日本腦膜炎IgG ELISA檢測試劑盒

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INTENDED USE

Japanese Encephalitis IgG ELISA test is for the exposure to Japanese Encephalitis Virus （JEV） is a assay system for the detection of antibodies in human serum to JEV derived recombinant antigen （JERA） （1-4）. This test is to aid in the diagnosis of human exposure to the Japanese Encephalitis Virus （JEV）. It is not intended to screen blood or blood components and is for professional in vitro diagnostic use only. This kit has not been optimized for vaccine induced seroconversion studies.

SUMMARY AND EXPLANATION OF THE TEST

Exposure to JEV causes a disease with a number of symptoms including encephalitis （5-8）. The Japanese Encephalitis IgG ELISA IgG assay employs a recombinant antigen called JERA, which can be used as a rapid serological marker for JEV infection. The JERA protein is a recombinant antigen, which consists of a stretch of peptides from different parts of the JEV antigens.

PRINCIPLE OF THE TEST

The Diagnostic Automation Inc. Japanese Encephalitis IgG ELISA consists of one enzymatically amplified "two-step" sandwich-type immunoassay. In this assay, JE IgG Positive Control （Represents reactive or equivocally reactive serum）, JE Negative Control （Represents non-reactive serum）, and unknown serum samples are incubated in microtiter wells. The serum samples are diluted with Sample Dilution Buffer for IgG. After incubation and washing, the wells are treated with an antibody specific for human IgG and labeled with the enzyme horseradish peroxidase （HRP）. After a second incubation and washing step, the wells are incubated with the Tetramethyl Benzidine （TMB） substrate.

An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined by absorbance measurement at 450 nanometers. Above a certain threshold, the ratio of the absorbencies of the JERA and the control wells accuray determines whether antibodies to JEV are present.

MATERIALS PROVIDED

The Japanese Encephalitis IgG ELISA kit contains sufficient reagents for one plate of 96 wells （12 x 8 strips） each. The kit contains the following reagents:

Japanese Encephalitis IgG ELISA Assay-specific materials:

1. Coated Microtiter Strips for JEV Human IgG: Strip holder in ziplock foil, containing 96 polystyrene microtiter wells coated with monoclonal antibody bound to recombinant JERA （Rows A, B, C, and D） and Control antigen NCA （Rows E, F, G and H）. Store at 2-8ºC until ready to use.

2. Sample Dilution Buffer for IgG: One bottle, 25 mL, for serum sample dilution. Store at 2-8°C until ready to use.

3 JE IgG Positive Control: One vial, 50 μL or two vials, 30 μL each of heat–inactivated serum. The JE IgG Positive Control will aid in monitoring the integrity of the kit as well. Store at 2-8C until ready to use.

Note: For long-term storage （>1 week）, serum can be further aliquoted in a smaller volume and stored between -20C and -70C.

4. JE Negative Control: One vial, 50 μL or two vials, 30 μL each of heat–inactivated serum. The JE Negative Control will aid in monitoring the integrity of the kit as well. Store at 2-8C until ready to use.

Note: For long-term storage （>1 week）, serum can be further aliquoted in a smaller volume and stored between -20C and -70C.

5. Ready to Use Enzyme Conjugate-HRP for JE IgG:

One bottle, 6 mL of a pre-diluted goat anti-human IgG-HRP conjugate to be used as is in the procedure below. Store at 2-8C until ready to use.

6. 10X Wash Buffer: One bottle, 120 mL of Wash Buffer to be used in all the washing steps of this procedure. Store at 2-8C until ready to use.

7. Wash Solution: One bottle, 20 ml of wash solution to be used in between the washing steps after the addition of enzyme conjugate-HRP of this procedure. Store at 2-8°C until ready to use.

8. Liquid TMB Substrate: One bottle, 9 mL of liquid substrate to be used in this procedure. Store at 2-8C until ready to use.

Note: The substrate should be kept in a light -protected bottle at all times as provided.

9. Stop Solution: One bottle, 6 mL to be used to stop the reaction. Store at 2-8C until ready to use.

Caution: Stop Solution contains strong acid. Wear protective gloves, mask and safety glasses. Dispose of all materials according to safety rules and regulations.

MATERIALS REQUIRED BUT NOT PROVIDED

1. Micro titer plate reader capable of absorbance measurement at 450 nm （DAX 800）

2. Biological or High-Grade Water

3. Vacuum Pump, Plate Washer （DAX 50）

4. Humidified Incubator or Water Bath

5. 1-10 μL Single-Channel Pipettor, 50-200 μL Single-and Multi-Channel Pipettors.

WARNINGS AND PRECAUTIONS

1. A thorough understanding of this package insert is necessary for successful use of the product. Reliable results will only be obtained by using precise laboratory techniques and accuray following the package insert.

2. Do not mix various lots of any kit component within an individual assay.

3. Do not use any component beyond the expiration date shown on its label.

4. Avoid exposure of the reagents to excessive heat or direct sunlight during storage and incubation.

5. Some reagents may form a slight precipitate, mix gently before use.

6. Incomplete washing will adversely affect the outcome and assay precision.

7. To minimize potential assay drift due to variation in the substrate incubation time, care should be taken to add the stopping solution into the wells in the same order and speed used to add the TMB solution.

8. Avoid microbial contamination of reagents, especially Ready to Use Enzyme Conjugate-HRP. Avoid contamination of the TMB Substrate Solution with the Enzyme Conjugate-HRP as well.

9. Wear protective clothing, eye protection and disposable gloves while performing the assay. Wash hands thoroughly afterwards.

10. Do not eat, drink, smoke or apply cosmetics where immunodiagnostic materials are being handled.

11. Do not pipette by mouth.

12. Use a clean disposable pipette tip for each reagent, Standard, Control or specimen.

13. Cover working area with disposable absorbent paper.

WARNING: POTENTIAL BIOHAZARDOUS MATERIAL

This kit may contain reagents made with human serum or plasma. The serum or plasma used has been heat inactivated unless otherwise stated. Handle all sera and kits used as if they contain infectious agents. Observe established precautions against microbiological hazards while performing all procedures and follow the standard procedures for proper disposal of specimens.

CHEMICAL HAZARD

Material Safety Data Sheets （MSDS） are available for all components of this kit. Review all appropriate MSDS before performing this assay. Avoid all contact between hands and eyes or mucous membranes during testing. If contact does occur, consult the applicable MSDS for appropriate treatment.

SPECIEM COLLECTION AND PREPERATION

Human serum must be used with this assay. Whole blood or plasma cannot be tested directly.

Remove serum from the clot of red cells as soon as possible to avoid hemolysis.

Testing should be performed as soon as possible after collection. Do not leave sera at room temperature for prolonged periods.

Serum should be used and the usual precautions for venipuncture should be observed. The samples may be stored at 2-8C for up to 7 days, or frozen at -20C or lower for up to 30 days. To maintain long-term longevity of the serum, store at -70C. Avoid repeated freezing and thawing of samples.

Frozen samples should be thawed to room temperature and mixed thoroughly by gentle swirling or inversion prior to use. Always quick spin before use.

If sera are to be shipped, they should be packed in compliance with Federal Regulations covering transportation of infectious agents.

Do not use sera if any indication of growth is observed.

PROCEDURE

Bring all kit reagents and specimens to room temperature （~25C） before use. Thoroughly mix the reagents and samples before use by gentle inversion.

Note: For long-term storage, all serum, including the experimental, cannot be repeatedly thawed and frozen. Sera should be further aliquoted in a smaller volume and stored at –70°C. Always quick spin serum samples contained in vials or tubes to collect sample at the bottom.

Preparation for Assay:

Preparation of 1X Wash Buffer

Dilute the 10X Wash Buffer to 1X using Biological or High-Grade Water. To prepare a 1X wash buffer solution, mix 120 ml 10X Wash buffer with 1080 ml distilled （or deionized） water and rinse out any crystals. Swirl until well mixed and all crystals are dissolved. After diluting to 1X, store at room temperature for a maximum of 6 months. Use 300 μL/well for each wash cycle.

Note: Determine if any precipitate, microbial growth, or turbidity is found in the 1X Wash Buffer solution before use. Do not use the 1X Wash Buffer If such contamination is found.

Coated Micro titer Strips select the number of Coated Microtiter Strips required for the assay. The remaining unused strips should be quickly placed back into the pouch with desiccant, sealed, and stored at 2-8C until ready to use or expiration.

Assay Procedure

Important: Carefully review the table below to understand the JERA and NCA organization. Rows A, B, C, and D are coated with JERA while rows E, F, G and H have been coated with NCA.

Positive, negative, and unknown sera to be tested should be assayed in duplicate. Refer to flow chart at the end of this section for illustration of this procedure.

1. Mark the Coated Microtiter Strips to be used.

2. Dilute your test sera, the JE Negative Control, and the JE IgG Positive Control to 1/300 using the provided Sample diluent.

Note: You may use small polypropylene tubes for these dilutions and use at least 3 L of test sera and JE Negative Control and JE IgG Positive Control; for example add 3 L serum to 897 L of “Sample Dilution Buffer” for IgG）.

3. Apply the 50 L/well of 1/300 diluted test sera, JE Negative Control, and JE IgG Positive Control to the plate by multi-channel pipettor.

An exemplary arrangement for twenty-two test serum samples in duplicate is shown below.

Note: Rows A-D is pre-coated with Japanese Encephalitis Recombinant Antigen （JERA）. Rows E-H is pre-coated with Normal Cell Antigen （NCA）.

4. Cover the plate with parafilm just on the well opening surface and both sides, so the bottom of the plates is not covered.

CALCULATION

Calculation of the Immune Status Ratio （ISR）: Compute the average of the unknown sample replicates with the JERA, and the replicates with the NCA, then calculate the JERA/NCA ratio （ISR）. . An ISR of less than 2.0 for the IgG assay should be presumed negative. An ISR of greater than 5.0 for the IgG assay should be presumed positive. The table below summarizes how results should be interpreted.

ISR Results Interpretation

<2.0 Negative No detectable IgG antibody by the ELISA test

2.0-5.0 Equivocal Need confirmatory test

>5.0 Positive Indicates presence of detectable IgG antibody. Recommend supplemental confirmatory testing.

LIMITATIONS

For export use only.

Since this is an indirect screening method, the presence of false positive and negative results must be considered.

All reactive samples must be evaluated by a confirmatory test.

The reagents supplied in this kit are optimized to measure JERA reactive antibody levels in serum.

Repeated freezing and thawing of reagents supplied in the kit and of specimens must be avoided.

Serological cross-reactivity across the flavivirus group is common. Certain sera from patients infected with Dengue, West Nile, and Saint Louis, and Rheumatoid sera may give false positive results. Therefore any JE positive sera must be confirmed with other tests.

This kit has not been optimized for vaccine induced seroconversion studies.

PERFORMANCE CHARACTERISTICS

Sensitivity: The sensitivity of this assay has not been established.

Specificity: All well confirmed JE sera were positive by the Japanese Encephalitis IgG ELISA IgG ELISA System. As a control, a number of normal sera and sera infected with unrelated agents such as CMV, EBV and VZV were tested. All produced ISRs that were below the cut-off value.

REFERENCES

1. Martin, D.A., Muth, D.A., Brown, T., Johnson, A.J., Karabatsos,R, Roehrig, J.T. 2000. Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections. J. Clin. Microbiol. 38（5）:1823-1826.

2. Cardosa MJ, Wang SM, Sum MS, Tio PH. Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses. BMC Microbiol. 2002 May 5;2（1）:9

3. Pandey B, Yamamoto A, Morita K, Kurosawa Y, Rai S, Adhikari S, Kandel P, Kurane I. Serodiagnosis of Japanese encephalitis among Nepalese patients by the particle agglutination assayEpidemiol Infect. 2003 Oct;131（2）:881-5.

4. Thakare JP, Gore MM, Risbud AR, Banerjee K, Ghosh SN. Detection of virus specific IgG subclasses in Japanese encephalitis patients.Indian J Med Res. 1991 Sep;93:271-6.

5. Lowry PW, Truong DH, Hinh LD, Ladinsky JL, Karabatsos N, Cropp CB, Martin D, and Gubler DJ. Japanese encephalitis among hospitalized pediactric and adult patients with acute encephalitis syndrome in Hanoi, Vietnam 1995. Am. J. Trop. Med. Hyg, 1998;58（3）:324-329.

6. Tsai TF. Factors in the changing epidemiology of Japanese encephalitis and West Nile fever. In: Saluzzo JF ed., Factors in the Emergence of Arboviral Diseases. Amsterdam: Elsevier, 1997;179-189.

7. Tsai TF. Japanese encephalitis. In: Feigin RD and Cherry JD （eds.）, Textbook of Pediatric Infectious Diseases, 4th edition, Philadelphia: W.B. Saunders, 1997;1993-2001.

8. Rosen L. The natural history of Japanese encephalitis. Annu. Rev. Microbiol., 1986;40:395-414.

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