Technical Review
June 2015
Species-Independent Measurement of
Complement Activation in Animals
PATENT PENDING
Author:
Janos Szebeni, M.D., Professor
Nanomedicine Research and Education Center
Semmelweis University
SeroScience Ltd
Budapest, Hungary
INTRODUCTION
The complement system (C-system) is a protein cascade of the immune system consisting of more than 30 plasma
and cell membrane proteins. It provides first line defense against microbial attacks against the body by way of cell
lysis and orchestration of their clearance by macrophages and recognition by lymphocytes for long-term, specific
immunity. As revealed more recently, the C-system also has important activities in non-immune physiological
processes, such as conception and tissue regeneration. Besides its essential role in maintaining health, the
importance of the C-system in medicine lies in the large number of diseases that are associated with abnormal
Complement function. Accordingly, quantitative analysis of different Complement proteins and their function are
of great practical importance in experimental and clinical medicine. There are numerous immunological methods
for Complement testing, including ELISA-based kits measuring cleavage products as markers of Complement
activation (1). However, most of the commercially available ELISAs are human specific (1), leaving the expanding
need for animal testing of complement activation largely unmet.
DESCRIPTION OF THE METHOD AND ASSAY REAGENTS
The present Pan-Specific C3 (PS-C3) Assay (MicroVueTM Pan-Specific C3 Reagent Kit) represents a novel approach
to measure Complement activation in animals. The C3 Complement Matrix (CCM) and the C3 Converter Reagent
(CCR) in the kit convert the activity of C3 in the animal specimen to human SC5b-9 that is detectable with Quidel’s
human SC5b-9 Plus EIA kit. Thus the method allows sensitive and quantitative measurement of C3 in animal
blood, plasma or serum, which, to the extent C3 had been consumed prior to the assay, also provides a measure
of prior Complement activation. The MicroVue Pan-Specific C3 Reagent Kit expands the methodical arsenal of
Complement analysis in animals, enabling, among many applications, preclinical immune toxicology testing of
C-mediated (pseudoallergic) adverse drug effects (2, 3, 4, 5).
The PS-C3 method is a three-step procedure. In the first step animals are treated – or their sera or plasma
incubated – with the test drugs, agents, or devices to explore possible activation. Samples are collected at
appropriate times for the second step: conversion of animal C3 to human SC5b-9. C3 conversion is performed as
specified in the MicroVue Pan-Specific C3 Reagent Kit.
In the third step SC5b-9 is measured using the human SC5b-9 Plus EIA kit. Table 1 lists the steps and reagents and
equipment applied at each step.
Table 1. Procedure of C3 Conversion by the PS-C3 Kit
I.
Step
Take blood samples from treated (and control)
animals and prepare plasma, or incubate test drug
with animal serum or plasma
Reagent/Equipment
Use 10-15 IU/mL heparin or 50 µg/mL hirudin (lepirudin) for
anticoagulation. No EDTA or citrate
II.
Prepare CCM by reconstituting with distilled water
CCM: C3 Complement Matrix, Cat. #1254400
III.
Mix CCM with animal sample, add Specimen Diluent
and CCR
Specimen Diluent, Cat. #1254700
CCR: C3 Converter Reagent, Cat. #1254500
IV.
Incubate at 37°C for 60 minutes
Water bath
V.
Stop reaction in ice bath by adding stop solution
Stop Solution, Cat. #1254600
VI.
Measure SC5b-9 in all aliquots
MicroVue SC5b-9 Plus Enzyme Immunoassay
Cat. #A020
Cat. #A029 (IVD, CE Mark for EU only)
Page 2 of 12
Examples for Measuring C-activation in Animals Using the PS-C3 Kit
Figure 1 highlights the dynamic range of animal C3 “convertibility” to human SC5b-9 for 11 animal species (dog,
sheep, minipig, pig, rat, mouse, cat, donkey, goat, horse, and rabbit). Serum samples from these animals were
incubated with CCR and SC5b-9 was measured before (black bars) and after (red bars) the incubation. Thus, the
experiment measured the total available, or “convertible,” C3 in these sera, quantifying the seras’ response to
CCR. Based on the post/pre incubation ratio of SC5b-9 values, the CCR-induced rises of SC5b-9 were in the
10-33-fold range, with cat giving the highest (33x) activation factor. Taken together with the absence of SC5b-9
changes in all controls (i.e. where the animal sample, or the CCM, or CCR were replaced by PBS), or if EDTA was
added to the sample before incubation), these data provide evidence that the PS-C3 kit measures the formation
of human SC5b-9 only in the presence of functional animal C3.
Figure 1: Total convertible C3 in the sera of different animals. Sera from different animals were incubated with the C3 converting reagent (CCR) of the PS-C3
kit, and human SC5b-9 was measured in the samples. Pre-conversion and post-conversion mean stopping the reaction before and after the incubation step
with CCR. Replacing CCM or CCR by equivical volume PBS in the reaction mixture, or adding to it 10 mM EDTA led to readings below the pre-conversion
baseline in all samples (not shown). Panel A and B show the results of 2 independent experiments using different animal sera. Number above the bars in A
are the number of animals tested, except rats, where pooled serum was used. In B, bars are individual measurements.
EXAMPLES IN DOGS
Table 2 demonstrates the use of the PS-C3 kit to measure the effects of anticancer drugs, vincristin and
carboplatin, on blood C3 levels in dogs in vivo. In all three (3) dogs treated with the indicated doses of drugs
(“drug treatment”), the post-conversion SC5b-9 values were lower after treatment than those obtained at
baseline, indicating C3 consumption. The extent of C3 consumption ranged between 20%-57%.
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Table 2. C3 Consumption in Dog Blood Caused by Reactogenic Anticancer Drugs
Drug
Dose
Vincristin
0.5 mg/m
Carboplatin
Vincristin
Dog
2
1
2
2
3
250 mg/m
0.5 mg/kg
Baseline
Drug Treatment
Preconversion
Postconversion
Preconversion
Postconversion
SC5b-9 ng/mL
5
79
3
35
5
5
52
124
3
3
24
98
C3
Consumption
(%)*
57*
56
20
*Calculation of C3 consumption: ((74-32)/74 )x 100 = 56.8
(Data : B.Kohn et al. 2013, Clinic of Small Animals, Faculty of Veterinary Medicine, Freie Universität Berlin – Germany)
Calculation of Percent C3 Consumption:
Baseline Postconversion – Baseline Preconversion =X
Drug Treatment Postconversion – Drug Treatment Preconversion = Y
C3 Consumption % =
𝑋−𝑌
𝑋
∗ 100
*Example: ((79-5) – (35-3) / (79-5)) * 100 = 56.8% C3 consumed
EXAMPLES IN RATS
In further studies the PS-C3 kit was validated in vivo by measuring plasma C3 levels in rats injected with known
activators of Complement; Zymosan and liposomal Amphotericin B (AmBisome) (6, 7). The question was whether
C3 consumption, measured by the PS-C3 assay, could be correlated with the adverse physiological effects that
Complement activation causes in many animals, including rats, as well as with the changes in hemolytic activity in rat
plasma, which reflects Complement consumption. Figures 2-4 demonstrate our results from these experiments. We
administered Zymosan (Figure 2) and AmBisome (Figure 3) to rats in an i.v. bolus and measured the changes of
systemic arterial pressure (SAP), white blood cell (WBC) and platelet counts, plasma level of thromboxane B2
(TXB2), plasma hemolytic activity (on sheep red blood cells (SRBC), and plasma C3 levels with the PS-C3 assay.
As shown in Figure 2, Zymosan (10 mg/kg) caused major, highly significant decrease of SAP (hypotension, A), drop
of WBC count (leukopenia, B), drop of platelet count (thrombocytopenia (C)) and rise of plasma TXB2 level (D), all
changes being consistent with major Complement activation. Similarly, even more expressed changes were
obtained after bolus administration of AmBisome (Figure 3, A-D). In both cases, the time courses of these
changes were highly correlated, even in minute resolution, and were associated with a major decline of plasma
hemolytic Complement activity (on SRBC) and C3 consumption measured by the PS-C3 assay. The changes of the
latter two parameters proceeded hand-in-hand as mirror images, providing remarkable proof that the PS-C3
assay is equally as sensitive in measuring plasma C3 as the SRBC assay. Nevertheless, the two assays differ from
each other in that the PS-C3 assay is specific for C3, while the SRBC assay measures total Complement activity.
Figure 4 shows the correlations of SAP with SRBC hemolytic activity (A) and C3 consumption (B), as well as the
correlation of latter two parameters with each other (C), during CARPA induced by several different Complement
activator drugs and agents. The pooled data indicate significant linear (A) and inverse correlations (B, C) among
these parameters, providing additional evidence for the use of the PS-C3 assay as a quantitative measure of in
vivo Complement activation.
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Figure 2: Physiological changes caused by Zymosan in rats and their correlations with C hemolytic activity in blood and C3 consumption, measured by the
PS-C3 assay. Rats were injected with 10 mg/kg Zymosan i.v. in bolus, and their SAP (A), WBC and platelet counts (B,C), plasma TXB2 (D), plasma hemolytic
activity on SRBC and C3 levels (E) were measured at the indicated times. Points and error bars on the curve are means  S.E.M for n=6 rats. Further details of
the experiment and assays are described in Ref. 11.
Figure 3: Physiological changes caused by AmBisome in rats and their correlations with C hemolytic activity in blood and C3 consumption, measured by
the PS-C3 assay. Rats were injected with 22 mg/kg AmBisome i.v. in bolus, and their SAP (A), WBC and platelet counts (B,C), plasma TXB2 (D), plasma
hemolytic activity on SRBC and C3 levels (E) were measured at the indicated times. Points and error bars on the curve are means  S.E.M for n=8 rats.
Further details of the experiment and assays are described in Ref. 11.
Page 5 of 12
Figure 4: Relationships among SAP changes and plasma hemolytic activity (A), C3 consumption (B) in rats following induction of CARPA with bolus
injection of different agents (see key). C, Correlation between C3 consumption, measured by the PS-C3 assay, and plasma hemolytic activity, measured in
the SRBC assay. Data pooled from experiments carried out with the following CARPA inducers (key): Zymosan 10, 10 mg/kg Zymosan; AmBisome 2.2, 2.2
mg/kg AmBisome; AmBisome 22, 22 mg/kg AmBisome; PEG Chol, liposomes consisting of 2K-PEG conjugated to cholesterol (Ref 12); AmBisombo 22,
AmBisome equivalent liposomes lacking amphotericin-B, applied at 22 mg/kg. The regression line and specified R2 indicate highly significant correlation
between C3 consumption and hemolytic activity. Further details of the experiment and assays are described in Ref. 11.
Similar studies were conducted with cobra venom factor (CVF) and liposomal doxorubicin (Doxil), which
essentially showed identical correlations. Taken together, these experiments provide strong evidence for the
utility of the PS-C3 assay for monitoring in vivo Complement activation in rats.
EXAMPLES IN PIGS
Figure 5 shows the use of the PS-C3 kit for measuring Complement activation in vitro in pig serum. Incubation of
the serum with AmBisome at 37oC led to progressive consumption of C3, indicating major activation. The
paralleling slower consumption of C3 in the “PBS” control (i.e. in the absence of AmBisome, can be explained with
spontaneous Complement activation at 37oC).
Figure 5: C3 consumption by AmBisome in-vitro. Pig serum was incubated with AmBisome or its vehicle (PBS) at 37oC, and samples were taken for the PS-C3
assay at the indicated times. Values are means for duplicate determinations.
Page 6 of 12
Figure 6 shows the C3 consumption in pigs in vivo following Zymosan-induced Complement activation. The rise of
pulmonary arterial pressure (PAP) shows, again, remarkable parallelism with C3 consumption.
Zymosan: 0.1 mg/kg
70
PAP mmHg /
SC5b-9 mg/mL
60
C3
50
40
30
PAP
20
10
0
0
5
10
15
Minutes
Figure 6: C3 consumption and rise of pulmonary arterial pressure (PAP) by Zymosan in a pig. A pig was injected with 0.1 mg/kg Zymosan i.v., and
heparinized plasma was analysed for C3 levels by the PS-C3 assay. PAP was measured via a Swan-Ganz catheter. The y-axis shows absolute values for both
PAP and C3 levels, the latter expressed as human SC5b-9.
Figure 7 shows a similar experiment in another pig, which obtained 1 mg/kg Zymosan. In addition to PAP and
SC5b-9, this Figure also shows the changes in SAP and HR; all data are expressed as a percent of baseline.
Figure 7: Time courses of the changes of PAP, SAP, HR and plasma C3 following i.v. administration of Zymosan in a pig at 1 mg/kg dose. The plasma
applied for the PAN-assay was anticoagulated with hirudin (lepirudin). The y-axis shows relative values for all parameters, expressed as % of baseline.
As in figure 6, there was good correlation between the rise of PAP, up to 35%, and fall of SC5b-9, by about 30%40%. The other hemodynamic changes were erratic.
Page 7 of 12
Figure 8 shows an experiment wherein a pig was injected with a multilamellar liposome (MLV) preparation used
in the clinic. The dramatic cardiopulmonary imbalance was reflected by roller-coaster changes in PAP and HR,
along with massive hypotension that led to shock and death of the animal within 20 minutes. The changes were
associated with gradual (up to 30%) fall of SC5b-9, hence, C3 consumption.
Figure 8: Time courses of the changes of PAP, SAP, HR and plasma C3 following i.v. administration of MLV in a pig at 5 mg (phospholipid)/kg dose. Other
conditions are similar to those described for Figure 7.
Figure 9 shows an exceptional result that we obtained in a pig injected with 1 mg/kg Zymosan. This pig reacted
differently compared to the one shown in Figure 7, inasmuch as the PAP reaction was maximal, and the SAP, HR
and SC5b-9 values also rose to unprecedented heights. An elevation of SC5b-9 implies increased C3, which finding
is counterintuitive, nevertheless explainable by hemoconcentration due to anaphylatoxin-induced capillary
leakage, and/or C3 release from the liver or other organs, as an acute phase reaction. Further studies are needed
to reproduce this phenomenon and dissect its cause(s). It should be noted though that rises of C3 are also
observed in mice following treatment with Zymosan and liposomes (9).
Figure 9: Time courses of the changes of PAP, SAP, HR and plasma C3 following i.v. administration of Zymosan in a pig at 1 mg/kg bolus. Other conditions
are similar to those described for Figures 7 and 8.
Page 8 of 12
EXAMPLES IN MICE
Figure 10 shows the maximal inducible rise of SC5b-9 in the sera of 3 mouse strains, providing evidence that the
assay also works in mice. It is important in this regard to emphasize that the dilutions which should be used for
the SC5b-9 assays after the conversion step in the PS-C3 assay differ in different species, e.g. 1:100 – 1:200-fold in
the case of dogs, rats and pigs, while only 1:10 – 1:20 with mice. It is recommended to test different dilutions for
each use of the kit, to find the effective dynamic range of the assay.
Figure 10: Feasibility of the PS-C3 assay to measure C3 consumption in mouse sera. Mouse sera were treated with the C3 Converter Reagent (Part 1254500)
of the PS-C3 assay and the sera were subjected to SC5b-9 testing by Quidel’s SC5B-9 Plus ELISA. The bars show the maximal measured SC5b-9 in each strain,
mean  SD for 3 animals in each group.
SUMMARY
The PS-C3 assay represents a new approach to measuring C3 levels and consumption in blood, plasma and serum
of a variety of animals, which allows for the estimation of Complement activation in vitro or in vivo. To date, the
method has been shown to work in the sera of the following species: bovine, chicken, dog, goat, guinea pig,
horse, mini pig, mouse, pig, rabbit, rat, sheep and turkey. The assay provides an unprecedented universal tool for
clinical and experimental immunologists, physiologists and many others for whom quantitative analysis of animal
Complement is essential.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
Quidel. Assays for Complement Antigens. http://www.quidel.com/research/elisa-kits. 2013.
Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs. U.S. Department
of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and
Research (CDER).
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm07
9239.pdf. 2002.
Hastings K.L., Implications of the new FDA/CDER immunotoxicology guidance for drugs. Int
Immunopharmacol. 2002;11:1613-8.
ICH-S8. Immunotoxicity studies for human pharmaceuticals. Step 2: Note for guidance on
immunotoxicity studies for human pharmaceuticals EMEA/CHMP/167235/2004. 2004.
Szebeni J., Complement activation-related pseudoallergy: a new class of drug-induced immune
toxicity. Toxicology. 2005;216:106-21.
Chanan-Khan A., et al. Complement activation following first exposure to pegylated liposomal
doxorubicin (Doxil): possible role in hypersensitivity reactions. Ann Oncol. 2003;14:1430-7.
Szebeni J., et al. A porcine model of complement-mediated infusion reactions to drug carrier
nanosystems and other medicines. Advanced drug delivery reviews. 2012;64(15):1706-16.
Proctor L.M., et al. Complement inhibitors selectively attenuate injury following administration of
cobra venom factor to rats. International immunopharmacology. 2006;6(8):1224-32.
Page 9 of 12
9.
10.
11.
Mizuno M., et al. Zymosan, but not lipopolysaccharide, triggers severe and progressive peritoneal
injury accompanied by complement activation in a rat peritonitis model. J Immunol.
2009;183(2):1403-12.
Vangadesan Krishnan, et al. The Crystal Structure of Cobra Venom Factor, a Co-factor for C3 and C5convertase CVF Bb. Structure. 2009 April15; 17(4): 611-619.
Dézsi, L., et al. Features of complement activation-related pseudoallergy to liposomes with
different surface charge and PEGylation: comparison of the porcine and rat responses. J Control
Release 2014;195:2-10.
EXPERIMENTAL SETUP EXAMPLE
In Vivo:




Apply drug or device to test animal (i.e. the concentration which will be applied for treatment later in
humans + 10 times more).
Animal testing always shows significant individual variation in response; therefore it is generally
recommended to test 6-10 animals. Ten or more individual animals should be evaluated when using the
rat model.
Blood Collection:
o Collect samples from treated and non-treated animals. Suggested sampling pattern at time 0 and
after application at 2,5,10, 15, 30, 60 and 120 minutes.
o Immediately prepare plasma or serum and assay. Alternatively, store samples at –70°C for testing
at a later time.
Apply the MicroVue Pan-Specific C3 Reagent Kit procedure for all samples, followed by measurement
using the MicroVue SC5b-9 Plus EIA.
In Vitro:
 Add different concentrations of drug or material of device to animal serum. Store one sample untreated
as a negative control and include a positive control by spiking a sample with HAGG or Zymosan. CVF is not
recommended for use as an in-vitro activator as residual CVF can bind to fragment Bb and form a C5
convertase, which can generate elevated levels of SC5b-9 in the absence of animal C3 protein.(10)
 Incubate at 37°C and collect samples after 15, 30, 60 and 120 minutes. Immediately place samples on ice.
 Apply the MicroVue Pan-Specific C3 Reagent Kit procedure for all samples, followed by measurement
using the MicroVue SC5b-9 Plus EIA.
SPECIMEN COLLECTION AND STORAGE
Sample type:


Serum
10-15 IU/mL heparin plasma or 50 g/mL hirudin plasma for anticoagulation
EDTA or citrate plasma cannot be used
If testing is not to be performed immediately, aliquot samples into appropriate volumes and store frozen at –70°C
For previously frozen specimens, thaw samples rapidly in a 37°C water bath until just thawed. Immediately place
samples in an ice bath until use.
REAGENTS AND MATERIALS PROVIDED
1. C3 Complement Matrix (Part 1254400) – Lyophilized power. Reconstitute powder with 0.9 mL deionized H2O
and mix thoroughly. Place in ice bath or prepare aliquots and store unused C3 Complement Matrix
immediately at –70°C or below.
2. Specimen Diluent (Part 1254700) – 10 mL. Store at 2°C to 8°C. Ready to Use.
Page 10 of 12
3. C3 Converter Reagent (Part 1254500) – 0.5 mL suspension. Vortex the C3 Converter Reagent immediately
before and during use, approximately every 10 samples. Store at 4°C. Ready to Use.
4. Stop Solution (Part 1254600) – 0.5 mL 0.2M EDTA. Store at 2°C to 8°C. Ready to use.
5.
Positive Control (Part 1267200) – Lyophilized Powder. Reconstitute powder with 200 L deionized water and
mix thoroughly. Place in ice bath for the assay or prepare aliquots and store unused Positive Control
immediately at –70°C or below.
MICROVUE PAN-SPECIFIC C3 REAGENT KIT
WORKFLOW
MicroVue Pan-Specific C3 Reagent Kit Package Insert, PN 20261en v2015FEB02
Page 11 of 12
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0164AI0614D-2 (06/15)
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