Components

Coxsackievirus B6 Monoclonal Antibody - (Catalog No. 3309).One 1 mL dropper vial containing mouse IgG(2a) monoclonal antibody against coxsackievirus B6, protein stabilizer and 0.1 % sodium azide (preservative).

Disclaimer

For in vitro Diagnostic UseCE Mark

General description

Light Diagnostics Coxsackievirus B6 Monoclonal Antibody is a type-specific reagent intended for use in indirect immunofluorescence screening for the presumptive identification of coxsackievirus B6 obtained in cell culture and not intended for testing directly on human specimens.

For in vitro diagnostic use.

Test Principle:

Light Diagnostics Coxsackievirus B6 Monoclonal Antibody (MAb Cox B6) can be used to identify a coxsackievirus B6 viral isolate in cell culture using an indirect immunofluorescence assay (IFA). The monoclonal antibody provided will bind to the type-specific coxsackievirus B6 antigen present in 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. Uninfected 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, such as coxsackieviruses, 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 (1). 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 (1).

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 (1): Three polioviruses, twenty-three coxsackieviruses A, six coxsackieviruses B, thirty-one echoviruses, and four other enteroviruses.

Enteroviruses, including echoviruses and coxsackieviruses, have been reported as the major etiologic agents of aseptic meningitis (2). Clinical syndromes associated with infections by each type of enterovirus have also been reported (3). Coxsackievirus B6 can cause aseptic meningitis, severe systemic infection in infants, 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 principles 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 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 (11). 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 is potentially more rapid and less expensive (12 - 18).

Diagnostics Kit

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Physical form

Format: Purified

Materials Required But Not Provided:
⋅ Acetone, reagent grade; stored in glass

⋅ Distilled water or deionized 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 such as RPMI or Eagle′s Minimum Essential Medium (EMEM) with fetal bovine serum (FBS) 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

⋅ Mounting Fluid (Catalog No. 5013)

⋅ Coxsackievirus B Control Slides (Catalog No. 5075)

⋅ Anti-Mouse IgG:FITC Conjugate (Catalog No. 5008)

⋅ Normal Mouse Antibody (Catalog No. 5014) as negative control

⋅ Phosphate buffered saline (PBS, 0.01 M pH 7.1-7.4 with 0.085% NaCl and 0.1% sodium 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)

⋅ Forceps

⋅ Humid chamber

⋅ Incubator, 37 * 1*C

⋅ Syringe filter, 0.45 micron

⋅ Ultrasonic water bath

⋅ Vortex mixer or sonicator

Storage and Stability

When stored at 2-8°C, the monoclonal antibody is stable up to the expiration date printed on the label. Do not freeze or expose to elevated temperatures. Discard any remaining reagent after the expiration date.

Warning and Precautions:

* The performance of Light Diagnostics Coxsackievirus B6 MAb has not been determined on direct specimens.

⋅ Sodium azide, present in the reagent, may react with lead and copper plumbing to form potentially explosive metal azides. When disposing of solutions that contain sodium azide, flush plumbing with a large volume of water to prevent build-up.

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

* 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.

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

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

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

* Mounting Fluid (Catalog No. 5013) contains a fluorescence enhancer that may be destructive to mucous membranes. Avoid direct skin or mucous membrane contact. If contact occurs, flush with large volumes of water.

* Slides prepared too early (﹤25% CPE) or too late (>95% CPE) can be difficult to read and can lead to false negatives.