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Complement CH50 Kit Insert November HaemoScan BV

COMPANY WITH

QUALITY MANAGEMENT SYSTEM

CERTIFIED BY DNV GL

= ISO 9001 =

Complement CH50

Assay

Kit Insert

Version: November 2016

Summary

Complement hemolytic activity is a functional test of the classical and

alternative pathway of complement in plasma or serum. The classical

pathway method (CH50) is based on lysis of sensibilized sheep

erythrocytes in the presence of Ca++ and Mg++. This method is suited

to evaluate the haemocompatibility of biomaterials and medical devices

according to the international standard ISO 10993-4:2002 after blood,

plasma, or serum contact with biomaterials and to assess the effects of

pharmaceuticals on inhibition or consumption of complement

components.

Introduction

Interactions between blood and biomaterials may activate the

complement system. Particularly during prolonged contact or during

contact of blood with large surfaces, this may induce adverse events, 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 CH50 or AP50 level (i.e. the dilution of 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 37 ºC.

After incubation samples are centrifuged to obtain supernatant,

containing free hemoglobin. The hemoglobin concentration is measured

by means of a spectrophotometer.

Positive reference is total lysis induced by lysis fluid, negative reference is

obtained after incubation with buffer.

The kit is designed to determine hemolytic activity of small samples

(50 µL or less). And can be performed in a 96 well microtiter plate.

The same template can also be used with microcentrifuge tubes.

Precautions

• The kit is intended for research use only.

• The kit should not be used beyond its expiry date.

• Do not combine reagents from Complement CH50 kits with

different lot numbers.

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• The erythrocytes are of ovine origin and these animals have been

tested and approved for consumption.

• 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 tubes

• 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, without cap,

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 0,800 and 0,900, 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 4, 8, 16, 32,

64 and 128 times, made by serial dilution in a round bottom plate,

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 4 to 128 times.

Assay Procedure

1. Pipette test samples or reference dilutions and controls in a round

bottom plate (see plate 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 incubator at 37 °C.

5. Pipette 100 µL stop solution to all wells.

6. Centrifuge the plate (or microcentrifuge tubes) 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 CH50 = 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

4x

1

8x

1

16x

1

32x

1

64x

1

128x

9

4x

9

8x

9

16x

9

32x

9

64x

9

128x

B

2

4x

2

8x

2

16x

2

32x

2

64x

2

128x

10

4x

10

8x

10

16x

10

32x

10

64x

10

128x

C

3

4x

3

8x

3

16x

3

32x

3

64x

3

128x

11

4x

11

8x

11

16x

11

32x

11

64x

11

128x

D

4

4x

4

8x

4

16x

4

32x

4

64x

4

128x

12

4x

12

8x

12

16x

12

32x

12

64x

12

128x

E

5

4x

5

8x

5

16x

5

32x

5

64x

5

128x

13

4x

13

8x

13

16x

13

32x

13

64x

13

128x

F

6

4x

6

8x

6

16x

6

32x

6

64x

6

128x

14

4x

14

8x

14

16x

14

32x

14

64x

14

128x

G

7

4x

7

8x

7

16x

7

32x

7

64x

7

128x

15

4x

15

8x

15

16x

15

32x

15

64x

15

128x

H

8

4x

8

8x

8

16x

8

32x

8

64x

8

128x

neg

con

trol

neg

con

trol

neg

con

trol

pos

con

trol

pos

con

trol

pos

con

trol

Figure 1. Suggested 96-well template for the CH50 assay.

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**Complement CH50 Kit Insert November HaemoScan BV**

**Complement CH50 Kit Insert November HaemoScan BV**

**Complement CH50 Kit Insert November HaemoScan BV**