RD-Ratio Diagnostics E-CVM-K02 Cytomegalovirus IgM Elisa test

CMV IgM Elisa

INTENDED USE

The RD-Ratio Diagnostics E-CVM-K02 Cytomegalovirus IgM Elisa test system is an Enzyme-Linked Immunosorbent Assay ( ELISA ) kit providing material for the detection of IgM-class antibodies to Cytomegalovirus in human serum or plasma. This assay is intended for in vitro use only.

SUMMARY AND EXPLANATION

Cytomegalovirus (CMV) belongs to Herpes virus family and is transmitted through saliva, sexual contact, perinatally, or through blood transfusions or organ transplantation. Infection with CMV appears to be worldwide and common despite the relative rarity of clinical disease. CMV causes most of the congenital virus infections in humans, with an incidence ranging from 0.2 to 2.2% of live births in different populations. Intrauterine transmission of the virus can occur at any time during gestation, but most infants are probably infected during birth or after birth from ingesting CMV-infected maternal milk. Disease of newbornwith CMV infection is an often severe, fatal illness, usually affecting the salivary glands, brain, kidneys, liver and lungs.

After the primary infection, CMV can persist in a dormant state as a latent infection. During immunosuppressive treatment of patients (e.g. recipients of organ transplants), latent infection can be activated and appear as a secondary infection.
CMV is one of the most serious and frequent pathogens in AIDS patients. CMV pneumonia, a life threatening infection may occur in about 20% cases BMT (Bone Marrow Transplant) patients. The ability to distinguish primary from latent infection is of great importance inasmuch as primary maternal infections have greater pathological potential for fetus.

Diagnosis is made mainly by serological findings of antibodies (IgG and IgM classes) to CMV. However, it is necessary to test the specimen for specific IgM; presence of specific IgM antibodies indicates the primary infection, whereas presence of specific IgG antibodies indicates the immune status of patients.

PRINCIPLE OF THE TEST

The CMV IgM assay is based on the principle of the capture of these immunoglobulins and subsequent identification of those, which are specific, making use of their ability to bind an antigen conjugated to peroxidase. The capture is performed using monoclonal antibodies bound to the solid phase with microtitration strips. The antigen is composed of purified and inactivated Cytomegalovirus antigen.

REAGENTS

The RD-RatioDiagnostics Cytomegalovirus IgM ELISA kit contains sufficient reagents for 96 wells. Each kit contains the following reagents:

MATERIAL PROVIDED 

• Anti-human IgM Antibody-Coated Microtitration Strip
• Wash Concentrate
• Sample Diluent
• TMB-Substrate
• Negative Control
• CutOff Control
• Positive Control
• CMV-HRP Conjugate
• Stopping Solution
• One Plate
• One Bottle
• One Bottle
• One Bottle
• One Vial
• One Vial
• One Vial
• One Bottle
• One Bottle
• E-ABM-10
• E-WSL-30
• E-DLB-40
• E-TMB-08
• E-CVM-01
• E-CVM-02
• E-CVM-03
• E-CVM-20u
• E-STP-09

MATERIAL NOT PROVIDED

• Microtitration plate reader capable of absorbance measurement at 450 nm
• Deionized/Distilled water
• Precision pipette to deliver 10 µl, 100 µl and 1 ml
• Semi-automatic pipette to deliver 100 µl
• Automatic microtitration plate washer
• Absorbent material for blotting the strips
• Incubator

E-CVM-K02 ucap

REAGENTS PROVIDED:

Antibody-Coated Microtitration Strips:
One stripholder containing 12x8 (96) microtitration wells coated with monoclonal Anti-human IgM antibodies. Store at 2-8°C until expiration date. 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. Once opened, the product is stable for 4 weeks at 2-8°C.
Wash Concentrate:
One bottle, 100 ml, containing a phosphate buffered saline, concentrated 10-fold containing 0.5% Brij weight by volume (w/v). Dilute with deionized/distilled
water prior to use. Store at 2-8°C until expiration date.
Sample Diluent:
One bottle, 100 ml, containing a BSA solution with 0.09% sodium azide as a preservative. Store at 2-8°C until expiration date.
Cytomegalovirus IgM Controls
Three vials, each 2 ml of human serum in a 0.01 M phosphate buffer with BSA containing 0.09% sodium azide as a preservative. Store at 2-8°C until
expiration date.
CMV-HRP-Conjugate:
One bottle, 12 ml, containing purified CMV antigen conjugated with peroxidase, in a phosphate buffer solution with 0.02% Proclin. Store at 2-8°C until
expiration date.
TMB-Substrate:
One bottle, 12 ml, containing tetramethylbenzidine (TMB) and hydrogen peroxide stabilized in citrate buffer, pH 3.8. Store at 2-8°C until expiration date.
Stopping Solution:
One bottle, 15 ml, containing 0.3 M H2
SO4
in solution. Store at 2-8°C until expiration date.

PRECAUTIONS

For in vitro use
The following universal Good Laboratory Practices should be observed:
Do not eat, drink, smoke or apply cosmetics where immunodiagnostic material is being handled. Do not pipet by mouth. Wear lab coats and
disposable gloves when handling immunodiagnostic material. Wash hands thoroughly afterwards. Cover working area with disposable absorbent
paper. Wipe up spills immediately and decontaminate affected surfaces. Avoid generation of aerosols. Provide adequate ventilation. Handle and
dispose all reagents and materials in compliance with applicable regulations.
WARNING: POTENTIAL BIOHAZARDOUS MATERIAL
This kit may contain some reagents made with human ad animal source material (e.g. serum, plasma or bovine albumin) or used in conjunction with human
source or animal material. The material in this kit has been tested by CE recommended methods and found to be non-reactive for HIV-1/2 Antibodies, HCV
and HbsAg; the animal source material has no record of any infection. No available test method can offer complete assurance of eliminating potential
biohazardous risk. Handle all reagents and patient samples at a Biosafety Level 2, as recommended for any potentially infectious human material in the
Centers for Disease Control/National Institutes of Health manual "Biosafety in Microbiological and Biomedical Laboratories," 4th Edition, April 1999.

WARNING AND PRECAUTION:

Some of the reagents in this kit contain sodium azide as a preservative at concentrations below the regulatory limit of < 0.1%. Although significantly diluted,
concentrated sodium azide is an irritant to skin and mucous membranes, and may react with lead and copper plumbing to form explosive metal
azides, especially if accumulated. Additionally, TMB and Sulfuric Acid, in concentrated amounts are also irritants to skin and mucous membranes.
These substances are in diluted form and therefore may minimize exposure risks significantly but not completely. Provide adequate ventilation.
Avoid contact with skin, eyes and clothing. In case of contact with any of these reagents, wash thoroughly with water and seek medical advice.
Dispose all nonhazardous reagents by flushing with large volumes of water to prevent buildup of chemical hazards in the plumbing system.Sample
diluent and controls contain diluted BSA.
For further information regarding hazardous substances in the kit, please refer to the component specific MSDS by request.

SPECIMEN COLLECTION AND HANDLING

Serum should be used, and the usual precautions for venipuncture should be observed. Specimens may be stored at 2-8°C for 2 days. For longer periods,
store at –20°C. Do not use hemolyzed or lipemic specimens. Avoid repeated freezing and thawing of samples.

PREPARATION FOR ASSAY

A thorough understanding of this package insert is necessary for successful use of the product. Reliable results will only be obtained by using precise
laboratory techniques and accurately following the package insert. Bring all kit reagents and specimens to room temperature (~25°C) before use.
Thoroughly mix the reagents and samples before use by gentle inversion. Do not mix various lots of any kit component within an individual assay. Do not
use any component beyond the expiration date shown on its label. Incomplete washing will adversely affected the outcome and assay precision. To
minimize potential assay drift due to variation in the substrate incubation time, care should be taken to add the stopping solution into the wells in the same
order and speed to add the TMB Chromogen Solution. Avoid microbial contamination of reagents, especially of the conjugate, wash buffer and diluent.
Avoid contamination of the TMB Chromogen Solution with the Conjugate. Use a clean disposable pipette tip for each reagent. Avoid pipettes with metal
parts. Containers and semi-automatic pipette tips used for the Conjugate and TMB can be reused provided they are thoroughly rinsed with
deionized/distilled water and dried prior to and after each usage. The enzyme used as the label is inactivated by oxygen, and is highly sensitive to microbial
contamination, sodium azide, hypochlorous acid and aromatic chlorohydrocarbons often found in laboratory water supplies. Use high quality water. Avoid
exposure of the reagents to excessive heat or sunlight during storage and incubation.

PREPARATION OF REAGENTS:

Wash Solution:
Dilute 1:10 with deionized/distilled water prior to use. If crystals are present, they should be dissolved at 37°C before dilution. Pour 100 ml of the
Wash Concentrate into a clean container and dilute by adding 900 ml of deionized/distilled water. Mix thoroughly by inversion. The wash solution is
stable for 5 days at room temperature and 2 weeks at 2-8°C when stored in a tightly sealed bottle.
Microtitration Strips:
Select the number of coated strips required for the assay. The remaining unused wells should be placed in the resealable pouch with a desiccant
pack.

The pouch must be resealed to protect from moisture.
E-CVM-K02 ucap/ 10-11
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Assay Procedure:

All specimens and reagents to reach room temperature (∼25°C) before use.
1. Mark the microtitration strips to be used.
2. Dilute serum samples 1:101 distributing 10 µl of serum into 1 ml of Sample Diluent.
3. Pipette 100 µl of each diluted serum sample and ready-to-use Controls to the appropriate wells. Leave one well for the substrate blank.
4. Incubate for 45 minutes at 37°C.
5. Aspirate and wash each well four (4) times for 30 seconds with Washing Solution using an automatic microplate washer or manually using a
dispenser. Blot and dry by inverting plate on absorbent material.
NOTE: Use of an automatic microplate washer is strongly recommended. Incomplete washing will adversely affect assay precision. If a microplate
washer is not available, (a) completely aspirate the liquid from each well, (b) dispense 0.35 ml of the Wash Solution into each well, and (c) repeat step
(a) and (b) four times.
6. Add 100 µl of CMV-HRP Conjugate into each well.
7. Incubate for 45 minutes at 37°C.
8. Aspirate and wash each well four times for 30 seconds with Washing Solution using an automatic microplate washer or manually using a
dispenser.Blot and dry by inverting plate on absorbent material.
9. Add 100 µl of TMB Chromogen Solution to each well using a dispenser.
10. Incubate for 15 minutes at room temperature. Avoid exposure to direct sunlight.
11. Add 100 µl of Stopping Solution to each well using a dispenser.
12. Read the absorbance of the solution in the wells within 30 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set
the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 600 or 620 nm.

RESULTS

Calculate the mean absorbance for each control and unknown.
Qualitative results:
If the absorbance 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 average OD value of the sample and that of the Cut-Off. The sample is considered:
Positive: if the ratio is > 1.1.
Doubtful: if +/- 10% of the Cut-Off.
Negative: if the ratio is < 0.9.
If the result is doubtful, repeat the test. If it remains doubtful, collect a new serum sample.

LIMITATIONS OF THE PROCEDURE

• A serum sample obtained during the late phase of infection, when only IgG antibodies are present, may be negative by this procedure.
• The test result should be used in conjunction with information available from the evaluation of other clinical and diagnostic procedures.
• Avoid repeated freezing and thawing of reagents and specimens.
• Grossly hemolyzed, icteric or lipemic specimens should be avoided.
• Heat inactivated sera should be avoided.
• Serological data of immunocompromised patients and newborn children have restricted value.

QUALITY CONTROL

Subtract the value of the blank from all the other readings. The OD values of Cut off Control must be at least 0.2. Positive Control must have an OD at least
1.5 times that of CutOff.

PERFORMANCE CHARACTERISTICS

1. Sensitivity and Specificity
90 well selected human sera, collected from a clinical laboratory in Frankfurt, were analyzed by this Cytomegalovirus IgM Elisa and reference Elisa method.
Out of 90 samples, 12 were positive for the presence of IgM antibodies to CMV by RD-RatioDiagnostics (RD-labs) Elisa, and reference method showed 12
of them as positive. The results indicate that the RD-RatioDiagnostics CMV IgM ELISA has 100% sensitivity and specificity comparing to commercial ELISA
Test A as reference method.The results are summarized below.
Assay Comparison RD -RatioDiagnostics
Test A Positive Negative Brd. Line
Positive 12 0 0
Negative 0 78 0
Brd line 0 0 0
2. Precision
E-CVM-K02 ucap/ 10-11
2. Inter-assay Study 3. Intra-assay study
No of
Replicates 16 Serum 1 Serum 2 Serum 3
No of
Replicates 16 Serum 1 Serum 2 Serum 3
Mean 0.72 0.516 0.0266 Mean 1.89 0.737 0.029
SD 0.056 0.025 0.005 SD 0.038 0.044 0.004
CV% 7.8 4.8 20.9 CV% 1.99 5.9 12.2
4-4
3. Interference study
Interferences with lipemic, hemolytic or icteric sera are not observed up to a concentration of 5 mg/ml hemoglobin, 5 mg/ml triglycerides and 0.2 mg/ml
bilirubin.

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. M. Musiani et al.: Rapid detection of antibodies against Cytomegalovirus induced immediate early and early antigens by an enzyme linked
immunosorbent assay. J. Clin. Pathol. 37: 122 (1984).
5. F. de Ory et al.: Serological diagnosis of Cytomegalovirus infections: comparison of six commercial methods of ELISA. Serodiagnosis and
Immunotherapy 2: 423 (1988).
6. Lazzarotto T, Gabrielli L, Guerra B, Congenital cytomegalovirus infection: recent advances in the diagnosis of maternal infection, Hum Immunol, 2004
May; 65( 5 ) 410-415