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3245 | LIGHT DIAGNOSTICS™ CMV DFA Kit, ~250 tests

3245
10 mL  
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      Overview

      Replacement Information

      Key Spec Table

      Key ApplicationsFormatHostDetection Methods
      IF FITC M Fluorescent
      Description
      Catalogue Number3245
      Replaces3255
      Brand Family Chemicon®
      Trade Name
      • LIGHT DIAGNOSTICS
      • Chemicon
      DescriptionLIGHT DIAGNOSTICS™ CMV DFA Kit, ~250 tests
      OverviewThe Light Diagnostics Cytomegalovirus (CMV) Direct Immunofluorescence Assay (DFA) is intended for pre-CPE detection and identification of immediate early antigen of human CMV in cell culture.

      This product is not FDA approved for use in testing blood or plasma donors and is not intended for use in direct detection of cytomegalovirus in clinical specimens.

      Test Principle:

      Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay utilizes a direct immunofluorescence technique for identifying immediate early antigen of human CMV in infected tissue cultures. The FITC-labeled monoclonal antibody provided will bind to CMV immediate early antigen present in the cell culture. Unbound FITC-labeled monoclonal antibody is removed by rinsing with Phosphate Buffered Saline (PBS). FITC exhibits an apple-green fluorescence when illuminated by ultraviolet light, allowing visualization of the antigen antibody complex by fluorescence microscopy. Cell fluorescence indicates a positive specimen. Uninfected cells stain a dull red due to the presence of Evans blue in the FITC-labeled monoclonal antibody.

      Summary and Explanation :

      CMV is a member of the family Herpesviridae. Synthesis of viral DNA and assembly of capsids occur in the nucleus where infective progeny are released by budding through the nuclear envelope. Also characteristic of herpes viruses, CMV undergoes periods of latency and reactivation.

      CMV is species-specific but has been isolated from many animal species.1 The first human CMV was isolated in 1956 from embryonic fibroblasts of adenoidal tissue.2 The terminal morphology of CMV-infected permissive cells is that of a large cell with a prominent intranuclear inclusion (Cowdry Type A, or "owl eye" cell). Such cells were identified in tissues of fatally infected infants, which gave rise to the name "cytomegalic inclusion disease (CID)."3

      Humans are believed to be the only reservoir of human CMV, and postnatal infections are acquired by close contact with individuals shedding virus. Transmission may be through saliva, urine, cervical and vaginal secretions, semen, breast milk, tears, feces, and blood.4,5,6 Infection rates vary with geographic location and socioeconomic conditions.

      CMV infection has been detected in newborn infants and is the most commonly identified cause of congenital infection. Those infants who develop symptoms may exhibit severe disease with jaundice, hepatosplenomegaly, petechiae, and central nervous system abnormalities. The risk of infections is probably the same throughout pregnancy; CID occurs most often in fetuses infected during the first half of gestation. CMV may be transmitted to about 50% of the fetuses after primary maternal infection; about 10% of these will be clinically affected.7,8 Many congenitally infected infants appear normal at birth but may develop neurologic sequelae. The route of transmission of CMV from mother to fetus has not been well elucidated. It is possible that the spread is hematogenous through cord blood or placental tissue and amniotic cells.

      During the past decade, an increased population of immunosuppressed individuals has resulted in a resurgence of CMV. Induced immunosuppression via chemotherapy and transplant regimens has occurred more frequently. CMV infection is common in patients receiving renal,9 bone marrow,10, heart,11 lung,12 and liver transplants.13 The rapid spread of acquired immunodeficiency syndrome (AIDS) is also related to the CMV resurgence. CMV is the most common viral opportunistic infection14,15 and may be a cofactor in the pathogenesis of the Human Immunodefiency Virus.16 CMV infection in AIDS has been implicated in pneumonitis,17 colotis,18,19 retinitis,20 and dementia.21

      Light Diagnostics Cytomegalovirus Direct Immunofluorescence Assay provides Pre-CPE detection of CMV in centrifugation-enhanced shell vials with an early detection option at 24 hours after specimen inoculation. The kit provides sufficient reagents to process 80 shell vials.
      Materials Required but Not Delivered· Acetone, reagent grade; stored in glass

      · Methanol, reagent grade, stored in glass

      · Distilled water

      · Negative and positive controls (representative CMV strains available from ATCC, Rockville, MD)

      · Sodium hypochlorite solution, 0.05% (1:100 dilution of household bleach)

      · Sterile shell vials with 12 mm coverslips for growth of MRC-5 or CMV permissive human fibroblasts

      · Tissue culture media (RPMI or Eagle's Minimum Essential Medium with Fetal Bovine Serum and antibiotics, or equivalent)

      · Vial transport medium that is non-inhibitory to CMV (Hanks balanced salt solution with antibiotics, or equivalent)

      · Phosphate buffered saline (PBS)

      · Phosphate buffered saline, 0.1% Sodium azide (PBS/NaN3)

      · 0.1N NaOH

      · 0.1N HCl

      · Microscope slides, non-fluorescing

      · Aspirator device with disposable sterile Pasteur pipettes (glass pipettes are required for use with acetone)

      · Centrifuge capable of 700 to 950 x g with biohazard buckets and adapters for shell vials

      · Fluorescence microscope with appropriate filter combination for FITC (excitation peak = 490 nm, emission peak = 520 nm) with 100x, 200x, and 400x magnification (dry objective)

      · Forceps

      · Humid chamber

      · Incubator, 37° ± 1°C

      · Syringe and needle or other implement for removing coverslip from shell vial

      · Syringe filter, 0.45 micron

      · Ultrasonic water bath

      · Vortex mixer or sonicator
      References
      Product Information
      Components
      • CMV FITC-Labeled Monoclonal Antibody - (Catalog No. 5090). One
      • 10 mL dropper vial containing FITC conjugated monoclonal antibody against CMV immediate early antigen IE1 and IE2 (MW 68 to 72 kDa, CMV AD169 derived protein). The monoclonal is an IgG 2a isotype, complete molecule antibody. Conjugate contains protein stabilizer, Evans blue, Tween-20 and 0.1% sodium azide (preservative).
      • CMV Control Slides - (Catalog No. 5027). Two slides containing one well of CMV-infected cells (positive) and one well of uninfected cells (negative).
      • Phosphate Buffered Saline (PBS) - (Catalog No. 5087). One packet of phosphate buffered saline salts.
      • Tween 20/Sodium Azide Solution (100X) - (Catalog No. 5037). One
      • 10 mL vial containing Tween 20/sodium azide concentrate.
      • Mounting Fluid - (Catalog No. 5013). One 10 mL dropper vial containing tris-buffered glycerin (pH 9.0 ± 0.5), a fluorescence enhancer and 0.1% sodium azide (preservative).
      Detection methodFluorescent
      FormatFITC
      Applications
      Key Applications
      • Immunofluorescence
      Biological Information
      HostMouse
      Antibody TypeMonoclonal Antibody
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • For in vitro Diagnostic Use
      • CE Mark
      Storage and Shipping Information
      Storage ConditionsWhen stored at 2-8°C, the CMV kit is stable up to the expiration date printed on the kit label. Do not freeze or expose to elevated temperatures. Discard any remaining reagents after the kit expiration date.

      Warnings and Precautions:

      * Sodium azide, present in the conjugate, PBS, and mounting fluid, can form potentially explosive metal azides with lead and copper pipes. Upon disposal, flush with large volumes of water to prevent build-up in plumbing.

      * Pooling, substitution, or alteration of any reagent may cause erroneous results.

      · Do not mix or substitute reagents from different kit lots.

      * Do not allow the shell vials or slides to dry at any time during the staining procedure.

      * Handle all specimens and materials coming in contact with them as potentially infectious materials. Decontaminate with 0.05% sodium hypochlorite.

      * Acetone and methanol are extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks or flame. Avoid breathing vapor. Use adequate ventilation.

      * Avoid contact with Evans blue (present in the Anti-CMV FITC-labeled monoclonal antibody) as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.

      * Avoid contact with the Mounting Fluid, which contains a fluorescence enhancer that may be destructive to the tissue of mucous membranes. If contact occurs, flush with large volumes of water.

      * Performance of the fluorescence microscope is of critical importance in achieving satisfactory test results. Microscope objectives, bulb intensity and wattage, and filters may affect results.

      * PBS with Tween 20 and/or sodium azide should not be used in viral isolation procedures.

      * Do not mouth pipette reagents.
      Packaging Information
      Material Size10 mL
      Transport Information
      Supplemental Information
      Specifications