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RDT EBV IgM Assay Detection of IgM antibodies directed against EBV VCA and IEA-Zebra antigens in human serum by rapid immunofiltration assay

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RDT EBV IgM Assay Detection of IgM antibodies directed against EBV VA and IEA-Zebra antigens in human serum by rapid immunofiltration assay 1. INTENDED USE Bio-Rad RDT EBV IgM Assay is an immunofiltration
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RDT EBV IgM Assay Detection of IgM antibodies directed against EBV VA and IEA-Zebra antigens in human serum by rapid immunofiltration assay 1. INTENDED USE Bio-Rad RDT EBV IgM Assay is an immunofiltration test for the qualitative detection of IgM antibodies directed against EBV VA recombinant antigen and IEA-ZEBRA peptide in human serum. The test Bio-Rad RDT EBV IgM Assay may be used in conjunction with other EBV Serology (Bio-Rad RDT EBV IgG Assay) and clinical patient history as an aid in the diagnosis of EBV infection and mononucleosis. FOR IN VITRO DIAGNOSIS USE. 2. SUMMARY AND EXPLANATION OF THE TEST Epstein-Barr virus was first described in1964 in patients with Burkitt lymphoma. It is one of the common viruses found in Humans. It belongs to the Herpesvirinae family which includes other human pathogenic viruses (Herpes simplex 1 and 2, Varicella zona virus (VZV), cytomegalovirus (MV), HHV6, HHV7 and HHV8). After the primary infection, EBV remains for the life in B-lymphocytes and epithelia of the nose and throat. When transmitted in saliva, EBV leads to infections in infants which are however often asymptomatic or sub-clinical. In young people between 15 and 25 years of age there is a second peak of contagion. In 2/3 of the cases the disease infectious mononucleosis (Pfeiffer s glandular fever, kissing disease ) can develop. linical symptoms include loss of appetite, fatigue, fever, rash, pharyngitis, tonsillitis, lymphangitis, leucocytis, headache and rheumatic pains and hepatic disorders. These can be attributed to the infection and the lytic life cycle of the virus in B-lymphocytes and the subsequent immune response. In rare cases serious complications may occur such as hemolytic anemia, pneumonia, neurological occurrences or cardiological disorders. Over 90 % of adults are seropositive and are virus carriers, since EBV persists throughout lifetime in B-lymphocytes in certain epithelium cells. As in the case of other herpes viruses, reactivation can occur in patients with immune defects or when immune suppressive treatments are given; here again IgG/M/A antibody titer can rise against early EBV antigens. B-lymphocytes infected by EBV immortalize, i. e. they can proliferate infinitely. This property, normally controlled or suppressed by the immune system can result in a series of malignant diseases: Lympho-proliferative diseases due to immune deficiencies caused by illness (e. g. AIDS) or by immune suppressive treatment (polyclonal lymphoma) or the Burkitt lymphoma together with chromosomal changes. Together with further cofactors, EBV causes nasopharyngeal carcinoma, which has a very high incidence in southern provinces of hina. During the lytic life cycle of the virus, there is a cascade-like formation of various regulating proteins in the early phase ( Immediate Early Antigens, IEA; Early Antigens , EA) and later of structural proteins ( Virus apsid Antigens ; VA and Membrane Proteins; MA). In the latent status, only a few antigens are formed, including EBNA 1-6 (Epstein-Barr Nuclear Antigens). In some cases of EBV primary infection, a called Heterophile humoral response occurs leading to a polyclonal stimulation of IgM. Those Heterophile IgM are directed in particular against antigenic components of animal cells (in particular bovine). They are non specific markers of EBV infection but can be found also in other types of infection (MV, toxoplasma). The protein called ZEBRA (BamH1 Z Epstein-Barr Replication Activator also called EB1 or Zta) performs a fundamental role in the switch from viral latency to lytic cycle. This protein is a transcription factor which performs, in particular, an activating role in the transcription of certain viral genes and in its own synthesis. The test for anti ZEBRA antibodies has been studied as an indirect marker of a viral reactivation, in particular in HIV seropositive immunosuppressed subjects. The anti ZEBRA antibodies appear very early during primary infections and detection of the presence of anti ZEBRA IgM antibodies enables primary EBV infections to be diagnosed very early. Humoral response to primary EBV infection is rapid. Antibodies directed against EBV target different viral proteins with antibodies correlating to the state of the disease. In case of primary infection, IgM and IgG appear sequentially as follows: IEA (Immediate Early antigen), Early antigen diffuse (EA-D), VA (viral capsid antigen) and EBNA (nuclear antigen). A Primary infection is characterized by the presence of IgM against IEA (protein ZEBRA), EA-D and VA whereas IgG against EBNA are absent. In old infection, there are typically anti VA IgG and anti EBNA IgG. In the serological EBV diagnosis by means of immune fluorescence tests using infected EBV producing cells, antibodies are mainly differentiated against three antigen classes: EA - early antigens, VA capsid/structure antigens and EBNA nuclear antigens, which are expressed during the latent phase. Immunofluorescence is now largely replaced by easier to perform and read methods (ELISA, blot) which use recombinant protein or synthetic peptides. The IgM response to IEA-ZEBRA antigen is very early and highly intense which allows detection of specific antibodies earlier than to other markers. This detection can occur 2 weeks before the appearance of early antigen (EA) tests, enabling early detection of infected patients. Association of ZEBRA protein and VA p18 antigen allows detection of all stages of primary infection whilst enhancing clinical sensitivity of the test. 1 3. PRINIPLES OF THE PROEDURE Bio-Rad RDT EBV IgM Assay is an immunofiltration assay which uses recombinant VA p18 antigens and ZEBRA synthetic peptide for detection of anti EBV IgM in human serum. The test comprises of a membrane solid phase, which is held in a plastic envelope containing wicking material. The membrane is visible to the user through a test window on the front of the device. ZEBRA and VA p18 antigens are immobilized on the membrane as test band at the level of zone. Protein A has been bound to the membrane as a control band at the level of the zone. When a pre-diluted serum sample is passed through the membrane, any (IgM) anti ZEBRA and anti-va p18 antibodies present bind to the corresponding antigen at the level zone. When the onjugate (Anti-human IgM mouse monoclonal antibody labeled with blue latex particles) is added, it reacts with any human IgM antibodies captured on the Antigens present on the membrane, and a blue colour develops. The onjugate also reveals the Protein A immobilized on the control zone, which demonstrated that the reagents are correctly functioning. The cassette is designed to completely absorb the volume of added reagents. 4. REAGENTS Supplied quantities of reagents have been calculated to allow 25 tests. All reagents are exclusively for in vitro diagnostic use. SORB DIL ONJ WASH Label Nature of reagents Presentation assette Sample Diluent onjugate Washing Solution assette (Ready-to-use) Recombinant VA p18 and ZEBRA synthetic proteins and Protein A immobilized on a membrane Sample Diluent (Ready-to-use) 20% Tween 20 and PBS buffer solution, 1% Bovine Serum Albumin, 0.1% sodium azide, 1.5% Prolin 300 onjugate (Ready-to-use) Anti-human IgM mouse monoclonal antibody labeled with blue latex particles, 0.1% sodium azide, 1% Bovine Serum Albumin, 1.5% Prolin 300 Washing Solution PBS buffer solution, 0.1% sodium azide, 1.5% Prolin 300, 10% Tween x 1 cassette 2 x 21 ml 2 x 21 ml 2 x 21 ml For storage conditions and expiration date, please refer to the indications mentioned on the box. 5. WARNINGS AND PREAUTIONS 5.1. Health and safety instructions Wear disposable labcoat, gloves and ocular protection when handling samples and reagents. Do not pipette by mouth. Do not eat, drink or smoke during the handling of the samples and the test. The samples of blood and serum must be regarded as potentially infectious. When performing the test, necessary precautions for handling of infectious products should be taken. The various elements of the test and the samples should be handled according to the procedure reserved for potentially infectious waste. Any material, including washing solution, that comes directly in contact with samples should be considered capable of transmitting infectious diseases. Avoid spilling samples or solutions containing samples. Spills must be rinsed with bleach diluted to 10 % and dried with adsorbent paper. The material used for cleaning must be discarded in a contaminated residue container. Patient samples, as well as contaminated material and products should be discarded after decontamination only: - either by immersion in bleach at the final concentration of 5 % of sodium hypochlorite during 30 minutes, - or by autoclaving at 121 for 2 hours at the minimum. Do not introduce solutions containing sodium hypochlorite into the autoclave. aution: Some of the reagents contain Prolin 300 1.5% For risks and security recommendations refer to the table at the end of the package insert 5.2. Precautions related to the procedure The reliability of the results depends on correct implementation of the following Good Laboratory Practices: Do not use expired reagents. Do not mix or associate within a given run reagents from different lots. Before use, to allow reagents to reach room temperature ( ). Use glassware thoroughly washed and rinsed with deionized water or, preferably disposable material. Use a new pipette tip for each sample. heck the pipettes and other equipments for accuracy and correct operations. 2 6. STORAGE AND VALIDITY OF OPENED AND / OR REONSTITUTED REAGENTS The kit must be stored at When the kit is stored at +2-8 before opening, each component can be used until the expiration date indicated on the outer label of the kit. The cassette (SORB) should be kept in the pouch. If so, the cassette is stable until the expiry date indicated on the pouch. Open the pouch before just before running the assay. All vials have to be stored at +2-8 immediately after each use. In the absence of contamination, they are stable until the expiration date indicated on the label. DO NOT FREEZE THE REAGENTS 7. SAMPLES It is recommended to perform the test immediately after collection and centrifugation of the sample. Otherwise, the serum can be stored at 2-8 for 7 days maximum. For longer storage, serum has to be frozen at 20. Frozen samples have to be brought to room temperature before performing the test and thoroughly mixed. Three freeze/thaw cycles should not affect results. Further freezing and thawing of samples should be avoided. In case of sample shipment, proceed according to regulation for shipment of products of human origin. For lyophilized preparation, it is recommended to carefully follow the corresponding reconstitution procedure and wait until the reconstituted serum reaches room temperature before running the test Do not heat the samples. 8. MATERIAL REQUIRED 8.1. Materials provided Refer to section 4, REAGENTS 8.2. Materials required but not provided Blood collection tube Disposable tubes. entrifuge Timer Disposable gloves. Goggles or safety glasses. Automatic or semi-automatic, adjustable or preset, pipettes or multi-pipettes, to measure and dispense 10 µl to 1000 µl, and 1,5 ml. Sodium hypochlorite (bleach) and sodium bicarbonate. ontainer for biohazard waste. 9. INSTRUTION FOR USE 9.1. Reagents preparation All the reagents are ready-to-use Specimen preparation Serum collected on dry tubes is the recommended sample type. Observe the following recommendations for the handling, processing, and storing serum samples: ollect all serum samples observing routine precautions. Allow samples to clot completely before centrifugation Keep tubes stoppered at all times. After centrifugation separate the serum and store it in a tightly stoppered storage tube Procedure Strictly follow the assay procedure and Good Laboratory Practices. Before use, allow reagents to reach room temperature ( ). 1. Take the cassette (SORB) out of the aluminum pouch for immediate use. 2. Drop 25 µl of serum in the bottom of a disposable tube and add 1.5 ml of dilution buffer (DIL). Thoroughly mix by pipetting back and forwards. 3. Use a laboratory pipette or directly drop the diluted sample from the tube into the cassette window. Allow the diluted sample to completely drain into the cassette window (about 15 sec.) 4. Thoroughly mix the onjugate reagent suspension (ONJ) by gentle agitation before testing. Pipette 1.5 ml of conjugate (ONJ) and drop it fully into the cassette window. Allow the developing reagent to completely drain into the cassette window (about 15 sec.) 5. Pipette 1.5 ml of Washing Solution (WASH) and drop it fully into the cassette window. Allow the washing buffer (WASH) to completely drain into the cassette window (about 15 sec.) 6. Read the result as soon as washing buffer is fully drained into the device window. Read within 20 minutes 3 10. QUALITY ONTROL An internal control procedure is incorporated into the test (control band). This insures that the reagents are correctly functioning. Absence of filtration or abnormal slow filtration ( 1 min.) of any of the 3 reagents as well as presence of a strong blue background on the membrane after washing step are indicative of instability of the device. In this case results should be considered as invalid and the sample should be retested. It is recommended to test a positive and negative controls in following circumstances: When opening a new lot When opening the first kit of new delivery. When used by a new operator All manufactured reagents are prepared according to our Quality System, starting from reception of raw material to commercialization of the final product. Each lot is submitted to quality control assessments and is released to the market only after conforming to pre-defined acceptance criteria. The records related to production and controls of each single lot are kept within Bio-Rad. 11. INTERPRETATION OF RESULTS Presence of a band is considered when a clear blue band appears on the membrane. In case of faint or shadow band, it is recommended to repeat the test on a new sample. POSITIVE: Anti EBV IgM positive (ZEBRA and/or VA p18) Presence of blue band (even of weak intensity) and blue control band. ZEBRA and/or VA p18 IgM antibodies presence is indicative of primary EBV infection or reactivation. NEGATIVE: Anti VA p18 and anti IEA-ZEBRA IgM negative. Absence of blue band and presence of blue control band. Anti ZEBRA and anti VA p18 IgM antibodies are missing which corresponds to a seronegative reaction or very early stages of a primary infection. INVALID: Absence of blue control band A wrong procedure is the most frequent cause of invalid results. The test has to be repeated with a new cassette Presence of dark blue background A strong blue background may appear on the membrane if one of the filtration steps takes longer than 1 min. In this case the test is invalid. Run a second test with the second set of vials supplied in the kit 12. TEST LIMITATIONS Diagnosis of EBV infection can only be established on the basis of a combination of clinical and biological data. 1. Bio-Rad RDT EBV IgM Assay is intended for screening of IgM directed against EBV VA p18 and ZEBRA antigens in serum. The test is for in vitro diagnostic use only. Bio-Rad RDT EBV IgM Assay does not allow quantitative detection of anti VA p18 and anti ZEBRA IgM antibodies nor the monitoring of antibodie level in known patients. 2. Bio-Rad RDT EBV IgM Assay indicates the presence of anti VA p18 and/or anti ZEBRA IgM antibodies in serum and cannot be used as single criteria for the diagnostic of primary EBV infection. A negative Bio-Rad RDT EBV IgM Assay result cannot completely exclude an early EBV primary infection. 3. As for all in vitro diagnosis tests, the result should be interpreted by the physician based on all other available clinical and biological data (mainly EBV IgG and Heterophile antibodies). Testing of two paired sera may also help to get accurate results. 4. ontrol will only validate the good functioning of the assay reagents but won't insure that the serum was added.. 5. Results from immunosuppressed patients should be considered with caution. 6. Bio-Rad RDT EBV IgM Assay can be used with serum only. Whole blood and plasma validation has not been carried out. 4 13. PERFORMANES HARATERISTIS Relative Specificity and Sensitivity Relative Performance of Bio-Rad RDT EBV IgM Assay was evaluated on a total of 158 samples from blood donors that were characterized using commercial VA IgM assay. Following results were observed: VA IgM ommercial EIA VA IgM assay Total samples Positive Negative Bio-Rad RDT Positive EBV IgM Assay Negative Total samples All equivocal results were excluded from the calculation. Total VA IgG concordance: 94.3% (149/158). 1. Bio-Rad RDT EBV IgM Assay Relative Specificity was determined comparatively to the negative results obtained with the commercial assays. VA IgM relative specificity: 94.9% (87.5% %). The four discordant results were tested an Immunoblot IgM assay; one was confirmed negative and three positive. The Bio- Rad RDT EBV IgM Assay specificity was then redefined at 98.7% (92.9% 100.0%). 2. Bio-Rad RDT EBV IgM Assay Relative Sensitivity was determined comparatively to the positive results obtained with the commercial assays. VA IgM relative sensitivity: 93.7% (85.9% %). None of the discrepant results was tested with a third method due to insufficient sample volume Precision 1. Repeatability Bio-Rad RDT EBV IgM Assay was evaluated for repeatability by testing four sera (one negative, one weak positive, one positive and one strong positive) 30 times on one lot. No changes in sample status were observed demonstrating repeatability of 100%. 2. Reproducibility Bio-Rad RDT EBV IgM Assay was evaluated for reproducibility by testing four sera (one negative, one weak positive, one positive and one strong positive) four times on three different lots. No changes in sample status or in band intensity were observed demonstrating reproducibility of 100% ross Reactivity Sera containing IgM antibody to potential cross-reacting pathogens were tested with Bio-Rad RDT EBV IgM Assay. Following results were observed: Pathology Total number of sera Positive results with Bio-Rad RDT EBV IgM Assay Lupus 6 0 Rhumatoid Polyarthritis 6 1 Hepatitis A 11 3 Hepatitis B 5 0 Hepatitis 5 0 Hepatitis E 3 0 HIV MV HSV 6 2 VZV 6 1 Parvovirus B Measles 4 0 Rubella 4 0 Mumps 3 0 Dengue 3 0 hikungunya 3 0 False IgM positive sample on other commercial automated assay 2 0 Total On 108 potentially cross-reacting pathogen tested sera, 23 were IgM positive with Bio-Rad RDT EBV IgM Assay, 11 corresponding to MV primary infection patients, 4 to primary Parvovirus B19 infection and 3 to acute hepatitis A. 5 14. BIBLIOGRAPHY REFERENES 1. Brousset, P. et al. Epstein-Barr virus (EBV) replicative gene expression in tumour cells of AIDS-related non Hodgkins lymphoma in relation to D4 cell number and antibody titres to EBV, AIDS, vol. 8, 1994, pp han, K.H. et al. Development and evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M enzyme-linked immunosorbent assay based on the 18-kilodalton matrix protein for diagnosis of primary EBV infection, Journal of clinical microbiology, vol 36, 1998, pp heng, Hwez-ming et al. Epstein-Barr Virus Nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors. Journal of clinical microbiology, vol. 29, N 10, Oct. 1991, pp heng, Hwee-Ming. Linear epitopes of the replication-activator protein of Epstein-Barr virus recongised by specific serum IgG in nasopharyngeal carcinoma. ancer immunol immunother, vol. 40, 1995, pp Epstein, M.A., B.B. Achong, and Y.M. Barr Virus particles in ultured Lymphoblasts from Burkitt s Lymphoma. In: Lancet 1: Epstein, M.A., Y.M. Barr, and B.G. Achong Studies wi
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