Complement AP50 Assay Kit HAEMOSCAN BV
COMPANY WITH
QUALITY MANAGEMENT SYSTEM
CERTIFIED BY DNV GL
= ISO 9001 =
Complement AP50
Assay
Kit Insert
Version: February 2016
Summary
Complement haemolytic activity is a functional test of the classical and
alternative pathway of complement in plasma or serum. The alternative
pathway method (AP50) is based on haemolysis of rabbit erythrocytes in
the presence of Mg++. This method is suited to evaluate the
haemocompatibility of biomaterials and medical devices according to the
international standard ISO 10993-4:2002 and to assess the effects of
pharmaceuticals on inhibition or consumption of complement proteins.
Introduction
Interactions between blood and a biomaterial may activate the
complement system. Particularly during prolonged contact or during
contact of blood with large surfaces, this may induce adverse events. This
is due to generation of an inflammatory reaction and loss of host defense
mechanism. Similarly, pharmaceuticals or their carriers may affect the
complement system. This results in consumption of complement proteins,
which reduces the AP50 level (i.e. the dilution of plasma/serum to obtain
50% lysis of erythrocytes).
Principle of the Test
An erythrocyte suspension is incubated for 30 minutes with serial diluted
serum or plasma at 37ºC. It is known that human complement is able to
activate the alternative pathway. After incubation samples are centrifuged
to obtain supernatant, containing free hemoglobin. The hemoglobin
concentration is measured by means of a spectrophotometer.
The positive reference is obtained by total lysis induced by lysis fluid and
the negative reference is obtained after incubation with buffer.
The kit is designed to determine haemolytic activity of small samples
(50 µL or less) and can be performed in a 96 well microtiter plate.
Precautions
• The kit is intended for research purposes only.
• The kit should not be used beyond its expiry date.
• Do not combine reagents from Complement AP50 kits with
different lot numbers.
• The erythrocytes are of rabbit origin and these animals have been
tested and approved for consumption.
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• Chemicals and reagents have to be treated as hazardous waste
according to biohazard safety guidelines or regulations.
• Wear disposable (latex) gloves when handling specimens and
reagents.
• Never pipette by mouth and avoid contact of skin and mucous
membranes
• Use disposable pipette tips throughout the procedure to avoid
contamination of reagents.
Contents of the Kit
• Erythrocyte concentrate 1,1 mL 1 tube
• Dilution Buffer 100 mL 1 bottle
• Lysis fluid 3,5 mL 1 bottle
• Stop Solution 100 mL 1 bottle
• Reference 1, low complement plasma
• Reference 2, normal complement plasma
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.
• Spectrophotometer capable of measuring at 415 nm.
• Micro-centrifuge + vials (1.5 mL) or centrifuge for microtiter
plates.
Test Procedure
Reagent Preparation
• Erythrocyte suspension: Add slowly, while mixing, the
erythrocyte suspension to 5 mL Dilution Buffer. Mix gently by endover-end
tumbling of the tube. Centrifuge the tube for 10 minutes
at 400xg. Remove the supernatant. Repeat this procedure if the
OD415 of the supernatant is >0,500. Resuspend the pellet in 40
mL Dilution buffer, this is sufficient for 7 microtiter plates. Dilute
25 µL of erythrocyte suspension in 75 µL lysis fluid, this should
give an OD415 between 0,400 and 0,500, when measured in a
microtiter plate in a spectrophotometer. Prewarm the suspension
at 37°C just before use.
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• Test samples: Human plasma is used in dilutions of 2, 3, 4,5,
6,8, 10,1, 15,2 times, made by serial dilution in a round bottom
plate of 2 parts in 1 part dilution buffer, resulting in 50 µL per well.
The positive control is lysis fluid instead of plasma, the negative
control is dilution buffer instead of plasma.
• Reference plasma: Both reference plasma samples are
reconstituted with 250 µL distilled water and are also used in
dilutions ranging from 2 to 15,2 times.
Assay Procedure
1. Pipette test sample or reference dilutions and controls in a round
bottom plate (see Fig. 1 for format example).
2. Add 50 µL of the erythrocyte suspension to each well.
3. Cover the plate with a microplate sheet.
4. Incubate for 30 minutes in an oven at 37°C.
5. Pipette 100 µL stop solution to all wells.
6. Centrifuge the plate at 400xg for 10 minutes.
7. Transfer 100 µL of supernatant to a well of a flat bottom microtiter
plate.
8. Measure OD 415 nm.
Calculations
1. Correct, for all test and reference materials, for the OD415 of the
negative control.
2. Calculate the amount of haemolysis for each sample and dilution.
3. Plot for each sample the haemolysis (%) on the x-axis and sample
concentration (%) on the y-axis.
4. Fit a linear curve (y=ax+b) through the data points.
5. Calculate AP50 = a * 0,5 +b.
Upon request an example calculation spreadsheet is available.
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Characteristics
1 2 3 4 5 6 7 8 9 10 11 12
A
1
2x
1
3x
1
4,5x
1
6,8x
1
10,1x
1
15,2x
9
2x
9
3x
9
4,5x
9
6,8x
9
10,1x
9
15,2x
B
2
2x
2
3x
2
4,5x
2
6,8x
2
10,1x
2
15,2x
10
2x
10
3x
10
4,5x
10
6,8x
10
10,1x
10
15,2x
C
3
2x
3
3x
3
4,5x
3
6,8x
3
10,1x
3
15,2x
11
2x
11
3x
11
4,5x
11
6,8x
11
10,1x
11
15,2x
D
4
2x
4
3x
4
4,5x
4
6,8x
4
10,1x
4
15,2x
12
2x
12
3x
12
4,5x
12
6,8x
12
10,1x
12
15,2x
E
5
2x
5
3x
5
4,5x
5
6,8x
5
10,1x
5
15,2x
13
2x
13
3x
13
4,5x
13
6,8x
13
10,1x
13
15,2x
F
6
2x
6
3x
6
4,5x
6
6,8x
6
10,1x
6
15,2x
12
2x
14
3x
14
4,5x
14
6,8x
14
10,1x
14
15,2x
G
7
2x
7
3x
7
4,5x
7
6,8x
7
10,1x
7
15,2x
15
2x
15
3x
15
4,5x
15
6,8x
15
10,1x
15
15,2x
H
8
2x
8
3x
8
4,5x
8
6,8x
8
10,1x
8
15,2x
neg
cont
rol
neg
cont
rol
neg
cont
rol
pos
contr
ol
pos
control
pos
control
Figure 1. Suggested 96-well template for the AP50 assay.
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Complement AP50 Assay Kit HAEMOSCAN BV
Complement AP50 Assay Kit HAEMOSCAN BV
Complement AP50 Assay Kit HAEMOSCAN BV