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3137 | LIGHT DIAGNOSTICS™ Respiratory Viral Screen and Identification DFA Kit

3137
1 kit  10 mL Screen & 2 mL each viral spec. abs.
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      Key ApplicationsFormatHostDetection Methods
      IF FITC M Fluorescent
      Description
      Catalogue Number3137
      Brand Family Chemicon®
      Trade Name
      • LIGHT DIAGNOSTICS
      • Chemicon
      DescriptionLIGHT DIAGNOSTICS™ Respiratory Viral Screen and Identification DFA Kit
      OverviewThe Light Diagnostics Respiratory DFA Viral Screening and Identification Kit is intended for in vitro diagnostic use for the screening and qualitative detection and identification of influenza A, influenza B, adenovirus, respiratory syncytial virus (RSV), parainfluenza 1, parainfluenza 2 and parainfluenza 3 in pateint samples following amplification in cell culture.

      For In Vitro Diagnostic Use

      Test Principle:

      Light Diagnostics Respiratory DFA Viral Screening & Identification Kit utilizes direct immunofluorescence to screen for the presence of influenza A, influenza B, RSV, adenovirus and parainfluenza 1, 2 and 3 viruses in infected cell cultures The fluorescein-labeled monoclonal antibody will bind to the appropriate viral antigen on the slide, if present. Unbound monoclonal antibody is removed by washing with a phosphate-buffered saline (PBS)/Tween Solution. The antigen-antibody complex will show apple-green fluorescence by fluorescence microscopy. Uninfected cells stain a dull red due to the presence of Evans Blue in the reagents.

      Summary and Explanation:

      Respiratory viruses are responsible for a significant proportion of illness in human populations. Some viruses (e.g. influenza) are seasonal while others (e.g. respiratory syncytial virus (RSV), adenovirus) predominantly affect different age groups. Antiviral therapy is available for some and not all respiratory viruses; rapid screening to identify these organisms allows for early institution of therapy and minimizes the inappropriate use of antibiotics. The two most widely used antiviral agents available for respiratory viruses are amantadine/rimantadine for influenza A, and ribavirin for RSV bronchiolitis. Both agents have differing modes of action, although most affect viral penetration or uncoating (1,2). At physiological concentrations, amantadine and rimantadine specifically inhibit replication of influenza A (3). Ribavirin has broad spectrum activity in vitro against a host of viruses such as RSV, measles, influenza A and B, and parainfluenza (4,5).

      Human adenoviruses belong to the family Adenoviridae, genus Mastadenovirus. They are icosahedral, non-enveloped, double stranded DNA viruses 70-90 nm in diameter (6). They have a protein coat of 240 hexon and 12 penton capsomeres. Adenoviruses are responsible for a significant number of clinical respiratory illnesses, particularly in children.

      Upper respiratory diseases in infants and young children can include colds, pharyngitis, and tonsillitis, while lower respiratory illnesses include bronchitis and bronchiolitis (6), and approximately 10% of childhood pneumonia (7). In children under 5, adenovirus is responsible for about 5% of cases of acute respiratory disease (ARD), which is manifested by nasal congestion, coryza, cough and, at times, tonsillitis, fever, and myalgia. The appearance of conjunctivitis with ARD constitutes pharyngoconjunctival fever.

      In addition to respiratory infections, adenoviruses can be a cause of other significant illnesses. Ocular diseases resulting from adenovirus infection include: epidemic keratoconjunctivitis (EKC), acute hemorrhagic conjunctivitis, and acute follicular conjunctivitis. In addition, adenovirus is related to several gastrointestinal disorders and is probably evident in 7-17% of all childhood gastroenteritis. Types 40 and 41 have been associated with diarrhea and acute gastroenteritis (8). Adenovirus has also been linked with intussusception (9), acute hemorrhagic cystitis (10), and meningoencephalitis.

      Research indicates that the incidence of adenovirus infection in immuno-compromised patients is probably no higher than in normal individuals; however, the severity and probability of death may be greater (11).

      With the exception of types 40 and 41 which grow only in Graham 293 cells, adenovirus can be cultured and isolated in a variety of cell lines such as Hep-2, A549, KB, and HEK. Infection is usually confirmed by immunofluorescence or enzyme immunoassay (EIA). Typical adenovirus cytopathic effects (CPE) manifest as grape-like clusters of rounded, refractive cells with intranuclear inclusions, which appear in 3 to 10 days post-infection (12).

      Influenza viruses are pleomorphic, enveloped viruses, 80-120 nm in diameter containing a segmented, single-stranded, negative-sense RNA genome, and are members of the family Orthomyxoviridae (13). Infections with influenza viruses cause highly contagious respiratory disease, which typically results in epidemics (27). There are three types, A, B, and C, in which specificity is conferred by internal nucleoprotein and matrix protein antigens (14).

      Adult infection with influenza viruses may display manifestations ranging from no symptoms to fatal pneumonia (13). More characteristic infections result in tracheobronchitis and small airway involvement (1), with rhinitis and/or pharyngitis. The same spectrum of clinical response can be seen in children with some distinct differences.

      In children, influenza infections usually cause higher fevers, which may be accompanied by febrile convulsions (15-17). Influenza viruses are responsible for 14% of childhood fevers with respiratory tract symptoms severe enough to warrant a physician's attention (15,17). In addition, children are more likely to experience more gastrointestinal involvement than adults (18) and they develop myositis, otitis media, and croup more frequently (19). Neonatal infection may result in unexplained fever (20) and is potentially fatal (20,21).

      Lower respiratory tract disease in children and adults associated with influenza infection is manifested in three forms of pneumonia: primary viral pneumonia, combined viral-bacterial pneumonia, and influenza infection followed by bacterial pneumonia (22). Non-pulmonary clinical responses to influenza infection include viremia, cardiac and CNS involvement, Reyes syndrome, and toxic shock.

      Influenza types A and B cause essentially the same spectrum of disease. However, type A infection results in hospitalization approximately four times more often than type B (23), and type B more commonly results in myositis and gastrointestinal involvement (24,25). Influenza type C rarely results in lower respiratory tract illness but causes sporadic upper respiratory tract disease (23,26). By adulthood, almost all individuals have antibody to type C (27).

      Detection of influenza A or B in culture provides an easy, sensitive and relatively rapid technique for identifying influenza infections, The addition of trypsin to cells makes it possible to culture Influenza viruses in a variety of cell lines such as Madin Darby canine kidney (MDCK), A549 lung carcinoma and primary monkey kidney (PMK) (28,29). Influenza virus can be detected in culture by immunofluorescence, hemadsorption or hemagglutination techniques using chicken or guinea pig erythrocytes. Cytopathic effect is evident in 3-7 days post-inoculation as vacuolation and cell degradation.

      Parainfluenza viruses, combined with RSV, represent the most significant upper respiratory pathogens in infants and young children (30). Parainfluenza viruses belong to the genus Paramyxovirus of the family Paramyxoviridae. They are enveloped viruses with a single-strand RNA genome of negative polarity and range in diameter from 150-200 nm (31). Four types of parainfluenza virus have been identified.

      Parainfluenza types 1 and 2 are major causes of laryngotracheobronchitis (croup). The severity of illness is greatest in children aged 2-4 years (32). Parainfluenza type 3 infection can lead to croup but, most notably, type 3 is second only to RSV as a cause of infant bronchiolitis and pneumonia (33,34). Illness from type 3 infection is most severe in infants less than 1 year old.

      Severe croup in early childhood or infancy may result in bronchial hyperactivity in older children or adolescents after exercise. However, it remains undetermined whether bronchial hyperactivity was a preexisting condition, which played a role in the pathogenesis of croup, or whether it develops as a complication of severe croup (25). In older children and adults, illness may be asymptomatic or mimic the common cold (36).

      Parainfluenza viruses grow well in primary monkey (RMK) or human kidney cell lines and in LLC-MK2, a rhesus kidney heteroploid cell line (38). Trypsin is needed in the medium for the recovery of types 1 and 2 but not type 3. Virus infection of tissue culture can be recognized by hemadsorption of guinea pig erythrocytes. Types 2 and 3 can be recognized by syncytium formation.

      Respiratory Syncytial Virus (RSV) belongs to the family Paramyxoviridae and the genus Pneumovirus. It is an enveloped pleomorphic virus ranging from 150-300nm in diameter (37,38) with a single-stranded RNA genome (39). By age 2, most children have experienced RSV infections, making it the most important viral cause of childhood lower respiratory tract illness.

      RSV infection usually results in colds with profuse rhinorrhea, but in first-time infections among infants 6 weeks to 6 months old, 25-40% will develop lower respiratory tract illness (37). RSV was responsible for more pneumonia and bronchiolitis than all other microbial pathogens (40). Studies suggest that childhood RSV pneumonia and bronchiolitis may result in long term respiratory abnormalities such as abnormal pulmonary function, asthma, and recurrent cough and bronchitis (41).

      RSV may be detected in specimens by immunofluorescence, EIA, following amplification in culture (41,43). A variety of cell lines are suitable for RSV cultivation. For primary isolation, Hep-2 (40) is the most commonly used cell line although others such as Vero, LLC-MK-2, MRC-5 and CV-l have been used. The virus produces characteristic cytopathic effects of syncytium formation and cell destruction.

      Light Diagnostics Respiratory DFA Viral Screening and Identification Kit utilizes fluorescein-labeled monoclonal antibodies for the detection and identification of RSV, influenza A, influenza B, adenovirus, parainfluenza 1, 2 and 3 following amplification in culture.
      Materials Required but Not Delivered· Cell culture for isolation of respiratory viruses: Each laboratory must maintain viable stocks of cells at appropriate passage-state that will efficiently allow replication of respiratory viruses from processed patient specimens. These cells must be checked periodically for ability to support growth of respiratory viruses. Appropriate cell lines can be obtained from the American Type Culture (ATTC), 12301 Parklawn Drive, Rockville, MD 20852.

      · Viral transport medium (VTM), which is non-inhibitory to the respiratory viruses and the tissue culture cells, used for viral isolation: Hank's balanced salt solution (HBSS) with antibiotics and a protein stabilizer is a suitable medium. Avoid use of animal sera (except precolostral fetal bovine serum (FBS)) as protein stabilizer to prevent interference from inherent antibody.

      · Acetone, reagent grade; stored in glass.

      · Deionized or distilled water.

      · Negative and positive controls (representative respiratory virus strains available from ATCC, Rockville, MD).

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

      · Shell vials with 12 mm coverslips or culture tubes for growth of cell culture.

      · Tissue culture media: RPMI or Eagle's Minimum Essential Medium (EMEM) with fetal bovine serum (FBS) and antibiotics or equivalent.

      · Acetone-cleaned microscope slides with wells in a hydrophobic mask.

      · Aspirator device with disposable sterile Pasteur pipettes.

      · 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, 400x magnification (dry objective).

      · Forceps.

      · Humid chamber.

      · Incubator, 37 ± 1°C.

      · Syringe and needle or other implement to remove coverslip from shell vial.

      · Syringe filter, 0.45 micron.

      · Ultrasonic water bath.

      · Vortex mixer or sonicator.
      References
      Product Information
      Components
      • DFA Respiratory Screen - (Catalog No. 5307). One 10 mL dropper vial containing a primary component specific for adenovirus, influenza A, influenza B, RSV, and parainfluenza 1, 2 and 3, protein stabilizer, Evans blue and 0.1% sodium azide (preservative).
      • DFA Identification Reagents - (Adeno - 5016, Influenza A - 5017, Influenza B - 5018, Para 1 - 5019, Para 2 - 5020, Para 3 - 5021, RSV - 5022). Seven (7) x 2 mL dropper vials containing fluorescein-labeled monoclonal antibody directed against: adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and RSV with protein stabilizer, Evans blue and 0.1% sodium azide (preservative).
      • Antigen Control Slides - (Catalog No. Adeno - 5009, Influenza A & B - 5010, Para 1,2,3 - 5011, RSV - 5012). Four (4) control slides containing wells with positive and negative control cells. Each positive control well is infected with one of the following: adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and RSV. Negative control wells contain uninfected cells.
      • Respiratory Control Slide - (Catalog No. 5071). One Control slide containing seven positive wells each infected with a different respiratory virus (Adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and respiratory syncytial virus) and one uninfected well.
      • Phosphate-Buffered Saline - (Catalog No. 5087). One (1) packet of PBS salts.
      • Tween 20 / Sodium Azide Solution (100X) - (Catalog No. 5037). One (1) 10 mL vial containing Tween 20 / Sodium Azide concentrate.
      • Mounting Fluid - (Catalog No. 5013). One (1) 10 mL dropper vial containing Tris-buffered glycerin, 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 2-8ºC, the Respiratory DFA Viral Screening & Identification 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 labeled monoclonal antibodies, PBS, and Mounting Fluid is toxic is ingested. Sodium Azide can form potentially explosive metal azides with lead and copper pipes. Upon disposal, flush with large amounts of water to prevent azide build-up.

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

      · Do not substitute reagents from other manufacturers.

      · 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 prior to disposal.

      · Acetone is 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 DFA Reagents ) as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.

      · PBS with Tween 20/Sodium Azide should not be used in viral isolation procedures.

      · Do not mouth pipette reagents.

      · Do not expose reagents to bright light during storage or incubation.

      · Alteration of protocol provided may cause erroneous results.

      · When staining multiple samples on a slide, avoid cross contamination between samples.
      Packaging Information
      Material Size1 kit
      Material Package10 mL Screen & 2 mL each viral spec. abs.
      Transport Information
      Supplemental Information
      Specifications