing some of the mAbs. The editorial assistance of Ms.
M. K. Occhipinti and Ms. Phyllis Minick is gratefully acknowledged.
This work was supported by National Institutes ofHealth grants Al-
07007, AM31023, and AM33826.
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