DRG Cytomegaly Virus CMV IgG Enzyme Immunoassay Kit User Manual

Instructions for Use
CMV IgG ELISA

Cytomegaly Virus CMV IgG Enzyme Immunoassay Kit

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| EIA-3468
| 96
| 0197

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The DRG CMV IgG ELISA provides materials for the quantitative and qualitative determination of IgG-class antibodies to Cytomegalovirus (CMV) in human serum and plasma (EDTA, lithium heparin or citrate plasma).
This assay is intended for in vitro diagnostic use only.

Summary and Explanation

Cytomegalovirus (CMV) is a member of the herpesvirus group (Beta subfamily, DNA virus of 150 nm – 200 nm). These viruses share a characteristic ability to remain dormant within the body over a long period. Initial CMV infection, which may have few symptoms, is always followed by a prolonged, inapparent infection during which the virus resides in cells without causing detectable damage or clinical illness. Severe impairment of the body´s immune system by medication or disease consistently reactivates the virus from the latent or dormant state.
CMV is found universally throughout all geographic locations and socioeconomic groups, and infects between 50 % and 85 % of adults.
CMV infection is more widespread in developing countries and in areas of lower socioeconomic conditions.
For the vast majority of people, CMV infection is not a serious problem, but it is to certain high-risk groups: the unborn baby during pregnancy, people who work with children, and immunocompromised persons, such as organ transplant recipients and persons infected with HIV.
The presence of virus resp. infection may be identified by Microscopy, PCR, Serology: CBR and detection of antibodies by ELISA.
IgM antibodies are the first to be produced by the body in response to a CMV infection. They are present in most individuals within a week or two after the initial exposure. IgM antibody production rises for a short time period and then declines. After several months, the level of CMV IgM antibody usually falls below detectable levels. Additional IgM antibodies are produced when latent CMV is reactivated.
IgG antibodies are produced by the body several weeks after the initial CMV infection and provide protection from primary infections. Levels of IgG rise during the active infection, then stabilize as the CMV infection resolves and the virus becomes inactive. After a person has been exposed to CMV, he or she will have some measurable amount of CMV IgG antibody in their blood for the rest of their life. CMV IgG antibody testing can be used, along with IgM testing, to help confirm the presence of a recent or previous CMV infection.

PRINCIPLE OF THE TEST

The DRG CMV IgG ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA).
Microtiter wells as a solid phase are coated with inactivated grade 2 Cytomegalovirus (CMV) antigen (strain AD-169).
Diluted patient specimens and ready-for-use controls are pipetted into these wells. During incubation Cytomegalovirus (CMV)-specific antibodies of positive specimens and controls are bound to the immobilized antigens.
After a washing step to remove unbound sample and control material horseradish peroxidase conjugated anti-human IgG antibodies are dispensed into the wells. During a second incubation this anti-IgG conjugate binds specifically to IgG antibodies resulting in the formation of enzyme-linked immune complexes.
After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid.
The intensity of this color is directly proportional to the amount of Cytomegalovirus (CMV)-specific IgG antibody in the patient specimen. Optical density at 450 nm is read using an ELISA microtiter plate reader.

WARNINGS AND PRECAUTIONS

• This kit is for in vitro diagnostic use only. For professional use only.
• Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
• All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
• Avoid contact with Stop Solution containing 0.2 mol/L H2SO4. It may cause skin irritation and burns.
• TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
• The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided
• Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
• Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
• Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
• Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
• Allow the reagents to reach room temperature (20 °C to 25 °C) before starting the test. Temperature will affect the optical density readings of the assay. However, values for the patient samples will not be affected.
• Never pipette by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
• Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
• Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
• Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
• Do not use reagents beyond expiry date as shown on the kit labels.
• All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
• Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
• Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guideline or regulation.
• For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG.

REAGENTS

Reagents provided

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells;
   Wells coated with inactivated grade 2 Cytomegalovirus (CMV) antigen (strain AD-169). (incl. 1 cover foil)

2. Sample Diluent *, 1 vial, 100 mL, ready to use, colored yellow; pH 7.2 ± 0.2.

3. Standard (Standard 1 – 3)*, 3 vials, ready to use; Concentrations: 10, 40, 80 DU/mL, Standard 1, 2.0 mL, colored green, green cap Standard 2, 1.0 ml, colored blue, blue cap Standard 3, 1.0 mL, colored red, red cap

4. Pos. Control *, 1 vial, 1.0 mL, ready to use; colored purple, black cap.

5. Neg. Control *, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.

6. Enzyme Conjugate *, 1 vial, 20 mL, ready to use, colored red, antibody to human IgG conjugated to horseradish peroxidase.

7. Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).

8. Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/L H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.

9. Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 6.5 ± 0.1 see ,,Preparation of Reagents”.

• contain non-mercury preservative

Equipment and material required but not provided

• A microtiter plate calibrated reader (450 nm/620 nm ± 10 nm)
• Calibrated variable precision micropipettes
• Incubator 37 °C
• Manual or automatic equipment for rinsing wells
• Vortex tube mixer
• Freshly distilled water
• Timer
• Absorbent paper

Storage Conditions and stability of the Kit

When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for 8 weeks if stored as described above.

Reagent Preparation

Allow all reagents and required number of strips to reach room temperature (20 °C to 25 °C) prior to use.

Wash Solution
Dilute Wash Solution 1 + 19 (e.g. 10 mL + 190 mL) with fresh and germ free redistilled water. This diluted wash solution has a pH value of 7.2 ± 0.2. Consumption: ~ 5 mL per determination. Crystals in the solution disappear by warming up to 37 °C in a water bath. Be sure that the crystals are completely dissolved before use. The diluted Wash Solution is stable for 1 week at 2 °C to 8 °C.

Disposal of the Kit

The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Safety Data Sheets.

Damaged Test Kits

In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations.

SPECIMEN COLLECTION AND PREPARATION

Serum or plasma (EDTA-, lithium heparin- or citrate plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens. For further information refer to chapter “Interfering Substances”

Specimen Collection Serum:

Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

Plasma:
Whole blood should be collected into centrifuge tubes containing anti- coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

Specimen Storage and Preparation

Specimens should be capped and may be stored for up to 5 days at 2 °C to 8 °C prior to assaying. Specimens held for a longer time (up to 12 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Specimen Dilution

Prior to assaying dilute each patient specimen 1 + 100 with Sample Diluent; e.g. 10 µL of specimen + 1 mL of Sample Diluent mix well, let stand for 15 minutes mix well before use.
For patients with concentrations greater than Standard 3 a second 1:10 dilution of this 1+100 diluted patient sample should be performed; e.g. 20 µL of first sample dilution + 180 µL Sample Diluent (mix well).
Please note: Standards and Controls are ready for use and must not be diluted!

ASSAY PROCEDURE

General Remarks

• It is very important to bring all reagents, samples and controls to room temperature before starting the test run!
• Once the test has been started, all steps should be completed without interruption.
• Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination
• Optical density is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
• As a general rule the enzymatic reaction is linearly proportional to time and temperature.
• Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
• To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells.
• During 37 °C incubation cover microtiter strips with foil to avoid evaporation.

Test Procedure

Prior to commencing the assay, dilute Wash Solution, prepare patient samples as described in point 5.3, mix well before pipette and establish carefully the distribution and identification plan supplied in the kit for all specimens and controls.

1. Select the required number of microtiter strips or wells and insert them into the holder. Please allocate at least:
   1 well| (e.g. A1)| for the substrate blank,
   ---|---|---
   1 well| (e.g. B1)| for the Neg. Control
   4 wells| (e.g. from C1 on)| for Standard 1-3
   1 well| (e.g. G1)| for the Pos. Control.

It is left to the user to determine standards, control, and patient samples in duplicate.

2. Dispense****
   100 µL of Neg. Control|  into well B1
   ---|---
   100 µL of Standard 1| into well C1 + D1
   100 µL of Standard 2| into well E1
   100 µL of Standard 3| into well F1
   100 µL of Pos. Control| into well G1 and
   100 µL of each d I l u t e d|  sample with new disposable tips into appropriate wells. Leave well A1 for substrate blank!

3. Cover wells with foil supplied in the kit. Incubate for 60 minutes at 37 °C.

4. Briskly shake out the contents of the wells.
   Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.
   Important note: The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

5. Dispense 100 µL Enzyme Conjugate into each well, except A1.

6. Incubate for 30 minutes at room temperature (20 °C to 25 °C). Do not expose to direct sunlight!

7. Briskly shake out the contents of the wells. Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.

8. Add 100 pL of Substrate Solution into all wells.

9. Incubate for exactly 10 minutes at room temperature (20 °C to 25 °C) in the dark.

10. Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well. Any blue color developed during the incubation turns into yellow.
    Note: Highly positive patient samples can cause dark precipitates of the chromogen!

11. Read the optical density at 450 nm/620 nm with a microtiter plate reader within 30 minutes after adding the Stop Solution.

6.3 Measurement
Adjust the ELISA microplate or microstrip reader to zero using the substrate blank in well A1. If – due to technical reasons – the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the optical density (OD) value of well A1 from all other OD values measured in order to obtain reliable results!
Measure the optical density (OD) of all wells at 450 nm and record the OD values for each control and patient sample in the distribution and identification plan.
Dual wavelength reading using 620 nm as reference wavelength is recommended.
Where applicable calculate the mean OD values of all duplicates.

CALCULATION OF RESULTS

7.1 Validation of the Test Run
The test run may be considered valid provided the following criteria are met:

7.2 Calculation of quantitative Results
In order to obtain quantitative results in DU/mL (DU = DRG Units) plot the (mean) OD values of Neg. Control and the 3 Standards 1, 2, and 3 on (linear/linear) graph paper in a system of coordinates against their corresponding concentrations (0, 10, 40 and 80 DU/mL) and draw a standard calibration curve (OD values on the vertical y-axis, concentrations on the horizontal x-axis). Read results from this standard curve employing the (mean) OD values of each patient specimen and control. All validated computer programs can be used, that provides the following function: 4 PL (4 Parameter Logistics). We use the DRG regression program for Windows (4 parameter Rodbart regression). If other regression software is used, the obtained values have to be validated by the user.
NOTE: Values of additionally (1:10, in total 1:1000) diluted patient samples must be multiplied by the appropriate dilution factor in order to obtain correct results! (Dilution: 1:10 = Dilution factor: 10). (See chapter “5.3 Specimen Dilution”).
7.3 Interpretation of quantitative Results
The following values should be considered as a guideline:

7.4 Calculation of qualitative Results
OD value of Standard 1 (Cut-off) = CO
Example: 0.4 = CO
7.5 Interpretation of qualitative Results

later – with new patient samples Results in the second test again in the grey zone NEGATIVE
POSITIVE| Mean ODpatient > ODCO + 10 %

QUALITY CONTROL

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.
It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results.
If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above-mentioned items without finding any error contact your distributor or DRG directly.

PERFORMANCE CHARACTERISTICS

9.1 Assay Dynamic Range
The range of the assay is between 1.18 ­ 80 DU/mL.
9.2 Specificity of Antigen (Cross-Reactivity)
No cross-reactivity was found for Herpes-simplex Virus 1 and 2, Varicella zoster Virus, and Epstein-Barr Virus (VCA).
9.3 Analytical Sensitivity
The analytical sensitivity of the DRG ELISA was calculated by adding 2 standard deviations to the mean OD of 20 replicate analyses of the negative control and was found to be 1.18 DU/mL (OD450nm 0.075).
9.4 Diagnostic Specificity
Diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. (Detected by method comparison with NovaTec ELISA with three lots of DRG ELISA, 109 samples, therefrom 43 negative samples are assayed.) It is 100% (for all three DRG production lots).
9.5 Diagnostic Sensitivity
Diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. (Detected by method comparison with NovaTec ELISA with three lots of DRG ELISA, 109 samples, therefrom 66 positive samples are assayed.) It is 100% (for all three DRG production lots).
9.6 Method Comparison
The DRG ELISA was compared with the NovaTec CMV IgG ELISA. 109 serum samples are assayed.

Agreement: 100%
9.7 Reproducibility
9.7.1 Intra-assay
The intra-assay (within-run) precision of the DRG CMV IgG ELISA was determined by 20 x measurements of 12 serum samples covering the whole measuring range.

9.7.2 Inter-assay
The inter-assay variation of the DRG CMV IgG ELISA was determined with 3 samples with 2 production kits in 10 independent runs with 2 replicates per run.

9.8 Recovery
Samples have been spiked by adding 3 solutions with known concentrations in a 1:1 ratio. The % recovery has been calculated by multiplication of the ratio of the measurements and the expected values by 100 (expected value = (endogenous value + added value) / 2; because of a 1:2 dilution of serum with spike material).

| Sample 1| Sample 2| Sample 3
---|---|---|---
Concentration [DU/mL]| 71.92| 2.62| 3.49
Average Recovery                  •| 95.6| 101.6| 98.6
Range of Recovery [%]| from| 87.6| 91.3| 82.2
to| 109.4| 118.6| 119.8

9.9 Linearity
Three samples (serum) containing different amounts of analyte were serially diluted with sample diluent and assayed with the DRG ELISA. The percentage recovery was calculated by comparing the expected and measured values for the analyte.

| Sample 1| Sample 2| Sample 3
---|---|---|---
Concentration [DU/mL]| 165.38| 155.95| 127.75
Average Recovery| 99.7| 100.6| 98.8
Range of Recovery [%]| from| 89.6| 91.8| 95.0
to| 114.4| 108.0| 105.7

LIMITATIONS OF USE

Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the optical density values. In immunocompromised patients and newborns, serological data only have a restricted value.
10.1 Interfering Substances
Haemoglobin, bilirubin, and triglyceride have an influence on the assay results already in low concentrations.

LEGAL ASPECTS

11.1 Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover, the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test. The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.
11.2 Therapeutic Consequences
should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient. Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis should take into consideration clinical history, symptomatology as well as serological data. Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived. The test result itself should never be the sole determinant for deriving any therapeutic consequences.
11.3 Liability
Any modification of the test kit and/or exchange or a mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement. Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during the transportation is not subject to the liability of the manufacturer.

Symbol|
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| European Conformity
| Consult instructions for use •
| In vitro diagnostic medical device •
| Catalog number •
| Batch code •
| Contains sufficient for tests •
| Temperature limit •
| Use-by date *
| Manufacturer •
| Biological risks•
| Caution •
| For research use only
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Distributed by| Distributed by
 Content|  Content
 Volume/No.| Volume/Na
Microtiter wells| Microtiter wells
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Enzyme Conjugate| Enzyme Conjugate
Substrate Solution| Substrate Solution
Stop Solution| Stop Solution
Zero Standard| Zero Standard
Standard| Standard
Control| Control
Pos. Control| Positive Control
Neg. Control| Negative Control
Cut-off Control| Cut-off Control
Wash Solution| Wash Solution
IgG-RF Sorbent| Rheumatoid factor absorbent
Sample Diluent| Sample Diluent
Conjugate Diluent| Conjugate Diluent

DRG Instruments GmbH, Germany
Frauenbergstralle 18, D-35039 Marburg
Phone: +49 (0)6421-1700 0,
Fax: +49 (0)6421-1700 50
Website: www.drg-diagnostics.de
E-mail: [email protected]

Distributed by:

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081
Phone: 973-564-7555,
Fax: 973-564-7556
Website: www.drg-international.com
E-mail: [email protected]

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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