HEV IgM EIA

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

SUMMARY

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 (Antibody capture principle)

This kit is a two-steps incubation, solid-phase antibody capture ELISA assay in which polystyrene microwell strips are pre-coated with antibodies directed to human immunoglobulin M proteins (anti-µ chain). The patient’s serum/plasma is added and during the first incubation stepd, any IgM-class antibodies will captured in the wells. After washing out all the other substances of sample and in particular IgG-class antibodies, the specific HEV IgM capture on the solid phase is detected by the addition of recombinant HEV ORF2 antigens conjugated to the enzyme horseradish peroxidase (HRP-conjugate). During the second incubation step, these HRP-conjugated antigens will specifically react only with HEV IgM antibodies. After washing to remove the unbound HRP-conjugate, chromogen solutions are added into the wells. In presence of (anti-µ) – (anti-HEV-IgM) – (HEV Ag-HRP) immunocomplex, the colorless chromogens are hydrolyzed by the bound HRP-conjugate to a blue-colored product. The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of color intensity can be measured and is proportional to the amount of antibody captured in the wells, and to the antibody in the sample respectively. Wells containing samples negative for HEV IgM remain colorless.

COMPONENTS

Microwell plate 1 x 96 well plate

Blank microwell strips fixed on a white strip holder. The plate is sealed in aluminium pouch with desiccant. 8x12/12x8-well strips wells per plate. Each well contains anti- IgM antibodies. The microwell strips can be broken to be used separately. Place unused wells or strips in the plastic sealable storage bag together with the desiccant and return to 2-8°C.

Negative control 1 vial (0.5 ml) Yellow-colored liquid / vial with green screw cap

Protein-stabilized buffer tested non-reactive for HEV IgM. Preservatives: 0.1% ProClin 300.

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

Positive control 1 vial (0.5 ml) Red-colored liquid / vial with red screw cap

Purified HEV IgM-class antibodies diluted in protein-stabilized buffer. Preservatives: 0.1% ProClin 300.

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

Specimen diluent 1 vial (12 ml) Blue-colored liquid / white vial with blue screw cap

Protein-stabilized buffer, casein, and sucrose solution.

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

HRP-conjugate reagent 1 vial (12 ml) Red-colored liquid / white vial with red screw cap

Horseradish peroxidase-conjugated recombinant HEV antigens.

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

Stock wash buffer 1 bottle (50 ml) Colorless liquid / clear bottle with white screw cap

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.

Cardbord plate cover 2 sheets

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

ADDITIONAL MATERIALS AND INSTRUMENTS REQUIRED BUT NOT PROVIDED

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.

7.) Dry incubator or water bath, 37±0.5°C.

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.

SPECIMEN COLLECTION/TRANSPORTATION AND STORAGE

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.

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.

SPECIAL INSTRUCTIONS FOR WASHING

A good washing procedure is essential to obtain correct and precise analytical data.
No less than 5 automatic washing cycles of 350-400μl/well are sufficient to avoid false positive reactions and high background.
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.
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.
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.
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.
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.

STORAGE AND STABILITY

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 IgM ELISA kit, protect the reagents from contamination with microorganism or chemicals during storage.

PRECAUTIONS AND SAFETY

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.

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.
Never use reagents beyond the expiry date stated on reagents labels or on the kit box.

CAUTION - CRITICAL STEP: Allow the reagents and samples to stabilize at room temperature (18-30°C) before use.
Shake reagent gently before, and return to 2-8°C immediately after use.
Use only sufficient volume of sample as indicated in the procedure steps. Failure to do so, may cause in low sensitivity of the assay.
Do not touch the bottom exterior of the wells; fingerprints or scratches may interfere with microwell reading.
When reading the results, ensure that the plate bottom is dry and there are no air-bubbles inside the wells.
Never allow the microplate wells to dry after the washing step. Immediately proceed to the next step.
Avoid assay steps long time interruptions. Assure same working conditions for all wells.
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.
Assure that the incubation temperature is 37°C inside the incubator.
When adding samples, avoid touching the well’s bottom with the pipette tip.
When reading the results with a plate reader, it is recommended to determine the absorbance at 450nm or at 450nm with reference at 630nm.
All specimens from animal origin should be considered as potentially infectious.
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.
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.
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.
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.
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.
Materials Safety Data Sheet (MSDS) available upon request.
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.

ASSAY PROCEDURE

Step1 Reagents preparation: Allow the reagents and samples to reach room temperature (18-30° C) for at least 15-30 min. Check the Wash buffer concentrate for the presence of salt crystals. If crystals have formed, resolubilize by warming at 37°C until crystals dissolve. Dilute the stock Wash Buffer 1 to 20 with distilled or deionized water.

Step2 Numbering wells: Set the strips needed in strip-holder and number sufficient number of wells including three Negative control (e.g. B1, C1, D1), two Positive control (e.g. E1, F1) and one Blank (A1, neither samples nor HRP-Conjugate should be added into the Blank well). If the results will be determined by using dual wavelength plate reader, the requirement for use of Blank well could be omitted. Use only number of strips required for the test.

Step3 Adding Diluent: Add 100m l Specimen Diluent into each well.

Step4 Adding Sample: Add 10m l of Positive control, Negative control, and Specimen into their respective wells. Use a separate disposal pipette tip for each specimen, Negative Control, Positive as to avoid cross-contamination.

Step5 Incubating Sample: Cover the plate with the plate cover and incubate for 30minutes at 37° C. It is recommended to use thermostat-controlled water tank to assure the temperature stability and humidity during the incubation. If dry incubator is used, do not open the door frequently.

Step6 Washing: At the end of the incubation, remove and discard the plate cover. Wash each well 5 times with diluted Wash buffer. Each time allow the microwells to soak for 30-60 seconds. After the final washing cycle, turn down the plate onto blotting paper or clean towel, and tap the plate to remove any remainders.

Step7 Adding HRP-Conjugate: Add 100m l HRP-Conjugate to each well except the Blank.

Step8 Incubating: Cover the plate with the plate cover and incubate the plate for 30 minutes at 37° C

Step9 Washing: At the end of the incubation, remove and discard the plate cover. Washing as in Step6.

Step10 Coloring: Dispense 50m l of Chromogen A and 50m l Chromogen B solution into each well including the Blank and mix by tapping the plate gently. Incubate the plate at 37°C for 15min avoiding light. The enzymatic reaction between the Chromogen A/B solutions produces blue color in Positive control and HEV-Ab positive sample wells.

Step11 Stopping Reaction: Add 50m l Stop Solution into each well and mix gently by tapping the plate. Intensive yellow color develops in Positive control and HEV-Ab positive sample wells.

Step12 Measuring the Absorbance: Calibrate the plate reader with the Blank well and read the absorbance at 450nm. If a dual filter instrument is used, set the reference wavelength at 630nm. Calculate the Cut-off value and evaluate the results. (Note: read the absorbance within 15 minutes after stopping the reaction)

INTERPRETATION OF RESULTS AND QUALITY CONTROL

Each microplate should be considered separately when calculating and interpreting results of the assay, regardless of the number of plates concurrently processed. The results are calculated by relating each sample’s optical density (OD) value to the Cut-off value (C.O.) of the plate. If the Cut-off reading is based on single filter plate reader, the results should be calculated by subtracting the Blank well OD value from the print report values of samples and controls. In case the reading is based on Dual filter plate reader, do not subtract the Blank well OD from the print report values of samples and controls.

Calculation of Cut-off value (C.O.) = *Nc + 0.26 (*Nc = the mean absorbance value for three negative controls.)

If one of the Negative control values does not meet the Quality control range specifications, it should be discarded and the mean value is calculated again using the remaining two values. If more than one negative control OD value does not meet the Quality control range specifications, the test is invalid and must be repeated.

Quality control range: The test results are valid if the Quality Control criteria are verified. It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient sample being analyzed.

1. The OD value of the Blank well, which contains only Chromogens and Stop solution, is less than 0.080 at 450 nm.

2. The OD value of the Positive control must be equal to or greater than 0.800 at 450/630nm or at 450nm after blanking.

3. The OD value of the Negative control must be less than 0.100 at 450/630nm or at 450nm after blanking.

3. Interpretations of the results: (S = the individual absorbance (OD) of each specimen)

Negative Results (S/C.O. <1): samples giving absorbance less than the Cut-off value are negative for this assay, which indicates that no HEV IgM-class antibodies have been detected with this HEV IgM ELISA kit.

Positive Results (S/C.O. ≥1): samples giving an absorbance greater than or equal to the Cut-off value are considered initially reactive, which indicates that HEV IgM-class antibodies have probably been detected using this HEV IgM ELISA kit. Retesting in duplicates of any reactive sample is recommended. Repeatedly reactive samples could be considered positive for HEV IgM. Therefore there are serological indications for current infection with hepatitis E virus.

Borderline (S/C.O. =0.9-1.1): Samples with absorbance to Cut-off ratio between 0.9 and 1.1 are considered borderline and retesting of these samples in duplicates is recommended to confirm the results. Repeatedly positive samples could be considered positive for HEV IgM antibodies.

TEST PERFORMANCE AND EXPECTED RESULTS

Specificity: The specificity of HEV-IgM ELISA was determined in 11 non-hepatitis E groups (included hepatitis A cases, hepatitis B cases, hepatitis C cases, inoculated with HBV vaccine groups, population with routine virus testing, blood donors and healthy individuals). Each sample group was no less than 150 members, with 7113 participants for the largest testing groups. The specificity ranged of 95.3%~100.0%.

Testing Center	Testing Groups	Samples	Pos. No.	Specificity (%)
1	Acute hepatitis A	168	1	99.4
	Acute hepatitis B	164	1	99.4
	Hepatitis C	168	4	97.6
	HBV vaccinated	871	25	97.1
	Routine virus testing group	445	3	99.3
2	Healthy individuals	273	0	100.0
2	Acute hepatitis A	298	14	95.3
3	Blood donors	7113	124	98.3
4	Healthy individuals	883	1	99.9
5	Nature population	388	1	99.7
5	Blood donors	355	3	99.2
Total	Healthy individuals	9012	129	98.6
	Other groups	2114	48	97.7
	Total	11126	177	98.4

- No interference was observed from rheumatoid factors up to 2000U/ml.

- No high dose hook effect observed during clinical testing.

- The assay performance characteristics are unaffected from elevated concentrations of bilirubin (up to 210mg/l), hemoglobin (up to 25mg/l), and albumin (up to 100g/l).

- Frozen positive/negative specimens have been tested to check for interferences due to collection and storage. The performance characteristics of HEV-IgM ELISA were not affected.

- Panels of specimens with elevated levels of anti-E.coli antibodies, specimens from pregnant women and individuals with auto-immune diseases were tested. The performance characteristics of Axiom HEV IgM were not affected.

Sensitivity: In the evaluation of 126 serial sera samples obtained from 31 hepatitis E patients (Testing Center 3), the sensitivity of Axiom HEV-IgM for the first serum sample and serial samples were 100% and 97.62% respectively, while the sensitivity of the reference HEV-IgM test was 90.32% and 86.51% respectively.

Parallel comparison testing was performed with 202 non-A-non-B hepatitis samples (Testing Center 1). Since no golden standard for hepatitis E is available, reference HEV IgM assay served as control reagent. The concordance rate between Axiom HEV-IgM and the reference IgM assay was 83.66%. If the true positive status was defined as positive for any of the two tests used in this study, the sensitivity of Axiom HEV IgM and the reference HEV IgM was 96.43% and 80% respectively.

Sensitivity Summary: The sensitivity of Axiom HEV IgM was 97.1%（95% confidence interval: 94.6%~98.5%）in the parallel testing of a total of 314 serum samples of acute hepatitis E, which was significantly higher than the reference HEV IgM tests (81.5%，95% confidence interval: 76.9%~85.4%）and the reference HEV IgM test (93.8%, 95% confidence interval: 83.1%~97.7%）

Testing Center	Samples	Axiom HEV-IgM		Reference HEV IgM ELISA		Reference HEV IgM ELISA
Testing Center	Samples	Pos.	Pos. Rate	Pos.	Pos. Rate	Pos.	Pos. Rate
1	140	135	96.4%	112	80.0%
2	48	47	97.9%	35	72.9%	45	93.8%
3	126	123	97.6%	109	86.5%
Total	314	305	97.1%	256	81.5%	45/48	93.8%

Precision	Dilution	N	Mean	SD	CV
Negative control		70	0.194	0.1180	-
Positive control	Undiluted	53	20.941	1.962	9%
	1:10	17	8.288	1.018	12%
	1:5	17	13.029	2.033	16%
	1:3	17	16.678	2.224	13%
	1:1	17	19.988	2.269	11%
Positive internal control		17	18.873	1.640	9%

LIMITATIONS

Non-repeatable positive result may occur due to the general biological of the ELISA assays. This assay is designed to achieve very high performance characteristics of sensitivity and specificity and the "sandwich model" minimizes the unspecific reactions due to interference with unknown matters in sample. A negative result with an antibody detection test does not preclude the possibility of infection.
If, after retesting of the initially reactive samples, the assay results are negative, these samples should be considered as non-repeatable (false positive) and interpreted as negative. As with many very sensitive ELISA assays, false positive results can occur due to the several reasons, most of which are related but not limited to inadequate washing step.
Positive results must be confirmed with another available method. Any positive result must be interpreted together with the patient clinical information and other laboratory results.
Common sources for mistakes: Kits beyond the expiry date, bad washing procedures, contaminated reagents, incorrect assay procedure steps, insufficient aspiration during washing, failure to add samples or reagents, equipment, timing, volumes, sample nature and quality.
The prevalence of the marker will affect the assay’s predictive values.
This kit is intended ONLY for testing of individual serum or plasma samples. Do not use it for testing of cadaver samples, saliva, urine or other body fluids, or pooled (mixed) blood.
This is a qualitative assay and the results cannot be use to measure antibodies concentrations.

INDICATIONS OF INSTABILITY OR DETERIORATION OF THE REAGENTS

Values of the Positive or Negative controls, which are out of the indicated Quality control range, are indicator of possible deterioration of the reagents and/or operator or equipment errors. In such case, the results should be considered as invalid and the samples must be retested. In case of constant erroneous results classified as due to deterioration or instability of the reagents, immediately substitute the reagents with new ones, or contact AXIOM technical support for further assistance.
If after mixing of the Chromogen A and B solutions into the wells, the color of the mixture turns blue within few minutes, do not continue carrying out the testing and replace the reagents with fresh ones.

VALIDITY

Please do not use this kit beyond the expiry date indicated on the kit box and reagent labels.

REFERENCES:

Reyes GR, Purdy MA, Kim JP, et al. Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 1990; 247: 1335–1339.

Clayson E, Innis B, Myint K, et al. Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am J Trop Med Hyg,1995,53:228–232.

Meng XJ, Purcell RH, Halbur PG, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA, 1997, 94: 9860–9865.

Tei S, Kitajima N, Takahashi K, et al. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 2003; 362(9381):371

Zheng YJ, Zhang J, Xia NS. A debate about that hepatitis E is a zoonosis. Chinese J Zoonosis (in press)

Wang YC, Zhang HY, Xia NS, et al. Prevalence, Isolation, and Partial Sequence Analysis of Hepatitis E Virus From Domestic Animals in China. J Med Virol 2002,67:516–521

Zhang JZ, Ng MH, Xia NS, et al. Conformational Antigenic Determinants Generated by Interactions Between a Bacterially Expressed Recombinant Peptide of the Hepatitis E Virus Structural Protein. J Med Virol 2001,64:125-132

Stanley WK, Zhang JZ, Zhuang H, et al. A bacterially expressed p a recombinant peptide of virus capsid protein. J Virol 2003(in press)

Zhang J, Ge SX, Huang GY, et al. Evaluation of antibody based and nucleic acid based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model. J Med Virol 2003 (in press)

He ZQ, Ge SX, Qiu Y, et al. Establishment and primary application of double antigen sandwich ELISA for detection of hepatitis E virus antibody. Chinese J Zoonosis 2002，18(6) 18-21

04/09 W/kd