Enzyme linked-immunosorbent assay for in vitro qualitive detection of total antibodies to hepatitis E virus in human or animal serum or plasma

Hepatitis E virus (HEV) is a non-enveloped, single-stranded RNA virus identified in 1990. Infection with HEV induces acute or sub-clinical liver diseases similar to hepatitis A. HEV infections, endemic and frequently epidemic in developing countries, is seen also in developed countries in a sporadic form with or without a history of traveling to endemic area. The overall case-fatality is 0.5~3%, and much higher (15~25%) among pregnant women. A hypothesis that HEV infection is a zoonosis was presented in 1995. Then a swine HEV and later an avian HEV were identified and sequenced separately in 1997 and 2001. Since then, HEV infection include anti-HEV, viremia and feces excretion of HEV was seen in a wide variety of animals, i.e., swine, rodents, wild monkeys, deer, cow, goats, dogs and chicken in both the developing and developed countries. A direct testimony was reported that the consumption of uncooked dear meat contaminated with HEV led to acute hepatitis E in human, and HEV genome sequences can be detected in pork livers available in the supermarkets in Japan. With the discovery of conformational epitopes in HEV, HEV serology was further explored and understood. The phenomenon of long-lasting and protective antibodies to HEV was observed which greatly enhance the understanding to the diagnosis, epidemiology, zoonosis-related studies and vaccine development.

PRINCIPLE OF THE ASSAY (Double antigen sandwich ELISA)

Ready to use as supplied. Once open, stable for one month at 2-8¡C.

Protein-stabilized buffer, casein, and sucrose solution.

Ready to use as supplied. Once open, stable for one month at 2-8°C.

Horseradish peroxidase-conjugated recombinant HEV antigens.

Ready to use as supplied. Once open, stable for one month at 2-8°C

pH 7.4; 20 × PBS. (Containing Tween-20 as a detergent). DILUTE BEFORE USE 1 to 20 with distilled/deionized water. Once diluted, stable for one week at RT or for two weeks when stored at 2-8°C.

• Chromogen solution A 1 vial (7 ml) Colorless liquid / white vial with green screw cap

Urea peroxide solution. Ready to use as supplied. Once open, stable for one month at 2-8°C

• Chromogen solution B 1 vial (7 ml) Colorless liquid / black vial with black screw cap

TMB solution (Tetramethylbenzidine dissolved in citric acid).

Ready to use as supplied. Once open, stable for one month at 2-8°C.

• Stop solution 1 vial (7 ml) Colorless liquid / white vial with white screw cap

Diluted sulfuric acid solution (2.0 M H₂SO₄). Ready to use as supplied.

• Plastic sealable bag 1 unit

For enclosing the strips not in use.

• Cardbord plate cover 2 sheets

To cover the plates during incubation and prevent evaporation or contamination of the wells.

• Package inserts 1 copy

1.) Freshly distilled or deionized water.

2.) Disposable gloves and timer.

3.) Appropriate waste containers for potentially contaminated materials.

4.) Disposable V-shaped troughs.

5.) Dispensing system and/or pipette (single or multichannel), disposable pipette tips.

6.) Absorbent tissue or clean towel.

8.) Microshaker for dissolving and mixing conjugate with samples.

9.) Microwell plate reader, single wavelength 450nm or dual wavelength 450nm and 630nm.

10.) Microwell aspiration/wash system.

1. Sample Collection: Either fresh serum or plasma samples can be used for this assay. Blood collected by venipuncture should be allowed to clot naturally and completely – the serum/plasma must be separated from the clot as early as possible as to avoid hemolysis of the RBC. Care should be taken to ensure that the serum samples are clear and not contaminated by microorganisms. Any visible particulate matters in the sample should be removed by centrifugation at 3000 RPM for at least 20 minutes at room temperature, or by filtration on 0.22µ filters. Plasma samples collected into EDTA, sodium citrate or heparin may be tested, but highly lipaemic, icteric, or hemolized samples should not be used as they could give erroneous results in the assay. Do not heat inactivate samples. This can cause sample deterioration.
2. Transportation and Storage: Store samples at 2-8°C. Samples not required for assaying within 3 days should be stored frozen (-20°C or lower). Multiple freeze-thaw cycles should be avoided. For shipment, samples should be packaged and labeled in accordance with the existing local and international regulations for transport of clinical samples and ethological agents.

1. A good washing procedure is essential to obtain correct and precise analytical data.
2. It is therefore recommended to use a good quality ELISA microplate washer, maintained at the best level of washing performances. In general, no less than 5 automatic washing cycles of 350-400μl/well are sufficient to avoid false positive reactions and high background.
3. To avoid cross-contaminations of the plate with sample or HRP-conjugate, after incubation do not discard the content of the wells but allow the plate washer to aspirate it automatically.
4. Anyway, we recommend calibrating the washing system on the kit itself in order to match the declared analytical performances. Assure that the microplate washer liquid dispensing channels are not blocked or contaminated and sufficient volume of Wash buffer is dispensed each time into the wells.
5. In case of manual washing, we suggest to carry out 5 cycles, dispensing 350-400μl/well and aspirating the liquid for 5 times. If poor results (high background) are observed, increase the washing cycles or soaking time per well.
6. In any case, the liquid aspirated out the strips should be treated with a sodium hypochlorite solution at a final concentration of 2.5% for 24 hours, before liquids are wasted in an appropriate way.
7. The concentrated Washing solution should be diluted 1 to 20 before use. For one plate, mix 50 ml of the concentrate with 950ml of water for a final volume of 1000ml diluted Wash Buffer. If less than a whole plate is used, prepare the proportional volume of solution.

The components of the kit will remain stable through the expiration date indicated on the label and package when stored between 2-8°C, do not freeze. To assure maximum performance of this HEV-Ab ELISA kit, protect the reagents from contamination with microorganism or chemicals during storage.

This kit is intended for in vitro use only. For professional use only. The ELISA assay is a time and temperature sensitive method. To avoid incorrect result, strictly follow the test procedure steps and do not modify them.

1. Do not exchange reagents from different lots, or use reagents from other commercially available kits. The components of the kit are precisely matched as to achieve optimal performance during testing.
2. Never use reagents beyond the expiry date stated on reagents labels or on the kit box.
3. CAUTION - CRITICAL STEP: Allow the reagents and samples to stabilize at room temperature (18-30°C) before use.
4. Shake reagent gently before, and return to 2-8°C immediately after use.
5. Use only sufficient volume of sample as indicated in the procedure steps. Failure to do so, may cause in low sensitivity of the assay.
6. Do not touch the bottom exterior of the wells; fingerprints or scratches may interfere with microwell reading.
7. When reading the results, ensure that the plate bottom is dry and there are no air-bubbles inside the wells.
8. Never allow the microplate wells to dry after the washing step. Immediately proceed to the next step.
9. Avoid assay steps long time interruptions. Assure same working conditions for all wells.
10. Calibrate the pipette frequently to assure the accuracy of samples/reagents dispensing. Always use different disposal pipette tips for each specimen and reagents as to avoid cross-contaminations. Never pipette solutions by mouth. The use of automatic pipettes is recommended.
11. Assure that the incubation temperature is 37°C inside the incubator.
12. When adding samples, avoid touching the well’s bottom with the pipette tip.
13. When reading the results with a plate reader, it is recommended to determine the absorbance at 450nm or at 450nm with reference at 630nm.
14. All specimens from animal origin should be considered as potentially infectious.
15. Therefore, handle reagents and specimens with extreme caution as if capable of transmitting infectious diseases. Strict adherence to GLP (Good Laboratory Practice) regulations can ensure the personal safety. Never eat, drink, smoke, or apply cosmetics in the assay laboratory.
16. Bovine derived sera may have been used in this kit. Bovine serum albumin (BSA) and fetal calf sera (FCS) are derived from animals from BSE/TSE free-geographical areas.
17. The pipette tips, vials, strips and sample containers should be collected and autoclaved for 1hour at 121°C or treated with 10% sodium hypochlorite for 30minutes to decontaminate before any further steps for disposal.
18. The Stop solution (2M H₂SO₄) is a strong acid. Corrosive. Use it with appropriate care. Wipe up spills immediately or wash with water if come into contact with skin or eyes. ProClin 300 used as a preservative can cause sensation of the skin.
19. The enzymatic activity of the HRP-conjugate might be affected from dust, reactive chemical, and substances like sodium hypochlorite, acids, alkalins etc. Do not perform the assay in the presence of such substances.
20. Materials Safety Data Sheet (MSDS) available upon request.
21. If using fully automated microplate processing system, during incubation, do not cover the plates with the plate cover. The tapping out of the remainders inside the plate after washing can also be omitted.