Original Clinical Science

C1q Binding Activity of De Novo Donor-specific

HLA Antibodies in Renal Transplant Recipients
With and Without Antibody-mediated Rejection
Maggie Yell, MD,1 Brenda L. Muth, RN, MS,2 Dixon B. Kaufman, MD, PhD,3 Arjang Djamali, MD,2
and Thomas M. Ellis, PhD1

Background. Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated
damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and
nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA.
The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal
transplant recipients and to define the properties of DSA that confer C1q binding ability. Methods. The DSA-positive sera from
34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR−, were assayed in C1q-
binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of
AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. Results. Fifty-three per-
cent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR− patients contained
C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR− patients
(16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q − when diluted to a comparable MFI level as the
C1q − DSA from AMR− patients, and some C1q − antibodies converted to C1q + when concentrated to MFI levels comparable
to those observed for AMR+/C1q + sera. Conclusions. The C1q binding activity by de novo DSA in patients with AMR largely
reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical
significance.
(Transplantation 2015;99: 1151–1155)

C omplement fixation by donor-specific HLA antibodies

is a primary mechanism for the induction of inflamma-
tion and organ damage during AMR. Early methods for
detecting HLA antibodies measured complement-fixing anti-
bodies exclusively, but these methods lacked sensitivity and
specificity.1 Single-antigen bead testing greatly improved the
sensitivity and accuracy of HLA antibody detection, but
was regarded to be too sensitive by some and, moreover, pro-
vided no information regarding the complement-fixing abili-
ties of the detected antibodies. Consequently, assessment of
Received 21 August 2014. Revision requested 17 October 2014.
Accepted 21 October 2014.
1
Department of Pathology and Laboratory Medicine, University of Wisconsin,
Madison, WI.
2 tle is known about the requirements for an antibody to fix
Department of Medicine, University of Wisconsin, Madison, WI.
3
Department of Surgery, University of Wisconsin, Madison, WI.
The authors declare no funding or conflicts of interest.
M.Y. participated in research design, writing of the paper, performance of the research,
and data analysis. B.L.M. participated in research design, writing of the paper, and
performance of the research. D.B.K. participated in writing of the paper and data
analysis. A.D. participated in research design, writing of the paper, performance of
the research, and data analysis. T.M.E. participated in research design, writing of the
paper, performance of the research, and data analysis.
Correspondence: Thomas M. Ellis, PhD, D4/212 Clinical Sciences Center, 600
Highland Ave, Madison, WI. (tellis2@wisc.edu).
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
ISSN: 0041-1337/15/9906-1151
DOI: 10.1097/TP.0000000000000699
immunologic transplant risk during the single antigen bead
era has been limited largely to measures of relative antibody
strength using mean fluorescence intensity (MFI) values.2-4
There has been recent interest in technical modifications
of the single antigen bead assay to allow discrimination of
complement fixing and noncomplement-fixing HLA anti-
bodies. Luminex solid-phase C1q binding assays detect the
ability of bead-bound HLA antibodies to fix complement
and detect the products of complement activation, such as
C3d, C4d, and C1q.5,6 Recent studies in renal and cardiac
transplant recipients support the clinical utility of a bead-
based C1q binding assay for distinguishing clinically signifi-
cant, complement-fixing HLA antibodies from those that
are noncomplement fixing and possess a lower clinical risk
for rejection and graft loss.7-11 Despite these observations, lit-
C1q in a solid phase assay compared to complement activa-
tion under physiologic conditions, and which of these charac-
teristics confer a higher risk of antibody-mediated rejection
(AMR). Further, the relationship between C1q binding activ-
ity and other measures of DSA known to contribute to rejec-
tion risk, such as antibody strength, has not been independently
examined. We study a population of 34 renal transplant re-
cipients who developed de novo DSA to determine the rela-
tionship between antibody C1q binding activity, strength,
and isotype composition with the presence of biopsy-
confirmed AMR. The objectives of the study are to assess
the ability of the solid phase C1q assay to distinguish DSA-
positive recipients with AMR from those without rejection

Transplantation ■ June 2015 ■ Volume 99 ■ Number 6 www.transplantjournal.com 1151

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

1152 Transplantation ■ June 2015 ■ Volume 99 ■ Number 6
and to determine whether C1q binding defines a unique sub-
set of antibodies capable of fixing complement and with a
higher risk for AMR. This approach offers a unique assess-
ment of the diagnostic value of measuring C1q-binding
DSA at the time of biopsy-proven AMR.
MATERIALS AND METHODS
Study Population
The study population comprised 34 recipients of deceased
(n = 28) or live donor (n = 6) kidney transplants performed
between 2004 and 2011. Patients received Campath,
Basiliximab, or thymoglolulin induction followed by combi-
nation maintenance immunosuppressive therapy using tacro-
limus, mycophenate, and prednisone. Patients underwent
biopsy based on clinical indications (increase in serum creat-
inine or proteinuria) 2 months to 8 years after transplanta-
tion. All patients had de novo DSA that were first identified
at the time of biopsy. Antibody-mediated rejection was de-
fined using Banff 2007 criteria.
HLA Antibody Testing
Testing for HLA antibodies and C1q binding was per-
formed using dithiothreitol (DTT)-treated serum samples
collected at the time of biopsy. Donor-specific HLA antibod-
ies were detected using Luminex single antigen bead assays
(One Lambda, Inc., Canoga Park CA) performed according
to the manufacturer's instructions with the single modifica-
tion in which a reduced volume of beads (3 vs 5 μL) was
used. Antibody specificities were identified using interpretive
criteria published previously, and all positive specificities
had MFI values above 600.12 The DSAs were classified as
de novo if they appeared after transplantation and were not
detected in pretransplant samples, based on single-antigen
bead testing. C1q binding activity was determined using the
C1qScreen assay (One Lambda, Inc.). To avoid the inhibitory
effects of DTT, the C1q binding assay procedure was modi-
fied to remove DTT by first incubating single-antigen beads
with test serum and washing once with PBS before the addi-
tion of recombinant C1q.
Concentration of serum samples was accomplished by cen-
trifugation through Amicon Ultracel centrifugal 100 k mem-
brane filters (Millipore Corp. Billerica, MA) at 12,000  g
for 5 to 20 minutes, until achieving an approximate 4-fold re-
duction in volume.
Immunoglobulin G Isotyping of Donor-Specific HLA
Antibodies
The relative amount of immunoglobulin (Ig)G1, 2, 3, and
4 comprising each DSA specificity was determined using
a modified single antigen bead assay and IgG isotype-
specific monoclonal antibodies. Twenty microliters of test se-
rum was incubated with 3 μL of single antigen beads for
30 minutes at room temperature, the beads were washed
3 times with wash buffer and then incubated for 30 minutes
at room temperature with saturating amounts of either
FITC-conjugated monoclonal antibodies specific for hu-
man IgG1 (8c/6-39), IgG2 (HP-6014), IgG3 (HP-6050), or
IgG4 (HP-6025) (Sigma-Aldrich, St. Louis, MO). Beads were
then washed 3 times, incubated with saturating amounts
of PE-conjugated anti-FITC for 30 minutes and then washed
3 times before analysis using a Luminex 100 analyzer that
was calibrated using standard fluorescence intensity (SFI)
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beads (One Lambda, Inc.). The MFI values for each isotype
were converted to SFI units, and the SFI value for each
isotype was adjusted to the same normalized scale based on
the F:P ratios of each anti-isotype antibody. The relative
abundance of each isotype comprising a given DSA was cal-
culated as the percentage of its normalized SFI value of the to-
tal normalized SFI for all isotypes.
Statistical Analysis
A χ2 analysis was performed using MedCalc statistical
software (MedCalc, Ostend, Belgium).
RESULTS
C1q + DSA Correlate With the Presence of AMR
Assessment of de novo DSAs in 34 renal transplant recipi-
ents for C1q-binding activity showed that the presence
of biopsy-confirmed AMR (P < 0.04; Table 1). Ten of
19 patients with AMR (53%) were positive for C1q + DSA,
whereas only 2 of 15 (13%) patients without AMR had
C1q + DSA. Additionally, a majority of patients with
C1q + DSA had AMR (10/12; 83%), whereas only 9 of
22 patients with C1q − DSA were diagnosed with AMR. All
9 of the AMR + C1q − patients had a single C1q − DSA (data
not shown). The diagnostic specificity and sensitivity of C1q-
binding DSA for AMR were 87% and 53%, respectively.
C1q + DSA Exhibit Higher MFI Values Than C1q DSA
The relationship between MFI value and C1q-binding ac-
tivity for all individual DSAs is shown in Figure 1 and sum-
marized in Table 2. The number of antibodies depicted
in Figure 1 is greater than the number of patients in the study
because some patients had multiple de novo DSA. C1q + DSAs
had significantly higher MFI values than C1q − DSAs regard-
less of whether the DSAs were from patients with or without
AMR. The mean MFI values for C1q + and C1q − de novo
DSA were 16,118 ± 6698 and 6429 ± 4003, respectively
(P < 0.0001). 2 C1q + DSA were identified in 2 patients with-
out AMR, with MFI values of 4377 and 19,721.
The majority of DSAs with MFIs greater than an arbitrary
threshold of 7000 were associated with AMR (21/26; 81%),
although there was considerable overlap in the distribution
of DSA MFI values between rejecting and nonrejecting recip-
ients (Figure 1). Analysis of DSAs from patients with and
without AMR showed a significant association between
DSAs with MFI values greater than 7000 and the presence
of AMR (P <0.017; Table 3). When compared to C1q bind-
ing activity, high DSA MFI values exhibited higher diagnostic
sensitivity (74% vs 53%) for the detection of AMR, but
lower specificity (73% vs 87%) (Table 4).
TABLE 1.
Correlation between the presence of C1q + donor-specific
antibodies and antibody-mediated rejection
AMR (No. Cases)
Positive Negative P
C1q Positive 10 2 0.04
C1q Negative 9 13
© 2015 Wolters Kluwer
FIGURE 1. Relationship between DSA MFI value and C1q-binding
activity in patients with AMR and without AMR.
Effect of Serum Dilution and Concentration on
C1q-Binding Activity
The correlation between DSA MFI value and C1q-binding
activity was further investigated by determining the depen-
dence of C1q-binding activity of a given antibody on its
MFI value. When sera from the 12 patients with C1q + DSA
were diluted to achieve MFI values comparable to levels ob-
served for 22 patients with C1q − DSAs (6784 ± 3386 vs
5864 ± 2686, respectively), all C1q + DSAs converted to
C1q − (Table 5).
To assess whether HLA antibodies acquired C1q-binding
activity when their MFI values increased, sera containing
C1q − HLA alloantibodies from 6 allosensitized transplant
candidates were concentrated 4-fold and then retested in
C1q-binding assays. Before concentration, all antibodies
had MFI values ranging from 3794 to 6985 and were
C1q − (Table 5). After concentration, 4 of 6 sera exhibited
an increase in MFI level accompanied by a conversion to a
C1q + phenotype. The antibodies in 2 sera that failed to show
an increase in MFI value after concentration also remained
C1q − (Table 6).
Composition of IgG Isotypes in C1q + and C1q DSA
The relative abundance of complement-fixing (IgG1,
IgG3) and nonfixing IgG isotypes (IgG2, IgG4) comprising
C1q + and C1q − antibodies was determined for all DSA de-
tected in our study population. We observed no difference
between C1q + and C1q − DSA in the relative abundance
(%) of the complement fixing isotypes IgG1 (82 ± 14 vs
TABLE 2.
MFI values for C1q + and C1q − DSA in patients with and
without AMR
MFI value
Luminex-C1q AMR+ AMR−
+ 18,233 ± 4268 n = (19) 11,784 ± 11,224 (n = 2)
− 5864 ± 2686 (n = 18) 5381 ± 2352 (n = 20)
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Yell et al 1153
TABLE 3.
Correlation between DSA MFI value and AMR
AMR No. Cases
MFI value + − P
>7000 14 4 0.017
<7000 5 11
78 ± 27) and IgG3 (8 ± 15 vs 9 ± 17) or the nonfixing isotypes
IgG2 (3 ± 3 vs. 6 ± 16) and IgG4 (7 ± 3 vs 7 ± 3).
DISCUSSION
The introduction of solid phase C1q binding assays of-
fered a new approach for stratifying HLA antibody risk by
distinguishing potentially harmful complement-fixing anti-
bodies from those that do not fix complement and exert
lower risk for mediating AMR. Consistent with previous re-
ports, our results indicate that DSA in patients with AMR
are predominantly C1q+, whereas DSA from patients with-
out AMR are predominantly C1q − .7‐11 However, this asso-
ciation can be explained by the fact that C1q + DSA in
recipients with AMR have significantly higher MFI values
than C1q − DSA in AMR− recipients. That these higher
MFI values are not simply a consequence of early inflamma-
tion precipitated by low strength C1q + DSA is indicated by
the fact that these antibodies revert become C1q − when their
MFI values are normalized to the levels observed for
C1q − DSA. In fact, the diagnostic accuracy of C1q binding
activity and elevated MFI value for detecting AMR were
comparable, with the MFI value showing considerably
higher diagnostic sensitivity than C1q binding ability for
the detection of AMR. Therefore, DSA strength measured
as MFI value and C1q-binding activity may provide equiva-
lent measures of AMR risk. Despite the fact that the size of
our study population is limited, there was no evidence that
C1q binding assays discriminate high- and low-risk de novo
DSA in individual renal transplant recipients.10 Further, the
observation that DSA in 9 of 19 patients were C1q − despite
the presence of C4d deposition in the allograft biopsy is con-
sistent with recent reports documenting the existence of
complement-fixing antibodies that are undetectable in the
standard C1q-binding assay.13
Some investigators suggest that the C1q-binding assay de-
tects differences in DSA functional capabilities, possibly re-
lated to composition of complement-fixing IgG isotypes.10
Although such differences in functional properties cannot
be ruled out, our data indicate that C1q-binding activity is
predominantly determined by antibody strength. The obser-
vation that a C1q + DSA converts to C1q − after normaliza-
tion of antibody MFI values to levels comparable to those
TABLE 4.
Diagnostic sensitivity and specificity of a positive Luminex-C1q
and MFI greater than 7000 for AMR detection
Detection of AMR
DSA Sensitivity,% Specificity, %
Luminex C1q+ 53 87
MFI > 7000 74 73
1154 Transplantation ■ June 2015 ■ Volume 99 ■ Number 6
TABLE 5.
Effects of normalization of C1q + DSA MFI values to levels
comparable C1q − on Luminex-C1q activity
N MFI
C1q + DSA 12 18,233 + 4268
C1q + DSA-diluted 12 6784 + 3386
C1q − DSA 22 5864 + 2686
observed for C1q − DSA demonstrate that C1q-binding ac-
tivity is not an inherent property of specific DSAs but more
likely is determined by a critical number or orientation of an-
tibodies binding to the target antigen. This is further sup-
ported by our ability to convert some C1q − antibodies to
C1q + after concentration, but this only held true for antibod-
ies whose MFI values were increased to levels greater than
10,000. We speculate that the 2 sera that failed to convert
to C1q + and exhibit similar increases in MFI values after
concentration may be comprised of antibodies with more
limited epitope specificities, which are at or near saturation.
Finally, C1q binding activity could not be explained by differ-
ences between the isotype composition of C1q + and
C1q − DSA, as both were comprised of similar relative
amounts of the complement-fixing isotypes, IgG1 and
IgG3. In this study, as well as in our unpublished experience,
IgG1 is the predominant isotype comprising HLA alloanti-
bodies, with only rare exceptions (data not shown).
Previous studies showing a relationship between C1q bind-
ing ability and elevated pre- and posttransplant risk in both
kidney and heart transplantation did not evaluate DSA MFI
value as an independent variable.7‐10 Although Chen et al6 re-
ported that C1q-binding activity does not correlate with anti-
body MFI value, statistical analyses of the data were not
performed to support this conclusion. A retrospective 2-by-2
statistical analysis of the data presented in Chen et al supports
a significant association between C1q-binding activity and
DSA MFI value, consistent with our findings (data not
shown). In a large study, Loupy et al11 showed that patients
with C1q + DSA after transplantation had significantly lower
rates of graft survival, and this association was independent of
DSA MFI value. However, the sera in the Loupy study were
not treated to avoid prozone effects, which have previously
been shown to mask an otherwise strong correlation between
MFI value and C1q-binding ability.14
A number of previous reports support the finding that the
solid phase C1q assay is strongly correlated with the amount
of antibody bound to each bead.13‐16 Zeevi et al14 showed a
TABLE 6.
Effects of serum concentration on C1q-binding activity of
C1q − DSA
Neat Concentrated
Sample MFI Luminex-C1q MFI
1 5489 Neg 12,243
2 4924 Neg 10,125
3 6985 Neg 13,112
4 5573 Neg 11,832
5 6323 Neg 7125
6 3794 Neg 5793
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significant relationship between C1q-binding activity and anti-
body strength measured by titer and MFI value is a study
of over 800 serum samples. The recent observation that
C1q − antibodies convert to C1q + when the assay is aug-
Luminex-C1q mented with antihuman globulin indicates that the C1q − assay
+ may not detect all antibodies that fix complement.13 Yet, de-
− spite the correlation between DSA strength and C1q binding
− ability, there were some reactivities that could not be explained
based on antibody strength alone. Three patients with AMR
and 1 patient without AMR had C1q + DSA with MFIs below
6000. These cases might reflect antibodies that bind beads
in low abundance but are directed against distinct epitopes
on the HLA molecule located in close enough proximity to
accommodate the spatial requirements for C1q binding.
Using monoclonal antibodies against defined HLA epitopes,
Duquesnoy et al. showed that a given monoclonal antibody
may or may not bind C1q depending on the spatial configura-
tion of the epitope on a given HLA molecule.17 More extensive
examination of cases where there is an apparent lack of corre-
lation between DSA C1q-binding activity and MFI value
might provide more valuable insights into the diagnostic and
clinical significance of the C1q assay. Nevertheless, it is clear
that the antibody MFI value must be considered a confound-
ing variable in studies designed to assess the clinical signifi-
cance of C1q + DSA,18 and a better understanding of the
mechanisms and clinical significance of the C1q-binding assay
is needed before routine use of this assay is warranted.
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