CAT.NO: IA-0001

GENERAL DESCRIPTION

This kit is a chromatographic immunoassay for qualitative detection of hemagglutinin (HA) of Influenza Type-A viruses, H5 strain (also known as highly pathogenic avian influenza) in avian excretes, human nasopharyngeal aspirates,swabs, nasal wash, chicken embryo whole virus inoculation or viral lysates, etc.

It is intended for clinical identification of specific H5, type-A influenza viruses.The use of this chromatographic immunoassay provides rapid reliable and safe diagnostic method for detection of H5 avian influenza within 30 minutes. Simple, single use device that has integrated quality control band and do not require any additional laboratory equipment.

SUMMARY

Avian influenza is an infectious disease of birds caused by type A strains of the influenza virus. The disease, first identified in Italy more than 100 years ago, now occurs worldwide. Infection triggers a wide spectrum of symptoms in birds, ranging from mild illness to a highly contagious and rapidly fatal disease resulting in severe epidemics. In the H5N1 bird flu in Hong Kong in 1997, patients had developed symptoms of fever, sore throat, cough and, in several of the fatal cases, severe respiratory distress secondary to viral pneumonia. Previously healthy adults and children, and some with chronic medical conditions, were affected.More recently, outbreaks of avian influenza H5N1 occurred among poultry in eight countries in Asia (Cambodia, China, Indonesia, Japan, Laos, South Korea, Thailand, and Vietnam) during late 2003 and early 2004. At that time, more than 100 million birds in the affected countries either died from the disease or were killed in order to try to control the outbreak.

By March 2004, the outbreak was reported to be under control. Beginning in late June 2004, however, new deadly outbreaks of influenza H5N1 among poultry were reported by several countries in Asia (Cambodia, China, Indonesia, Malaysia [first-time reports], Thailand, and Vietnam ). It is believed that these outbreaks are ongoing. Human infections of avian influenza H5N1 however, have been reported in Cambodia (1case/1 death) Thailand (17cases/1 deaths) and Vietnam (51cases/ 33deaths) during both of these outbreak periods. Viral Strains

Hemagglutinin (HA) is a surface glycoprotein on Influenza A responsible for binding to N-AcetylNeuraminic Acid (NeuNAc) or commonly Sialic acid on host cell surface receptors. The Influenza viruses form the A virus group have principally similar morphological, chemical and biological features. The differentiation of the types is possible by the different antigenicity of their nucleo- and matrix proteins that have type-specific antigenicity. However, the essential immunodominant antigens and primary targets in diagnosis are the hemagglutinin (HA) and the neuraminidase (NA) antigens.

PRINCIPLE OF ASSAY

The H5-HA(Ag) Rapid Test employs chromatographic lateral flow device with double antibody

When the sample is added, it migrates by capillary diffusion trough the strip rehydrating the gold conjugate. If present, HA will bind with anti-HA-Au forming particles. These particles will continue to migrate along the strip until the Test Zone (T) where they are captured by monoclonal anti-HA antibodies previously immobilized there, and a visible red line will appear. If there is no H5 specific HA in sample, no red line will appear in the Test Zone (T). The anti-HA-Au will continue to migrate alone until is captured in the Control Zone (C) from immobilized goat anti-mouse IgG antibodies aggregating in a red line, which indicates the validity of the test. "sandwich" –like principle for detection of hemagglutinin (HA) of H5 influenza in sample. Colloidal gold conjugated monoclonal antibodies (anti-HA-Au) reactive to H5 HA, are dry-immobilized onto a nitrocellulose membrane strip.

COMPONENTS

• Twenty Cards/kit
• Rapid H5-HA(Ag) card in aluminium pouch with desiccant:
• Virus Lysis Buffer - one bottle of 30ml.
• Package Insert -1 copy
• Materials required but not provided: clock or timer, disposable pipettes, specimen collection container, centrifuge, biohazard waste container

SPECIMEN COLLECTION

For liquid specimens

In case that solid specimens will be tested (e.g. fecal samples), such sample must be dissolved 1:10 with lysis buffer ( for example 0.1g of sample mixed with 1ml of lysis buffer).

For specimens collected into cotton swabs, elute the samples with 1ml of virus lysis buffer. Shake thoroughly at room temperature for 30 minutes and than centrifugate. Use only the supernatant for testing.

Samples that should not be tested within 24 hours after collection, should be stored frozen at –mix each specimen (1:1) with virus lysis buffer. –80°C.

STORAGE AND STABILITY

This test can be stored at room temperature (18-30℃, do not freeze!) for 18 months from the date of manufacture (see label on strip pouch). Use immediately after opening.

PRECAUTIONS AND SAFETY

This test is for In Vitro Use only

FOR PROFESSIONAL USE ONLY

All the waste and sample should be treated in case of transmitting disease and must be properly disinfected (autoclaving is preferred) before disposal.

Once taking the strip out of the pouch, carry out your testing as early as possible (no more than 20minutes) to avoid moisture. The nitrocellulose membrane can absorb water, which can affect the test chromatography performance.

The performance characteristics of the test depend on sample quality and preparations. For strong positive samples, the red line corresponding to the Test Zone (T) may appear in 3-5 minutes after sample loading, but for weak positive samples, the red line may appear in the Test Zone(T) in 15 minutes. Results obtained after 30 minutes can lead to incorrect interpretation.

• Make sure that the test is within the validity indicated.
• Calibrate the pipette frequently to assure the accuracy. Use different disposal pipette tips for each specimen in order to avoid cross-contaminations.
• Do not modify the test procedure.
• Avoid moisture.
• A test giving an invalid result should be repeated.
• Do not reuse lancets, test strips, pipettes. Autoclave before disposal.

All specimens from human origin should be considered as potentially infectious. Strict adherence to GLP (Good Laboratory Practice) regulations can ensure the personal safety. Never eat, drink, smoke, or apply cosmetics in the assay laboratory.

ASSAY PROCEDURE

Allow the strip to reach room temperature (appropriately 30min). Open the pouch and slowly pipette 80μl of virus lysate buffer pre-treated sample into the sample window (S). Avoid dropping solutions in the observation window. Place the strip on flat surface and read the results within 30minutes.

INTERPRETATION OF RESULTS AND QUALITY CONTROL

Negative Result

Quality Control: One red line always appears next to Control Zone (C) If no red line appears in the Control Zone (C), the test is invalid - discard the test and repeat with new sample and new strips: Only one red line appears in the Control Zone (C), indicating that no hemagglutinin of Influenza A, viruses H5 strain have been detected with this Rapid Test. However, this does not exclude the possibility for infection with H5 influenza virus.

Positive Results

Two red lines appear, indicating that hemagglutinin of Influenza A viruses, H5 strain has been detected using this Rapid Test.

Positive negative invalid

The positive result obtained with this H5-HA(Ag) Rapid Test alone cannot be the final diagnosis of infection with H5 strain avian influenza. Any positive result must be interpreted in conjunction with the patient clinical history and another laboratory testing results. Follow-up and supplementary testing with other analytical system is required to confirm any positive results.

I. SPECIFICITY EVALUATION ON H5 STRAINS FROM VARIOUS SOURCES:

Virus                         H5 Strain Avian Influenza                               Non-H5 Strains
 
Source Hong Kong Indonesia Vietnam Malaysia Europe Mainland China  H3 H9 H7 NDV
 
Pos/total 6/6             7/7          5/5         1/1        1/1       24/24              0/2 0/5 0/2 0/2

 II. DETECTION OF NON-H5 STRAINS:

III. DETECTION OF H5 STRAINS:

RESULTS

• ELISA false positive rate:     

          – 0% among human and other influenza virus strains (10 samples)

          - 0.25% (1/400) bird excrements samples.

• Rapid test false positive rate: 

          – 0% among human and other influenza virus strains (10 samples)

PRODUCTION

• ISO 9001:2000; ISO 13485:2003, GMP certificated

REFERENCES

• Fouchier RAM, Schneeberger PM, Rozendaal FW, et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci 2004. Published online before print January 26, 2004
• Fouchier RAM, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls J Virol 2005 Mar;79(5):2814-22
• Horimoto T, Kawaoka Y. Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev 2001;14(1):129-49
• Keawcharoen J, Oraveerakul K, Kuiken T, et al. Avian influenza H5N1 in tigers and leopards. Emerg Infect Dis 2004 Dec;10(12)
• Luschow D, Werner O, Mettenleiter TC, et al. Protections of chickens from lethal avian influenza A virus infections by live-virus hemagglutinin (H5) gene. Vaccine 2001 Jul 20;19(30):4249-59
• Monto AS. The threat of an avian influenza pandemic. (Perspective) N Engl J Med 2005 Jan 27;352(4):323-4
• OIE. Highly pathogenic avian influenza. Technical disease card database. Apr 22, 2002

For ordering please go to DR.WANG, Fax: 0049 7071 792022, E-Mail:info@normae.de

• For more Ordering Information
• Asking for more information or protocol: info@normae.de