PLATELIA™ CMV IgM

96 TESTS 72681
IMMUNOENZYMATIC CAPTURE METHOD FOR THE QUALITATIVE DETERMINATION OF IgM-CLASS
ANTIBODIES TO CYTOMEGALOVIRUS IN HUMAN SERUM

Ref. 91011/BRD English 1/10

 TABLE OF CONTENTS

1. INTENDED USE

2. SUMMARY AND EXPLANATION OF THE TEST

3. PRINCIPLE OF THE TEST

4. KIT CONTENTS AND REAGENT PREPARATION

5. STORAGE AND STABILITY OF REAGENTS

6. PRECAUTIONS

7. TYPE AND STORAGE OF SAMPLE

8. TEST PROCEDURE

9. SCHEME OF TEST PROCEDURE

10. TEST VALIDATION

11. INTERPRETATION OF RESULTS

12. LIMITATIONS OF THE PROCEDURE

13. ANALYTICAL SPECIFICITY

14. DIAGNOSTIC SENSITIVITY AND SPECIFICITY

15. PRECISION

16. “TROUBLE SHOOTING”

17. REFERENCES

Ref. 91011/BRD English 2/10

1. INTENDED USE
IMMUNOENZYMATIC CAPTURE METHOD FOR THE QUALITATIVE DETERMINATION OF IgM-CLASS
ANTIBODIES TO CYTOMEGALOVIRUS IN HUMAN SERUM

2. SUMMARY AND EXPLANATION OF THE TEST

Cytomegalovirus is a herpes virus transmitted by close human contact. No symptoms of infection are
apparent in the majority of cases. However, the virus is very dangerous and may be fatal in
immunodepressed patients. Serum-negative female patients who become infected during pregnancy may
transmit the disease to the fetus. In 95% of cases this occurs without symptoms, but some neonates may
present jaundice, hepato-splenomegaly and retarded psycho-motorial development. For this reason it is of
great importance to determine the immunitary state of the patient before the start of pregnancy, if possible,
and check for serum conversion.
The assay of specific IgM is of great importance in the diagnosis of primary infection.

3. PRINCIPLE OF THE TEST

The test for the assay of Cytomegalovirus IgM is based on the principle of the capture of these
immunoglobulins by anti-human IgM monoclonal antibodies found on the solid phase. A subsequent
incubation with cytomegalovirus antigen in a complex with monoclonal antibodies conjugated to horse radish
peroxidase selects the IgM antibodies specific for the antigen and is revealed by the addition of the
peroxidase substrate. When the enzymatic reaction is stopped by the addition of a sulphuric acid solution, a
yellow colouring forms. The colour, which is proportional to the amount of specific antibodies present in the
sample, can be read in an ELISA microplate reader.

4. KIT CONTENTS AND REAGENT PREPARATION

- Reagents are sufficient for 96 determinations.
Bring to room temperature before use.

MT PLATE MICROPLATE. 12 x 8 wells coated with anti-human IgM monoclonal antibodies.

Use: open the package on the opposite side from the code (M followed by the lot number)
which is useful for identification purposes, remove the support and strips to be used from the
foil package, and place the unused strips in the polythene bag with the silica gel, expel the
air and seal by pressing the closure.

CONTROL + POSITIVE CONTROL (1 x 1.6 mL)

Contents: Diluted human serum (containing anti-CMV IgM antibodies) in Phosphate buffer
0.01 mol/L with BSA 1% and sodium azide 0.09%, liquid, ready for use without further
dilution.
Colour: the colour of the controls is proportional to the relative antibody titer.

CONTROL CUT OFF CUT OFF CONTROL (1 x 2.5 mL)

Contents: Diluted human serum (containing anti-CMV IgM antibodies) in Phosphate buffer
0.01 mol/L with BSA 1% and sodium azide 0.09%, liquid, ready for use without further
dilution.
Colour: the colour of the controls is proportional to the relative antibody titer.

Ag ANTIGEN. Freeze-dried powder x 6 vials.

Contents: Partially purified Cytomegalovirus, inactivated by treatment with Beta-
propiolactone, in Phosphate buffer containing mouse ascitic fluid and lactose.
Preparation: reconstitute with the conjugate volume shown on the label, mixing by inversion.

CONJ CONJUGATE (1 x 18 mL)

Contents: monoclonal antibodies labelled with peroxidase, in phosphate buffer with phenol
0.05% and Bronidox 0.02%.
Preparation: ready for use.
The immunocomplex should be prepared about 45 min. before use.

CONTROL IgM - IgM NEGATIVE CONTROL (PF93900) (1 x 1.6 mL)

INTERCHANGEABLE BETWEEN LOTS
Contents: Diluted human serum in Phosphate buffer 0.01 mol/L with BSA 1% and sodium
azide 0.09%, liquid, ready for use without further dilution.

Ref. 91011/BRD English 3/10

WASH BUF 10x WASH BUFFER 10X (PF93603). 1 x 100 mL. INTERCHANGEABLE BETWEEN LOTS
Contents: Phosphate buffered saline, concentrated 10 times; contains Brij 0.5% .
Preparation: dilute the required volume 1:10 with distilled water in order to obtain the
washing buffer ready for use. If crystals are present, they should be dissolved at 37°C
before dilution.

SAMP DIL DILUENT 2 (PF93611). 1 x 100 mL. For dilution of serum samples. Ready for use.
INTERCHANGEABLE BETWEEN LOTS
Contents: Proteic solution in phosphate buffer with sodium azide 0,09% containing methyl
orange as dye.

SUBS TMB SUBSTRATE (PF93619). 15 mL. Ready for use. INTERCHANGEABLE BETWEEN LOTS
Contents: Tetramethylbenzidine 0.26 mg/mL and hydrogen peroxide 0.01% stabilised in
citrate buffer 0.05 mol/L (pH 3.8).

H2SO4 0.3 M STOP SOLUTION (PF93602). 1 x 16 mL. INTERCHANGEABLE BETWEEN LOTS

H2SO4 0.3 mol/L, in solution ready for use.

ADHESIVE FILMS (2)

POLYTHENE BAG (1)

MATERIALS REQUIRED BUT NOT PROVIDED.

- Incubator at 37°C
- Microplate reader (wave length 450 or 450/620 nm, with linearity up to OD >= 2000)
- Microplate washer (preferable) able to dispense volumes in the range 225-375 µL
- Distilled or deionised water
- Normal laboratory glassware: cylinders, test-tubes etc.
- Micropipettes for the accurate collection of 10, 100, 1000 µl solution
- Disposable gloves
- Timer
- Sodium Hypochlorite solution (5%)
- Containers for collection of potentially infectious materials
- Absorbent tissue

5. STORAGE AND STABILITY OF REAGENTS

Reagents must be stored at 2/8°C.
The expiry date is printed on each component and on the box label.

Reagents have a limited stability after opening and/or preparation

REAGENT CONDITIONS
Microplate 5 weeks at 2/8°C, polythene bag
Controls 5 weeks at 2/8°C
Conjugate 5 weeks at 2/8°C
Reconstituted antigen 15 days at 2/8°C,
Substrate until the expiry date at 2/8°C, 1 week at 15-30°C in the dark
Sample Diluent until the expiry date at 2/8°C
Wash Buffer 2 weeks at 2/8°C, 5 days at 15/30°C
Stop Solution until the expiry date at 2/8°C

6. PRECAUTIONS
FOR IN VITRO DIAGNOSTIC USE ONLY.

Caution: This kit contains materials of human origin which have been tested and gave a negative
response by FDA-approved methods for the presence of HbsAg and for anti-HIV-1, anti-HIV-2 and
anti-HCV antibodies. As no diagnostic test can offer a complete guarantee regarding the absence of
infective agents, all material of human origin must be handled as potentially infectious. All
precautions normally adopted in laboratory practice should be followed when handling material of
human origin.

Ref. 91011/BRD English 4/10

Health and Safety Information
1. Do not pipette by mouth. Wear disposable gloves and eye protection while handling specimens and
performing the assay. Wash hands thoroughly when finished.
2. The following reagents contain low concentrations of harmful or irritant substances:
a) The Wash Buffer contains detergents
b) The conjugate contains phenol
c) The substrate is acid
d) The controls contain 0.09% Sodium Azide which can react with lead and copper in plumbing
forming highly explosive deposits of metal azides; dilute with large amounts of water to eliminate.
If any of the reagents come into contact with the skin or eyes, wash the area extensively with water.
3. Non-disposable apparatus should be sterilized after use. The preferred method is to autoclave for 1 h at
121°C; disposables should be autoclaved or incinerated.
4. Sulphuric acid required for the Stop Soution and hydrochloric acid used for washing glassware are
corrosive and should be handled with appropriate care. If they come into contact with the skin or eyes,
wash thoroughly with water.
5. Neutralized acids and other liquid waste should be decontaminated by adding a sufficient volume of
sodium hypochlorite to obtain a final concentration of at least 1.0%. A 30 minute exposure to 1% sodium
hypochlorite may be necessary to ensure effective decontamination.
6. Spillage of potentially infectious materials should be removed immediately with adsorbent paper tissue
and the contaminated area swabbed with, for example, 1.0% sodium hypochlorite before work is
continued. Sodium hypochlorite should not be used on acid-containing spills unless the spill area is first
wiped dry. Materials used to clean spills, including gloves, should be disposed of as potentially
biohazardous waste. Do not autoclave materials containing sodium hypochlorite.

Analytical precautions
1. Allow all reagents and samples to come to room temperature (18-30°C) before use. Immediately after
use return reagents to the recommended storage temperature. It is important to work at the correct
temperature. Check that the thermostat does not go below 35°C or over 39°C. Open the envelope
containing the strips after at least ½ hr at room temperature.
2. Do not use the reagents beyond the stated expiry date. Microbiological contamination of reagents must
be avoided as this may reduce the life of the product and cause erroneous results.
3. Do not modify the Test Procedure or substitute reagents from other manufacturers or other lots unless
the reagent is stipulated as interchangeable. Do not reduce any of the recommended incubation times.
4. Any glassware to be used with the reagents should be thoroughly washed with 2M hydrochloric acid and
then rinsed with distilled water or high quality deionized water.
5. Avoid the use of self-defrosting freezers for the storage of samples.
6. Do not expose reagents to strong light or hypochlorite fumes during storage or during incubation steps.
7. Do not allow wells to become dry during the assay procedure.
8. Care must be taken not to cross-contaminate reagents. It is important that pipettes are dedicated for
exclusive use with the various reagents.
9. Care should be taken to avoid touching or splashing the rim of the well with conjugate. Do not "blow-out"
from microplates.
10. Enzyme immunoassays can occasionally exhibit an "edge effect" which must be minimised by
increasing the humidity during incubation steps. Plates must be covered with their covers and incubated
at 37°C either in a water bath with a rack or float to support the plates if necessary, or in an incubator.
Alternatively, plates can be incubated in an approved analyser. See the appropriate operating manual
for further details. CO2 incubators must not be used.
11. Ensure that the bottom of the plate is clean and dry, and that no bubbles are present on the surface of
the liquid before reading the plate.
12. Use of highly hemolyzed samples, incompletely clotted sera, or samples with microbial contamination
may give rise to erroneous results.
13. For each instrument used, read the manufacturer's instructions manual carefully to obtain additional
information on the following points:
- installation and particular requisites
- operating principles, instructions, precautions and risks
- manufacturer's specifications and instrument performance
- servicing and maintenance.

Ref. 91011/BRD English 5/10

7. TYPE AND STORAGE OF SAMPLE
The sample is composed of serum collected in the normal manner from the vein and handled with all
precautions dictated by good laboratory practice. The fresh serum may be stored for 4 days at 2/8°C, or
frozen for longer periods at –20°C, and can be thawed a maximum of 3 times. Defrosted samples must be
carefully mixed before performing the test. Heat inactivation can lead to erroneous results. The quality of the
sample can be seriously affected by microbial contamination which leads to erroneous results.
Strongly lipemic, icteric or contaminated samples should be avoided. If a new sample cannot be obtained,
such samples should be clarified by filtration (0.45 µm) or centrifugation (3000 rpm x 10').
The test is not applicable to human plasma.

8. TEST PROCEDURE
Manual Technique
- Prepare the required number of strips.
- Prepare the washing buffer by diluting the Wash Buffer 10x (100 mL + 900 mL H2O).
- Prepare the antigen by reconstituting the freeze-dried product with the conjugate (volume reported on
the label).

Dilute samples 1:101 distributing 10 µL of serum into 1 mL of diluent. Dispense 100 µL of each diluted
sample per well (duplicate testing is recommended). Place UNDILUTED calibrators (if possible, in duplicate)
in a strip (100 µL in each well). Leave one well for the blank, performed using 100 µL of the substrate
mixture.
Wells are covered with protective film and incubated for 45 minutes at 37°C. After washing four times for 30
seconds (300 µl ± 75 µl) , add 100 µL of conjugate to each well and incubate again for 45 minutes at 37°C,
covering the wells with the protective film. The plate is washed again 4 times, as described above. Finally,
the substrate is distributed, 100 µL/well.
After 15 minutes at room temperature the enzymatic reaction is stopped with 100 µL of Stop Solution.
The absorbance (O.D.) is read at 450 nm or 450/620 nm within 30 min.

9. SCHEME OF TEST PROCEDURE FOR Platelia™ CMV IgM

STEP 1 Place 100 µL of diluted samples / controls in the wells of the strips.

Incubate for 45 min. at 37°C

Wash 4 times (300 µl )

STEP 2 Add 100 µL of immunocomplex to each well

Incubate for 45 min. at 37°C

Wash 4 times (300 µl)

STEP 3 Add 100 µL of Substrate to each well

Incubate for 15 min. at R.T.

STEP 4 Add 100 µL of Stop Solution

Read absorbance at 450 nm within 30 min.

10. TEST VALIDATION

Subtract the value of the blank (<= 0.150) from all the other readings. The O.D. values of the control Cut-off
serum when tested in triplicate must be within 25% of the mean value. Disregard any abnormal value and
recalculate the mean. The Positive control must have an O.D. at least 1.5 times that of the Cut-Off serum.
The ratio between Negative Control and Cut-off must be less than 0.6. The O.D. of the cut-off must be >=
0.2 at 450 nm and >= 0.16 at 450/620 nm.

Ref. 91011/BRD English 6/10

11. INTERPRETATION OF THE RESULTS
Qualitative results
If the adsorbance of the sample is higher than that of the Cut-Off, the sample is positive for the presence of
specific IgM.
Calculate the ratio between the O.D. value of the sample and that of the Cut-off (INDEX).
The sample is considered:

Positive: if the ratio is > 1.2

Doubtful: ± 20% of the Cut-Off
Negative: if the ratio is < 0.8
If the result is doubtful, repeat the test. If it remains doubtful, collect a new serum sample.

12. LIMITATIONS OF THE PROCEDURE

All positive test results require careful interpretation since false positive reactions or heterotypic IgM
responses may occur with sera from patients with heterophile-positive mononucleosis, or Varicella Zoster.
A specific IgM response may be observed in reactivation and reinfection as well as with primary infections
with CMV.
Because of all the complications of serologic diagnosis of congenital infection, virus isolation from urine in
the first week of life remains the best way to diagnose intrauterine involvement. Absence of CMV specific
IgM does not exclude the possibility of CMV infection. It has been reported that 10-30% of infants may fail to
develop CMV IgM antibody response despite congenital infection with CMV.
The test results should be used in conjunction with information available from the clinical evaluation and
other diagnostic procedures.
Samples which are strongly positive for the presence of anti-Varicella Zoster (VZV) and Epstein Barr IgM
antibodies can give false positive results.

13. ANALYTICAL SPECIFICITY

44 serum samples containing potential interfering substances were tested:
- Rheumatoid factor (n=8)
- Heterophile antibodies (n=4)
- Bilirubin (n=8)
- Triglycerides (n=9)
- Varicella Zoster IgM positive (n=2)
- Hypergammaglobulinemia (n=13).

Two doubtful results were found with the VZV and heterophyl-positive samples, while no interference was
noted in the other cases.

14. DIAGNOSTIC SENSITIVITY AND SPECIFICITY

In a clinical trial, 105 samples taken from patients whose clinical picture was compatible with a CMV
infection, were analysed with the present kit in parallel with the method taken as reference to establish the
sensitivity. With the reference method, 62 samples were effectively positive. The results are reported in the
following table.

REFERENCE METHOD
+ -
+
61 5
Platelia™ CMV IgM
-
1 217

The Platelia™ CMV IgM kit has a sensitivity of 98.4% and a specificity of 97.8%.

Ref. 91011/BRD English 7/10

15. PRECISION OF THE PLATELIA™ CMV IgM KIT

In-run Precison:
Sample CMM 1 CMM 2 CMM 3 Cut Off Positive Control
(Negative<Cut (Positive>Cut (Positive)
off) Off)
n (replicates) 24 24 24 12 12
O.D. 0.15 0.37 0.55 0.27 0.76
CV% 5.26 7.42 7.93 5.6 9.46

Between-run Precision:
Normal Ratio
Sample Average CV%
Positive Control 5.2 7.4
CMM1 0.52 9.3
CMM2 1.44 15
CMM3 2.9 20

Between-batch Precision:
INDEX
Sample Batch n. 144 Batch n. 145 Batch n. 146 Average CV%
Positive Control 5,52 5,59 4,66 5,25 9,85
CMM1 0,54 0,54 0,59 0,55 4,83
CMM2 1,60 1,34 1,58 1,51 9,48
CMM3 3,04 2,86 3,61 3,17 12,42

Ref. 91011/BRD English 8/10

16. TROUBLE SHOOTING GUIDE

PROBLEM POSSIBLE SOURCE TEST OR ACTION

Invalid run (all negative) One or more reagents not added Recheck procedure
or added in wrong sequence Check for unused solutions. Repeat test.
Unreactive plate Check the code on the package
containing the plate (see package insert
paragraph 4 for correct code).
Check for moisture in unused plate.
(Silica gel desiccant must be pale
yellow).Repeat test.
Invalid run (all positive) Contamination of substrate Take new aliquot of substrate.
Inadequate washing Ensure that wash apparatus works well.
Poor precision Incomplete washing of wells Ensure that wash apparatus works well.
Inadequate aspiration of wells Ensure that wash apparatus works well.
Pipetting error Check pipette function.
Reagent addition too slow Avoid drying of the plate after washing
step. Add reagents immediately.
Presence of bubbles Avoid air bubbles during pipetting.
Optical pathway not clean Check instrument light source and
detector for dirt. Wipe bottom of plate
with soft tissue.
Inadequate Color development Incorrect incubation times or Check for temperature control and time
temperature monitoring.
Adhere to recommended instruction for
use.
Inadequate volume of substrate Check pipette function.
added to the plate

17. REFERENCES
1. G.B. Wisdom: Enzyme-Immunoassay. Clin. Chem. 22: 1243 (1976).
2. H.O. Kangro: Cytomegalovirus serology: does it give the answer? Serodiagnosis and Immunotherapy 1:
91 (1987).
3. Grint P.C.A. et al.: Screening tests for antibodies to cytomegalovirus: an evaluation of five commercial
products. J. Clin. Pathol. 38: 1059 (1985).
4. Van Loon A.M. et al.: Direct enzyme-linked immunosorbent assay that uses peroxidase-labelled antigen
for dtermination of immunoglobulin M antibody to cytomegalovirus. J. Clin. Microbiol. 13: 416
5. M. Musiani et al.: Rapid detection of antibodies against cytomegalovirus induced immediate early and
early antigens by an enzyme linmed immunosorbent assay. J. Clin. Pathol. 37: 122 (1984).
6. F. de Ory et al.: Serological diagnosis of cytomegalovirus infections: comparison of six commercial
methods of ELISA. Serodiagnosis and Immunotherapy 2: 423 (1988).
7. P. Dal Monte et al.: Cytomegalovirus umano. Diagnosis 2: 67 (1990).
8. R. Ziegalmaier et al.: ELISA. la Ricerca Clin. Lab. 10: 83 (1980).

Ref. 91011/BRD English 9/10

 - CE marking (European directive 98/79/CE on in vitro diagnostic medical devices)

- EG Markierung (Europäische Richtlinie 98/79/EG über In-vitro-Diagnostika)

- For in vitro diagnostic use

- In vitro-Diagnostikum

- Manufacturer

- Hersteller

- Storage temperature limitation

- Lagerungstemperatur

- Consult Instruction for use

- Siehe Gebrauchsanweisung

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Ref. 91011/BRD 10/10