Light Diagnostics type specific monoclonal antibody Coxsackievirus B1 is intended for use in indirect fluorescence screening for the presumptive identification of Coxsackie B1 virus obtained in cell culture and not intended for testing directly on human specimens.
Light Diagnostics Coxsackievirus B1 Monoclonal Antibody (MAb Cox B1) can be used to identify a Coxsackie B1 viral isolate in cell culture using an indirect immunofluorescence assay (IFA). The monoclonal antibody provided will bind to the type specific Coxsackie B1 isolate present on the cell culture slide. Unbound monoclonal antibody is removed by rinsing with phosphate buffered saline (PBS). A secondary FITC (fluorescein isothiocyanate) labeled antibody is then added which will bind to the antigen-antibody complex. Unbound secondary antibody is removed by rinsing with PBS. FITC exhibits an apple green fluorescence when illuminated by ultraviolet light allowing visualization of the complex by microscopy. A positive result is indicated by cell fluorescence. Non-infected cells stain a dull red if Evans Blue counterstain is used in the FITC-labeled secondary antibody or used elsewhere in the procedure.
Background and Clinical Significance:
Enteroviruses are classified to be in the picornavirus family, pico [small] + RNA [ribonucleic acid] + virus. Picornaviruses are among the smallest and simplest ribonucleic acid containing viruses known . The RNA for many enteroviruses have now been cloned and complete genomic sequences have been obtained. The RNA from all the sequenced enteroviruses are similar in length, about 7400 nucleotides, and have identical organization .
The human alimentary tract is the predominant site of enterovirus replication and the viruses were first isolated from enteric specimens. These viruses are the causes of paralytic poliomyelitis, aseptic meningitis-encephalitis, myocarditis, pleurodynia, hand-foot-and-mouth disease, conjunctivitis, and numerous other syndromes associated with extra-intestinal target organs. There are 67 numbered types of enteroviruses in the enteroviruses family : Polioviruses (3), Coxsackieviruses A (23), Coxsackieviruses B (6), Echoviruses (31), and other Enteroviruses (4)
Enteroviruses, including Echoviruses and Coxsackieviruses, have been reported as the major etiologic agents of aseptic meningitis . Clinical syndromes associated with infections by each type of enterovirus have also been reported . Coxsackievirus B1 can cause pleurodynia, aseptic meningitis, severe systemic infection in infants, meningoencephalitis, pericarditis, myocarditis, and undifferentiated febrile illness.
Establishing an association between an enterovirus and a particular disease in a patient requires laboratory confirmation of infection, usually by either isolation of the virus, or documentation of a specific serologic response in a properly timed specimen. Detailed descriptions of principals and procedures for diagnosis of enterovirus infections have been published [4-7]. Cell culture techniques have made the accurate detection of enteroviruses possible [8-10]. The identification of the enterovirus isolates will help prevention, treatment and understanding of the infectious diseases, and even discovery of new virus isolates. The typing of enterovirus isolates is generally accomplished by neutralization with type specific pools of immune sera . This method is time consuming (7 days or more) and expensive. As an alternative, typing of enteroviruses with type specific monoclonal antibody and/or group specific monoclonal antibody pool(s) by the indirect immunofluorescence assay (IFA) is potentially more rapid and less expensive [12 - 18].
Coxsackievirus B1 Monoclonal Antibody - (Catalog No. 3304).
One dropper vial containing 1 mL, sufficient for 25 tests, ready to use, mouse IgG(2a) monoclonal antibody against the Coxsackie B1 virus, protein stabilizer and 0.1 % sodium azide (preservative).
Materials Required But Not Provided:
· Acetone, reagent grade; stored in glass.
· Distilled water.
· Sodium hypochlorite solution, 0.05% (1:100 dilution of household bleach).
· Sterile shell-vials with 12 mm coverslips containing monolayer of cell line appropriate for growth of Enteroviruses.
· Tissue culture media (RPMI or Eagle′s Minimum Essential Medium with fetal bovine serum and antibiotics, or equivalent).
· Viral transport medium which is non-inhibitory to Enterovirus.
· 0.1N NaOH.
· 0.1N HCl.
· Microscope slides, non-fluorescing.
· No. 1 cover slips.
· Negative and positive control slides.
· Anti-Mouse IgG/FITC Conjugate (Catalog No. 5008).
· Normal Mouse Antibody to be used as negative control.
· Phosphate Buffer Saline (PBS, 0.01 M pH 7.1-7.4 with 0.085% NaCl and 0.1% Azide), (Catalog No. 5087).
· 0.05% Tween 20 /0.1% Sodium Azide Solution (optional), (Catalog No. 5037).
· Aspirator device with disposable sterile Pasteur pipettes.
· Centrifuge capable of 700-950 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, 400x, magnification (dry objective).
· Humid chamber.
· Incubator, 37 * 1*C.
· Syringe filter, 0.45 micron.
· Ultrasonic water bath.
· Vortex mixer or sonicator.
· Mounting Media (Catalog No. 5013).
· Coxsackievirus B Control Slides (Catalog No. 5075).
Almacenamiento y estabilidad
When stored at 2-8°C, the monoclonal antibody is stable up to the expiration date printed on the label. Avoid multiple freeze and thaw.
Warning and Precautions:
* For in vitro diagnostic use.
* The performance of Light Diagnostics Coxsackievirus B1 MAb has not been determined on direct specimens.
* Sodium azide, present in the reagents, can form potentially explosive metal azides with lead and copper pipes. As a precaution, flush with large amount of water to prevent azide build-up.
* Do not allow the slides to dry at any time during the staining procedure
* Slides prepared too early (<25% CPE) or too late (>95% CPE) can be difficult to read and can lead to false negatives.
* Handle all specimens and materials coming in contact with them as potentially infectious materials. All samples should be handled at the Biosafety Level 2 as recommended for any potentially infectious material in the Center for Disease Control/National Institute of Health Manual, "Biosafety in Microbiological and Biomedical Laboratories," (1984). Decontaminate with 0.05% sodium hypochlorite.
* Avoid contact with Evans Blue if present in any reagent as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.
* Do not mouth pipette reagents.
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Cláusula de descargo de responsabilidad
For in vitro Diagnostic Use