HSV-1 IgM ELISA Test

Cat. 4014

For In Vitro Diagnostic Use STORAGE CONDITION AND STABILITY

Store the kit at 2-8°C. Keep the microwell strips sealed with desiccants in the
aluminum bag. All kit components are stable until the expiration date printed
INTENDED USE on the label if the recommended storage conditions are strictly followed.
VicTorch HSV-1 IgM ELISA Test is a microwell ELISA test designed for the
qualitative detection of IgM antibodies to HSV-1 in human serum. PREPARATION FOR ASSAY
1. Bring all reagents to room temperature and gently mix well.
SUMMARY AND CLINICAL SIGNIFICANCE
Herpes Simplex virus type one (HSV-1) is usually associated with infections 2. Dilute the wash buffer (30x) with deionized or distilled water. Mix well.
in the oropharyngeal area and eyes while Herpes Simplex virus type two
(HSV-2) causes most genital and neonatal infections (1,2). However, the ASSAY PROCEDURE
tissue specificity is not absolute (3). HSV-2 can be isolated occasionally from 1. Prepare 1:51 dilution of test samples by adding 5 l of test samples to
the oropharyngeal area and 5 to 10% of primary genital infections may be 250l of sample diluent in the separate tubes (Note: Negative Control,
caused by HSV-1. Positive Control, and Calibrator are prediluted. No dilution is
Infants infected with HSV appear normal at birth but almost invariably necessary.).
develop symptoms during the newborn period (1,4,5). Neonatal HSV 2. Using a multichannel pipette, transfer 100 l of Negative Control,
infection may remain localized or become disseminated (1,4,5). Localized Positive Control, Calibrator, and each diluted sample from the tubes to
infection may involve one or a combination of sites. These are skin, eyes, the wells.
mouth or central nervous system. Disseminated infection is manifested by
pneumonitis, hepatitis, disseminated intravascular coagulopathy and 3. Cover the wells and incubate the wells at 37°C for 30 minutes.
encephalitis (1,4,5). Of the infants with neonatal HSV, about one half of the 4. Vigorously shake out the liquid from the wells. Fill each well with
surviving infants will develop severe neurological or ocular sequelae (3). diluted wash buffer. Wash 5 times.
A number of serologic procedures have been developed to detect antibodies 5. Add 100 l of HRP conjugate solution to each well and incubate for 30
to HSV. These include complement fixation, indirect immunofluorescent minute at 37°C.
antibody, plaque neutralization, and ELISA (2,4,6). When compared to other
serologic tests, ELISA may be a very specific, sensitive and reliable method
6. Repeat step 4.
for detection of antibodies to HSV (6,7). The ELISA procedure allows an 7. Add 100 l of the TMB substrate solution to each well and incubate for
objective determination of antibody status to be made on a single dilution of 10 minutes at room temperature.
the test specimen and is suitable for screening large numbers of patient 8. Add 100 l of stop solution to each well. Gently shake wells.
samples. 9. Set the microplate reader wavelength at 450 nm and measure the OD of
Antibody of the IgM class is produced during the first 2-3 week of infection
each well. A filter of 620-690 nm can be used as a reference wavelength
with HSV and exists only transiently in most patients. Serologic procedures
to optimize the assay result.
which measure the presence of IgM antibodies help discriminate between
primary and recurrent infections since IgM antibodies rarely found in
QUALITY CONTROL
recurrent infections.
High affinity IgG antibodies to HSV, if presence in a sample, may interfere 1. Each time the assay is run, the calibrator must be run in duplicate or
with the detection IgM specific antibody (9). High affinity IgG antibody may triplicate. A positive and negative control must also included in each
preferentially bind to HSV-1 antigen leading to false negative IgM results. assay.
Also, rheumatoid factor, if present along with antigen specific IgG, may bind 2. The mean OD value for the controls should fall within the following
to IgG causing false positive IgM results. Both of the problem can be ranges:
eliminated by deactivating IgG in the sample before testing for IgM. OD Range
Calibrator > Negative Control
PRINCIPLE OF THE ASSAY Negative Control  0.200
In the VicTorch HSV-1 IgM ELISA test, the wells are coated with HSV-1 Positive Control  0.500
antigens. Diluted patient serum and control are incubated in the wells. The
HSV-1 IgM specific antibodies, if present, will bind to the solid phase If above conditions are not met, the test should be considered invalid and
antigens. All unbound antibodies are washed off. Horse Radish Peroxidase should be repeated.
conjugated anti human IgM is added. The unbound HRP conjugate is washed
off. Upon addition TMB substrate, the bound enzyme generates color. The INTERPRETATION OF RESULTS
intensity of the color is directly proportional to the concentration of anti HSV-
1 IgM in the samples.
A. Calculations
Calculate an OD ratio for each specimen by dividing its OD value by the
calibrator OD as follows
KIT COMPONENTS
Specimen OD
1. Twelve 1x 8-well strips coated with inactivated HSV-1 antigen. The Specimen OD ratio = 
strips are packaged in a strip holder. Calibrator OD
2. Sample diluent (25 ml). B. Interpretations
3. Prediluted Negative Control, Positive Control and Calibrator, 1.5 ml for OD ratios are interpreted as follows:
each. OD Ratio
4. HRP-anti-human IgM conjugate (10 ml). Negative (Neg.) Specimens  0.90
5. Wash buffer (25 ml) 30x concentrate. Positive (Pos.) Specimens  1.00
Equivocal (Eq.) Specimens 0.91-0.99
6. TMB substrate solution (10 ml).
7. Stop solution (10 ml). 1. An OD ratio  0.90 indicates no detectable antibody to HSV-1. A
negative result indicates no current infection with HSV-1.
ADDITIONAL MATERIALS REQUIRED BUT NOT PROVIDED
Microtiter plate reader capable of measuring the optical density (OD) at 450
2. An OD ratio  1.00 is positive for IgM antibody to HSV-1. A positive
nm either with or without a reference filter of 620-690 nm. Microtiter pipettes value indicates a current infection with HSV-1.
capable of delivering 5-200 l, pipette tip and deionized or distilled water. 3. Specimens with OD ratio values in the equivocal range (0.91-0.99)
should be retested.
SPECIMEN COLLECTION
Collect blood by venipuncture. Allow blood to clot and separate the serum by LIMITATION OF THE ASSAY
centrifugation. If samples can not assayed immediately, they must be stored at 1. Test results should be interpreted in conjunction with the clinical
2-8°C or frozen. evaluation and the results of other diagnostic procedures.
2. Samples collected too early in the course of an infection may not have
detectable levels of IgM. In such cases, a second sample may be
collected within 3 weeks and tested concurrently with the original
specimen to look for seroconversion.
3. HSV-1 and HSV-2 share many cross-reacting antigens and a majority of
the antibody produced in response to an initial infection is to common
antigens.
4. Low levels of HSV-1 IgM antibodies may be detectable for up to one
year following primary infection in some patients. Measurement of IgG
antibodies may be of some value in the serological assessment of these
results.
5. The OD ratio of a single serum specimen cannot be used to determine
recent infection. Pared samples(acute and convalescent) should be
collected and tested concurrently to demonstrate seroconversion.
6. The negative results from immunosuppressed patients must be
interpreted with caution.

REFERENCES
1. Sunstrum J. Herpes simplex infections: a review. J Clin Immunoassay
1989; 12:175-8.
2. Benedetti JK Zeh J, Selke S, Corey L. Frequency and reactivation of
nongenital lesion amoung patiens with genital herpes simplex virus. Am
J Med 1995;98:237-42.
3. Nahmais A.J, Dannenbarger J, Wickliffe C, and Muther J: Clinical
aspects of infection with Herpes simplex viruses I and II. In: AJ
Nahmias, WR Dowdle, and RF Schinzai, eds., The Human Herpes
Viruses, an Interdisciplinary Perspective, Elsevier/North-Holland
Publishing co., New York, pp 2, 1980.
4. Whitley RJ and Hutto C: Neonatal Herpes Simplex virus infections. Ped.
Rev. 7:119, 1985.
5. Whitley RJ. Herpes Simplex virus infections. In:Remington JS, Klein
JO, editors. Infectious Disease of the Fetus and Newborn Infant.
Philadelphia: WB Saunders, 1990:282-305.
6. Denoyel GA, Gaspar A, and Novyrigat C: Enzyme immunoassay for
measurement of antibodies to Herpes Simplex virus infection:
Comparison and complement fixation, immunofluorescent antibody, and
neutralization techniques. J. Clin. Micro. 11:114-119, 1980.
7. Van Loon AM, van der Logt JTM, Heessen FWA, Postma B, Peeters
MF. Diagnosis of herpes simplex virus encephalitis by detection of
virus-specific immunoglobulins A and G in serum and cerebrospinal
fluid by using an antibody-capture enzyme-linked immunosorbent
assay. J Clin Microbiol 1989;27:1983-7.
8. Salonen E-M, Vaheri A, Suni J, and Wager O: Rheumatoid factor in
acute viral infections: Interference with determination of IgM, IgG, and
IgA antibodies in an enzyme immunoassay. J. Infect. Dis. 142:250-255,
1980.
9. Frser KB, Shirodaria PV, and Stanford CF:Fluorescent staining and
human IgM Br.Med. J. 3:707, 1971.

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