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3291 | LIGHT DIAGNOSTICS™ HSV 1&2 DFA Typing Kit, ~50 tests

3291
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      Key ApplicationsFormatHostDetection Methods
      IF FITC M Fluorescent
      Description
      Catalogue Number3291
      Brand Family Chemicon®
      Trade Name
      • LIGHT DIAGNOSTICS
      • Chemicon
      DescriptionLIGHT DIAGNOSTICS™ HSV 1&2 DFA Typing Kit, ~50 tests
      OverviewIntended Use

      The Light Diagnostics HSV 1/2 Typing DFA Kit is intended for the detection and identification of herpessimplex viruses 1 and 2 in direct specimens and for use in culture confirmation with standard tube cultures and shell vials.

      Test Principle

      Light Diagnostics HSV 1/2 Typing DFA Kit utilizes specific reagents for the detection and identification of HSV 1 and HSV 2. The HSV 1 Typing Reagent consists of two fluorescein-labeled monoclonal antibodies specific for HSV 1 glycoprotein C and ICP35 respectively (14). HSV 2 Typing Reagent consists of two fluorescein-labeled monoclonal antibodies that specifically bind HSV 2 polypeptides. In Western blots these appear as two major bands with molecular weights of between 110-120 Kd and between 78-82 Kd and are consistent with the monoclonal antibodies recognizing epitopes within glycoprotein G of HSV 2.

      The typing reagents will bind to HSV 1 or HSV 2-infected cells specifically. Unbound reagent is removed by rinsing with phosphate-buffered saline (PBS). Illumination with ultraviolet light allows visualization of the antigen-antibody complexes by fluorescence microscopy. HSV-infected cells will exhibit apple-green fluorescence with the specific reagent while cells stain a dull red due to the presence of Evans blue in the typing reagents.

      Summary and Explanation:

      Herpessimplex virus (HSV) is a member of the Herpesviridae family, alphaherpesvirus subfamily. HSV is a large, enveloped viruses, about 190 nm in diameter containing a linear, double-stranded DNA. The capsid is enclosed within a baggy, phospholipoprotein envelope. There are two biologically distinct serotypes of HSV, classified as type 1 (HSV 1) and type 2 (HSV 2); these are also known as Human Herpes Virus 1 (HHV-1) and 2 (HHV-2). The serotypes are closely related, with extensive sequence homologies of their DNAs.

      HSV causes a multitude of human diseases (1-5). Type 1 causes gingivostomatitis, intense pharyngitis, tonsillitis, and occasionally encephalitis in infants and children during their primary infections, and ocular, nasal, orolabial, and oropharyngeal lesions in children and adults. Due to the ubiquity of HSV 1 and its ease of spread by aerosolized droplets, fomites, and direct contact, most adults experience HSV 1 infection during their lifetime. Type 2 is more frequently associated with painful genital lesions, urethritis, and cervicitis in adults, and is a formidable STD agent. If the virus is present in the birth canal at the time of delivery, either from primary or recurrent infection, the result will likely be a severe generalized infection in the neonate. Thus, maternal genital HSV infections pose a substantial risk to the fetus and newborn. Recurrent infection is the most common form of infection during gestation. Shedding of virus at the time of delivery is the usual route of transmission from mother to neonate. Neonatal HSV infection is generally symptomatic and often lethal, with a mortality rate in untreated cases of 70%. The clinical presentation may be localized infection of the skin, eyes and mucosa, encephalitis, or disseminated disease.

      Most HSV infections are characterized by infection of mucocutaneous surfaces and transport to the dorsal root ganglia where further viral replication occurs followed by a period of latency. Reactivation is accompanied by viral excretion at, or close to, the original site of infection, with or without the associated clinical signs and symptoms. Recurrent lesions are usually less severe than the primary infection. In immunocompromised patients such as AIDs cases and persons recovering from cancer or organ transplant treatments, HSV can cause painful recurrent lesions or overwhelming disseminated disease.

      Acyclovir, famciclovir, foscarnet, and other nucleoside analogs can reduce clinical symptoms and virus shedding in oral and genital herpes, encephalitis, neonatal herpes, and herpetic keratitis (6-8). Identification of the virus in conjunctival specimens allows prompt treatment with acyclovir to reduce the chance of blindness. Vaccines prepared with viral gycoproteins have been studied but not proven effective (9).

      HSV can be readily recovered from clinical specimens by passage in many cells lines, such as HEp2, HEK, NCI-H292, RD, primary rabbit kidney, human diploid fibroblast cells, and others. Incubation time in stationary or roller cultures is from 1 to 5 days, and the cytopathology is quite evident. Tests on direct specimens, including IFA, EIA, latex agglutination, histopathology, DNA hybridization, and PCR are often used for CSF specimens and allow immediate serotyping of specific proteins or DNA sequences (1, 2, 12). Adequate specimens include eye swabs, swabs of vesicular lesions, saliva, throat swabs, cerebrospinal fluid, and tissues, as dictated by the clinical symptoms. Urine may also be a valid specimen..

      The initial finding of HSV 1 in ocular and respiratory infections and HSV 2 in genital infections is still relatively true, but sufficient cases of HSV 2 in nongenital lesions and HSV 1 in genital lesions occur as to make testing for both types necessary. It is important to identify the particular herpesvirus and to differentiate between them in order to institute the proper epidemiological control measures and treatment. Both serotypes can constitute a serious nosocomial problem for immunocompromised patients. Since recurrent episodes are more common in HSV 2 infected patients than in HSV 1 infected patients, typing of HSV 1 can be of prognostic value (13). In addition, specific identification will differentiate HSV from other viruses, mycoplasmas, and bacteria which can cause similar clinical findings but would require different treatment strategies.
      Materials Required but Not DeliveredAcetone, reagent grade; stored in glass

      Deionized or distilled water

      Positive controls, for culture isolation procedures (reference HSV strains available from ATCC, Rockville, MD.)

      Sodium hypochlorite solution, 0.05% (1:100 dilution of household bleach)

      Sterile 1 dram shell vials with 12 mm coverslips for growth of MRC-5 or other HSV-permissive cell line

      Tissue culture media, RPMI or Eagle's Minimum Essential Medium (EMEM) with precolostral bovine serum and antibiotics or equivalent

      Dacron or cotton swabs (not alginate) for specimen collection

      Viral transport medium (VTM) which is non-inhibitory to HSV, Hanks Balanced Salt Solution (HBSS) with antibiotics or equivalent

      Sterile PBS (pH 7.0 - 7.6)

      Microscope slides, non-fluorescing

      No. 1 coverslips

      Aspirator device with disposable sterile Pasteur pipettes

      Centrifuge capable of 700-950 x g with biohazard buckets and adapters for shell vials

      Fluorescence microscope with 100 watt mercury or halogen lamp, appropriate filter combination for FITC (excitation peak = 490 nm, emission peak = 520 nm), 100x, 200x and 400x magnification (dry objective)

      Forceps

      Humid chamber

      Incubator, 37 ± 1°C

      Syringe and needle or other implement to remove coverslip from shell vial

      Ultrasonic water bath

      Vortex mixer or sonicator
      References
      Product Information
      Components
      • Mounting Fluid - (Catalog No. 5013). One 10 mL dropper vial containing Tris-buffered glycerin, a fluorescence enhancer and 0.1% sodium azide (preservative).
      • Tween 20/Sodium Azide Solution (100X) - (Catalog No. 5037). One 10 mL vial containing Tween 20 /sodium azide concentrate.
      • Phosphate-Buffered Saline (PBS) - (Catalog No. 5087). One packet of phosphate-buffered saline salts.
      • HSV Control Slides - (Catalog No. 5093). Two slides containing one well of HSV type 1-infected cells, one well of HSV type 2 infected cells and one well of uninfected cells.
      • HSV 2 Typing Reagent - (Catalog No. 5234). One 2 mL dropper vial containing a fluorescein-labeled monoclonal antibodies specific for HSV 2, protein stabilizer, Evans blue and 0.1% sodium azide (preservative).
      • HSV 1 Typing Reagent - (Catalog No. 5233). One 2 mL dropper vial containing a fluorescein-labeled monoclonal antibodies specific for HSV 1, protein stabilizer, Evans blue and 0.1% sodium azide (preservative).
      Detection methodFluorescent
      FormatFITC
      Applications
      Key Applications
      • Immunofluorescence
      Biological Information
      ImmunogenHerpessimplex antigen specific to type 1(5233) or type 2 (5234) depending on the kit reagent used
      HostMouse
      SpecificityHerpessimplex viral antigen for type 1(5233) or type 2 (5234) depending on the kit reagent used
      Species Reactivity
      • Human
      Antibody TypeMonoclonal Antibody
      Radioactive IsotypeFITC labeled for DFA use
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • For in vitro Diagnostic Use
      Storage and Shipping Information
      Storage ConditionsWhen stored at 2-8°C, the Light Diagnostics HSV 1/2 Typing DFA 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.

      Warnings and Precautions:

      · The sodium azide (NaN3) used as a preservative in the HSV 1 Typing Reagent, HSV 2 Typing Reagent, PBS/Tween and Mounting Fluid is toxic if ingested. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides (15,16). Upon disposal, flush with large volumes of water to prevent build-up in plumbing.

      · Pooling or alteration of any reagent may cause erroneous results.

      · Do not substitute reagents from other manufacturers.

      · Incubation times or temperatures other than those specified may give erroneous results. Any such change must be validated by the user.

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

      · Handle all specimens and materials coming in contact with them as potentially infectious and dispose of with proper precautions. Decontaminate with 0.05% sodium hypochlorite.

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

      · Do not mouth pipette reagents.

      · Avoid contact with Evans blue (present in Typing Reagents) as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.

      · 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.
      Packaging Information
      Material Size1 kit
      Transport Information
      Supplemental Information
      Specifications