Download Hantavirus (Hantaan)

3.
Hantavirus Dobrava /
Hantaan IgG/IgM
ELISA
4.
Art. No.:
Contents:
Store at:
PR59165
96 Tests
2 - 8°C
IVD
Enzyme Immunoassay for the Detection of Human
Antibodies against Hantavirus DOB/HTN in Serum
Instruction sheet / Gebrauchsanweisung / Carnet
d’instruction / instrucciones de uso / gebruiksaanwijzing
/ istruzioni per l'uso / σελίδα οδηγιών / bruksanvisning /
modo de emprego: www.progen.de
1.
Hantavirus – Reservoir, Infection
and Diseases
Hantaviruses are single-stranded enveloped RNA viruses in the Bunyaviridae family, are widespread and
are strictly associated with their serotype-specific reservoir hosts. They cause hemorrhagic fever with renal
syndrome (HFRS) in Europe and Asia and lead to hantavirus cardiopulmonary syndrome (HCPS) in America.
The severity of the disease depends on the infecting
virotype with case fatality rates from 0.1% - 10% for
HFRS and 25-35% for HCPS (13). Natural reservoir
hosts are rodents, the virus is transmitted to humans
via aerosol derived from rodent urine, feces or saliva.
HFRS is caused by Hantaan virus and the closely related Dobrava virus as well as Seoul type and Puumala
virus. Hantavirus serotypes show the same geographical distribution pattern as their reservoir hosts.
Hantaan virus (host: Apodemus agrarius) is endemic in
Asia, Dobrava virus (hosts: different Apodemus sp.) is
distributed in the Balkans, Central, Eastern and Southern Europe, the form of the disease ranges from moderate to severe. Puumala serotype (host: Myodes
clareolus) is predominant in Scandinavia and Western
Europe and causes a mild infection, also called
Nephropathia epidemica (NE). In Germany more than
90% of Hantavirus infections are caused by Puumala
Virus.
People at high risk are farmers, soldiers, campers.
2.
Application of the Test
The Hantavirus Dobrava/Hantaan (DOB/HTN) IgG/IgMELISA is suited for the detection of specific IgG and
IgM antibodies against DOB/HTN in cases of suspected hemorrhagic fever with renal syndrome (HFRS).
20-168 PR59165 Hanta (DOB/HTN) IgG+IgM Elisa en_V12
Principle of the Test
The microtiter plate is coated with recombinant nucleocapsid protein of Hantaan virus. For determination of
IgM antibodies, patient sera must be incubated with
rheumatoid factor IgG absorbent before starting the test
procedure in order to eliminate unspecific reactions
caused by IgG antibodies or rheumatoid factor. During
the incubation period specific antibodies against the
recombinant Hantaan antigen are bound to the solid
phase. After washing, the specific IgG and IgM antibodies are detected with peroxidase-conjugated secondary
antibody. Addition of substrate solution results in a
color reaction, which is proportional to the bound specific antibody content. The absorbance is then measured photometrically.
Material and Reagents Required
but not Provided
Distilled Water
Graduated cylinder
Tubes for dilution of samples
Precision pipettes (5 µl, 20 µl, 50 µl, 200 µl, 1000 µl)
Multichannel- or dispensing pipettes (100 and 200 µl)
Pipette tips
ELISA reader, 450 nm filter
Gloves
Timer
5.
Material and Reagents Provided
5.1.
Contents of Kit (PR59165)
MTP, 96-well microtiterplate, coated with recombinant
Hantaan antigen, sealed in an aluminum bag with desiccant. If required, individual wells can be broken off
from each strip. Ready-to-use!
PG, Positive control IgG, human sera with stabilizers
and preservatives; 1 vial, 1.5 ml (green lid). Ready-touse!
RG, Reference control IgG, human sera with stabilizers
and preservatives; 1 vial, 1.5 ml (red lid). Ready-touse!
PM, Positive control IgM, human sera with stabilizers
and preservatives; 1 vial, 1.5 ml (yellow lid). Ready-touse!
RM, Reference control IgM, human sera with
stabilizers and preservatives; 1 vial, 1.5 ml (blue lid).
Ready-to-use!
NEG, Negative control, human sera with stabilizers
and preservatives; 1 vial, 1.9 ml (colorless lid). Readyto-use!
SB 20x , Sample buffer (20x), PBS pH 7.4, contains
preservatives (red lid) 1 bottle, 15 ml. Dilute before
use!
WASH 20x, Wash buffer (20x), PBS pH 7.5, contains
preservatives, 1 bottle, 50 ml. Dilute before use!
CG 20x, Anti IgG peroxidase conjugate (20x), 1 vial,
750 µl (black lid). Dilute before use!
CM 20x, Anti IgM peroxidase conjugate (20x), 1 vial,
750 µl (white lid). Dilute before use!
S, Substrate, tetramethylbenzidine (TMB); 1 bottle, 12
ml. Ready-to-use!
1
STOP, Stop solution, 0.5 M sulfuric acid, 1 bottle,
12 ml. Ready-to-use!
ABS, Rheumatoid factor IgG absorbent; anti human
IgG with stabilizers and preservatives; 1 vial, 1.5 ml.
Ready-to-use!
Adhesive foil for covering ELISA test strips; 2 pieces.
6.
Preparation
6.1.
Sample Material and Storage
Human serum must be used as sample material for the
Hantavirus DOB/HTN IgG/IgM ELISA. Samples can be
stored at 2-8°C up to 6 weeks. Samples can be stored
undiluted for several months at -20°C. Avoid repeated
freezing/thawing.
6.2.
Preparation of Reagents
Allow kit to reach room temperature (20-26°C). Buffer
concentrates may contain salt crystals which dissolve
quickly at 37°C. Let buffer cool to room temperature
(20-26°C) before starting the test.
Dilute required volumes of reagents immediately before
use!
Ready-to-use sample buffer, 1+19
Example for 8 wells: Add 1 ml SB 20x to 19 ml distilled water.
Ready-to-use wash buffer, 1+19
Example for 8 wells: Add 1 ml Wash 20x to 19 ml
distilled water.
Ready-to-use conjugates, 1+19
Example for 8 wells: Add 50 µl CG 20x or CM 20x to
950 µl ready–to-use wash buffer.
Dilution of patient samples, 1+200
IgG: Add 5 µl serum to 1000 µl ready-to-use sample
buffer.
IgM: Dilution as described for IgG. Add 15 µl ABS to
250 µl diluted serum, incubate 30 min at room temperature (20-26°C).
7.
Stability
Store the test kit and components at 2-8°C. The unopened reagents are stable until the expiry date indicated.
Stability after opening:
After opening all components are stable for 3 months at
2-8°C. Place the unused microassay strips in the aluminum bag provided in the kit.
Stability after dilution:
The ready-to-use wash buffer and sample buffer are
stable for 1 week at 2-8°C. Ready-to-use conjugate
solution must be used within 60 min.
8.
Test Procedure
Sample incubation: Pipette 100 µl undiluted negative, positive, and reference controls as well as diluted
(and, if required pretreated with ABS) patient sera per
well. Cover strips with adhesive foil. Incubate at 37°C
for 45 min.
Wash: Empty microassay strips and fill each well with
200 µl ready-to-use wash buffer. Empty wells again
and repeat this wash step three times. Remove excess
liquid by tapping the strips onto absorbent paper.
20-168 PR59165 Hanta (DOB/HTN) IgG+IgM Elisa en_V12
Conjugate incubation: Pipette 100 µl ready-to-use
conjugate (IgG or IgM) per well. Cover strips with adhesive foil. Incubate at 37°C for 45 min.
Wash: Empty microassay strips and carry out wash
steps as described above (4 x 200 µl per well).
Substrate reaction: Pipette 100 µl ready-to-use substrate per well. Incubate 10 min at room temperature
(20-26°C).
Stop: Add 100 µl STOP to each well.
Measure color within 20 min at 450 nm (reference
wavelength at 650 nm).
9. Notes for the User
For professional use.
Precision and recovery depend on the following
critical factors:
Hemolytic, lipemic, icteric or microbially contaminated
samples could cause erroneous results.
Perform the incubations at 37°C and room temperature
(20-26°C), as required.
Maintain an exact pipetting sequence.
Run test in duplicate.
Incubation periods should not be exceeded by more
than 10%. Incubation period starts after the last pipetting step.
The time to pipette the samples should not exceed
60 sec for each ELISA test strip.
The time to pipette conjugate, substrate and stop solution should not exceed 10 sec for each ELISA test strip.
Security notes:
Stop solution (sulfuric acid) and components of substrate may cause skin irritations. If acid or substrate
should come into contact with eyes, rinse out immediately with plenty of water and consult a physician!
The human serum used in this product has been tested
for Human Immunodeficiency Virus, Hepatitis B and C
and found to be negative (not repeatedly reactive).
However, all human blood products should be considered to be potentially infectious. Observe general precautions concerning the handling of potentially infectious material.
Some of the reagents contain preservatives. Do not
swallow! Avoid any contact with skin or mucous membranes!
Safety data sheet is available on request!
Disposal considerations
Product: Chemicals must be disposed of in compliance
with the respective national regulations. Disposal of
biological components should be in accordance with
existing disposal practices employed for patient serum
samples or infectious waste.
Packaging: Packaging must be disposed of in compliance with the country-specific regulations. Handle contaminated packaging in the same way as the product
itself. If not officially specified differently, noncontaminated packaging may be treated like household
waste or recycled.
Measures after damage on transport
If a kit is considerably damaged, please contact the
manufacturer or local distributor. Do not use considerably damaged components for a test procedure. Store
such components or kits until the complaint is processed.
2
10. Quality Control
12. Evaluation of the Test
The following ratios and absorbent values represent
quality control parameters of the kit control. The ratios
have to be calculated and met for a valid test run (absorbance 450 nm, A):
a) IgG
Evaluation of the original Hantavirus (Hantaan) IgG
ELISA in parallel with the Hantavirus (Puumala) IgG
ELISA as well as Hantavirus Hantaan Antibody IF Test,
Hantavirus Puumala Antibody IF Test, Hantavirus
Seoul Antibody IF Test, revealed a diagnostic efficiencya as follows:
A positive control IgG or
A positive control IgM
PG
RG
> 0,8
A positive control IgG
=
A reference control IgG
NEG = A negative control IgG
A reference control IgG
RG
PM
Seoul IFT
PUU IFT
HTN IFT
n = 38
n = 29
n = 56
95%
88%
99%
75%
98%
84%
> 1.8
< 0.5
A positive control IgM
=
RM
NEG
A reference control IgM
RM
A reference control IgM
< 0.5
If the above criteria are not met, the test run is invalid
and has to be repeated.
11. Calculation and Interpretation of
Results
For calculation of results, the ratio of the absorbance of
the patient sample and the reference control is determined:
A patient sample
A reference control
Hantaanb IgG
ELISA
Puumala IgG
ELISA
Sensitivity and Specificity was determined with
194 sera of healthy blood donors and a total of 123 IgG
positive sera (56 IgG positive for Hantaan virus in indirect immunofluorescence). Sensitivity was 98%, specificity for Hantaan virus was 99%.
b) IgM
Evaluation of the original Hantavirus (Hantaan) IgM
ELISA in parallel with the Hantavirus (Puumala) IgM
ELISA as well as the Hantavirus Hantaan Antibody
IF Test, Hantavirus Puumala Antibody IF Test, Hantavirus Seoul Antibody IF Test, revealed a diagnostic
efficiency as follows:
= Q
and interpreted as follows:
Seoul IFT
PUU IFT
HTN IFT
n = 47
n = 25
n = 50
95%
74%
100%
68%
99%
62%
a) For IgG antibodies
Q<1
ELISA
> 2
A negative control IgM
=
IFT
Negative: No IgG antibodies against
DOB/HTN virus detected.
No clear interpretation possible. The course
of the disease should be monitored after
10 days.
1  Q  1.5 In case of suspected Hantavirus infection, it
is recommended to test the sample also for
DOB/HTN IgM antibodies and/or antibodies
against the Puumala serotype.
IFT
ELISA
Hantaanc IgM
ELISA
Puumala IgM
ELISA
Sensitivity and specificity was determined with 194 sera
of healthy blood donors and a total of 122 IgM positive
sera (50 IgM positive for Hantaan virus in indirect immunfluorescence). Sensitivity was 100% and the specificity for Hantaan virus was 100%.
13. Upgrade of Test Application
Q<1
Negative: No IgM antibodies against
DOB/HTN detected.
1Q 2
No clear interpretation possible. The course
of the disease should be monitored after
10 days.
In case of suspected Hantavirus infection, it
is recommended to test the sample also for
antibodies against the Puumala serotype.
To analyze the performance of the PROGEN Hantaan
IgG/IgM ELISA for the detection of IgG and IgM antibodies against Dobrava Virus, microtiter plates were
coated with recombinant nucleocapsid protein (Nterminus) of Dobrava virus and compared with the
corresponding microtiter plate of the Hantaan Virus
ELISA kit. The test panel included human samples
derived from different geographic regions:
Belgium (mainly PUU serotype), Southern Europe/The
Balkans (mainly Dobrava serotype), Sweden (mainly
PUU serotype), China (mainly HTN serotype), Mecklenburg-Western Pomerania (mainly DOB serotype),
Lower Franconia (mainly PUU serotype), BadenWuerttemberg (mainly PUU serotype), and different
Q>2
Positive: Specific IgM antibodies against
DOB/HTN virus detected.
a
diagnostic efficiency = specificity/2 + sensitivity/2
b
New: Dobrava/Hantaan IgG ELISA (see §13)
c
New: Dobrava/Hantaan IgM ELISA (see §13)
Q > 1.5
Positive: Specific IgG antibodies against
DOB/HTN virus detected.
b) For IgM antibodies
20-168 PR59165 Hanta (DOB/HTN) IgG+IgM Elisa en_V12
3
serotype samples from external quality assessment
(EQA, Instand e.V.).
a) IgG
n = 92
PROGEN
Hantaan
MTPL
IgG
negative
PROGEN Dobrava MTPL
borderneg.
pos.
line
49
0
0
borderline
0
0
0
positive
0
1
42
Results from IgG antibody analysis of 92 samples with
both, DOB MTPL and HTN MTPL showed sensitivity
and a specificity of 100%, i.e. a diagnostic efficiency of
100%. One (1) sample was tested positive with the
HTN MTPL and was borderline with the DOB MTPL.
The remaining 91 samples revealed consistent results.
b) IgM
n = 123
PROGEN
Hantaan
MTPL
IgM
PROGEN Dobrava MTPL
borderneg.
pos.
line
negative
borderline
85
0
0
2
1
0
positive
0
1
34
Results from IgM antibody analysis of 123 samples with
both, DOB MTPL and HTN MTPL showed a sensitivity
of 100% and a specificity of 97.7%, i.e. a diagnostic
efficiency of 98.9%. One (1) sample was tested positive
with the HTN MTPL and was borderline with the DOB
MTPL, two (2) sera were borderline with the HTN MTPL
and found negative with the DOB MTPL.The remaining
120 sera revealed consistent results.
Results and Interpretation:
All tested HTN- and DOB-specific sera were found
positive with the HTN MTPL. From these conclusive
results the application of the Hantaan IgG/IgM ELISA
can be extended and upgraded to
Hantavirus Dobrava/Hantaan (DOB/HTN)
IgG/IgM-ELISA.
Due to the extensive cross-reactivity (6, 7, 8, 10, 11, 12) between Dobrava and Hantaan Virus, the most probable
serological diagnosis of the infecting hantavirus serotype can be made in connection with the patient's travel
history:
If the patient has no travel history outside Europe, a
Dobrava virus infection is most likely.
(Data/ Recommendations provided by Instand e.V.)
14. References
1. Gött P, Zöller L, Yang S, Stohwasser R, Bautz EKF,
Darai G. Antigenicity of Hantavirus nucleocapsid
proteins expressed in E. coli. Virus Res 19: 1-16
(1991)
2. Zöller L, Yang S, Gött P, Bautz EKF, Darai G. Use
of recombinant nucleocapsid proteins of the Puumala and Nephropathia epidemica serotypes of
Hantaviruses as immunodiagnostic antigens. J Med
Virol 39: 200-207 (1993)
20-168 PR59165 Hanta (DOB/HTN) IgG+IgM Elisa en_V12
3. Zöller L, Yang S, Gött P, Bautz EKF, Darai G. A
novel µ-capture ELISA based on recombinant proteins allows sensitive and specific diagnosis of
hemorrhagic fever with renal syndrome. J Clin Microbiol 31: 1194-1199 (1993)
4. Gött P, Zöller L, Darai G, Bautz EKF. A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral nucleocapsid protein. Virus Genes, 14 (1):31-40 (1997)
5. Clement J, Heyman P, McKenna P, Colson P, and
Avsic-Zupanc T. The Hantaviruses in Europe: from
the Bedside to the Bench. Emerg Infect Diseases
Vol. 3 No. 2 (1997)
6. Elgh F, Lundkvist A, Alexeyev OA, Stenlund H,
Avsic-Zupanc T, Hjelle B, Lee HW, Smith KJ, Vainionpää R, Wiger D, Wadell G and P Juto. Serological Diagnosis of Hantavirus Infections by an Enzyme-linked Immunosorbent Assay Based on Detection of Immunoglobulin G and M Responses to
Recombinant Nucleocapsid Proteins of Five Viral
Serotypes. J Clin Microbiol 35(5):1122-1130 (1997)
7. Brus-Sjölander K, Lundkvist A. Dobrava virus infection: serologic diagnosis and cross-reactions to other hantaviruses. J Virol Methods 80:137-143 (1999)
8. Araki K, Yoshimatsu KM, Ogino M, Ebihara H,
Lundkvist A, Kariwa H, Takashima I and J Arikawa.
Truncated Hantavirus Nucleocapsid Proteins for
Serotyping Hantaan, Seoul and Dobrava Hantavirus
Infections. J Clin Microbiol 2397- 2404 (2001)
9. Togni G u. a.: Präanalytik. Schweiz. Med. Forum. 6
113-120 (2002)
10. Aitichou M, Saleh S, McElroy A, Schmaljohn C,
Ibrahim MS. Identification of Dobrava, Hantaan,
Seoul and Puumala viruses by one-step real-time
RT-PCR. J Virol Methods 124:21-26 (2005)
11. Mertens M. Entwicklung serologischer Testverfahren zum Nachweis Hantavirus spezifischer Antikörper und deren Anwendung bei epidemiologischen Untersuchungen. Dissertation Universität
Greifswald (2010)
12. Papa A. Genetic Detection of Dobrava/Belgrade
Virus, Bulgaria. Letters, Emerg Infect Diseases, Vol.
17, No. 2 (2011)
13. Krüger, DH, Schönrich, G und Klempa, B Human
Vaccines 7:6, 685-693; June 2011
PROGEN Biotechnik GmbH
Maaßstraße 30
69123 Heidelberg
Germany
Telephone +49 (0) 6221 / 8278 0
Telefax +49 (0) 6221 / 827824
www.progen.de
[email protected]
Date of release: 19.07.2013
4
Test Procedure
Hantavirus DOB/HTN IgG and IgM
Sample Dilution, 1+200
1000 µl + 5 µl
Only for IgM ELISA:
250 µl diluted serum + 15 µl ABS
Incubate 30 min at room temperature (20-26°C)
Undiluted NEG, PG, RG, PM, RM, and
100 µl
Diluted Samples
Incubate 45 min at 37°C
Wash 4 x with 200 µl Wash Buffer
Diluted Conjugate
100 µl
Incubate 45 min at 37°C
Wash 4 x with 200 µl Wash Buffer
S
100 µl
Incubate 10 min at room temperature (20-26°C)
STOP
100 µl
Read absorbance at 450 nm
(reference wavelength 650 nm)
20-168 PR59165 Hanta (DOB/HTN) IgG+IgM Elisa en_V12
5