當(dāng)?shù)貢r(shí)間2014年8月7日，據(jù)英國(guó)《每日郵報(bào)》消息，75歲的西班牙牧師米格爾 帕拉斯，因感染埃博拉病毒從利比亞被軍用飛機(jī)遣返回馬德里。這使他成了第一個(gè)帶著致命的埃博拉病毒回歐洲治療的人。據(jù)說帕拉斯先生的情況在一夜之間發(fā)生惡化，據(jù)當(dāng)?shù)孛襟w報(bào)道他現(xiàn)在已經(jīng)不能獨(dú)立行走，而且病毒很有可能會(huì)轉(zhuǎn)移.參考試劑使用：

 Ebola virus test kit recommended as below:

Enzyme-linked immunosorbent assay (ELISA) for the
qualitative detection of human IgM class antibodies to
hantavirus
This package insert is for export only and not for
distribution in the United States.
Outside of the United States:
For in vitro Diagnostic Use.
INTENDED USE
Focus Diagnostics’ Hantavirus IgM DxSelectTM test is intended for the qualitative detection of human IgM class antibodies to Hantavirus in human sera.
SUMMARY AND EXPLANATION OF TEST
Hantavirus (HTV) is a novel genus in the Bunyaviridae family, in which it stands as the only non-arthropod-borne human pathogen. Transmission to man occurs
via inhalation of viral particles from aerosolized excreta from HTV-infected rodents. Phylogeny and epidemiology of HTV’s are closely linked to those of their
respective rodent reservoirs. To date, up to 30 different HTV strains have been isolated or characterized, of which at least 13 have pathogenic significance for
man. The clinically most important strains are murine Hantaan (HTN) and Seoul (SEO) in Asia, Puumala (PUU) and Dobrava (DOB) in Europe, and Sin Nombre
(SNV) in the America’s.1 Whereas HTN, PUU, SEO, and DOB all have the kidney as the main target organ in man, SNV and SNV-like agents primarily affect
the lung. The HTV strains typically have regional distribution; however, geographic overlap between strains does exist.
Over 150,000 cases of hemorrhagic fever with renal syndrome (HFRS) occur world wide each year.2 HFRS is characterized by fever, renal failure and, at times
hemorrhagic manifestations. The clinical manifestations of HFRS are more severe when caused by HTN and less severe when due to SEO and PUU. In central
Europe HFRS due to DOB may also cause a severe form of disease with mortality of up to 20%.3
Hantavirus pulmonary syndrome (HPS) is a disease of rapid onset characterized by fever and severe pulmonary dysfunction with mortality approaching 50%.
SNV in North America and the Andes virus in South America have been the causative agents of HPS3 to date.
The HTV are enveloped and have a tripartite, negative-stranded RNA which encodes several proteins including an RNA-dependent RNA polymerase, 2 envelope
glycoproteins and a nucleocapsid protein (NP). The small (S) segment of the genome encodes for the NP and the medium (M) segment encodes for 2 envelope
proteins, G1 and G2.4, 5 The NP is the predominant target of the antibody response; however, antibodies directed to the NP are highly cross-reactive between the
HTV species. The envelope proteins tend to induce a lower antibody response, but the antibody is species specific. Previously Rossi, et al.2 found that the HTN
NP was a potential candidate for the target antigen in an ELISA-based format to detect IgG.
Recently Elgh, et al.4 studied the kinetics of the antibody response to recombinant NP (rNP) in nephropathia epidemica patients caused by Puumala virus. They
found that IgG was detected within 2 to 8 days of disease onset, remained high for 2 to 5 months and gradually declined over 2 to 3 years. Nearly all patients
remained positive for IgG after 2 to 3 years. The IgM was detectable in most cases within 2 to 8 days of disease onset with nearly all patients positive at 5 to 15
days. The IgM titers declined rapidly and most patients are negative at 2 to 5 months after disease onset.
As is true with other viral diseases, it is difficult to diagnose HTV infection based on clinical grounds alone; thus, serologic methods to detect HTV have evolved
as the test of choice for aiding the diagnosis of HTV infection. The Focus Diagnostics Hantavirus DxSelectTM kit uses a cocktail of baculovirus-derived
recombinant NP of HTV strains. Using a rNP cocktail allows for detecting antibodies to a broad range of pathogenic species of HTV. The Focus Diagnostics
Hantavirus DxSelectTM will detect antibodies to the most clinically relevant pathogenic strains of Hantaviruses, i.e., SEO, HTN, PUU, DOB, and SNV.
TEST PRINCIPLE
In the Focus Diagnostics Hantavirus IgM DxSelectTM assay, the polystyrene microwells are coated with Hantavirus antigens. Patient sera and controls are diluted
in a solution containing hyper-immune anti-human IgG precipitating immunoglobulin to remove both free and complexed IgG from the sample. The diluted
serum samples and controls are incubated in the wells to allow any specific antibody present in the samples to react with the antigen. Nonspecific reactants are
removed by washing and peroxidase-conjugated anti-human IgM is added to react with the IgM present. Excess conjugate is removed by washing. Enzyme
substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a
spectrophotometric reading of optical density (OD), which is directly proportional to the amount of antigen-specific IgM present in the sample. Sample OD
readings are compared with reference cut-off OD readings to determine results.
MATERIALS SUPPLIED
The Focus Diagnostics Hantavirus IgM DxSelectTM Test kit contains sufficient materials to perform 96 determinations. Allow the supplied reagents to warm to
room temperature before use. All un-opened materials are stable at 2 to 8°C until the expiration date stated on the reagent label.
Antigen Wells, 96 wells REF EL1601 Ag
12 eight-well polystyrene microwell strips on a frame. Each well is coated with a blend of recombinant Hantavirus antigens. Each strip may be broken down into
individual wells for cost effective use. To avoid condensation, allow the antigen strips to warm to room temperature before opening the sealed packets.
IgM Conjugate, 16 mL REF EL1602 CONJ IgM
1 vial of affinity-purified and peroxidase-conjugated goat anti-human IgM (μ chain specific). Contains protein, buffer, and preservatives.
IgM Detectable Control, 0.30 mL REF EL1615 CONTROL >
1 vial of human serum. Contains 0.1% sodium azide as a preservative. Requires dilution before use (see Specimen, Controls and Calibrator Preparation,
below).
Non-Detectable Control, 0.30 mL REF EL1612 CONTROL <
1 vial of human serum. Contains 0.1% sodium azide as a preservative. Requires dilution before use (see Specimen, Controls and Calibrator Preparation,
below).
IgM Cut Off Calibrator, 0.30 mL REF EL1603 CONTROL CAL
1 vial of human serum. Contains 0.1% sodium azide as a preservative. Requires dilution before use (see Specimen, Controls and Calibrator Preparation, below).
Hantavirus IgM DxSelect?
Page 2
IgM Sample Diluent, 100 mL REF EL1613 DIL IgM
1 vial of goat anti-human IgG antibody, protein, surfactant, food coloring, and preservatives in PBS.
10X Wash Buffer, 100 mL REF EL0405 BUF WASH
1 vial of surfactant in PBS with preservatives.
To prepare a 1X wash buffer solution, mix 100 mL 10X Wash Buffer with 900 mL distilled (or deionized) water and rinse out any crystals. Use only the highest
grade purified water for reconstitution of the wash buffer. It has been observed that some sources of deionized water contain materials, which can interfere in the
assay. Swirl until well mixed and all crystals are dissolved.
Substrate Reagent, 16 mL REF EL0009 SUBS TMB
1 vial of tetramethylbenzidine (TMB) and hydrogen peroxide in buffer. A dark blue color indicates contamination with peroxidase; and, if this occurs, use a fresh
bottle.
Stop Reagent, 16 mL REF EL0105 SOLN STOP
One vial of 1 M sulfuric acid.
Sealing Tape
2 sheets of sealing tape.
MATERIALS REQUIRED, BUT NOT SUPPLIED
1. Distilled water
2. 250 or 500 mL wash bottle
3. 1 L graduated cylinder
4. 12 x 75 mm borosilicate glass test tubes or equivalent
5. 10 μL pipettors with disposable tips
6. 100 μL pipettors with disposable tips (100 μL 8- or 12-channel pipettor recommended for runs over 48 wells)
7. 1 mL pipet or dispenser
8. 5 mL pipet
9. Timer
10. Paper towels or absorbent paper
11. Sink
12. Vortex mixer or equivalent
13. ELISA plate spectrophotometer, wavelength = 450 nm
SHELF LIFE AND HANDLING
1. Kits and kit reagents are stable through the end of the month indicated in the expiration date when stored at 2 to 8°C.
2. Do not use test kit or reagents beyond their expiration dates.
3. Do not expose reagents to strong light during storage or incubation.
4. Allow reagents to warm to room temperature before use.
WARNINGS AND PRECAUTIONS
1. This package insert is for export only and not for distribution in the United States. Outside of the United States this kit is for in vitro diagnostic use.
2. All blood products should be treated as potentially infectious. Source materials from which this product (including controls) was derived have been
screened for Hepatitis B surface antigen, Hepatitis C antibody and HIV-1/2 (AIDS) antibody by FDA-approved methods and found to be negative.
However, as no known test methods can offer 100% assurance that products derived from human blood will not transmit these or other infectious agents, all
controls, serum specimens and equipment coming into contact with these specimens should be considered potentially infectious and decontaminated or
disposed of with proper biohazard precautions. CDC and the National Institutes of Health recommend that potentially infectious agents be handled at the
Biosafety Level 2.6,7
3. The Hantavirus antigen plates are produced with recombinant Hantavirus antigens; however, the plates should be considered potentially infectious and
handled accordingly.
4. Sodium azide at a concentration of 0.1% has been added to the controls as an antibacterial agent. To prevent buildup of explosive metal azides in lead and
copper plumbing, controls should be discarded into sewerage only if diluted and flushed with large volumes of water. Use copper-free and lead-free drain
systems where possible. Occasionally decontaminate the drains with 10% sodium hydroxide (CAUTION: caustic), allow to stand for 10 minutes, then flush
with large volumes of water.
5. The stop reagent contains sulfuric acid. Do not allow to contact skin or eyes. If exposed, flush with copious amounts of water.
6. Do not substitute or mix reagents from different kit lots or from other manufacturers.
7. Use only protocols described in this insert. Incubation times or temperatures other than those specified may give erroneous results.
8. Cross-contamination of patient specimens can cause erroneous results. Add patient specimens and handle strips carefully to avoid mixing of sera from
adjoining wells. Avoid contamination of the substrate reagent with traces of the enzyme conjugate.
9. Bacterial contamination of serum specimens or reagents can produce erroneous results. Use aseptic techniques to avoid microbial contamination.
10. Perform the assay at room temperature (approximate range 20 to 25°C).
11. Use proper pipetting techniques, maintaining the pipetting pattern throughout the procedure to ensure optimal and reproducible values.
SPECIMEN COLLECTION AND PREPARATION
Serum is the preferred specimen source. No attempt has been made to assess the assay’s compatibility with other specimens. Hyperlipemic, heat-inactivated,
hemolyzed, or contaminated sera may cause erroneous results; therefore, their use should be avoided.
Specimen Collection and Handling
Collect blood samples aseptically using approved venipuncture techniques by qualified personnel. Allow blood samples to clot at room temperature prior to
centrifugation. Aseptically transfer serum to a tightly closing sterile container for storage at 2 to 8°C. If testing is to be delayed longer than 5 days, the sample
should be frozen at –20°C or colder. Thaw and mix samples well prior to use.
Specimen, Controls and Calibrator Preparation
Dilute each specimen, control and calibrator 1:101 as follows: label tubes and dispense 1000 μL of IgM Sample Diluent into each labeled tube. Add 10 μL of
specimen, control or calibrator to each appropriate tube containing the 1000 μL IgM Sample Diluent and mix well by vortex mixing. Wait 10 minutes during
which time a fine precipitate will form in the tubes, sequestering IgG into an immune complex and preventing its interference in the IgM assay. The precipitate
will not interfere with the assay.
Hantavirus IgM DxSelect?
Page 3
TEST PROCEDURE
1. Bring all reagents to room temperature before use. Remove the Antigen Well packet from cold storage. To avoid condensation, allow micro-well strips to
reach room temperature before opening the foil packet. If less than a full plate is to be used, return unused strips to the foil packet with desiccant and reseal
completely. Store unused antigen wells at 2 to 8°C. (Note: At the end of the assay, retain the frame for use with the remaining strips.)
2. Fill wells with 1X Wash Buffer solution (see MATERIALS SUPPLIED, above) and allow to soak for 5 minutes. Decant (or aspirate) the antigen wells and
tap vigorously to remove Wash Buffer. Blot the emptied Antigen Wells face down on clean paper towels or absorbent paper to remove residual Wash
Buffer.
3. Dispense 100 μL of the IgM Sample Diluent into the “blank” wells and 100 μL of each diluted specimen, control or calibrator (see Specimen, Controls, and
Calibrator Preparation, above) into the appropriate wells. (Note: For runs with more than 48 wells it is recommended that 250 μL of each diluted sample
first be added to a blank microtiter plate in the location corresponding to that in the ELISA wells. The samples can then be efficiently transferred into the
Antigen Wells with a 100 μL 8- or 12-channel pipettor.)
4. Cover plates with sealing tape (or place in a humid chamber), and incubate for 60 ± 1 minutes at room temperature (20 to 25°C).
5. Remove sealing tape (or remove wells from the humid chamber), and empty the contents of the wells into a sink or a discard basin.
6. Fill each well with a gentle stream of 1X Wash Buffer solution from a wash bottle, then empty contents into a sink or a discard basin.
7. Repeat wash (step 6) an additional 2 times.
8. Tap the antigen wells vigorously to remove 1X Wash Buffer. Blot the emptied Antigen Wells face down on clean paper towels or absorbent paper to
remove residual 1X Wash Buffer.
9. Dispense 100 μL Conjugate to all wells, using a 100 μL 8- or 12-channel pipettor.
10. Cover plates with sealing tape (or place in a humid chamber) and incubate for 30 ± 1 minutes at room temperature (20 to 25°C).
11. Repeat wash steps 5 through 8.
12. Pipet 100 μL of Substrate Reagent to all wells, using a 100 μL 8- or 12-channel pipettor. Begin incubation timing with the addition of Substrate Reagent to
the first well. (Note: Never pour the substrate reagent into the same trough as was used for the conjugate.)
13. Incubate for 10 ± 1 minutes at room temperature (20 to 25°C).
14. Stop the reaction by adding 100 μL of Stop Reagent to all wells using a 100 μL 8- or 12-channel pipettor. Add the Stop Reagent in the same sequence and
at the same pace as the Substrate was added. In antibody-positive wells, color should change from blue to yellow.
15. Gently blot the outside bottom of wells with a paper towel to remove droplets that may interfere with reading by the spectrophotometer. Do not rub with the
paper towel as it may scratch the optical surface of the well. (Note: Large bubbles on the surface of the liquid may affect the OD readings.)
16. Measure the absorbance of each well within 1 hour of stopping the assay. Set the microwell spectrophotometer at a wavelength of 450 nm. Zero the
instrument on the blank wells.
QUALITY CONTROL
Each plate run (or strips or wells from a single plate) must include the Cut-off Calibrator and 2 controls. It is recommended that until the user becomes familiar
with the kit performance, all specimens, controls and the Cut-off Calibrator should be run in duplicate with the Cut-off Calibrator run twice for a total of 4 wells.
If single wells are used, the Cut-off Calibrator should be run in triplicate. Include a minimum of 1 blank well (containing sample diluent only) for instrument
calibration purposes.
The Cut-off Calibrator has been formulated to give the optimum differentiation between negative and positive sera. Although the absorbance value may vary
between runs and between laboratories, the mean value for the Cut-off Calibrator wells must be within the range of 0.150 to 0.450 OD units. All replicate Cutoff
Calibrator ODs should be within 0.10 absorbance units from the mean value.
Report results as index values relative to the Cut-off Calibrator. To calculate index values, divide specimen optical density (OD) values by the mean of the Cutoff
Calibrator absorbance values.
1. The Detectable Control index value should be between 1.5 and 3.0.
2. The Non-Detectable Control index value should be less than 0.8.
If the Calibrator or controls are not within these parameters, patient test results should be considered invalid and the assay repeated.
INTERPRETATION OF TEST RESULTS
Report all patient results as index values relative to the Cut-off Calibrator: to calculate index values, divide specimen optical density (OD) values by the mean of
the Cut-off Calibrator absorbance values.
> 1.10 Positive. Patient specimens, which exhibit an index value of > 1.10 indicate the presence of IgM antibodies to Hantavirus. This is indicative of an
early response to infection.
≥ 0.90
and
≤ 1.10
Equivocal. Patient specimens which exhibit an index value of ≥ 0.90 but ≤ 1.10 are considered doubtful or equivocal results. All equivoval results
should be retested. If, on retesting, the first sample remains equivocal, a second sample should be drawn several weeks later to identify a rise in
IgM antibody titer. If the second sample is either negative or equivocal, this indicates no IgM antibody to Hantavirus are detectable.
< 0.90 Negative. Patient specimens which exhibit an index value of < 0.90 indicate no detectable IgM antibodies to Hantavirus.
LIMITATIONS
1. All results from this and other serologies must be correlated with the clinical history, epidemiological data, and other data available to the attending
physician in making the diagnosis of Hantavirus infection.
2. Timing of the sample is critical in the serological demonstration of an IgM response. Patients with early Hantavirus may test IgM-negative. A negative
result does not rule out Hantavirus. If a negative test is obtained on a patient with signs and symptoms of Hantavirus, repeat testing on a second sample 2
weeks later.
3. Specimens from patients with IgM antibodies to cytomegalovirus, influenza virus, and mycoplasma may give positive or equivocal results. If these
diseases are suspected, specific serological testing should be carried out.
Hantavirus IgM DxSelect?
Page 4
REFERENCES
1. Clement, J., McKenna, P., et al. Epidemiology and laboratory diagnosis of hantavirus (HTV) infections. Acta Clinica Belgica (1995), 50:9 – 19.
2. Lundkvist, A., Hukic, M., Horling, J., Gilljam, M., Nichol, S., Niklasson, B.. Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in
Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera. J Med Virol (1997) 53:51.
3. Elgh, F., Linderholm, M., Wadell, G., Tarnvik, A., Juto, P. Development of humoral cross-reactivity to the nucleocapsid protein of heterologous
hantaviruses in nephropathia epidemica. FEMS Immunol Med Micriob (1998) 22:309.
4. Rossi, C.A., Schmaljohn, C.S. Meegan, J.M., LeDuc, J.W. Diagnostic potential of a baculovirus-expressed nucleocapsid protein for hantaviruses. Arch
Virol (1990)[Suppl 1]: 19-28.
5. Schuldt, C., Zoller, L., et al. Baculovirus expression of the nucleocapsid protein of a Puumala serotype hantavirus. Virus Genes (1994), 8: 143 – 149.
6. NCCLS. Procedures for the Handling and Processing of Blood Specimens; Approved Guideline (NCCLS H18-A2). 2nd ed. (1999).
7. CDC-NIH Manual. (1999) Biosafety in Microbiological and Biomedical Laboratories. 4th ed. And National Committee for Clinical Laboratory Standards
(NCCLS). Protection of Laboratory Workers from Instruments, Biohazards and Infectious Disease Transmitted by Blood, Body Fluids and Tissue (NCCLS
M29-A).

 angent:m.sdwandefu.com
PI.EL1600M.OUS
Rev. N
Date written: 31 March 2011
Cypress, California 90630, U.S.A