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  文件名称:  Complement Convertase Kit Insert February HaemoScan BV
  公司名称:  上海拜力生物科技有限公司
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Complement Convertase Kit Insert February HaemoScan BV

 COMPANY WITH
 QUALITY MANAGEMENT SYSTEM
 CERTIFIED BY DNV GL
 = ISO 9001 =
 Complement Convertase
 Assay
 Kit Insert
 Version: February 2018
Summary
Measurement of complement convertase is a sensitive and specific
method to determine complement activation by biomaterials. During
incubation with plasma, complement factors can bind to the surface of
the material followed by the formation of a complement convertase
complex. After washing, to remove unbound complement proteins, the
biomaterials are incubated in a medium with a specific chromogenic
substrate. Cleavage of the substrate is quantified by measuring the
optical density at 405 nm. Positive and negative controls are included in
the kit.
This method can be used to evaluate the biocompatibility of biomaterials
and medical devices according to the international standard ISO
10993/Part 4.
Introduction
Medical devices that come in contact with blood may activate the natural
host defense mechanism of blood by a foreign-body reaction. One of the
potential effects is activation of the complement system via the
alternative pathway. In view of the wide-reaching biologic effects of the
complement system, the consequences of uncontrolled complement
activation could be devastating. Continued activation of the complement
system attracts leukocytes that release lysosomal enzymes and oxygen
radicals, which in turn, lead to necrosis of normal tissue.
Normally, tight controls are in effect which regulate the complement
system to protect host tissue. The cascade is intrinsically moderated by
the instability of the enzymes (convertases) formed. Once a component is
activated, failure to rapidly combine with its substrate causes it to decay.
However, during the use of medical devices these regulatory mechanisms
often appear inadequate due to the foreign nature of the surface of these
devices. Therefore, testing of complement convertase activity by
materials used for the construction of medical devices is needed to
ensure the use of materials with as low complement activation as
possible.
This method was developed for in vitro testing of complement activation
by materials, based on the formation of convertases on the material
surface and chromogenic substrate conversion by the convertase
(Complement Convertase Assay, or CCA). Since C5 convertase is
biologically most relevant, a substrate with an amino-acid sequence
similar to the C5a cleavage site is incorporated.
2
Principle of the Test
A test material of interest (ideally the surface area should be known) is
incubated with plasma. During incubation, complement factors can bind
to the surface of the material followed by the formation of a complement
convertase complex. After incubation, samples are washed and analyzed
for complement convertase activity with a complement convertase￾specific chromogenic substrate. The rate of color development depends
on how much convertase has been generated on the surface of the
biomaterial.
Precautions
• The kit is intended for research use only.
• The kit should not be used beyond its expiration date.
• Do not combine reagents from CCA Kits with different lot nos.
• Plasma should always be treated as a potential biohazard during
use and for disposal.
• Chemicals and reagents have to be treated as hazardous waste
according to biohazard safety guidelines or regulations. For
information on hazardous substances included in the kit please
refer to the Material Safety Data Sheets, which are available upon
request.
• Wear disposable (latex) gloves when handling specimens and
reagents.
• Never pipette by mouth and avoid contact of skin and mucous
membranes with reagents containing sodium azide and DMSO.
• Use disposable pipette tips throughout the procedure to avoid
contamination of reagents.
Contents of the Kit
• CCA Plasma 20 mL 2 bottles
• Wash Buffer 10x, Borate-NaCl 32 mL 1 bottle
solution, 0,01% sodium azide
• Substrate, Convertase-specific 580 µL 2 vials
chromogenic substrate in DMSO
• Coloring Medium, Tris-Borate 32 mL 1 bottle
solution, 0.01% sodium azide
• Reference 1, Low-density 1 cm2 5 pcs
polyethylene (LDPE)
• Reference 2, Polydimethyl- 1,4 cm2 5 pcs
siloxane (PDMS)
• Reference 3, Medical steel (MS) 1,2 cm2 5 pcs
3
Additional Materials and Equipment
The following materials and equipment are required but are not provided
with the kit:
• (Calibrated) adjustable pipettes with disposable tips
• Incubator at 37 °C
• Timer
• 96-well plate
• Plate reader capable of measuring at 405 nm wavelength
• Tweezers
• Mixer
• Micro-centrifuge vials (0,5 mL and 1,5 mL)
• NaCl, 0,9%
• Optional, for cleaning of test samples: RBS (chlorinated trisodium
phosphate, sodium metasilicate) or a comparable detergent.
• Sonicator
• Ultra pure water
• Ethanol (70%)
Test Procedure
Preparation of Materials for Testing
The CCA test can be used for coated or uncoated biomaterials. It is
recommended that clean samples be tested. The following procedure is
recommended to clean biomaterials:
1. Sonicate the biomaterial for 15 min in 2% RBS and wash three
times with ultra pure water.
2. Incubate the biomaterial for 5 min in 70% ethanol and wash three
times with ultra pure water.
3. Dry the material in the air.
Notes:
• Certain materials might be affected by RBS, sonication and/or
ethanol, therefore this cleaning procedure is a recommendation
only; each user should determine an own optimal procedure.
• Cleaned materials should always be handled with tweezers.
• The reference materials provided in the kit are clean and ready for
use.
Reagent Preparation
Determine the volumes of reagents and number of multi-well strips
required.
4
Prepare reagents as follows:
• CCA Plasma: Defrost a vial with CCA plasma at 37 °C and store
at room temperature until use. Thawed plasma must be used
within 4 hours and cannot be refrozen. Do not unnecessarily
expose the plasma to 37 °C at this point.
• Wash buffer: Dilute the concentrated Wash buffer 10x with ultra
pure water. Diluted wash buffer can be stored for 48 h at 2-8 °C.
After thawing Wash buffer 10x can be refrozen for later use.
• Substrate solution: Add 14 mL of Coloring medium to a vial of
substrate. Prepare freshly prior to use; this reagent cannot be
stored beyond 1 day. Store at room temperature until use.
Assay Procedure
Notes in Advance:
• Never use pipettes or vials of glass since glass is a material that
may substantially activate the plasma.
• Select 2 or 3 types of reference materials to which the activities of
the test materials can be compared.
• The reference materials provided in the kit have been cleaned by
the protocol described above and are ready for use.
• The volumes given in this procedure are based on pieces of
material of 1.0 x 0.5 cm (two-sided surface area of approximately
1 cm2
). Test materials should be prepared in pieces of
approximately the same size.
1. Place the selected reference and (cleaned) test materials in 1.5
mL micro-centrifuge vials using tweezers. Fix the materials
vertically between the walls of the vials to prevent floating.
2. Place Reference 1 in an identical vial for the blank.
3. Add 650 µL CCA plasma to each vial with test or reference
material. Ensure that the biomaterial is completely immersed in
the plasma.
4. Add 650 µL 0,9% NaCl to the blank(s).
5. Incubate the vial(s) for 15 min at room temperature.
6. During this incubation, prepare an identical number of 1.5 mL
micro-centrifuge vials, each containing 1.5 mL wash buffer.
7. Add, after the 15 min incubation, 1 mL wash buffer to the vials
with specimens. Remove the specimens with a pair of tweezers,
briefly blot dry with a piece of tissue or filter paper and transfer to
vials with 1.5 mL of wash buffer. Do not mix or vortex.
8. Remove the specimens as previously and transfer to 0.5 mL vials.
Add 0.5 mL substrate solution to each vial.
9. Incubate up to 24 h at room temperature. The rate of color
development depends on how much convertase has been
5
generated on the surface of the biomaterial and is linear up to
OD405 nm = 0.75.
10. Transfer 225 µL of each tube to a 96-well plate. As a template,
Table 1 can be used.
11.Read the optical density within 30 min at 405 nm (OD405).
Calculations
1. Correct the determined OD405 values for the mean blank result
(BLK).
2. Calculate the CCA result of the test and reference materials in
OD405/24 h/cm2
.
Assay Criteria
The result of the blank (BLK) should be OD405 ≤ 0.075.
All measured OD405 values should be ≤ 0.75.
See the ‘Certificate of Analysis’ for recommended target ranges for the
reference materials.
Table 1. Suggested 96-well template for the CCA assay.
Characteristics
CCA results are classified as follows:
INACTIVE: OD405/24 h/cm2 ≤ 0.06
LOW: 0.06 <OD405/24 h/cm2 ≤ 0.20
MEDIUM: 0.20 <OD405/24 h/cm2 ≤ 0.60
HIGH: OD405/24 h/cm2 > 0.40
6
1 2 3 4 5 6 7 8 9 10 11 12
A BLK BLK
B
C
D
E
F
G
H
SPL
5
SPL
5
SPL
13
SPL
13
C TRL
1
C TRL
1
SPL
6
SPL
6
SPL
14
SPL
14
C TRL
2
C TRL
2
SPL
7
SPL
7
SPL
15
SPL
15
C TRL
3
C TRL
3
SPL
8
SPL
8
SPL
16
SPL
16
SPL
1
SPL
1
SPL
9
SPL
9
SPL
17
SPL
17
SPL
2
SPL
2
SPL
10
SPL
10
SPL
18
SPL
18
SPL
3
SPL
3
SPL
11
SPL
11
SPL
19
SPL
19
SPL
4
SPL
4
SPL
12
SPL
12
SPL
20
SPL
20

Complement Convertase Kit Insert February HaemoScan BV

Complement Convertase Kit Insert February HaemoScan BV

Complement Convertase Kit Insert February HaemoScan BV

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