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RABIES DFA REAGENT
详细描述
Description:
LIGHT DIAGNOSTICS™ Rabies DFA Reagent
Trade Name:
- LIGHT DIAGNOSTICS
- Chemicon (Millipore)
Qty/Pk:
5 mL
Product Overview:
The Light Diagnostics Rabies DFA Reagent is intended for the detection of rabies antigens in culture and in acetone-fixed brain and submaxillary tissues of infected animals. Thus the assay could be used as an aid in the indirect diagnosis of human rabies virus infection. All specimens that are negative or indeterminate by DFA testing should be further tested by cell culture or animal inoculation methods.
For in vitro diagnostic use.
Test Principle
The Light Diagnostics Rabies DFA Reagent uses a blend of three fluorescein-labeled monoclonal antibodies directed against the rabies nucleocapsid protein to detect the virus in infected tissue. The labeled antibody is incubated with suspect rabies-infected tissue and will bind to rabies antigen present. Unbound antibody is removed by washing and the antigen-antibody complex is visualized using fluorescence microscopy. Rabies protein in infected cells will fluoresce bright apple-green.
Summary and Explanation:
Rabies virus infections have been known and recognized since ancient times (1). All species of both wild and domestic mammals are susceptible and may be capable of transmitting the disease to other animals including humans. Human mortality associated with rabies is still widely reported in countries where rabies is enzootic in domestic dogs; rabies is still considered the most significant zoonotic disease by the World Health Organization (2). Even in areas where canine rabies has been controlled by widespread dog vaccination programs, the disease may persist in terrestrial wildlife populations and bats.
The virus is transmitted by the bite of an infected animal through the introduction of infectious saliva into the wound. Non-bite transmission may occur rarely by transfer of infectious saliva or nervous tissue from a rabid animal into an existing cut, open wound, or mucous membrane. Infection can also occur from large amounts of aerosolized virus or organ transplant from an infected patient (3).
A diagnosis of rabies depends on the visualization of viral protein in the brains of infected animals using direct immunofluorescence assay (DFA) . This test plays a critical role in the management of animal bites and the prevention of rabies. DFA examination of brain tissue from a rabies-suspect animal enables the laboratory to provide a reliable determination of rabies infection within a few hours of receipt of a specimen. This allows for a definitive diagnosis before administering rabies post-exposure prophylaxis (4). A negative diagnosis is of equal importance as it prevents unnecessary and expensive post-exposure anti-rabies treatment. DFA tests provide a sensitivity and specificity that approaches 100% and is the standard to which all other methods are compared (5).
DFA performed on brain tissue is capable of detecting the virus before Negri bodies are demonstrable and is as sensitive as virus isolation in mice or cell culture (6,7,8,9,10). Early rabies conjugates were produced utilizing serum antibodies from immunized animals of a variety of species (4). Recently, monoclonal antibodies specific for antigenic sites on the rabies nucleocapsid protein have been used to provide highly specific and uniform staining reactions (10), with reduced background staining (4).
For in vitro diagnostic use.
Test Principle
The Light Diagnostics Rabies DFA Reagent uses a blend of three fluorescein-labeled monoclonal antibodies directed against the rabies nucleocapsid protein to detect the virus in infected tissue. The labeled antibody is incubated with suspect rabies-infected tissue and will bind to rabies antigen present. Unbound antibody is removed by washing and the antigen-antibody complex is visualized using fluorescence microscopy. Rabies protein in infected cells will fluoresce bright apple-green.
Summary and Explanation:
Rabies virus infections have been known and recognized since ancient times (1). All species of both wild and domestic mammals are susceptible and may be capable of transmitting the disease to other animals including humans. Human mortality associated with rabies is still widely reported in countries where rabies is enzootic in domestic dogs; rabies is still considered the most significant zoonotic disease by the World Health Organization (2). Even in areas where canine rabies has been controlled by widespread dog vaccination programs, the disease may persist in terrestrial wildlife populations and bats.
The virus is transmitted by the bite of an infected animal through the introduction of infectious saliva into the wound. Non-bite transmission may occur rarely by transfer of infectious saliva or nervous tissue from a rabid animal into an existing cut, open wound, or mucous membrane. Infection can also occur from large amounts of aerosolized virus or organ transplant from an infected patient (3).
A diagnosis of rabies depends on the visualization of viral protein in the brains of infected animals using direct immunofluorescence assay (DFA) . This test plays a critical role in the management of animal bites and the prevention of rabies. DFA examination of brain tissue from a rabies-suspect animal enables the laboratory to provide a reliable determination of rabies infection within a few hours of receipt of a specimen. This allows for a definitive diagnosis before administering rabies post-exposure prophylaxis (4). A negative diagnosis is of equal importance as it prevents unnecessary and expensive post-exposure anti-rabies treatment. DFA tests provide a sensitivity and specificity that approaches 100% and is the standard to which all other methods are compared (5).
DFA performed on brain tissue is capable of detecting the virus before Negri bodies are demonstrable and is as sensitive as virus isolation in mice or cell culture (6,7,8,9,10). Early rabies conjugates were produced utilizing serum antibodies from immunized animals of a variety of species (4). Recently, monoclonal antibodies specific for antigenic sites on the rabies nucleocapsid protein have been used to provide highly specific and uniform staining reactions (10), with reduced background staining (4).
Key Applications:
Immunofluorescence
Usage Statement:
- For in vitro Diagnostic Use
- CE Mark
Components:
- Rabies DFA Reagent (concentrate) - (Catalog No. 5100) One vial containing 5 mL of a concentrated blend of FITC-labeled monoclonal antibodies directed against rabies virus in buffer with protein-stabilizer and 0.1% sodium azide as preservative.
- NOTE: The Working Dilution must be determined by titration of the Reagent by the user.
Brand Family:
Chemicon
Presentation:
Materials Required But Not Provided:
· Fluorescence microscope with appropriate filter combination for FITC (excitation peak = 490nm, emission peak = 515nm) with 100x, 200x, and 400x magnification (dry objective).
· Incubator (37°C ± 1°C)
· Humid chamber
· Coplin staining jars
· Coverslips ( No. 1)
· Phosphate-buffered saline (PBS) (Catalog No. 5087 or equivalent)
· Rabies Reagent Diluent (Cat. No. 5070) or equivalent (PBS containing 1.0% bovine serum albumin (BSA), fraction V).
· Deionized or distilled water
· Mounting fluid suitable for rabies immunofluorescence 10% buffered glycerol at pH 8.0 and above.
· Microscope slides with hydrophobic coating or acrylic pen or wax pencil to mark uncoated slides
· Homogenizer (aerosol-tight)
· Normal and infected mouse brain suspensions
· Fluorescence microscope with appropriate filter combination for FITC (excitation peak = 490nm, emission peak = 515nm) with 100x, 200x, and 400x magnification (dry objective).
· Incubator (37°C ± 1°C)
· Humid chamber
· Coplin staining jars
· Coverslips ( No. 1)
· Phosphate-buffered saline (PBS) (Catalog No. 5087 or equivalent)
· Rabies Reagent Diluent (Cat. No. 5070) or equivalent (PBS containing 1.0% bovine serum albumin (BSA), fraction V).
· Deionized or distilled water
· Mounting fluid suitable for rabies immunofluorescence 10% buffered glycerol at pH 8.0 and above.
· Microscope slides with hydrophobic coating or acrylic pen or wax pencil to mark uncoated slides
· Homogenizer (aerosol-tight)
· Normal and infected mouse brain suspensions
Format:
FITC
Host:
Mouse
Storage Conditions:
When stored at 2-8° C, the Reagent is stable up to the expiration date printed on the label.
The diluted Reagent may be aliquoted and stored at -20°C. Avoid repeated freezing and thawing.
Discard any remaining reagents after the kit expiration date.
Warnings and Precautions:
· For in vitro diagnostic use. Directions for use should be carefully followed.
· Sodium azide, present in the reagent, is toxic if ingested. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing of solutions, flush with large amounts of water to prevent azide build-up.
· Do not mouth pipette reagents.
· Handle all specimens, slides and materials coming in contact with them as potentially infectious. Decontaminate with 0.05% sodium hypochlorite. All test material should be inactivated by incineration or steam autoclaving after use.
· Pooling or alteration of any reagent may cause erroneous results.
· Do not allow the reagent to dry on the slides during the staining procedure.
· Acetone is extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks, or flames. Use adequate ventilation and avoid breathing vapor. Performance of the fluorescence microscope is critical in achieving satisfactory test results. Microscope objectives, bulb intensity and wattage, and filters may affect results.
· Working Dilution that is visibly cloudy should not be used.
CAUTION: All procedures with known or suspected rabies-infected tissues must be performed by rabies-immunized personnel, following accepted Biosafety Level 2 practices. In addition, any manipulations or procedures with the potential for production of infectious aerosols should be performed in a Class II biosafety cabinet. Suitable protective clothing should be worn.
The diluted Reagent may be aliquoted and stored at -20°C. Avoid repeated freezing and thawing.
Discard any remaining reagents after the kit expiration date.
Warnings and Precautions:
· For in vitro diagnostic use. Directions for use should be carefully followed.
· Sodium azide, present in the reagent, is toxic if ingested. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing of solutions, flush with large amounts of water to prevent azide build-up.
· Do not mouth pipette reagents.
· Handle all specimens, slides and materials coming in contact with them as potentially infectious. Decontaminate with 0.05% sodium hypochlorite. All test material should be inactivated by incineration or steam autoclaving after use.
· Pooling or alteration of any reagent may cause erroneous results.
· Do not allow the reagent to dry on the slides during the staining procedure.
· Acetone is extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks, or flames. Use adequate ventilation and avoid breathing vapor. Performance of the fluorescence microscope is critical in achieving satisfactory test results. Microscope objectives, bulb intensity and wattage, and filters may affect results.
· Working Dilution that is visibly cloudy should not be used.
CAUTION: All procedures with known or suspected rabies-infected tissues must be performed by rabies-immunized personnel, following accepted Biosafety Level 2 practices. In addition, any manipulations or procedures with the potential for production of infectious aerosols should be performed in a Class II biosafety cabinet. Suitable protective clothing should be worn.
Detection Methods:
Fluorescent
Product Name:
LIGHT DIAGNOSTICS™ Rabies DFA Reagent
Antibody Type:
Monoclonal Antibody
