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3105 | LIGHT DIAGNOSTICS™ Respiratory Panel I Viral Screening and Identification IFA

1 kit  10 mL Screen & 2 mL each viral spec. abs.
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      Key ApplicationsFormatHostDetection Methods
      IF FITC M Fluorescent
      Catalogue Number3105
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionLIGHT DIAGNOSTICS™ Respiratory Panel I Viral Screening and Identification IFA
      OverviewThe Light Diagnostics Respiratory Panel 1 Viral Screening and Identification Kit is intended for in vitro diagnostic use in the qualitative culture confirmation of adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and respiratory syncytial virus (RSV).

      Test Principle:

      Light Diagnostics Respiratory Panel 1 Viral Screening and Identification Kit utilizes an indirect immunofluorescence technique for identifying virus in infected cell cultures. The mouse monoclonal antibodies provided will bind to the appropriate viral antigen on the specimen slide. Unbound antibody is washed from the slide with phosphate buffered saline (PBS). This is followed by the addition of fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG which will bind to the antigen-antibody complex. Unbound labeled antibody is washed from the slide with PBS. FITC exhibits an apple green fluorescence when excited by ultraviolet light allowing visualization of the complex by fluorescence microscopy. Cell fluorescence indicates a positive specimen. Uninfected cells stain a dull red due to the presence of Evans blue counterstain in the FITC-labeled secondary antibody. The Respiratory Viral Screen reagent is used to confirm the indiscriminate presence of respiratory viruses. Individual adenovirus, influenza A & B, parainfluenza 1, 2, & 3, and RSV monoclonal antibody reagents are then used for specific virus identification.

      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. adenovirus) predominantly affect different age groups. In a given population, respiratory viruses may be responsible for a considerable amount of morbidity. The advent of appropriate antiviral therapy against some respiratory viruses makes rapid screening to identify these organisms imperative, allowing early institution of therapy. The two most widely used antiviral agents today are amantadine/rimantadine for influenza A and ribavirin for RSV bronchiolitis. Both agents have varying modes of action although most involve early infection steps such as penetration or uncoating (41, 63). At physiologically attainable concentrations, amantadine and rimantadine specifically inhibit replication of influenza A (13). Ribavirin has broad spectrum activity in vitro against a host of viruses such as RSV, measles, influenza A and B, and parainfluenza (46, 47, 53).

      The two commonly used modes of identifying viruses are: culture isolation/ confirmation, and direct detection. Culture isolation/confirmation is the standard method for most laboratories, and is the most sensitive method available for the detection of respiratory viruses. Light Diagnostics Respiratory Panel 1 utilizes monoclonal antibodies for culture confirmation to provide clear, easy to interpret results in only 60 minutes.

      By age 2, most children have experienced respiratory syncytial virus (RSV) infection making it the most important viral cause of childhood lower respiratory tract illness (64). 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 (64). In most areas researched, RSV was responsible for more pneumonia and bronchiolitis than all other microbial pathogens (48). 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 (52). RSV has also been implicated in sudden infant death syndrome (SIDS), although the nature of the association is undetermined (58).

      RSV belongs to the family Paramyxoviridae and the genus Pneumovirus. It is an enveloped pleomorphic virus ranging from 150-300nm in diameter (5, 31, 34) with a single-stranded RNA genome (64).

      RSV may be detected by immunofluorescence, EIA, neutralization, or culture isolation and confirmation (21, 32, 49). A variety of cell lines are suitable for RSV cultivation. For primary isolation, Hep-2 (33) or HeLa (34) are acceptable cell lines although others such as Vero, LLC MK-2, or CV-l have been used. The virus produces characteristic cytopathic effects of syncytium formation and cell destruction (48).

      Influenza viruses causes 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 (40).

      Adult infection with influenza virus characteristically results in tracheobronchitis and small airway involvement (43, 66) with possible development of rhinitis and/or pharyngitis. Infection can, however, display clinical manifestations ranging from no symptoms to fatal pneumonia (25). The same spectrum of clinical response can be seen in children with some distinct differences. Fever may be higher in children and be accompanied by febrile convulsions (23, 24, 62, 70, 71). Influenza viruses are responsible for 14% of childhood fevers with respiratory tract symptoms severe enough to warrant a physician's attention (23, 71). Children experience more gastrointestinal involvement than adults (14) and they develop myositis, otitis media, and croup more frequently (28, 38). Neonatal infection may result in unexplained fever (50) and is potentially fatal (1, 18, 50, 59). 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 (54). Non-pulmonary clinical responses to influenza infection include viremia, cardiac and CNS involvement, Reyes syndrome, and toxic shock (54).

      Influenza types A and B cause essentially the same spectrum of disease. Type A infection, however results in hospitalization approximately four times more often than type B (38), and type B more commonly results in myositis and gastrointestinal involvement (11, 16, 37, 42).

      Influenza type C rarely results in lower respiratory tract illness but causes sporadic upper respiratory tract disease (19, 38, 51). By adulthood, almost all individuals have antibody to type C (57).

      Influenza viruses are members of the family Orthomyxoviridae. They are pleomorphic, enveloped and contain a segmented, single-stranded, negative-sense RNA genome. They range in diameter from 80-120nm (40).

      Addition of trypsin 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) (20, 65) as well as the classic embryonated hen eggs or the allantoin-on-shell system (17).

      Influenza virus can be detected by immunofluorescence, hemadsorption or hemagglutination techniques using chicken or guinea pig erythrocytes. Isolation/culture confirmation provides an easy, sensitive and relatively rapid technique for identifying influenza infections. 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 (4, 6, 7, 39). Four types of parainfluenza viruses have been identified in children and adults. Types 1 and 2 are major causes of laryngotracheobronchitis (croup). The severity of illness is greatest in children aged 2-4 years (67). 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 (8, 9, 25, 60, 61). Illness from type 3 infection is most severe in infants less than 1 year old (67).

      In older children and adults, illness may be asymptomatic or mimic the common cold (68). 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 (26, 44).

      Parainfluenza type 4 has been associated only with mild upper respiratory illness in adults and children and is difficult to identify in cell cultures (67).

      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 150-200nm (10).

      Parainfluenza viruses grow well in primary simian or human kidney cell lines and in LLC-MK2, a rhesus kidney heteroploid cell line (20). 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.

      Adenoviruses are responsible for a significant number of clinical respiratory illnesses. Upper respiratory diseases caused by adenoviruses include colds, pharyngitis, and tonsillitis and occur mostly in infants and young children. Approximately 10% of childhood pneumonia is probably caused by adenovirus (45). Other adenovirus related lower respiratory illnesses include bronchitis and bronchiolitis (29). In children under 5, adenovirus is responsible for about 5% of cases of acute respiratory disease (ARD). ARD may be manifested by nasal congestion, coryza, cough and, at times, tonsillitis, fever, and myalgia. The appearance of conjunctivitis with ARD constitutes pharyngoconjunctival fever (3). Adenovirus has commonly been associated with pertussis syndrome, but recent studies suggest that the presence of adenovirus in these cases may be a reactivation of latent virus from tonsillar tissue during Bordetella pertussis infections (55).

      Ocular illnesses resulting from adenovirus infection include epidemic keratoconjunctivitis (EKC), acute hemorrhagic conjunctivitis, and acute follicular conjunctivitis. 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 (15, 22, 69). The virus has also been linked with intussusception (2), acute hemorrhagic cystitis (56), and meningoencephalitis.

      Research indicates that the incidence of adenovirus infection in immuno-compromised patients is probably no higher than in normal individuals; however, severity and probability of death may be greater (30).

      Human adenoviruses belong to the family Adenoviridae, genus Mastadenovirus. They are non-enveloped, double stranded DNA viruses icosahedral in shape ranging from 70-90 nm in diameter (29). They have a protein coat of 240 hexon and 12 penton capsomeres.

      Adenovirus can be cultured and isolated in a variety of cell lines and identified by several methods. Suitable cell lines include Hep-2, HeLa, KB:A549 and HEK. Graham 293 cells may be used for propagation of types 40 and 41. Confirmation of infection is usually achieved by immunofluorescence or enzyme immunoassay (EIA), but it can be done by complement fixation, hemagglutination-inhibition, and neutralization methods (35). Typical adenovirus cytopathic effects (CPE) manifest as grape-like clusters of rounded, refractive cells. These cells, when stained with hematoxylin and eosin have intranuclear inclusions which appear in 3 to 10 days (12).
      Materials Required but Not Delivered1. 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 Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852.

      2. Viral transport medium 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.

      3. Tissue culture media such as RPMI or Eagle's Minimum Essential Medium (EMEM) with appropriate amount of FBS can be used for maintenance after virus infection.

      4. Sterile tissue culture tubes, dram vials, or multi-well plates

      5. Acetone, 99.5%

      Note: Acetone is hygroscopic and should be kept in tightly stoppered bottles. Presence of moisture in the acetone may result in a hazy appearance on the substrate during fluorescence microscopy.

      6. Acetone-cleaned glass slides

      7. Sterile pipettes

      8. Humid chamber

      9. Sodium hypochlorite solution (0.05%)

      10. No. 1 coverslips

      11. Incubator with rheostat for temperature regulation

      12. Sterile swabs

      13. Forceps

      14. Vials for collection and transportation of specimens

      15. Fluorescence microscope with appropriate filter combination for FITC (excitation peak 490 nm, emission peak 520 nm)

      16. Sterile glass beads (1-3 mm diameter)

      17. Centrifuge

      18. Vortex mixer or sonicator

      19. Distilled water
      Product Information
      • Antigen Control Slide - (Catalog No. Adeno - 5009, Influenza A & B - 5010, Para 1,2,3 - 5011, RSV - 5012). Four control slides containing wells with positive and negative control cells. One positive control well each is infected with the following: adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and RSV. Negative control wells contain uninfected cells.
      • Monoclonal Antibodies - (Catalog No. Adeno - 5000, Influenza A - 5001, Influenza B - 5002, Para 1 - 5003, Para 2 - 5004, Para 3 - 5005, RSV - 5006). Seven x 2 ml dropper vials containing monoclonal antibody against: adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and RSV.
      • Respiratory Viral Screen - (Catalog No. 5007). One 10 ml dropper vial containing monoclonal antibody against: adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, and respiratory syncytial virus.
      • Normal Mouse Antibody - (Catalog No. 5014). One 10 ml dropper vial containing normal mouse antibody to be used as a negative control.
      • Anti-Mouse IgG:FITC Conjugate - (Catalog No. 5008). Two 10 ml dropper vials containing FITC labeled goat anti-mouse IgG.
      • PBS - (Catalog No. 5087). One packet of Phosphate Buffered Saline Salts yields 1 liter when dissolved in distilled water. Store in a clean closed container at room temperature.
      • Tween 20 / Sodium Azide Solution (100X) - (Catalog No. 5037). One 10 ml vial containing polyoxyethylene sorbitan monolaurate (Tween 20) and sodium azide (NaN3) concentrate to be diluted 1:100 in PBS.
      • Mounting Fluid - (Catalog No. 5013). One 10 ml dropper vial containing Tris- buffer, glycerin, fluorescence enhancer, and sodium azide as preservative. Store at room temperature.
      Detection methodFluorescent
      Key Applications
      • Immunofluorescence
      Biological Information
      Antibody TypeMonoclonal Antibody
      Physicochemical Information
      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 Light Diagnostics Respiratory Panel 1 Viral Screening and 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.

      A marked decrease in fluorescence may indicate conjugate or antibody deterioration. A positive control should be tested with each batch of specimens to ensure proper functioning of these reagents and proper staining procedure. If, after appropriate analysis, there is a decrease in staining intensity, discontinue use of the reagents.

      Warnings and Precautions:

      · Sodium azide (present in the conjugate, monoclonal antibodies, wash buffer, and mounting fluid) can react with lead or copper plumbing to form potentially explosive metal azides. When disposing of these materials, flush with large volumes of water to prevent azide build-up.

      · Pooling or diluting conjugates or monoclonal antibodies may cause erroneous results.

      · Do not allow slides to dry at any time during the staining procedure.

      · Handle all specimens and materials as if potentially infectious. Decontaminate with 0.05% sodium hypochlorite (a 1:100 dilution of household bleach) prior to disposal.

      · Avoid prolonged exposure to bright lights.

      · Avoid contact with Evans blue (present in the Anti-Mouse IgG : FITC Conjugate) as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.

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

      · Do not mouth pipette reagents.

      · Do not substitute reagents from other manufacturers.

      · Alteration of protocol provided may cause erroneous results.

      · When staining multiple samples on a slide, avoid cross contamination between samples.

      · Normal mouse antibody should be tested with each cell culture isolate. Fluorescence indicates a nonspecific reaction and the test is considered invalid.
      Packaging Information
      Material Size1 kit
      Material Package10 mL Screen & 2 mL each viral spec. abs.
      Transport Information
      Supplemental Information


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      Manuales del usuario

      Respiratory Panel Viral Screening & Identification Indirect Immunofluorescence Assay