Autokit CH50 Read this instruction sheet thoroughly before use.
In vitro Liposome Immunoassay
For the quantitative determination of Total Complement Activity in human Serum
995-40801
0413 D4
Intended use Classification according to Regulation (EC) No 1272/2008
The Autokit CH50 is an in vitro Liposome Immunoassay (LIA) for the quantitative determination of
total complement activity (CH50) in human serum, using an automated procedure.
Summary and explanation of the test
The complement cascade, consisting of ~ 20 serum proteins, plays an important role in the
body’s immunological defense system. Complement activity in human sera can provide important • Signal word Warning
information in the diagnosis of many diseases. Clinically, complement activity is a direct indicator • Hazard-determining components of labelling:
of abnormalities of the complement system, and is different from immunoreactive components Mixture containing: 5-Chloro-2-methyl-2H-isothiazol-3-one [EC No 247-500-7] and 2-Methyl-2H-
of the system. Complement activity has been correlated with the active stage of systemic lupus isothiazol-3-one [EC No 220-239-6] (3:1).
erythematosus, rheumatoid arthritis, cryoglobulinemia-vasculitis, some forms of nephritis, and • Hazard statements
inherited deficiencies of the complement system.1 Previously, the most commonly used assay for May cause an allergic skin reaction.
total complement activity was based on complement-mediated hemolysis of antibody-sensitized
• Precautionary statements
erythrocytes.2 In this method, appropriate serum dilutions are necessary to measure lysis of the
Avoid breathing dust/fume/gas/mist/vapours/spray.
indicator cells. A simpler method, which does not require serum dilution, has been developed.3
However, both methods are complicated and time-consuming, and the reagents are not stable Wear protective gloves/protective clothing/eye protection/face protection.
because of the use of erythrocytes. In addition, it is difficult to automate a hemolytic complement Contaminated work clothing should not be allowed out of the workplace.
assay because of the unstable nature of the erythrocyte dispersion. If skin irritation or rash occurs: Get medical advice/attention.
Liposomes, consisting of concentric shells of lipid bilayers separated by aqueous interspaces, Wash contaminated clothing before reuse.
have been used extensively to study complement-mediated immune damage to cell membranes.4,5 IF ON SKIN: Wash with plenty of soap and water.
A homogeneous assay for total complement activity based on immune lysis of liposomes has Specific treatment (see on this label).
been reported previously.6 The degree of liposome lysis is determined from entrapped alkaline Dispose of contents/container in accordance with local/regional/national/international
phosphatase activity, and the procedure, which is performed manually, cannot be applied to regulations.
automated laboratory analyzers.
This method requires adding many reagents to reaction lubes, a long reaction lime, and the
use of antibodies binding liposomes, which might induce aggregation and sedimentation of the
Standard procedure
liposomes in the prepared reagent. Temperature: 37°C (Hitachi®717)
Recently, we developed an automated homogeneous liposome-based assay for total complement Sample Blank Measurement
activity in human serum. We used a homogeneous population of small-size liposomes 0 5 9.4 10.0
(200 nm), which gave a stable dispersion, and glucose-6-phosphate dehydrogenase (G6PDH,
EC 1.1.1.49) as the entrapped enzyme (the optimum pH of G6PDH is neutral in comparison with (min)
that of alkaline phosphatase). Using these liposomes we developed a fully automated assay
λ main: 340 nm
system for total complement activity.7
Sample: 10 µL R2: 125 µL λ sub: 700 nm
R1: 250 µL
Principle of the method The above standard procedure is an example. Instrument applications are available upon request.
When a sample is mixed with the liposome and the substrate, the antibodies in the reagent combine
with dinitrophenyl (DNP) on the liposomes and then complements in the sample are activated by
the antigen-antibody complex. The activated complements break the membrane of the liposome.
Warnings and precautions
• For in vitro diagnostic use only.
The enzyme, G6PDH, contained in the liposome, reacts with NAD and glucose 6-phosphate
• The usage and application of this test is reserved for professional use only. Please
(G6P) in the reagent. During this enzyme reaction the NAD is reduced to NADH. As a result of
refer to respective national and local regulations and legislation.
this reduction, absorbance at 340 nm increases. The absorbance increase is proportional to the
• Not to be used internally in humans and animals.
complement activity in the sample.
• Operate the instruments according to operator’s manuals under appropriate conditions.
• Do not mix the reagents from one test unit with those of another test unit which has
Reactions a different lot number.
Enzyme reaction • Do not use the containers and other materials in the package for any purposes other
G6PDH than those described herein.
D-Glucose-6-Phosphate + NAD ß-Gluconolactone-6-Phosphate + NADH • Clinical diagnosis must be determined with clinical symptoms and other test results
(Detection at 340 nm) by a physician.
• Store the reagents under the specified conditions. Do not use reagents past the
Reagent preparation expiration date stated on each reagent container label.
Reagent 1: (R1) • After opening the reagents, it is recommended to use them immediately. When the
Use Liposome (R1) as supplied. This solution is stable until expiration date. opened reagents are stored, cap the bottles and keep them under the specified conditions.
Reagent 2: (R2) + (R2a) • Since all specimens are potentially infectious, they should be handled with
Reconstitute one bottle (for 20 mL) of Substrate (R2) with one bottle (20 mL) of Diluent (R2a) to appropriate precaution. Refer to respective good laboratory practice protocols for
prepare the Substrate Solution. The Substrate Solution is stable for 40 days at 2 - 10°C. preventing transmission of infection and handle samples in accordance with any
other local or national regulations relating to the safe handling of such materials.
• If the reagents come in contact with the mouth, eyes or skin, wash off immediately
Reagents with a large amount of water. Consult a physician if necessary.
Contents and storage conditions • When discarding the reagents, dispose of them according to local or national
R1: Liposome 2 bottles x 20 mL Store at 2 - 10°C regulations.
(Do not freeze)
R2: Substrate 1 bottle x for 20 mL Store at 2 - 10°C Physical or chemical Indications of instability
R2a: Diluent 1 bottle x 20 mL Store at 2 - 10°C The presence of precipitates in the reagents or values of control sera outside the manufacturer‘s
R1: Liposome Contains liposome G6PDH 4 U/mL acceptable range may be an indication of reagent instability.
R2: Substrate Contains anti-DNP antibody, goat 24 mmol/L G6P
9 mmol/L NAD Instruments
R2a: Diluent Contains maleate buffer, pH5.0 5 mmol/L The reagent is designed to be used on commercially available automated analyzers. Refer to
Mixture containing: the operating manual for a description of instrument operation and specifications. Performance
5-chloro-2-methyl-2H-isothiazol-3-one standards on alternative instrumentation must be established by the end user.
[EC No 247-500-7] and
2-methyl-2H-isothiazol-3-one
Specimen collection and preservation
[EC No 220-239-6] (3:1)
Use serum as a specimen.
This kit (R2a) contains components classified as following: It is recommended to measure the complement activity in the specimen immediately after
separation of serum. If needed, store specimens at -70°C or lower. Ascorbic acid, bilirubin,
hemoglobin, and lipemic turbidity do not have a significant effect on the measurement.
Manufacturer: Wako Chemicals GmbH
Fuggerstraße 12, D-41468 Neuss
GB.DE.FR
Telephon(e): +49-2131-311-0
Facsimile: +49-2131-311-100
1 www.wako-chemicals.de GB 0413 D4
Autokit CH50 Read this instruction sheet thoroughly before use.
In vitro Liposome Immunoassay
For the quantitative determination of Total Complement Activity in human Serum
Materials supplied Sensitivity: The minimum detectable level of CH50 is estimated to be 10 U/mL.
Refer to the section entitled „Reagents“.
Specifity (WAKO-30R)
Materials required but not supplied Additive Study
Automated analyzer
Ascorbic acid (mg/dL) none 10 20 30 40 50
CH50 Calibrator (Code No. 997-43801)
Complement Control (Code No. 991-43701) CH50 (U/mL) 36.0 36.0 36.0 35.0 35.5 35.5
Results Bilirubin (mg/dL) none 8 16 24 32 40
The final results are automatically calculated and printed in concentration. CH50 (U/mL) 35.0 36.0 36.0 36.0 37.0 37.0
Calibration Hemoglobin (mg/dL) none 100 200 300 400 500
The CH50 assay produces a calibration curve by plotting absorbance vs. concentration. It is
CH50 (U/mL) 40.0 40.0 40.0 40.0 40.0 39.5
recommended to perform calibration at least once a week.
Quality control References
A quality control program is recommended for all clinical laboratories. The analysis of control 1. Schur PH : Complement studies of sera and other biologic fluids. Hum Palhol 1983 ;
material in both the normal and abnormal ranges with each assay is recommended for monitoring 14 : 338-42.
the performance of the procedure. The values obtained for controls should fall within the 2. Mayer MM : Complement and complement fixation. In : Kabat EA, Mayer
manufacturer‘s acceptable ranges. If values are to be established for unassayed control material, MM , eds. Experimental immunochemistry, 2nd ed. Springfield, IL: Charles C Thomas.
the laboratory should assay each level of control material a sufficient number of times to generate 1967 ; 133-240.
a valid mean and acceptable range. 3. Kitamura H , Inai S , Nagaki K : A simple procedure for the titration of total hemolytic
complement activity. Jpn J Clin Chem 1983 ; 12 : 143-7.
Limitations of the procedure 4. Kinsky SC : Antibody-complement interaction with lipid model membranes. Biochim
The measurable range of Wako Autokit CH50 is 10 - 60 U/mL. Biophys Acta 1972 ; 265 : 1-23.
5. Akots G , Braman JC , Broeze RJ , Bowden DW : Rapid, homogeneous phase,
Expected values liposome-based assays for total complement activity. Complement 1984 ; 1 : 125-33.
Serum : 31,6 - 57,6 U/mL. 6. Bowden DW , Rising M , Akots G , Myles A , Broeze RJ : Homogeneous
The expected values are calculated by a normal value distribution. 243 persons, showing assay liposome-based assay for total complement activity in serum. Clin Chem 1986 ;
results within the expected values in 12 biochemicals parameters, were selected from 880 persons. 32 : 275-8.
These data were performed in Japan. (Internal data). 7. Yamamoto S , Kubotsu K , Kida M , Kondo K , Matsuura S , Uchiyama S ,
Since expected values are affected by age, sex, diet, geographical location and other factors, Yonekawa 0 , Kanno T : Clin Chem 1995 ; 41 : 586-90.
each laboratory should establish its own expected values for this procedure.
Ordering information
Interfering substances Code No. Product Package
Ascorbic acid concentrations up to 50 mg/dL, hemoglobin concentrations up to 500 mg/dL and
bilirubin concentrations up to 40 mg/dL do not have a significant effect on the Autokit CH50 995-40801 Autokit CH50 R1: 2 x 20 mL
assay. R2: 1 x for 20 mL
R2a: 1 x 20 mL
Performance characteristics 997-43801 CH50 Calibrator CAL: 5 conc. x for 0,5 mL
Accuracy (WAKO-30R) 991-43701 Complement Control H: 10 x for 0,5 mL (high)
No. Expected (U/mL) Observed (U/mL) Recovery (%) L: 10 x for 0,5 mL (low)
1 27.1 31.0 114.4
2 36.5 40.0 109.6
3 47.3 47.0 99.4
4 54.6 53.5 98.0
Precision (WAKO-30R)
Within-Run Precision
Run # Sample # Replicates Mean (U/mL) SD SV (%)
1 1 21 49.5 0.5 1.10
1 2 21 25.9 0.3 1.35
2 1 21 46.2 0.5 1.14
2 2 21 27.9 0.3 1.05
Total Precision
Concentration # of assay Mean SD CV Swr ST
level days (U/mL) (%)
Low 21 26.9 1.54 5.7 16.6 16.7
High 21 48.3 1.57 3.2 18.9 22.1
The data was collected according to NCCLS Guidelines.
Manufacturer: Wako Chemicals GmbH
Fuggerstraße 12, D-41468 Neuss
GB.DE.FR
Telephon(e): +49-2131-311-0
Facsimile: +49-2131-311-100
2 www.wako-chemicals.de GB 0413 D4
