Description

INTENDED USE
The anti-CMV (IgG) ELISA is intended for the qualitative detection of IgG antibodies to Cytomegalo-virus in human serum or plasma.
INTRODUCTION
Human cytomegalovirus (CMV) is a member of the Herpetoviridae family and is one of the seven human herpesviruses pathogenic for man. It is ubiquitous, species-specific and is spread by close human contact. The viral capsid, which has a DNA core, is icosahedral in shape and is formed of 162 capsomers. Surrounding the capsid, are one or more oval membranes containing lipids. CMV infection can be pri-mary or secondary. Primary infection may be acquired through different transmission routes and in different periods of life (i.e., congenital and post-natal infections). Following primary infection, CMV enters a latency phase during which the virus can be found in B lymphocytes. Subsequent reactivation of viral replication (secondary infection) may take place concomitantly with changes in the relationship between host and virus, such as pregnancy, serious illness, immunosuppressive therapy or stress. Congenital infection is transmitted transplacentally or at birth and can occur even if pregnant women already present antibodies to CMV (reinfection with exogenous virus), unlike rubella or toxoplasmosis. If seronegative women contract primary CMV infection during pregnancy, sequelae may be abortion, stillbirth or neonatal malformation. This is the case even if the birth of a normal, seronegative child is possible in almost 50% of maternal infections. The clinical picture of congenital CMV infection is always severe and includes psychomotor retardation, deafness, retinochoroiditis, microcephaly, hydrocephalus, cardiac disease, hepatitis, hepatosplenomegaly, thrombocytopoenia. The mortality rate is quite high. Most individuals (40-90%) acquire primary CMV infection during childhood or adulthood. Post-natal infections are transmitted through close contact with infected biological fluids (urine, saliva, breast milk, semen, cervical secretions, faeces), infected blood products and, occasionally, organ transplants. In immunocompetent individuals, the clinical picture of post-natal CMV infection is usually mild or asymptomatic. The commonest signs include fever, malaise, and increased serum transaminase levels without jaundice. By contrast in immunocompromized patients (organ transplant recipients, patients with AIDS, lymphoproliferative diseases or cancer), symptoms may be severe because of disseminated and/or visceral infection, and include splenomegaly, pneumonia, haemolyticanemia, myocarditis and encephalitis. In these patients the disease may be fatal. The immune response to CMV involves synthesis of antibodies of the IgM class some weeks after infection by CMV and, one week later, of antibodies of the IgG class. Levels of IgM to CMV usually increase for some weeks and decrease slowly thereafter, in four to six months. Occasionally, IgM may circulate for years. Specific IgM assay is instrumental in di-agnosing acute CMV infection, which remains difficult to identify on symptoms alone. However, it is not always possible to distinguish between primary and secondary infection, because reactivation may in-duce synthesis of IgM in immunocompromized patients. Specific IgG assay is useful in distinguishing subjects having acquired the disease from those who have not. This is particularly important in order to adopt suitable prophylaxis in susceptible individuals.
Determination of immune status to CMV is of particular importance (a) in immunocompromized patients, in whom the disease may have serious consequences; (b) in young fertile or pregnant women, so to avoid virus transmission to the foetus; (c) in organ transplant recipients and donors; and (d) in blood donors. White blood cells, namely polymorphonuclear leucocytes, may carry CMV which may infect blood or organ recipients. Detection of IgM to CMV allows adequate treatment to be administered, as needed. Prophylaxis of CMV infection may be achieved by administration of high titered virus specific immuno-globulin preparations. In addition, overt disease may be treated with specific antiviral agents (gancyclovir, foscarnet).
PRINCIPLE OF THE TEST
The anti-CMV (IgG) ELISA is based on indirect ELISA. Microwells are pre-coated with CMV antigens. Once the sample is added, anti-CMV (IgG), if present, binds to pre-coated antigens. After incubation and wash procedures, enzyme conjugate reagent is added, and the anti-human IgG inside binds to anti-CMV (IgG) attached to the solid phase in the previous step. After another incubation and wash procedures, add substrate solution and chromogen solution to initiate a chromogenic reaction. Once the color devel-opment is completed, add the stop solution, and then read the absorbance of each sample. The color intensity is directly proportional to anti-CMV (IgG) concentration.
MATERIALS PROVIDED
1. Coated Wells: microplate with CMV recombinant antigen coated wells (1 plate, 96 wells).
2. Enzyme Conjugate Reagent: horseradish peroxidase (HRP) labeled anti-human (IgG) MAb in stabi-lizing buffer (1 vial, 11.5 ml).
3. Negative Control: human serum/plasma non-reactive for anti-CMV IgG, diluted in buffer with preservatives (1 vial, 1.0 ml)
4. Positive Control: human serum/plasma reactive for anti-CMV IgG, diluted in buffer with preserva-tives (1
vial, 1.0 ml)
5. Wash Fluid Concentrate: PBS-Tween (1 bottle, 50.0 ml, 20×)
6. Substrate Solution: hydrogen peroxide (1 vial, 7.5 ml)
7. Chromogen Solution: tetramethylbenzidine (TMB) (1 vial, 7.5 ml)
8. Stop Solution: 1.0 M H2SO4 (1 vial, 7.5 ml)
9. Sample Diluent: buffer solution with preservatives (1 bottle, 11.5 ml)
MATERIALS REQUIRED BUT NOT PROVIDED
1. Micropipettes and multichannel micropipettes of appropriate volume (the use of accurate pipettes with disposable plastic tips is recommended)
2. Distilled water
3. Vortex mixer
4. Absorbent paper or paper towel
5. Incubator
6. Disposable reagent troughs
7. Instrumentation
1. Automated microplate strip washer
2. Microplate reader
STORAGE OF TEST KIT AND INSTRUMENTATION
1. Unopened test kits should be stored at 2 – 8℃ upon receipt. The test kit may be used throughout the
expiration date of the kit (1 year from the date of manufacture). Refer to the package label for the expiration date.
2. Microplate after first use should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened components will remain stable for at least 2 months, or until the expiry date, whichever is earlier, provided it is stored as prescribed above.
SPECIMEN COLLECTION, PREPARATION, TRANSPORT AND STORAGE
1. Plasma specimens may be used with this test but serum is the recommended specimen type for this assay.
2. When plasma specimen is used, it is recommended to use 1.5 g/L EDTA, 10.9 mmol/L sodium citrate or 20 – 30 U/ml herapin as the anticoagulant.
3. Collect all blood samples observing universal precautions for venipuncture.
4. Any turbidity and particulate matters might interfere with the test, hence must be removed by cen-trifugation before testing.
5. Allow samples to clot before centrifugation.
6. Specimens could be stored at room temperature for up to 8 hours. For specimens which are not to be assayed within 8 hours of collection, they must be stored at 2 – 8℃ for no more than 48 hours. Specimens to be transported or stored for a longer period should be stored frozen at – 20℃ or a lower temperature. Avoid multiple freeze-thaw cycles. After thawing, ensure specimens are thor-oughly mixed and brought to room temperature before being assayed.
7. Avoid grossly hemolytic and lipemic specimens.
8. Do not add sodium azide into the specimen as a preservative.
PRECAUTIONS AND WARNINGS
1. For in vitro diagnostic use only
2. This package insert must be fully understood prior to operation. The operation must be stringently in accordance with the instruction for use.
3. Micropipette tips are not interchangeable to eliminate cross contamination.
4. Specimens added must be mixed thoroughly. The presence of bubbles must be eliminated.
5. The microtiter plate must be washed completely. Each well must be fully injected with Wash Fluid. The strength of injection, however, is not supposed to be too intense to avoid overflow. In each wash cycle, liquids in each well must be dried. The microtiter plate should be stroked onto absor-bent paper to remove residual water droplets. It is recommended to wash the microtiter plate with an automated microplate stripwasher.
6. Wear disposable gloves when dealing with specimens and reagents. Wash hands after operations. All specimens must be regarded as potentially infectious. Waste material must be disposed of safely according to relevant local and national requirements.
7. Avoid any skin contact with all reagents. Stop Solution contains H2SO4, in case of contact, wash thoroughly with water.
8. Do not smoke, drink, eat or apply cosmetics in the working area. Do not pipette by mouth. Use protective clothing and disposable gloves.
REAGENT PREPARATION
1. Obtain the assays from the fridges. Place at room temperature (18 – 25℃) and equilibrate for at least 30 minutes.
2. Mix the reagents by gently inverting or swirling.
3. Dilute Wash Fluid Concentrate 20 folds with distilled water.
4. Calibrate the temperature of the incubator at 37℃. Only use after the temperature is stabilized.
IMPORTANT NOTES
1. Do not use reagents after expiration date.
2. Do not mix or use components from kits with different lot numbers.
3. Do no reuse the plate covers.
4. It is recommended that no more than 32 wells be used for each assay run, if manual pipetting is used, since pipetting of all specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
5. Replace caps on reagents immediately. Do not switch caps.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
ASSAY PROCEDURE
1. Secure the desired number of coated wells in the holder. Prepare data sheet with sample identifica-tion.
2. Leave 1 well for the blank, add 100 μl of Negative Control to the next 3 wells, then 100 μl of Positive Control to the following 2 wells. Add 100 μl of Sample Diluent into each of the rest of the wells, and then add 10 μl of specimen into each of the wells with added Sample Diluent.
3. Mix thoroughly by shaking on a vortex mixer for 10 seconds. Apply the plate seal, Incubate at 37℃ for 45 minutes.
4. Wash 6 times (an automated microplate strip washer is recommended); strike the microtiter plate onto absorbent paper at the end of the last wash cycle.
5. Add 100 μl of Enzyme Conjugate Reagent into each well except for the blank well.
6. Repeat steps 3, 4 and 5.
7. Add 50 μl of Substrate Solution, then 50 μl of Chromogen Solution into each well. Gently mix and incubate at 37℃ for 10 minutes without exposure to sunlight.
8. Add 50 μl of Stop Solution to each well. Mix thoroughly on a vortex mixer.
9. Immediately after mixing, read the absorbance of each well at 450 nm using 620 – 630 nm as the reference wavelength. Alternatively, the actual absorbance can be obtained by subtracting the ab-sorbance of each well at 450 nm with the absorbance of the blank well at 450 nm.
INTERPRETATION OF RESULTS
1. Test is valid only if absorbance of Positive Control ≥0.7, and absorbance of Negative Control ≤ 0.1.
2. Calculation of the cut-off value
Cut-off value = 0.1 + mean absorbance of Negative Control replicates (in case the mean absorbance of Negative Control replicates ＜0.05, use 0.05 instead of the actual mean)
INTERPRETATION OF RESULTS
The specimen is positive when the absorbance ≥ the cut-off value, otherwise, the specimen is negative.
PERFORMANCE CHARACTERISTICS
1. Sensitivity
The sensitivity reaches 99.28% (690/695)
2. Specificity
The specificity is 100% (25/25)
3. Precision
The coefficient of variance (CV) calculated from 10 replicate runs is not greater than 15%.
LIMITATIONS
1. This assay is only suited for aiding in the diagnosis of CMV infected patients, not to be used to screen blood sources.
2. This assay is not suited for monitoring the therapeutic treatment for CMV infections.
3. As with other sensitive immunoassays, there is a possibility that non-repeatable reaction may occur due to inadequate washing. So do aspirate the well or get rid of entire content of wells completely before adding the
wash solution.
4. As with all diagnostic tests, a definitive clinical diagnosis should not be made based only on the results of a single test. A complete evaluation by a physician is needed for a final diagnosis.
5. The test is for research use, further manufacturing and export only.
BATCH CODE
USE BY
MANUFACTURER
CONTAINS SUFFICIENT FOR <n> TESTS
IN VITRO DIAGNOSTIC MEDICAL DEVICE
TEMPERATURE LIMITATION
CATALOGUE NUMBER
CONSULT INSTRUCTIONS FOR USE
REFERENCES
1. Anet K., IDA O., et. al. J. Infect. Dis. 1985, 151, 772.
2. Revello,M. G. and Gerna, G. Clin. Microbiol. Rev. 2002, 15, 680.