APPLICATION NOTE

Complement TCC Flexi
A Gyrolab® system application

For research and professional use only. Not for use in diagnostic procedures.
This application note contains a suggested protocol and performance data.
Each individual laboratory must set up their own method and perform relevant validations.

Background
In all cases where determination of complement function is wanted, soluble terminal complement
complex (sTCC also known as TCC or sC5b-9) levels can be very informative as a supplement tofunctional assessment of the three complement pathways. It reflects the historical in vivo activity ofcomplement in a given sample.
The complement system plays an essential role in chronic, autoimmune and infectious disease. There
are three pathways of complement activation, namely the classical, the alternative and the lectin
pathway. The soluble terminal complement complex is a product of the terminal pathway and can be a
result from all three complement activation pathways. Since TCC reflects activation to the end of thefinal terminal pathway irrespective of the initial pathway involved, it is a particularly good candidate forgeneral evaluation of complement activation (1).
It is well-known that the complement system plays a key role in the development and amplification of
the inflammatory process at the tissue level in various pathological conditions. Increased levels of TCC
can be detected in for example hemolytic uremic syndrome (HUS) (2), Systemic lupus erythematosus(SLE) (3, 4) and rheumatoid arthritis (RA) (5). The complement system can also be activated by artificial
surfaces, for example during hemodialysis or cardiopulmonary bypass, resulting in increased levels of
TCC (6, 7). TCC is also well suited for studies of complement activation by biomaterials in medicaldevices (8).
TCC can like other activation products of complement be measured in assays using neo-epitope specificmonoclonal antibodies. Neo-epitopes are hidden in the native complement component and exposed
after complement activation (9, 10).

PurposeThe purpose of the Complement TCC assay and the Complement TCC Flexi reagents is to detect TCC
in human EDTA plasma. The Complement TCC Flexi application note allows users to independently setup, customize and optimize a Complement TCC Flexi assay based on Svar Life Science’s Complement
TCC assay on the Gyrolab® automated platforms (Gyros Protein Technologies) or another platform of
their choice using Svar Life Science’s Flexi products.
The product is intended for professional use. The results shall not be used for clinical diagnosis orpatient management. FOR RESEARCH USE ONLY.

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APPLICATION NOTE
Assay design
It is recommended to set up Complement TCC Flexi assays as 3-step
sandwich assays with the capture and detection antibodies used intheir designated functions to ensure optimal performance of the assay.
This 3-step sandwich assay design is also used in Svar Life Science’s
Complement TCC ELISA (COMPL TCC RUO). A recommended
Gyrolab® assay protocol based on the Complement TCC ELISA isdescribed below, allowing for automated TCC quantification in EDTA
plasma using Gyrolab® platforms.

Mouse anti-TCC
TCC
Mouse anti-TCC

Materials and methods
The following semi-quantitative Complement TCC Flexi assay was developed on a Gyrolab® xPlore
using the Flexi reagents listed in Table 1 combined with additional materials and equipment listed inTable 2. The assay has not been validated on a multi-disc instrument. This protocol may not be directlytransferrable to other platforms without further optimization and validation.
Table 1. Flexi reagents required.
Reagent
Complement TCC Flexi Capture antibody (CAP Ab)
Complement TCC Flexi Detection antibody (DET Ab)
Complement Flexi Diluent (DIL)
Complement TCC Flexi Calibrator stock (CAL)
Complement TCC Flexi Low control (CONTROL L)
Complement TCC Flexi High control (CONTROL H)

Volume
16 µL
16 µL
32 mL
70 µL
35 µL
35 µL

Concentration
1.25 mg/mL
1.25 mg/mL
400 ng/mL
CoACoAProduct code
FX1354
FX1355
FX1344
FX1356
FX1357FX1358Store all the Flexi reagents at 2–8°C. Once conjugated, follow the antibody conjugate storage
instructions from the supplier of the respective conjugation kits. Reagents of different lots or different
conjugation reactions shall not be mixed. Dispose of the reagents as hazardous waste.
Table 2. Materials or equipment required but not provided by Svar Life Science.
ItemSupplier®
Gyrolab platform (e.g. xPlore)
Gyros Protein Technologies
Biotinylation kit
See Gyrolab® User Guide®
Alexa Fluor 647-labelling kit
See Gyrolab® User Guide
TM
Bioaffy 1000 CD
Gyros Protein Technologies
PBS-T
See Gyrolab® User Guide
Rexxip® F
Gyros Protein Technologies®
Gyrolab wash buffer pH 11
Gyros Protein Technologies
PCR Plate 96
Gyros Protein Technologies
Microplate foil
Gyros Protein Technologies
Microplate foil adapter
Gyros Protein Technologies
Precision pipettes with disposable tips
-Product code
See Gyrolab® User Guide
See Gyrolab® User Guide
P0004253
See Gyrolab® User Guide
P0004825
P0020096
P0004861
P0003313
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APPLICATION NOTE
Complement TCC Flexi Gyrolab® protocol
Before starting a Complement TCC Flexi Gyrolab® assay, equilibrate all materials to room temperatureand perform the assay at room temperature. The assay protocol is summarized in Table 3.
Table 3. Complement TCC Flexi Gyrolab® protocol summary.
Capture100 µg/mL biotinylated* capture antibody in PBS-TDetection
10 µg/mL Alexa Fluor® 647-labeled* detection antibody in Rexxip® FAnalyte
TCC in EDTA plasmaStandard curve
Calibrator stock in diluent: 400, 200, 100, 50, 10, 0 ng/mL
CD-type
BioaffyTM 1000 CD
Method1000-3W-006-A
Wash buffer 1
PBS-T
Wash buffer 2Gyrolab® wash buffer pH 11
PMT-setting
PMT 5%
Standard fit model
5-parameter logistic (5PL) curve fit excluding the blank
Expected dynamic range
10–400 ng/mL
Sample dilution1:10 (MRD) to 1:80 in diluent.
*Conjugation of antibodies is required before use, see Gyrolab® User Guide.

Antibody conjugation
The anti-TCC capture antibody should be biotinylated according to the Gyrolab® standard protocols(Gyrolab® User Guide) prior to use in Gyrolab® assays. Centrifuge the antibody solution before
conjugation to ensure sufficient antibody recovery and mix carefully with a pipette, avoid formation offoam, before aspirating the solution. In the Gyrolab® assay, the conjugated capture antibody is diluted
to 100 µg/mL with PBS-T.
The anti-TCC detection antibody should be labeled with Alexa Fluor® 647 according to the Gyrolab®
User Guide prior to use in Gyrolab® assays. Centrifuge the antibody solution before conjugation toensure sufficient antibody recovery and mix carefully with a pipette, avoid formation of foam, before
aspirating the solution. In the Gyrolab® assay, the conjugated capture antibody is diluted to 10 µg/mL
with Rexxip® F.
Sample preparation
EDTA plasma samples should be equilibrated to room temperature and freshly diluted just before
running the assay as the samples may have residual complement activity. Undiluted samples can bekept at 2–8°C for up to 8 h. All samples should be centrifuged prior to dilution (Gyrolab® User Guide).
See Specimen collection for detailed information on collecting and storing specimen.
The centrifuged samples should be diluted with the Complement Flexi diluent to a minimum
recommended dilution (MRD) of 1:10 up to 1:80. Samples containing high levels of TCC may need tobe diluted further. When diluting samples, it is recommended to dilute at least 10 µL of sample (e.g.
10 µL sample + 90 µL diluent for 1:10 dilution). To calculate the concentration of TCC in the plasma
sample, the measured analyte concentration should be compensated for the dilution factor, e.g. 10x if
using the MRD above. It is recommended to measure all samples, calibrators and controls in duplicatesto ensure that results are precise.

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APPLICATION NOTEStandard curve
Purified human TCC is used as the assay standard. The standard curve should be diluted in a dilution
series from the calibrator stock (400 ng/mL) using the Complement Flexi diluent, see recommended
standard curve in Table 4. Analyte concentrations are interpolated from the recommended standard
curve using the PMT 5% response and a 5-parameter logistic (5PL) curve fit excluding the blank. It isrecommended to include the high and low control samples in each assay run to verify the performanceof the assay. Using the recommended assay protocol, the controls should measure within the limits
given on their respective certificate of analysis.
Table 4. Recommended standard curve preparation. Note: the graph shows an example of a semi-quantitative
standard curve and should not be used for actual subject sample interpretation.
Conc.
Calibrator
Calibrator
Diluent(ng/mL)
solutionA
40010 µL
B
200
10 µL A
10 µL
C10010 µL B
10 µL
D5010 µL C
10 µL
E1010 µL D
40 µL
FBlank
10 µL

Specimen collection
It is recommended to measure TCC levels in EDTA plasma. Blood samples are to be collected using
aseptic venipuncture technique and EDTA plasma is obtained using standard procedures. A minimumof 5 mL of whole blood is recommended. Centrifuge blood samples and transfer cell-free plasma to aclean tube. Plasma must be properly handled to prevent in vitro complement activation.
The centrifuged EDTA plasma may be kept at 4°C up to 8 hours and analysis should be performed
within this timespan. For longer storage, plasma should be frozen at -70°C or lower. Samples should
not be frozen and thawed more than once.
Matrices other than EDTA plasma have not been tested, and the user should carefully validate theassay’s performance in other matrices and pay special attention to matrix effects and false positives
derived from potential in vitro complement activation.

Assay performance
All results reported below were generated using the recommended Complement TCC Flexi Gyrolab®
assay protocol as described above.
Working range
Table 5. Assay working range. The lowest (LLOQ) and highest (ULOQ) concentrations of the standard curve thatgave a Total Error (% absolute bias + % CV) < 30 % in three repeated assessments were assigned as LLOQ andULOQ.
LOD
LLOQ
ULOQ(ng/mL) (ng/mL) (ng/mL)
1010400

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APPLICATION NOTE
Precision
Between-run precision was determined by analyzing 8 TCC-containing EDTA-plasma samples, someof which were spiked with calibrator material, at 1:5–1:10 dilution across the measuring range in 4
replicates at 3 occasions (Table 6). One of the test occasions was used to calculate within-run precision(Table 6).
Table 6. Within-run and between-run precision. Note: Results below are mean concentrations at the respective
dilutions used. 1n=4 replicates, 2n=3 runs
Within-run1
Between-run2
SampleMean conc.
CVMean conc.
CV(ng/mL)(%)(ng/mL)
(%)
24.0
1.5
24.4
4.4
1
26.7
9.4
29.1
11.7
2
49.6
5.3
48.1
3.9
3
86.6
4.2
83.6
3.0
4
117.5
3.0
119.1
1.9
5
195.8
5.8
206.6
4.8
6194.5
24.4
226.9
12.4
7261.8
11.3
267.0
2.1
8Linearity
Dilution series were prepared with four TCC-containing EDTA-plasma samples and were used to assesslinearity. As demonstrated here, dilutions between 1:10 and 1:80 yield accurate TCC results (Table 7).
Table 7. Linearity and recovery. Four TCC-containing EDTA-plasma samples were diluted so that they were in
range of the standard curve and the linearity of measured concentrations was assessed. Theoretical true
concentrations were calculated based on the 1:10 dilution. Dilutions outside the reported range may not be linear.
Mean measured conc.
Expected conc.
Recovery
SampleDilution factor(ng/mL)(ng/mL)
(%)
1:10
280.0
280.0
100.0
1:20
138.6
140.0
99.0
1
1:40
77.2
70.0
110.3

2

3

4

1:80
1:10
1:20
1:40
1:80
1:10
1:20
1:40
1:80
1:10
1:20
1:40
1:80

42.1
275.5
151.8
86.9
45.7
280.8
149.1
81.5
42.5
349.6
178.9
91.4
53.6

35.0
275.5
137.8
68.9
34.4
280.8
140.4
70.2
35.1
349.6
174.8
87.4
43.7

120.3
100.0
110.2
126.1
132.8
100.0
106.2116.1
121.1
100.0
102.3
104.6
122.7

Note: The use of different dilution factors may be required depending on assay setup, sample type as well as sample analyte
concentration. Samples measuring above the ULOQ should be diluted further. Plasma samples without complement activation
may be below the LOD at MRD.

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APPLICATION NOTE
Recommendations
It is recommended that users determine the concentrations of antibodies after conjugation and titrate
their signal against the standard curve and controls to ensure that the assay is functional.
Usage on other platforms or with altered protocols should be carefully validated by the user.
If customizing this assay, it is important to screen matrices and assess backgrounds, in particular for
disease-specific matrices. Assay parameters should be validated in-house. Data given in this document
should only be considered as a guidance.

References
1.

Harboe M, Thorgersen EB, Mollnes TE. Advances in assay of complement function and activation.
Adv Drug Deliv Rev. 2011 Sep 16;63(12):976-87. doi: 10.1016/j.addr.2011.05.010
2. Noris M, Mescia F, Remuzzi G. STEC-HUS, atypical HUS and TTP are all diseases of complementactivation. Nat. Rev. Nephrol. 2012 Nov;8(11):622-33. doi: 10.1038/nrneph.2012.195
3. Porcel JM, Ordi J, Castro-Salomo A, Vilardell M, Rodrigo MJ, Gene T, Warburton F, Kraus M,Vergani D. The value of complement activation products in the assessment of systemic lupus
erythematosus
flares.
Clin
ImmunolImmunopathol.
1995
Mar;74(3):283-8.
doi: 10.1006/clin.1995.1040
4. Mollnes TE, Haga HJ, Brun JG, Nielsen EW, Sjöholm A, Sturfeldt G, Mårtensson U, Bergh K,Rekvig OP. Complement activation in patients with systemic lupus erythematosus without nephritis.
Rheumatology (Oxford). 1999 Oct;38(10):933-40. doi: 10.1093/rheumatology/38.10.933
5. Struglics A, Okroj M, Swärd P, Frobell R, Saxne T, Lohmander LS, Blom AM. The complement
system is activated in synovial fluid from subjects with knee injury and from patients withosteoarthritis. Arthritis Res Ther. 2016 Oct 6;18(1):223. doi: 10.1186/s13075-016-1123-x
6. Deppisch R, Schmitt V, Bommer J, Hänsch GM, Ritz E, Rauterberg EW. Fluid phase generation ofterminal complement complex as a novel index of bioincompatibility. Kidney Int. 1990
Feb;37(2):696-706. doi: 10.1038/ki.1990.36
7. Ovrum E, Fosse E, Mollnes TE, Am Holen E, Tangen G, Abdelnoor M, Ringdal MA, Oystese R,Venge P. Complete heparin-coated cardiopulmonary bypass and low heparin dose reducecomplement and granulocyte activation. Eur J Cardiothorac Surg. 1996;10(1):54-60.
doi: 10.1016/s1010-7940(96)80266-1
8. Stang K, Krajewski S, Neumann B, Kurz J, Post M, Stoppelkamp S, Fennrich S, Avci-Adali M,Armbruster D, Schlensak C, Burgener IA, Wendel HP, Walker T. Hemocompatibility testingaccording to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic
activation. Mater Sci Eng C Mater Biol Appl. 2014 Sep;42:422-8. doi: 10.1016/j.msec.2014.05.070
9. Mollnes TE, Lea T, Frøland SS, Harboe M. Quantification of the Terminal Complement Complex in
Human Plasma by an Enzyme-Linked Immunosorbent Assay Based on Monoclonal Antibodies
against a Neoantigen of the Complex. Scand. J, Immunol. 22, 197-202. 1985. doi: 10.1111/j.13653083.1985.tb01871.x
10. Mollnes TE, Redl H, Høgåsen K, Bengtsson A, Garred P, Speilberg L, Lea T, Oppermann M, GötzeO, Schlag G. Complement activation in septic baboons detected by neoepitope-specific assays forC3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC). Clin Exp Immunol 1993:
91:295 300. doi: 10.1111/j.1365-2249.1993.tb05898.x

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