Ordering Information
- : 3299
- : 2 mL
LIGHT DIAGNOSTICS™ SimulFluor® Para 1,2,3/Adeno, ~50 tests
Description:
LIGHT DIAGNOSTICS™ SimulFluor® Para 1,2,3/Adeno, ~50 tests
Trade Name:
- LIGHT DIAGNOSTICS
- Chemicon (Millipore)
Qty/Pk:
2 mL
Product Overview:
Intended Use
Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay is intended for the simultaneous detection of parainfluenza 1, 2, and 3 and adenovirus following amplification in cell culture.
For In Vitro Diagnostic Use.
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 anti-viral therapy against some respiratory viruses makes rapid screening to identify these organisms imperative, allowing early institution of therapy. The two most widely used anti-viral agents today are amantadine/rimantadine for influenza A and ribavirin for respiratory syncytial virus bronchiolitis. Both agents have varying modes of action although most involve early infection steps such as penetration or uncoating (12,19). At physiologically attainable concentrations, amantadine and rimantadine specifically inhibit replication of influenza A (4). Ribavirin has broad spectrum activity in vitro against a host of viruses such as respiratory syncytial virus, measles, influenza A and B, and parainfluenza (7,13,14).
Parainfluenza viruses, combined with respiratory syncytial virus, represent the most significant upper respiratory pathogens in infants and young children (1,2,3,5). Four types of parainfluenza virus have been identified in children and adults. Types 1 and 2 are major causes of laryngotracheo-bronchitis (croup). The severity of illness is greatest in children ages 2 to 4 years (6). Parainfluenza type 3 infection can also lead to croup and is second only to respiratory syncytial virus as a cause of infant bronchiolitis and pneumonia (8,9,10,11,15). Illness from type 3 infection is most severe in infants less than 1 year old (6). In older children and adults, illness may be asymptomatic or mimic the common cold (16). 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 pre-existing condition, which played a role in the pathogenesis of croup, or whether it developed as a complication of severe croup illness (17,18).
Parainfluenza type 4 has been associated only with mild upper respiratory illness in adults and children and is difficult to identify in cell culture (6).
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 to 200 nm (20).
Parainfluenza viruses grow well in primary simian or human kidney cell lines and in LLC-¬MK2, a rhesus kidney heteroploid cell line (21). Trypsin is needed in the medium for the recovery of types 1 and 2, but not 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.
Human adenoviruses belong to the family Adenoviridae, genus Mastadenovirus. They are icosahedral, non-enveloped, double stranded DNA viruses 70 90 nm in diameter (23). 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 (23), and approximately 10% of childhood pneumonia (24). 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 disease 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 (25). Adenovirus has also been linked with intussusception (26), acute hemorrhagic cystitis (27), 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 (28).
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 (29).
Viruses can be identified in patient specimens and by culture isolation/ confirmation. Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay utilizes monoclonal antibodies to simultaneously detect parainfluenza 1, 2 and 3, and identify adenovirus following amplification in culture. Adenovirus can also be detected and identified in direct patient specimens.
Test Principle
Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay utilizes a single reagent for the simultaneous detection of parainfluenza 1, 2 and 3, and identification of adenovirus. The SimulFluor® Para 1, 2, 3/ Adeno Reagent contains two components. The primary component is directed against parainfluenza 1, 2 and 3; the second component is specifically directed against adenovirus. The monoclonal antibodies in the components will bind to the appropriate viral antigen on the specimen slide. Unbound antibody is washed from the slide with phosphate buffered saline (PBS). Parainfluenza 1, 2 and 3-infected cells will exhibit an apple-green fluorescence; while, adenovirus-infected cells will exhibit a yellow-gold fluorescence. The primary component staining the parainfluenza 1, 2 and 3-infected cells bright apple-green does not differentiate between these viruses. The secondary component staining the adenovirus-infected cells bright yellow-gold allows for the differentiation of adenovirus from parainfluenza 1, 2 and 3 in a single well. Uninfected cells stain dull red due to the presence of Evans blue counterstain in the reagent.
Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay is intended for the simultaneous detection of parainfluenza 1, 2, and 3 and adenovirus following amplification in cell culture.
For In Vitro Diagnostic Use.
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 anti-viral therapy against some respiratory viruses makes rapid screening to identify these organisms imperative, allowing early institution of therapy. The two most widely used anti-viral agents today are amantadine/rimantadine for influenza A and ribavirin for respiratory syncytial virus bronchiolitis. Both agents have varying modes of action although most involve early infection steps such as penetration or uncoating (12,19). At physiologically attainable concentrations, amantadine and rimantadine specifically inhibit replication of influenza A (4). Ribavirin has broad spectrum activity in vitro against a host of viruses such as respiratory syncytial virus, measles, influenza A and B, and parainfluenza (7,13,14).
Parainfluenza viruses, combined with respiratory syncytial virus, represent the most significant upper respiratory pathogens in infants and young children (1,2,3,5). Four types of parainfluenza virus have been identified in children and adults. Types 1 and 2 are major causes of laryngotracheo-bronchitis (croup). The severity of illness is greatest in children ages 2 to 4 years (6). Parainfluenza type 3 infection can also lead to croup and is second only to respiratory syncytial virus as a cause of infant bronchiolitis and pneumonia (8,9,10,11,15). Illness from type 3 infection is most severe in infants less than 1 year old (6). In older children and adults, illness may be asymptomatic or mimic the common cold (16). 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 pre-existing condition, which played a role in the pathogenesis of croup, or whether it developed as a complication of severe croup illness (17,18).
Parainfluenza type 4 has been associated only with mild upper respiratory illness in adults and children and is difficult to identify in cell culture (6).
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 to 200 nm (20).
Parainfluenza viruses grow well in primary simian or human kidney cell lines and in LLC-¬MK2, a rhesus kidney heteroploid cell line (21). Trypsin is needed in the medium for the recovery of types 1 and 2, but not 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.
Human adenoviruses belong to the family Adenoviridae, genus Mastadenovirus. They are icosahedral, non-enveloped, double stranded DNA viruses 70 90 nm in diameter (23). 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 (23), and approximately 10% of childhood pneumonia (24). 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 disease 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 (25). Adenovirus has also been linked with intussusception (26), acute hemorrhagic cystitis (27), 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 (28).
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 (29).
Viruses can be identified in patient specimens and by culture isolation/ confirmation. Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay utilizes monoclonal antibodies to simultaneously detect parainfluenza 1, 2 and 3, and identify adenovirus following amplification in culture. Adenovirus can also be detected and identified in direct patient specimens.
Test Principle
Light Diagnostics™ SimulFluor® Para 1, 2, 3/ Adeno Immunofluorescence Assay utilizes a single reagent for the simultaneous detection of parainfluenza 1, 2 and 3, and identification of adenovirus. The SimulFluor® Para 1, 2, 3/ Adeno Reagent contains two components. The primary component is directed against parainfluenza 1, 2 and 3; the second component is specifically directed against adenovirus. The monoclonal antibodies in the components will bind to the appropriate viral antigen on the specimen slide. Unbound antibody is washed from the slide with phosphate buffered saline (PBS). Parainfluenza 1, 2 and 3-infected cells will exhibit an apple-green fluorescence; while, adenovirus-infected cells will exhibit a yellow-gold fluorescence. The primary component staining the parainfluenza 1, 2 and 3-infected cells bright apple-green does not differentiate between these viruses. The secondary component staining the adenovirus-infected cells bright yellow-gold allows for the differentiation of adenovirus from parainfluenza 1, 2 and 3 in a single well. Uninfected cells stain dull red due to the presence of Evans blue counterstain in the reagent.
Key Applications:
Immunofluorescence
Usage Statement:
- For in vitro Diagnostic Use
- CE Mark
Components:
- SimulFluor® Para 1, 2, 3/ Adeno Reagent - (Catalog No. 5299).
- Para 1, 2 & 3 Control Slide - (Catalog No. 5011).
- Adeno Control Slide - (Catalog No. 5009).
- Phosphate Buffered Saline - (Catalog No. 5087).
- Tween 20 / Sodium Azide Solution (100X) - (Catalog No. 5037).
- Mounting Fluid - (Catalog No. 5013).
Brand Family:
Chemicon
Format:
SimulFluor
Storage Conditions:
When stored at 2° to 8°C, the SimulFluor® Para 1, 2, 3/ Adeno Reagent is stable up to the expiration date printed on the kit label. Do not freeze or expose to elevated temperatures. Discard any remaining reagent after the kit expiration date.
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.
• Do not expose reagents to bright light during storage or incubation.
• Avoid contact with Evans blue (present in the SimulFluor® Para 1, 2, 3/ Adeno Reagent) 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.
Mounting Fluid 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.
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.
• Do not expose reagents to bright light during storage or incubation.
• Avoid contact with Evans blue (present in the SimulFluor® Para 1, 2, 3/ Adeno Reagent) 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.
Mounting Fluid 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.
Host:
Mouse
Detection Methods:
Fluorescent
Product Name:
LIGHT DIAGNOSTICS™ SimulFluor® Para 1,2,3/Adeno, ~50 tests
Materials Required but Not Delivered:
1. 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 (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.
3. Tissue culture media such as RPMI or Eagle's Minimum Essential Medium (EMEM) with appropriate amount of precolostral FBS can be used.
4. Sterile tissue culture tubes, dram vials, or multi well plates
5. Acetone, reagent grade or better
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 with wells at least 6mm in diameter in a hydrophobic mask
7. Sterile pipettes
8. Humid chamber
9. Sodium hypochlorite solution (0.05%)
10. No. 1 coverslips
11. 37°C 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 fluorescein, excitation peak 490 nm, emission peak 520 nm
16. Sterile glass beads (1 3 mm diameter)
17. Centrifuge
18. Vortex mixer
19. Sonicator
20. Distilled or deionized water
2. 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.
3. Tissue culture media such as RPMI or Eagle's Minimum Essential Medium (EMEM) with appropriate amount of precolostral FBS can be used.
4. Sterile tissue culture tubes, dram vials, or multi well plates
5. Acetone, reagent grade or better
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 with wells at least 6mm in diameter in a hydrophobic mask
7. Sterile pipettes
8. Humid chamber
9. Sodium hypochlorite solution (0.05%)
10. No. 1 coverslips
11. 37°C 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 fluorescein, excitation peak 490 nm, emission peak 520 nm
16. Sterile glass beads (1 3 mm diameter)
17. Centrifuge
18. Vortex mixer
19. Sonicator
20. Distilled or deionized water
Antibody Type:
Monoclonal Antibody