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- : 1 kit
LIGHT DIAGNOSTICS™ Parainfluenza 1, 2, and 3 DFA, ~50 tests
Description:
LIGHT DIAGNOSTICS™ Parainfluenza 1, 2, and 3 DFA, ~50 tests
Trade Name:
- LIGHT DIAGNOSTICS
- Chemicon (Millipore)
Qty/Pk:
1 kit
Product Overview:
Light Diagnostics™ Parainfluenza 1, 2, and 3 DFA is intended for the detection and identification of parainfluenza 1, parainfluenza 2, and parainfluenza 3 following amplification in cell culture.
For in vitro diagnostic use.
Test Principle:
Light Diagnostics Parainfluenza 1, 2, and 3 DFA Kit uses fluorescein- conjugated monolconal antibodies to identify virus in infected cells. The labeled antibodies will bind to antigen present in the cell. Unbound antigen is removed by washing. The antigen-antibody complex can be visualized by fluorescent microscopy. Positive specimens exhibit apple-green fluorescence while un-infected cells are stained a dull red.
Summary and Explanation:
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).
Along with RSV, parainfluenza viruses represent the most significant upper respiratory pathogens in infants and young chil-dren, but in older children and adults may be asymptomatic or mimic the common cold (1-3,5,16). Severe croup in early childhood or infancy may result in bronchial hyperactivity in older children or adolescents after exercise. However, it has yet to be determined whether bronchial hyperactivity is a pre-existing condition which plays a role in the pathogenesis of croup or whether it develops as a complication of severe croup (17,18).
Four types of parainfluenza viruses have been identified. Types 1 and 2 are major causes of laryngotracheo-bronchitis (croup), with greatest severity in children ages 2 to 4 years (6). Type 3 infection can also lead to croup but, most notably, is a major cause of infant bronchiolitis, pneumonia, and hospitalization, second only to RSV (8-11,15). Infection from type 3 virus is most severe in infants less than 1 year old (6). Parainfluenza type 4 has been associated only with mild upper respiratory illness in adults and children.
The advent of anti-viral therapy against some respiratory viruses makes rapid identification of these organisms imperative, allowing early and proper institution of therapy. The two most widely used anti-viral agents today are amantadine/rimantadine and ribavirin, both effecting early infection steps such as penetration or uncoating (12,19). Ribavirin has broad spectrum activity in vitro against a host of viruses such as RSV, measles, influenza A and B, as well as parainfluenza (7,13,14).
Three common mechanisms of identifying parainfluenza viruses are: culture isolation/confirmation, direct detection, and serology. Of these, culture isolation/confirmation is the gold standard and the most sensitive method available for detection. Culture is used to confirm negative direct detection findings and to identify co-infections.
Parainfluenza viruses grow well in primary simian kidney cell lines (RMK) and in LLC-MK2, a rhesus kidney heteroploid cell line (21). Parainfluenza infection of tissue culture can be recognized by hemadsorption of guinea pig erythrocytes. Types 2 and 3 can be recognized by syncytium formation, while type 4 is difficult to identify in cell cultures (6).
For in vitro diagnostic use.
Test Principle:
Light Diagnostics Parainfluenza 1, 2, and 3 DFA Kit uses fluorescein- conjugated monolconal antibodies to identify virus in infected cells. The labeled antibodies will bind to antigen present in the cell. Unbound antigen is removed by washing. The antigen-antibody complex can be visualized by fluorescent microscopy. Positive specimens exhibit apple-green fluorescence while un-infected cells are stained a dull red.
Summary and Explanation:
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).
Along with RSV, parainfluenza viruses represent the most significant upper respiratory pathogens in infants and young chil-dren, but in older children and adults may be asymptomatic or mimic the common cold (1-3,5,16). Severe croup in early childhood or infancy may result in bronchial hyperactivity in older children or adolescents after exercise. However, it has yet to be determined whether bronchial hyperactivity is a pre-existing condition which plays a role in the pathogenesis of croup or whether it develops as a complication of severe croup (17,18).
Four types of parainfluenza viruses have been identified. Types 1 and 2 are major causes of laryngotracheo-bronchitis (croup), with greatest severity in children ages 2 to 4 years (6). Type 3 infection can also lead to croup but, most notably, is a major cause of infant bronchiolitis, pneumonia, and hospitalization, second only to RSV (8-11,15). Infection from type 3 virus is most severe in infants less than 1 year old (6). Parainfluenza type 4 has been associated only with mild upper respiratory illness in adults and children.
The advent of anti-viral therapy against some respiratory viruses makes rapid identification of these organisms imperative, allowing early and proper institution of therapy. The two most widely used anti-viral agents today are amantadine/rimantadine and ribavirin, both effecting early infection steps such as penetration or uncoating (12,19). Ribavirin has broad spectrum activity in vitro against a host of viruses such as RSV, measles, influenza A and B, as well as parainfluenza (7,13,14).
Three common mechanisms of identifying parainfluenza viruses are: culture isolation/confirmation, direct detection, and serology. Of these, culture isolation/confirmation is the gold standard and the most sensitive method available for detection. Culture is used to confirm negative direct detection findings and to identify co-infections.
Parainfluenza viruses grow well in primary simian kidney cell lines (RMK) and in LLC-MK2, a rhesus kidney heteroploid cell line (21). Parainfluenza infection of tissue culture can be recognized by hemadsorption of guinea pig erythrocytes. Types 2 and 3 can be recognized by syncytium formation, while type 4 is difficult to identify in cell cultures (6).
Key Applications:
Immunofluorescence
Usage Statement:
- For in vitro Diagnostic Use
- CE Mark
Components:
- Parainfluenza 1 Reagent- (Catalog No. 5019). One (1) 2 mL dropper vial containing FITC labeled Anti-Parainfluenza 1 antibody in a protein-buffered solution containing 0.02% Evans Blue counterstain, and sodium azide as a preservative.
- Parainfluenza 2 Reagent - (Catalog No. 5020). One (1) 2 mL dropper vial containing FITC labeled Anti-Parainfluenza 2 antibody in a protein-buffered solution containing 0.02% Evans Blue counterstain, and sodium azide as a preservative.
- Parainfluenza 3 Reagent - (Catalog No. 5021). One (1) 2 mL dropper vial containing FITC labeled Anti-Parainfluenza 3 antibody in a protein-buffered solution containing 0.02% Evans Blue counterstain, and sodium azide as a preservative.
- Parainfluenza Control Slide - (Catalog No. 5011). Two (2) control slides containing one infected (positive) well and one non-infected (negative) well for each parainfluenza 1, 2, and 3.
- Phosphate Buffered Saline (PBS) - (5087). One (1) packet of phosphate buffered saline (PBS) salts to yield 1 liter upon reconstitution with distilled water. Store in a clean closed container at room temperature.
- Tween 20 / Sodium Azide Solution (100X) - (5037). One (1) 10 mL of Tween 20 and sodium azide concentrate to be diluted 1:100 in PBS.
- Mounting Fluid - (Catalog No. 5013). One (1) 10 mL dropper vial containing tris buffer, glycerin, fluorescence enhancer, and sodium azide as preservative. Store at room temperature.
Brand Family:
Chemicon
Format:
FITC
Storage Conditions:
If stored at 2-8°C and protected from light, the Parainfluenza 1, 2, and 3 DFA kit is stable until the date indicated on vial. Do not freeze or expose to elevated temperatures. Discard any remaining reagents after the kit expiration date.
Warnings and Precautions:
1. Sodium azide (present in the conjugate diluent, PBS solution, and Mounting Fluid) is toxic if ingested. Sodium azide can form potentially explosive metal azides with plumbing. Upon disposal, flush with large volumes of water to prevent azide build up.
2. Pooling or diluting conjugates may cause erroneous results.
3. Do not use reagents past expiration date.
4. Avoid leaving reagents above 2-8°C for extended periods.
5. Do not allow slides to dry at any time during the staining procedure.
6. Handle all specimens and materials coming in contact with them with the same precautions as for potentially infectious material. Decontaminate with 0.05% sodium hypochlorite (a 1:100 dilution of household bleach).
7. Do not expose reagents to bright light during storage or incubation.
8. Acetone is extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks or flame. Avoid breathing vapor. Use adequate ventilation.
9. Avoid contact with Evans Blue as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.
10. Do not mouth pipette reagents.
11. Do not substitute reagents from other manufacturers.
12. Alteration of protocol provided may cause erroneous results.
13. When staining multiple samples on a slide, avoid cross-contamination between samples.
Warnings and Precautions:
1. Sodium azide (present in the conjugate diluent, PBS solution, and Mounting Fluid) is toxic if ingested. Sodium azide can form potentially explosive metal azides with plumbing. Upon disposal, flush with large volumes of water to prevent azide build up.
2. Pooling or diluting conjugates may cause erroneous results.
3. Do not use reagents past expiration date.
4. Avoid leaving reagents above 2-8°C for extended periods.
5. Do not allow slides to dry at any time during the staining procedure.
6. Handle all specimens and materials coming in contact with them with the same precautions as for potentially infectious material. Decontaminate with 0.05% sodium hypochlorite (a 1:100 dilution of household bleach).
7. Do not expose reagents to bright light during storage or incubation.
8. Acetone is extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks or flame. Avoid breathing vapor. Use adequate ventilation.
9. Avoid contact with Evans Blue as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.
10. Do not mouth pipette reagents.
11. Do not substitute reagents from other manufacturers.
12. Alteration of protocol provided may cause erroneous results.
13. When staining multiple samples on a slide, avoid cross-contamination between samples.
Host:
Mouse
Detection Methods:
Fluorescent
Product Name:
LIGHT DIAGNOSTICS™ Parainfluenza 1, 2, and 3 DFA, ~50 tests
Materials Required but Not Delivered:
1. Cell lines capable of supporting growth for isolation of parainfluenza 1, 2, and 3. Each laboratory must maintain viable stocks of cells at appropriate passage-state that will efficiently allow replication of parainfluenza 1, 2, and 3 from processed patient specimens. These cells must be checked periodically for ability to support growth of parainfluenza 1, 2, and 3. 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 parainfluenza 1, 2, and 3 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 the use of animal sera (except precolostral fetal bovine serum) 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 fetal bovine serum.
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 hydrophobic mask.
7. Sterile pipettes.
8. Humid chamber.
9. Sodium hypochlorite (0.05%) solution.
10. No. 1 coverslips.
11. Incubator with rheostat for temperature regulation.
12. Sterile swabs.
13. Forceps.
14. Vials for collection and transport 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 or sonicator.
19. Deionized or distilled water.
2. Viral transport medium which is non-inhibitory to parainfluenza 1, 2, and 3 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 the use of animal sera (except precolostral fetal bovine serum) 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 fetal bovine serum.
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 hydrophobic mask.
7. Sterile pipettes.
8. Humid chamber.
9. Sodium hypochlorite (0.05%) solution.
10. No. 1 coverslips.
11. Incubator with rheostat for temperature regulation.
12. Sterile swabs.
13. Forceps.
14. Vials for collection and transport 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 or sonicator.
19. Deionized or distilled water.
Antibody Type:
Monoclonal Antibody