You are on page 1of 4

King Fahad Medical City - P.O.

Box 59046, Code 11525, Riyadh, KSA


Pathology and Clinical Laboratory Medicine Administration - Immunology / Serology Section

IMMUNOLOGY / SEROLOGY STANDARD OPERATING PROCEDURES


TITLE / DESCRIPTION:

INDEX NUMBER(S):

Antibodies against gangliosides (IgG)


Test Instruction for the Ganglioside Profile 2 EUROLINE
EFFECTIVE DATE:

REVISION NUMBER / DATE:

May 2013

IM/SE-SP 0002
DISSEMINATION:

1st Revision May 2013

1.0TEST PRINCIPLE
The Euroline test kit provides a qualitative in-vitro assay for human antibodies of class IgG to
the seven Gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b and GQ1b in serum or plasma.
The test kit contains test strips coated with parallel lines of purified antigens. The blot strips will
be incubated in the first reaction step with diluted patient serum. In the case of positive
samples, specific antibodies of the class IgG (and IgA, IgM) will bind to the antigens To detect
the bound antibodies, a second incubation is carried out using an enzyme-labeled anti-human
IgG (enzyme conjugate), which is capable of promoting a colour reaction.
2.0

CLINICAL SIGNIFICANCE
Antibodies against the Monosialoganglioside GM1 are associated with multifocal motor
neuropathy (MMN) with a prevalency of 40-70%. These antibodies are in most cases of class
IgM. Furthermore elevated antibody titers against the Monosialoganglioside GM1 occur in
patients with Guillain-Barre syndrome in 22-30% of the cases. The titer correlates with disease
activity. In the acute phase the titer increases to a maximum value and decreases during the
disease.
Antibodies against the Disialongaglioside GD1b have been described in rare cases of patients
with sensory neuropathy.
Antibodies against the Tetrasialoganglioside GQ1b can be detected in more than 90% of the
patients with Miller-Fisher syndrome.

3.0SPECIMEN TYPE
3.1

Sample Material: Human serum or EDTA, heparin or citrate plasma.

3.2

Stability: Patient samples to be investigated can generally be stored at +2C to +8C


for up to 14 days. Diluted samples should be incubated within one working day.

3.3

Sample dilution: The patient samples for analysis are diluted 1:51 with ready for use
universal buffer. For example, add 30l of serum to 1.5ml ready for use universal
buffer and mix well by vortexing. Sample pipettes are not suitable for mixing.

4.0REQUIRED REAGENTS
4.1

NOTE: All reagents must be brought to room temperature (+18C to +25C) around
30 minutes before use. After first use, the reagents are stable until the indicated
expiry date if stored at +2C to +8C and protected from contamination, unless
stated otherwise below.
4.1.1

IM/SE-SP0002

Coated test strips: Ready for use. Open the package with the test
strips only when the strips have reached room temperature to prevent
condensation on the strips have reached room temperature to prevent
condensation on the strips. After removal of the strips the package
should be sealed tightly and stored at +2C to +8C.
Page 1 of 4

King Fahad Medical City - P.O. Box 59046, Code 11525, Riyadh, KSA
Pathology and Clinical Laboratory Medicine Administration - Immunology / Serology Section

IMMUNOLOGY / SEROLOGY STANDARD OPERATING PROCEDURES

5.0

4.1.2

Enzyme conjugate: The enzyme conjugate is supplied as a 10x


concentrate. For the preparation of the ready for use enzyme
conjugate the amount required should be removed from the bottle
using a clean pipette and diluted 1:10 with the ready for use universal
buffer. For 1 test strip dilute 0.15ml anti-human IgG concentrate with
1:35 ml universal buffer. The enzyme conjugate should be used on the
same working day.

4.1.3

Universal buffer: The universal buffer is supplied as a 10x concentrate.


For the preparation of the ready for use universal buffer shake the
bottle (50ml). The amount required should be removed from the bottle
using a clean pipette and diluted 1:10 with deionised or distilled water.
Due to the special membrane used for the patient EUROLINE the
ready for use universal buffer is used for the dilution of patient
samples, the dilution of the enzyme conjugate should be diluted with
18.0 ml deionised or distilled water. The ready for use universal buffer
should be used on the same working day.

4.1.4

Substitute solution: Ready for use. Close bottle immediately after use,
as the contents are sensitive to light.

4.1.5

Warning: Some of the reagents are poisonous (buffer, substrate


solution). Avoid contact with skin.

TEST CALIBRATION
5.1

Measurement Range: The EUROLINE is a qualitative method. No measurement


range is provided. The titer limit is given at a dilution of 1:51.

5.2

Cross Reactions: The high analytical specificity of the test system is guaranteed by
the quality of the antigen substrates used (antigens and antigen sources). This
EUROLINE specifically detects IgG class antibodies to GM1, GM2, GM3, GD1a,
GT1b and GQ1b. No cross reactions with other autoantibodies have been found.

5.3

Interference: Haemolytic, lipaemic and icteric sera up to a concentration of 5mg/ml


for haemoglobin, of 20 mg/ml for triglycerides and of 0.4 mg/ml bilirubin showed no
effect on the analytical results of the present EUROLINE.

5.4

Inter- and intra assay variation: The inter-assay variation was determined by multiple
analyses of characterized samples over several days. The intra assay variation was
determined by multiple analyses of characterized samples on one day. In every case,
the intensity of the bands was within the specified range. This EUROLINE displays
excellent inter-and intra assay reproducibility.

5.5

Sensitivity and specificity:

5.6

In the sera of clinically characterized patients with Guillain Barre syndrome (GBS,
n=71), chronic inflammatory demyelinating polyneuropathy (CIDP, n=13), multifocal
motor neuropathy (MMN, n=18) or Miller-Fisher syndrome (MFS, n=5) and in the
sera of healthy blood donors (n=60) antibodies gangliosides of class IgG and IgM
were investigated.

6.0 QUALITY CONTROL

7.0 PROCEDURE

IM/SE-SP0002

Page 2 of 4

King Fahad Medical City - P.O. Box 59046, Code 11525, Riyadh, KSA
Pathology and Clinical Laboratory Medicine Administration - Immunology / Serology Section

IMMUNOLOGY / SEROLOGY STANDARD OPERATING PROCEDURES


7.1 Pretreat:

Due to the special membrane used in the present EUROLINE a


pretreatment of the test strips as in other EUROLINE tests is not
necessary.

7.2 Incubate:

FIN each channel with 1.5 ml of the diluted samples and incubate at
room
temperature (+18C to +25C) for 60 minutes on a rocking shaker.

(1st step)
7.3 Wash:

Aspirate off the liquid from each channel and wash 3 x 5 minutes each
with 1.5 ml working strength universal buffer on a rocking shaker.

7.4 Incubate:
(2nd step)

Pipette 1.5 ml diluted enzyme conjugate (alkaline phosphatase


conjugated anti-human lgG) into each channel and incubate for 60
minutes at room temperature (+ 18C to +25C) on a rocking shaker.

7.5 Wash:

Aspirate off the liquid from each channel. Wash as described above.

7.6 Incubate:
(3rd step)

Pipette 1.5 ml substrate solution into the channels of the incubation tray.
Incubate for 10 minutes at room temperature (+ 18C to +25C) on a
rocking shaker.

7.7 Stop:
7.8

8.0

9.0

10.0

Evaluate:

Aspirate off the liquid from each channel and wash 3 x 1 minute each
with deionised or distilled water.
Place test strip on the evaluation protocol, air dry and evaluate.

CALCULATIONS

REFERENCE INTERVALS

INTERPRETATION OF RESULTS
10.1
Handling: Place the wet strips after incubation onto the plastic foil on the evaluation
protocol and align with the marks. Carefully dab the strips with absorbent paper (after
complete drying the strips stick to the plastic foil). The wet strips show a dark background
colour. This colour vanishes after the strips have dried completely. Only dried strips should
be evaluated. Distinctly recognizable bands on the blot strips which correspond with bands
of the blot strip picture drawn on the evaluation protocol are entered into the protocol.
If the evaluation is performed digitally using EUROLineScan, the blot strips should be
placed onto the corresponding work sheet as described above. For long term stability the
test strips can be coated with the adhesive plastic foil after scanning. The code for entering
the Test in the EUROLineScan is GP2_EL (Ganglioside Profile 2_EL).

Attention: At the bottom of the strips there is a control band. The incubation was performed
correctly if a strong colour reaction is visible at the band function control. Some samples
might give a dark background staining of the membrane and white bands at the position of
the antigens. These unspecific reactions have to be interpreted as negative.

Antigens: The following antigens have been coated on the membrane strips.

IM/SE-SP0002

Page 3 of 4

King Fahad Medical City - P.O. Box 59046, Code 11525, Riyadh, KSA
Pathology and Clinical Laboratory Medicine Administration - Immunology / Serology Section

IMMUNOLOGY / SEROLOGY STANDARD OPERATING PROCEDURES

11.0

Name

Gangliosidetype

Source

GM1

Monosialoganglioside GM1

Bovine brain

GM2

Monosialoganglioside GM2

Bovine brain

GM3

Monosialoganglioside GM3

Dog erythrocytes

GD1a

Disialoganglioside GD1a

Bovine brain

GD1b

Disialoganglioside GD1b

Bovine brain

GT1b

Trisialoganglioside GT1b

Bovine brain

GQ1b

Tetrasialoganglioside GQ1b

Bovine brain

REFERENCES
11.1 EUROIMMUN, Antibodies against gangliosides (IgG), Test Instruction for the
Ganglioside Profile 2 EUROLINE; Package Insert, (DL 1130-1601-2G); Version
07/05/2008.

12.0 REVISION HISTORY


REVISION DATE
May 2013
May 30, 2015

13.0

REVISION NUMBER

CHANGES

1st Revision

Signatory

2nd Revision

Minor Revision

DISTRIBUTION
13.1
13.2
13.3

Bench copy available to all staff (hardcopy)


Supervisor (Soft Copy on CD)
Head of Department (Soft copy on CD)

Written By:

Lab. Tech. 1
Immunology / Serology Section
Pathology and Clinical
Laboratory Medicine
Administration
Date: May 16, 2013

IM/SE-SP0002

Reviewed by:

Mrs. Maria Paz Rafael


Supervisor
Immunology / Serology
Section
Pathology and Clinical
Laboratory Medicine
Administration
Date: May 16, 2013

Approved By:

Dr. Aziz A. Chentoufi


Dept.Head
Immunology / Serology
Section
Pathology and Clinical
Laboratory Medicine
Administration
Date: May 16, 2013

Page 4 of 4