日本腦膜炎IgM檢測試劑盒 （乙型腦炎IgM檢測試劑盒）

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INTENDED USE

The Diagnostic Automation Inc. Japanese Encephalitis IgM ELISA test is for exposure to Japanese Encephalitis Virus （JEV） is an ELISA assay system for the detection of IgM 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 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 consists of one enzymatically amplified "two-step" sandwich-type immunoassay. In this assay, JE Detect Negative Control （Represents non-reactive serum）, JE Detect IgM Positive Control （Represents reactive serum）, and unknown serum samples are incubated in microtitre wells which have been coated with anti-human IgM antibodies, followed by incubation with both JEV derived recombinant JERA and Normal Cell Antigen （NCA） separay. The serum samples are diluted with Sample Dilution Buffer for JE Detect IgM. After incubation and washing, the wells are treated with a JERA-specific antibody labeled with the enzyme horseradish peroxidase （HRP）. After a second incubation and washing step, the wells are incubated with the tetramethylbenzidine （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 Kit contains sufficient reagents for one plate of 96 wells （12 x 8 strips） each. The kit contains the following reagents:

-specific materials:

1. Coated Micro titer Strips for Human IgM: Strip holder in foil pouch, containing 96 polystyrene microtiter wells coated with antibody to human IgM in each well. Store at 2-8C until ready to use. The anti-human IgM coated wells are used to capture IgM antibodies from human samples.

2. Sample Dilution Buffer for IgM Type A: One bottle, 25 mL, for serum dilution prior to use in assay. Store at 2-8C until ready to use.

Note: If any precipitate is seen, vortex the tube very well to obtain a homogeneous solution and then use.

3. JE Negative Control: One vial, 50 μL or two vials 30 μL each of heat–inactivated serum. The JE Detect Negative Control will aid in monitoring the integrity of the kit as well. Store at 2-8C untilready to use for up to 7 days. Quick spin the vial briefly before use to collect the content at the bottom.

Note: For long-term storage, serum can be further aliquoted in a smaller volume and stored at –70°C.

4. JE IgM Positive Control: One vial, 50 μL or two vials 30 μL each of heat–inactivated serum. The JE Detect IgM Positive Control will aid in monitoring the integrity of the kit. Store at 2-8C until ready to use for up to 7 days. Quick spin the vial briefly before use to collect the content at the bottom.

Note: For long-term storage, serum can be further aliquoted in a smaller volume and stored at –70°C.

5. Ready-to-use JE Antigen （JERA） for IgM: One tube （3 mL） of Ready-to-use JERA solution. Store at 2-8C until ready to use.

6. Ready-to-use normal cell antigen （NCA） for JE IgM: One tube （3 mL） of Ready-to-use NCA solution. Store at 2-8C until ready to use.

7. Ready to Use Enzyme Conjugate-HRP for JE IgM: One bottle, 6 mL of a pre-diluted HRP conjugated flavivirus reactive monoclonal antibody （mAb） to be used as is in the procedure below. Store at 2-8C until ready to use.

8. 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.

9. EnWash: One bottle, 20 mL of EnWash to be used in between the washing steps after the addition of enzyme conjugate-HRP of this procedure. Store at 2-8ºC until ready to use.

10. 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.

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

Caution: 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. ELISA Spectrophotometer capable of absorbance measurement at 450 nm （DAX 800）

2. Biological or High-Grade Water

3. Vacuum Pump

4. Plate Washer （ DAX 50）

5. Humidified Incubator or Water Bath

6. 1-10 μL Single-Channel Pipettors, 50-200 μL Single-and Multi-Channel Pipettors.

7. Polypropylene tubes

8. Parafilm

9. Timer

10. Vortex

WARNINGS AND PRECAUTIONS

1. All human source materials used in the preparation of controls have tested negative for antibodies to HIV 1&2, Hepatitis C and Hepatitis B surface antigen. However, no test method can ensure 100% efficiency. Therefore, all human controls and antigen should be handled as potentially infectious material. The Center for Disease Control and the National Institute of Health recommend that potentially infectious agents be handled at the Biosafety Level 2.

2. 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.

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

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

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

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

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

8. 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.

9. Avoid microbial contamination of reagents, especially of the Ready to Use Enzyme Conjugate HRP for IgM. Avoid contamination of the TMB Substrate Solution with the Enzyme Conjugate-HRP.

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

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

12. 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.

o Note: CSF can be used. However, our kit has not been tested or optimized with CSF. Before using the InBios kit, one has to optimize the CSF system.

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

Preparation of Reagents

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 up to 6 months. Check for contamination prior to use.

Microtiter Wells:

Select the number of coated wells required for the assay. The remaining unused wells should be placed back quickly into the pouch and stored at 2-8C until ready to use or expiration.

Assay Procedure

1. Positive, negative and unknown serum to be tested should be assayed in duplicate. Refer to flow chart at the end of this section for illustration of this procedure. Twenty-two test specimens can be tested in duplicate on one 96 well plate.

2. Mark the Microtiter strips to be used.

3. Dilute test sera, and controls to 1/100 using the provided Sample Dilution Buffer. Use small polypropylene tubes for these dilutions and at least 4 L of sera and positive and negative controls. For example: 4 L serum plus 396 L of Sample Dilution Buffer for JE Detect IgM to make 1/100 dilution.

4. Apply the 50 L/well of 1/100 diluted test sera, JE Detect Negative Control, and JE Detect IgM Positive Control to the plate by single or multi-channel pipettor as appropriate. An exemplary arrangement for twenty-two test serum samples in duplicate is shown below.

5. Cover the plate with parafilm just on the well opening surface, so the bottom of the plates is not covered.

Note: This is to make sure the temperature distribution is evenly spread out in all wells from bottom and sides; any extra parafilm can be cut-out once the top is sealed to block evaporation.

6. Incubate the plate at 37oC for 1hour in a humidified incubator with water container. Humidification can be achieved using a water tray at the bottom of incubator.

Note: Do not stack plates on top of each other. They should be spread out as a single layer. This is very important for even temperature distribution. Do not use CO2 or other gases. Do not place plates in contact with any wet substances such as wet paper towels etc.

CORRECT METHOD

7. After the incubation, wash the plate 6 times with automatic plate washer using 1x Wash buffer. Use 300 L per well in each wash cycle.

8. Add 50 L /well of JERA into row A-D and 50 L/well of NCA into row E-H by multi-channel pipettor.

11. After the incubation, wash the plate 6 times with automatic plate washer using 1x Wash buffer. Use 300 L per well in each wash cycle.

12. Add 50L/well of ready to use Enzyme-HRP conjugate into all wells by multi-channel pipettor.

13. Cover the plate with Para film just on the well opening surface, so the bottom of the plate should not be covered （see step 5）.

14. Incubate the plate at 37oC for 1hour in a humidified incubator （see step 6）.

15. After the incubation, wash the plate 6 times with automatic plate washer using 1x Wash buffer.

16. Add 150 L/well of EnWash into all wells by multi-channel pipettor.

17. Incubate the plate at room temperature for 5 minutes without any cover on the plate.

18. After the incubation, wash the plate 6 times with automatic plate washer using 1x Wash buffer.

19. Add 75 L/well of Liquid TMB substrate into all wells by multi-channel pipettor.

20. Place and incubate the plate at room temperature in a dark place （or container） for 10 minutes without any cover on the plate.

21. After the incubation, add 50 L /well of Stop solution into all wells by multi-channel pipettor and incubate at room temperature for 1 minute without any cover on the plate.

22. After the incubation, read the RAW OD 450 value with a Micro plate reader. Please make sure the micro plate reader does NOT subtract or normalize any blank values or wells.

CSF application: CSF should be run using undiluted （1:1） samples. If there is not enough volume to test, the CSF samples may be diluted out 1:2, or higher using the Sample Dilution Buffer for JE IgM provided. However, one needs to optimize the proper dilution factor. The rest of the process is the same as described for serum.

Note: It is necessary to validate the CSF system in a laboratory before using unknown samples.

QULAITY CONTROL

Each kit contains positive and negative control sera to ensure assay performance. The negative and positive controls are intended to monitor for substantial reagent failure. The positive control will not ensure precision at the assay cut-off. The test is invalid and must be repeated if the ISR value of either the controls do not meet the specifications. Acceptable Immune Status Ratio （ISR） values for these controls are found on specification table below. If the test is invalid, patient results cannot be reported. Quality control requirements must be performed in conformance with local, state, and/or federal regulations or accreditation requirements and your laboratory’s standard Quality Control procedures. It is recommended that the user refer to NCCLS C24-A and 42 CFR 493.1256 for guidance on appropriate QC practices. The results below are given strictly for guidance purposes only. Applicable for raw spectrophotometric readings only.

Calculation of the Negative Control

Calculate the mean JE Detect Negative Control values with JERA and with the Control antigen

Calculation of the Immune Status Ratio （ISR）: Compute the average of the two unknown sample replicates with the JERA, and replicates with the NCA, then calculate the JERA/NCA ratio （ISR）. The ISR for the positive control should be greater than 6.0, while the ISR for the negative control should be less than or equivalent to 4.0.Selection of the Cut-off: The cut-off was selected using values from a small set of field data and is an estimate only.

Interpretation of Results: The table below shows how the results should be interpreted.

ISR Results Interpretation

<4.0 Negative No detectable IgM antibody by the ELISA test

4-6 Equivocal Need confirmatory test

>6.0 Positive Indicates presence of detectable IgM 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

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

In areas where JE and dengue are co-existent, JE positive samples should also be assayed for dengue reactivity. Samples with borderline JE positivity and medium to high dengue reactivity could be suspected for dengue infection and require further confirmatory assays.

The assay performance characteristics have not been established for visual result determination.

Results from immunosuppressed patients must be interpreted with caution.

Assay results should be interpreted only in the context of other laboratory findings and the total clinical status of the patient.

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

The use of JE Detect IgM kit for vaccine induced seroconvesion studies may lead to many “Equivocal” results.

PERFORMANCE CHARACTERISTICS

Specificity Studies:

Note: Specificity panel includes normal and other disease sera, such as sera from patients with autoimmune diseases （ANA, RF, etc; not including Dengue, and WNV, and SLE sera）. Limited studies with dengue sera showed cross-reactivity with some sera. One convalescent serum did not show any IgM reactivity.

Serological Sensitivity: 31/31, or 100%

Serological Specificity: 0/196, or 100%

Cross-reactivity Studies

The table below shows the results for the cross-reactivity study performed with the kit.

Interference Study:

Eight plasma samples containing high levels （860 -5630 IU） of Rheumatoid factor gave negative results in the IgM assay.

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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