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The Diagnostic Hybrids, Inc. D3 DFA Herpes Simplex Virus Identification and Typing Kit is intended for use in the qualitative detection and typing of human herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) in cell cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). Negative results do not rule out an infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Performance using direct specimen testing has not been evaluated.

The Diagnostic Hybrids, Inc. D3 DFA Herpes Simplex Virus Identification and Typing Kit uses two separate blends of HSV Type-1 (HSV-1) and HSV Type-2 (HSV-2) antigen-specific murine MAbs that are directly labeled with fluorescein for the rapid detection and typing of HSV. The reagents can specifically detect and type either HSV-1 or HSV-2. The HSV-1 MAbs were developed using HSV-1(f) cell lysate as immunogen - one MAb has been determined to be directed against HSV-1 glycoprotein C1; the antigen to the other is undetermined. The HSV-2 MAbs were developed using a HSV-2 recombinant glycoprotein G as immunogen. Kit Components: • HSV-1 DFA Reagent - One dropper bottle containing a blend of two fluorescein labeled murine monoclonal antibodies directed against HSV-1 specific glycoproteins. The HSV-1 MAbs were developed using HSV-1(f) cell Ivsate as immunogen - one MAb has been determined to be directed against HSV-1 glycoprotein C1, the antigen to the other is undetermined. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. • HSV-2 DFA Reagent - One dropper bottle containing a blend of two fluorescein labeled murine monoclonal antibodies directed against HSV-2 specific glycoproteins. The HSV-2 MAbs were developed using a HSV-2 recombinant glycoprotein G immunogen. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative • HSV-1/HSV-2 Antigen Control Slides - Individually packaged control slides containing wells with cell culture derived positive and negative control cells. Each slide consists of four wells containing acetone fixed cells; two wells of noninfected cells and one well each of HSV-1 infected cells and HSV-2 infected cells. Each slide is intended to be stained only one time. • PBS Concentrate - A 40X concentrate consisting of 4% sodium azide in phosphate buffered saline (after dilution to 1X in water, the concentration of sodium azide in the solution is 0.1%). • Mounting Fluid - an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. The cells to be tested, on a slide prepared from a tube culture or on a monolayer of cells cultured in a multi-well plate or a coverslip in a shell vial, are fixed in acetone. The HSV-1 DFA Reagent or HSV-2 DFA Reagent is added to the cells to detect the presence of HSV specific viral antigens. After incubating for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied Phosphate Buffered Saline (PBS). To prepare the slide for examination, a drop of the supplied Mounting Medium is added to the stained cells and a coverslip is placed on the slide. To prepare the centrifuge enhanced cell cultures for examination, a drop of Mounting Fluid is placed on a clean microscope slide. The coverslip is removed from the shell vial and placed on to the Mountina Fluid. For multi-well plates, monolayers are fixed with an 80% aqueous acetone solution. The HSV DFA Reagent is added to the cells to detect the presence of any HSV specific viral antigens. After incubating for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied Phosphate Buffered Saline (PBS). Mounting Fluid is added to each well to cover the monolayers. The slides or wells are examined using a fluorescence microscope equipped with the correct filter combination for FITC at a magnification of 100-400X. Virus infected cells will be stained with bright apple-green fluorescence while uninfected cells will contain no apple-green fluorescence but will fluoresce red by the Evan's Blue counterstain which is included in the HSV DFA Reagent. If no fluorescent cells are found, report result as, "No herpes simplex virus detected." If fluorescent cells are found in the HSV-1 DFA Reagent stained monolayer, report result as "Herpes simplex virus type 1 isolated by cell culture". If fluorescent cells are found in the HSV-2 DFA Reagent stained monolaver, report result as "Herpes simplex virus type 2 isolated by cell culture." If fluorescent cells are found in both the HSV-1 and HSV-2 stained wells, the results should be reported as "Herpes simplex virus types 1 and 2 isolated by cell culture". Included in the kit are HSV-1/HSV-2 Antigen Control Slides. A Control Slide is intended to function as an indicator that the kit reagents are working properly in the test. [The slides are prepared with wells of HSV-1 infected cells, HSV-2 infected cells, and uninfected cells.] Positive and negative controls must demonstrate appropriate staining characteristics for specimen results to be valid. Controls may also aid in the interpretation of test results. It is recommended that cell culture positive (infected with known HSV isolate) and negative (uninfected cells) controls be run with each assay to provide a means to ensure adequate performance of the cell culture system used. If control cultures fail to perform correctly, results are considered invalid.

The Diagnostic Hybrids D3 DFA Herpes Simplex Virus Identification Kit is intended for use in the qualitative detection of human herpes simplex virus (HSV) in cell cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). Negative results do not preclude an infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Performance using direct specimen testing has not been evaluated.

The Diagnostic Hybrids D3 DFA Herpes Simplex Virus Identification Kit includes a DFA Reagent that contains a blend of four fluorescein-labeled murine monoclonal antibodies (MAbs), two directed against HSV type 1 (HSV-1) and two against HSV type 2 (HSV-2). The HSV-1 MAbs were developed using HSV-1(f) cell lysate as immunogen -- one has been determined to be directed against HSV-1 glycoprotein C1, the antigen to the other is undetermined. The HSV-2 MAbs were developed using a HSV-2 recombinant glycoprotein G immunogen. The kit includes the following components: • HSV DFA Reagent - A blend of fluorescein labeled murine monoclonal antibodies directed against antigens produced in HSV-infected cell culture. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. • HSV Antigen Control Slides - Individually packaged control slides containing wells with cell culture derived positive and negative control cells. Each HSV positive well is identified. The negative wells contain uninfected cells. Each slide is intended to be stained only one time. • PBS Concentrate - A 40X concentrate consisting of 4% sodium azide in phosphate buffered saline (after dilution to 1X in water, the concentration of sodium azide in the solution is 0.1%). • Mounting Fluid - an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. The cells to be tested, on a slide prepared from a tube culture or on a monolayer of cells cultured in a multi-well plate or a coverslip in a shell vial, are fixed in acetone. The HSV DFA Reagent is added to the cells to detect the presence of HSV specific viral antigens. After incubating for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied Phosphate Buffered Saline (PBS). To prepare the slide for examination, a drop of the supplied Mounting Medium is added to the stained cells and a coverslip is placed on the slide. To prepare the centrifuge enhanced cell cultures for examination, a drop of Mounting Fluid is placed on a clean microscope slide. The coverslip is removed from the shell vial and placed on to the Mounting Fluid. For multi-well plates, monolayers are fixed with an 80% aqueous acetone solution. The HSV DFA Reagent is added to the cells to detect the presence of any HSV specific viral antigens. After incubating for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied Phosphate Buffered Saline (PBS). Mounting Fluid is added to each well to cover the monolayers. The slides or wells are examined using a fluorescence microscope equipped with the correct filter combination for FITC at a magnification of 100-400X. Virus infected cells will be stained with bright apple-green fluorescence while uninfected cells will contain no apple-green fluorescence but will fluoresce red by the Evan's Blue counterstain' which is included in the HSV DFA Reagent. If no fluorescent cells are found, report result as, "No herpes simplex virus detected". If fluorescent cells are found, report result as, "Herpes simplex virus isolated by cell culture." Included in the kit are HSV Antigen Control Slides. A Control Slide is intended to function as an indicator that the kit reagents are working properly in the test. [The slides are prepared with wells of HSV infected cells and uninfected cells.] Positive and negative controls must demonstrate appropriate staining characteristics for specimen results to be valid. Controls may also aid in the interpretation of test results. It is recommended that cell culture positive (infected with known HSV isolate) and negative (uninfected cells) controls be run with each assay to provide a means to ensure adequate performance of the cell culture system used. If control cultures fail to perform correctly, results are considered invalid.

Light Diagnostics SimulFluor® HSV1/2 Immunofluorescence Assay is a direct immunofluorescence test intended for the detection and identification of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) following amplification in cell culture or by direct examination of clinical specimens prepared by cytocentrifugation. Specimens found to be negative on direct specimen examination should be tested by cell culture. For in vitro diagnostic use.

Light Diagnostics SimulFluor® HSV 1/2 Immunofluorescence Assay utilizes a single reagent for the simultaneous detection and identification of HSV-1 and HSV-2. The SimulFluor® HSV 1/2 Reagent consists of two components; the primary component specific for HSV-1 will bind to the glycoprotein C and a capsid-associated protein in HSV-1 infected cells, while the secondary component, specific for HSV-2, will bind to the glycoprotein G in HSV-2 infected cells. Unbound reagent is removed by rinsing with phosphate-buffered saline (PBS). Illumination with ultraviolet light allows visualization of the antigenantibody complexes by fluorescence microscopy. When an FITC filter set is used, HSV-1- infected cells will exhibit apple-green fluorescence and HSV-2infected cells will exhibit yellow-gold fluorescence. The uninfected cells will stain a dull red due to the presence of Evans blue in the SimulFluor® HSV 1/2 reagent. A blend of monoclonal antibodies directed against HSV-1 and HSV-2 is used in the Light Diagnostics SimulFluor® HSV 1/2 reagent. The use of monoclonal antibodies ensures increased specificity of reagent and reduces the risk of nonspecific background or interference.

The ELVIS® HSV ID/Typing Test System is a qualitative test indicated for use in the isolation and identification of HSV from lesions and body fluids suspected of containing viable HSV-1 and/or HSV-2. Both serotypes have been isolated from various parts of the body, particularly when HSV-associated disease is indicated. Performance of this assay has not been established for use with antiviral therapy or prenatal monitoring.

The subject device provides Cells, Replacement Medium and Test Reagents for the culture, identification and typing of HSV isolated from patient specimens. It consists of ELVIS™ HSV Cells in a tube configuration [ELVIS™ HSV Gold Test, 510(k) No. K960578] and in shell vial and multiwell plate configurations [ELVIS™ HSV Test, 510(k) No. K941924],and reagents to which have been added HSV-typing monoclonal antibodies to permit the user to directly type the HSV-positive specimens. ELVIS™ HSV Cells are genetically engineered Baby Hamster Kidney cells, which, when infected with either HSV-1 or HSV-2 are induced to generate and accumulate an endogenous, intracellular bacterial enzyme, β-galactosidase. Other related viruses are not capable of inducing this enzyme. In addition to the induction of this specific enzyme, HSV infection of cells results in the formation of HSV, type-specific proteins. The presence of these proteins can be detected microscopically by their fluorescence when HSV-type-specific, fluorescent labeled antibodies are used. Thus, when HSV-infected monolayers are fixed using Solution 1 and treated with Solution 2T, which contains the chromogenic substrate for the ß-galactosidase enzyme, those cells infected with HSV are stained an indigo blue while uninfected cells remain colorless. Both type-2-specific, fluorescein labeled monoclonal antibodies and type-1-specific, non-labeled monoclonal antibodies are also incorporated in Solution 2T. This allows for the monoclonal antibodies to react with their specific antigens in the HSV-infected ELVIS™ Cells at the same time as the enzyme is causing the deposition of blue stain. After a 1 hour incubation period, which allows for these reactions to proceed in monolayers whose specimens contained viable HSV, the monolayers are examined for blue cells using standard light microscopy: those which do not contain blue cells are negative for HSV; those which contain blue cells are positive for HSV and can now be examined with a fluorescence microscope to determine the HSV type. I E fluorescent cells are seen, the HSV in the specimen is type 2; if no fluorescent cells are seen, the HSV in the specimen is type 1. This can be confirmed by treating the monolayer with Solution 3 which contains fluorescein-labeled goat-antimouse antibodies which will react with the type 1-specific monoclonal antibodies in the HSV-1 infected cells. Thus, the major change in the composition of the above legally marketed devices is the incorporation of the monoclonal antibodies into the Solution 2 which contains the chromogenic substrate for the ß-galactosidase enzyme (which has been re-named Solution 2T. Additionally, two other reagents are included with the subject device, i.e., Solution 3, the fluorescein-labeled goat-antimouse antibody, and a Buffered Glycerol Mounting Medium used on the fixed and stained monolayers, to prevent them from drying before microscopic examination for fluorescence.

HSV-1 Antigen Control Slides are quality control materials used to confirm the accuracy of staining reagents used in cell culture confirmatory assays for the detection of HSV-1. Each individual slide contains two wells of fixed cells, one HSV-1 infected well and one uninfected well. The two wells of cells on the HSV-1 Antigen Control Slide are stained concomitantly with the cells inoculated with patient sample providing both a positive and negative control for the staining reagents and procedure. Lack of staining of the positive well indicates a failure of one or more components of the staining reagent.

Each individual slide contains two wells of fixed cells, one HSV-1 infected well and one uninfected well.

HSV-2 Antigen Control Slides are quality control materials used to confirm the accuracy of staining reagents used in cell culture confirmatory assays for the detection of HSV-2. Each individual slide contains two wells of fixed cells, one HSV-2 infected well and one uninfected well. The two wells of cells on the HSV-2 Antigen Control Slide are stained concomitantly with the cells inoculated with patient sample providing both a positive and negative control for the staining reagents and procedure. Lack of staining of the positive well indicates a failure of one or more components of the staining reagent.

Each individual slide contains two wells of fixed cells, one HSV-2 infected well and one uninfected well.

The Test Kit provides the cells and reagents necessary for detection and identification of HSV in patient specimens.

The subject device consists of a mixed cell monolayer comprised of MRC-5 (Human Fetal Lung) and genetically modified Baby Hamster Kidney Cells (ELVIS™ HSV cells) which, when infected with HSV-1 or -2, are activated to produce and accumulate intracellularly the bacterial enzyme, beta-galactosidase. As in standard tube culture procedures, inoculated monolayers are examined daily for CPE. When CPE is observed, the monolayers are fixed and stained for the presence of beta-galactosidase. If blue cells are detected, indicating the presence of the enzyme which was induced by HSV, then the specimen is confirmed as being positive for HSV. If CPE is not detected by day 7 after inoculation, the monolayers are stained for the presence of pre-CPE, blue, HSV-infected cells. If none are found, the specimens are negative for HSV.