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Published in final edited form as: Microbes Infect. 2021 Oct 7;24(2):104890. doi: 10.1016/j.micinf.2021.104890

Developing and validating a modified Enzyme linked Immunosorbent Assay Method for detecting HEV IgG antibody from Dried Blood Spot (DBS) samples in endemic settings

Rosy Sultana 1,3,4,#, Taufiqur Rahman Bhuiyan 2,#, Afsana Shirin Sathi 2, Salma Sharmin 2, Sharmina Yeasmin 5, Muhammad Ikhtear Uddin 2, Md Saruar Bhuiyan 2, Kaiissar Mannoor 1, Muhammad Manjurul Karim 3, K Zaman 2, Firdausi Qadri 1,2,4,*
PMCID: PMC8960178  NIHMSID: NIHMS1782943  PMID: 34628012

Abstract

Serological analysis is an integral part of laboratory practice nowadays. The present study was aimed to develop and validate a modified Enzyme linked Immunosorbent Assay (ELISA) for determination of IgG antibody against Hepatitis E Virus (HEV) using dried blood spots (DBS) and corresponding plasma samples. A total of 65 samples (45 HEV patients, 20 healthy controls) were analyzed.

DBS and plasma samples demonstrated equivalent optical densities for detecting anti-HEV IgG. A highly significant correlation was observed between plasma and DBS sample absorbances (R2 = 0.98; p <0.001) at dilution 1:200, indicating true agreement between the two procedures. The assay exhibited decent linearity and showed no effect of physiological hematocrit on assay performance.

Data suggested recommendable promise in using DBS as a suitable alternative to plasma samples to determine HEV IgG antibody evidenced by significant correlation with plasma results. Therefore, identical method for processing DBS specimens including it’s proper storage is recommended for implementation of a modified ELISA in different settings.

Keywords: HEV IgG antibody, ELISA, DBS

1. Introduction

Hepatitis E virus often causes epidemic forms of acute viral hepatitis that is one of the major public health concern for low and middle income countries including Bangladesh [1]. It is a small non-enveloped, single stranded, positive sense, RNA virus commonly transmitted through feco-oral route [2, 3]. Among the four major HEV genotypes (HEV1, HEV2, HEV3, and HEV4), HEV1 and HEV2 are prevalent in developing countries and outbreaks resulting from sewerage contamination of drinking water supplies are the common scenario in those regions. In contrast, the transmission of HEV3 and HEV4 genotypes to humans occurs mainly through consumption of contaminated or undercooked meat from infected animals. Pig is supposed to be the main reservoir, although wild boars, rabbits, goats, sheep, deer can be considered as potential zoonotic sources of transmission to humans and they are mostly encountered in developed countries [4–7]. Globally, the virus is found to affect > 20 million individuals annually with symptomatic cases (three million) and HEV-related (~56,000) deaths [8]. A diverse spectrum of clinical manifestations are being observed with HEV infection including acute and self-limiting hepatitis, acute-on-chronic liver disease, chronic hepatitis, cirrhosis, and liver failure though chronic HEV infection may occur among immunocompromised patients (e.g., solid-organ transplant recipients) [9]. This infection is generally self-limiting in both men and non-pregnant women with very rare reports of fatality (<0.1%) [10]. However, the most striking feature is the aggressiveness of HEV infection during pregnancy; the severity of morbidity of pregnant mothers resulting in very high mortality (5–25%) distinguishes it from the infection caused by other hepatitis viruses [11]. The acute phase of the disease can be detected by sero diagnosis of HEV IgM with or without HEV IgG, whereas presence of HEV IgG only is indicative of previous exposure to infection and also a state of immunity [12]. The proposition of collecting blood specimens on a filter paper and subsequently using the dried blood spot (DBS) for diagnosis of infectious diseases started more than a century ago [13]. DBS regarded as a minimally invasive sampling method that enables researchers’ suitable collection, storage and testing procedures with reduced risk of bacterial contamination and hemolysis [14]. DBS samples eliminate the need for venipuncture, especially in infant, young children and hard-toreach individuals. Moreover, amount of required volume of blood is less, also suitable for newborn screening for inherited metabolic disorders in remote settings [15].

ELISA protocols were initially optimized by using serum or plasma specimens although need to be optimized and validated for DBS specimen [16]. Optimal performance of DBS-based ELISA method need to be ensured with a standard validation protocols that include: (1) the type of filter paper used to prepare the DBS, (2) the size of the DBS punch, (3) the quality of the DBS specimens used for testing, and (4) the appropriate elution procedure (including choice of elution buffer, right dilution of eluted material, elution temperature, and time) to minimize background without compromising the sensitivity of the assay [17]. Actual use of DBS in laboratory diagnosis of a number of infectious diseases is now recognized as a reliable sampling strategy for conducting many epidemiological surveys. An outstanding diagnostic performance of HCV screening in DBS samples has appeared as a promising approach reported by a meta-analysis study [18]. For HIV screening, determination of HIV antibody by ELISA is used as first conventional technique where western blotting (WB) as a complementary assay for confirmation of positive result also uses DBS[19]. DBS offer a highly sensitive and specific sampling technique for detecting HIV viral load and early infant diagnosis, that seems to be more accessible in remote settings [15]. Validation of a commercially-available ELISA to assess Epstein-Barr virus (EBV) antibody in dried blood spots (DBS) was demonstrated by another group of researchers [20].

Since, in different countries, dried blood spot based assay has been used to support in the diagnosis of infectious diseases, metabolic disorders, screening for systemic diseases, it can also be used to monitor the level of immunity (IgG) in naturally infected as well as in vaccinated individuals [21]. Therefore, this study has demonstrated the practicability of adapting an ELISA by validating DBS as a suitable method for detecting HEV IgG antibody instead of using plasma/serum specimens. It will again focus on the usefulness of DBS-based ELISA methods for diagnosis of human diseases of public health significance and also help to determine the feasibility of using DBS specimens for accurately detecting the antibody level in pre and post vaccination phases in urban people of Bangladesh.

2. Materials and Methods

2.1. Study Participants

Female patients (45 cases) with confirmed acute viral hepatitis, age range 18–45 years from Dhaka Medical College Hospital and apparently healthy 20 female subjects of similar age range through personal contact were recruited (within a week) in this study. Clinical symptoms of hepatitis were manifested with nausea, vomiting, low-grade fever, upper abdominal pain, and yellow colored urine. Patients revealing increased inflammatory markers for acute hepatitis (serum bilirubin and alanine transaminase) and HEV antibody (IgM only or both IgM and IgG) by ELISA (Wantai, China) were included as cases. The study was approved by the institutional ethical review committee of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and Bangladesh University of Health Sciences (BUHS) and Dhaka Medical College Hospital (DMCH), Dhaka, Bangladesh.

2.2. Sample collection, processing and preservation

Blood sample (5 ml from each patient/ healthy control) was collected aseptically by venipuncture:4 ml was taken in a plain Vacutainer (Red-top tube) and 1ml in an EDTA tube. Collected blood samples were secured in cold chain box and transported to the Institute for developing Science and Health initiatives (ideSHi) Laboratory. Blood in EDTA tubes mixed thoroughly by gentle shaking and transferred to the Mucosal Immunology and Vaccinology Laboratory (MIVL), icddr,b. Blood in Red-top Vacutainer was allowed to clot and centrifuged at 3000 rpm for 10 minutes. Separated serum aliquoted in microcentrifuge tubes for biochemical tests (eg, serum bilirubin and alanine transaminase) and determination of HEV IgM and HEV IgG. All these biochemical and serological analysis were done under an approved HEV viral load and genotype study.

In the MIVL, icddr,b DBS card (Whatman 903) was prepared from EDTA sample tubes by pipetting 50 μl blood in each of the 5 circles, left in biosafety cabinet air for 4 hours for air-drying. And rest of the blood sample centrifuged to prepare plasma, aliquoted in microcentrifuge tube and along with DBS card secured in Zipper bag and stored at −80°C until analysis (for long term storage).

2.3. Laboratory methods

Biochemical liver function tests e.g., estimation of serum bilirubin, alanine transaminase (ALT) were carried out instantaneously after collection by using biochemistry autoanalyzer (Dimension EXL 200, Siemens, USA). HEV IgM and HEV IgG antibodies in serum samples were initially determined by ELISA (Wantai Biological, China) to confirm the presence or absence of hepatitis E antibodies in both the patients and healthy controls. Later, HEV IgG antibodies in plasma and DBS were determined by minor modification of manufacturer’s procedure using HEV IgG ELISA Kit (Wantai Biological, China).

2.4. Elution of DBS samples

A semi-automated DBS hole-punch machine (1296–071DELFIA®, Perkin Elmer, USA) for excision of blood spots was used for obtaining DBS disc diameter of 3.2 mm that contained 1.5 μl plasma [16]. Calculated volume of Wantai ELISA sample diluent was taken in microcentrifuge tubes according to manufacturer instructions. From DBS preparation desired numbers of 3.2 mm circle DBS disc were punched out and added into the microcentrifuge tubes containing required volume of diluent buffers that yielded different ranges (10, 20, 30, 40, 50, 60, 80, 100, 200 and 500) of sample dilution. Microcentrifuge tubes containing diluent and DBS disc were incubated at 4°C for overnight. On the next morning, the contents were mixed thoroughly using shaker (Bioshaker XP, QInstruments, Germany) followed by centrifugation at 1677g (Sigma, Germany) for 5 minutes at room temperature and supernatant was removed.

Corresponding plasma sample was subjected to similar serial dilution to that of DBS elution. DBS elute and plasma preparations were subjected to ELISA in duplicate for the detection of anti-HEV IgG antibodies using Wantai HEV IgG, Beijing, China kits.

2.5. Validation of the modified ELISA procedure

Sixty five plasma samples (45 hepatitis cases and 20 healthy controls) were validated for anti-HEV IgG antibody detection by ELISA (Wantai, China) as per the manufacturer’s instructions. DBS specimens were analyzed using the same kit with minor modification in the manufacturer’s procedure. For each assay, negative control in triplicates and positive controls in duplicates and one blank were used. In order to validate the present study and to obtain quantitative HEV IgG results, samples absorbance values calibrated against the standard curve of WHO reference serum (WHO 95/584, 100WHO units/ml). A serial dilution of 125, 250, 500, 1000, 2000, 4000, 8000, 16000 of the reference serum was prepared which had following concentration 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.006 WU/ml. The WHO reference reagent serves as a biological reference preparation for antibodies to hepatitis E virus and free from HBsAg, anti-HIV and HCV-RNA.

HEV IgG antibody was determined from DBS elutes and serial dilution preparation of corresponding plasma samples by indirect ELISA method using Wantai Kits. In the assay procedure 100 μl of diluted WHO reference serum, DBS elute preparations and serially diluted plasma samples were used. Plasma specimens were diluted at 1:100; however, if the absorbances were beyond the range of the standard curve, they were run again at dilutions ranging from 1:200 to 1:500. Plates were read at a wavelength of 450 nm by EON (BioTek) ELISA reader.

2.6. Calculation of Cut-off Values (COV)

In conventional ELISA methods, Cut-off values are established for plasma or serum samples that may not be compatible for DBS elutes. It is very much crucial to determine the COV, basically applicable for use with DBS specimens. Earlier investigators have determined the mean Cut-off values for DBS-based ELISA and calculated assay accuracy using ODs of positive and negative DBS controls in multiple runs [17, 22]. However, the absorbance value for the DBS samples above which samples were considered positive in this study was calculated by the kit manufacturer’s recommendation for the calculation of Cut-off value (COV). The results are calculated by comparing each specimen absorbance value to the Cut-off value of the assay plate. The Cut-off value is calculated by adding a kit recommended factor (0.16) with the mean absorbance value of three negative controls (eg, COV= NC+0.16).

Sensitivity

The test sensitivity was determined by the analysis of 37 HEV-positive DBS samples (31 strongly reactive and 6 weakly reactive), confirmed with clinical and laboratory based diagnosis of HEV infection.

Specificity

In order to verify specificity of the test, 20 HEV IgG-negative DBS samples (including 12 healthy controls) of the HEV project for viral load and genotype study were used.

Limit of detection (LOD)

In order to determine the assay limit of detection, three HEV IgG positive DBS was diluted in series (1:11 to 1: 500) in sample diluents. Similarly, corresponding plasma specimens were diluted. All the specimens were run in parallel to minimize the assay variation. The LOD was set as the highest plasma dilution that has given the absorbance to meet the positivity criteria for IgG antibodies to HEV.

2.7. Statistical analyses

Linear regression analysis has been done for performing correlation in assays between DBS and plasma specimens. All the statistical analyses were carried out using the software SPSS (SPPSS Inc.; version 15.0 for Windows) and Graph Pad Prism (version 6.0).

3. Results

3.1. Demographic information

Blood samples of 45 women with acute HEV infection and 20 healthy controls women were analyzed in the study. Of the 45 hepatitis subjects, 37 were positive for both anti-HEV IgM and anti-HEV IgG antibodies and 8 were positive for anti-HEV IgM antibody only but negative for anti-HEV IgG antibody. Of the 20 healthy controls, 8 were positive for anti-HEV IgG only and 12 were negative for both the HEV antibodies (IgM and IgG) (Fig. 1). Age, distribution of hepatitis and healthy control subjects, serum bilirubin and alanine transaminase levels of the participating women were shown in table 1.

Figure 1:

Figure 1:

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Stratification of HEV IgG positive and negative samples of the hepatitis cases and healthy controls. Y axis shows the numbers. Numbers over the bars indicate number of subjects in each group.

Table 1:

Age, serum bilirubin and ALT of the subjects on the basis of anti-HEV IgG antibody status

Variables HEV IgG + ve HEV IgG -ve
Hepatitis/Healthy control woman
 Numbers (n) 45 20
 Hepatitis 37 8
 HC 8 12
Age (median, years)
 Hepatitis 28 27
 HC 26 27
Serum bilirubin (mg/dl)
 Hepatitis 9 (1–37) 9 (2–20)
 HC 0.7 (0.4–0.9) 0.3 (0.2–0.5)
Serum ALT (U/l)
 Hepatitis 614 (19–4730) 83 (10–277)
 HC 22 (13–29) 19 (15–25)
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HEV, hepatitis E virus; IgG, Immunoglobulin G; HC, healthy control; ALT, alanine transaminase; HEV IgG +ve, positive for IgG antibodies to HEV; HEV IgG-ve, negative for IgG antibodies to HEV.

Data were shown in mean (range) for serum bilirubin and ALT.

3.2. Cut-off Value (COV), Sensitivity, Specificity, Limit of detection (LOD)

A standard curve was generated in each assay by using WHO reference serum (WHO 95/584, 100 WHO units/ml) as assay control for accepting unknown specimen OD values as well as for quantification of HEV specific antibodies in unknown samples. We have determined the result of each specimen by considering the COV for assay according to the specification (WE-7296, Wantai, China). COV was calculated by taking mean absorbance of three negative controls OD that was added with kit recommended factor 0.16. Analyses of sensitivity and specificity between ELISA from plasma and DBS specimens showed 100% similarity in the assay. LOD of the assay equipment was between 0.00 to 3.99 (data not shown). Sample optical densities (SOD) for measurement of anti-HEV IgG in DBS and corresponding plasma of HEV IgG positive and negative participants showed comparable responses including healthy controls (Fig. 2).

Figure 2:

Figure 2:

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Comparison of optical densities for measurement of anti-HEV IgG antibody in DBS and corresponding plasma of the patients with HEV and healthy control subjects. Y axis showed the mean absorbance (optical densities) at 450 nm with SEM.

3.3. Interpretation of the results

Results were interpreted by following manufacturer’s instructions (WE-7296, Wantai, China). Samples were defined as negative, positive and borderline if absorbance (A) value to Cut-off (CO) ratio(A/CO) was <1, ≥1 and 0.9–1.1 respectively. Samples giving a value less than the cut-off value are negative for the assay, which indicates that no IgG antibody to HEV (8/45 and 12/20) have been detected by Wantai HEV-IgG ELISA kit. Therefore, there are no serological indications for development of IgG antibody for HEV. Similarly, HEV-IgG positive (37/45 and 8/20) and borderline (0/65) specimens were detected.

3.4. Agreement between analysis of DBS and corresponding plasma samples in determination of HEV IgG antibody

The mean (range) HEV IgG titers for DBS and plasma samples were 39.9 WU/ml (1.25–160) and 46.09 WU/ml (1.25–160) respectively at 1:200 dilution which were statistically comparable. Correlation between DBS and corresponding plasma samples (n= 65) was evaluated. Optical densities for measurement of anti-HEV IgG between DBS and corresponding plasma showed significantly high linear correlation (R2= 0.98; p <0.001) at the same dilution with a slope of 1, indicating true agreement between the two procedures (Fig. 3).

Figure 3:

Figure 3:

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Correlation between DBS and corresponding plasma samples for determination of HEV IgG antibody of the study subjects. Optical densities (OD) of DBS are shown in X-axis and optical densities (OD) of corresponding plasma are shown in Y-axis that were measured at 450 nm.

4. Discussion

DBS samples have been used successfully for many years for screening of metabolic diseases in neonates and also for the diagnosis of infectious diseases [23], surveillance of disease, molecular studies, determination of resistance pattern, investigation and strain typing in viral hepatitis outbreak [24]. Since reliable laboratory diagnosis primarily depends on its pre-analytic stage, the choice of the correct sample and the conditions at which sample are being transported to the laboratory should be emphasized [19]. Collection of venous blood from targeted populations, researchers/technicians biosafety, processing of blood and preservation of specimens may be major obstacles in field settings where there is scarcity of skilled laboratory personnel (e.g., phlebotomist, technician) [16, 25, 26]. However, the DBS preparation method is relatively economic requiring less quantity of blood, easy to transport from one place to another keeping in ziplock plastic bags and cold chain is not mandatory during shipping. The stability of DBS in extreme environmental conditions (i.e., elevated ambient temperature and high humidity) and loss of infectivity of viruses after drying of blood spot on filter paper matrix makes it an ideal specimen type for antibody assays such as ELISA [27]. A DBS study for HEV-RNA amplification during a HEV epidemic has shown 90.6% concordance with serum samples [28]. However, it lacks validation of DBS samples for anti-HEV IgG. The present study reports a modified ELISA method that has been optimizing at 1:200 dilution of DBS elutes and corresponding plasma samples in parallel and thus validating the modified protocol for using dried blood spots, so that quantitative detection of anti-HEV IgG antibody can be done for assessment of immunogenicity and diagnosis of Hepatitis E virus. However, challenges with developing the ELISA method by using DBS samples and validating the result with plasma in parallel (by comparing the sample absorbance/ optical density of both sample types) for remote settings have impacted testing outcome in certain populations. To alleviate these challenges, dried blood spot (DBS) samples have been validated for use in a variety of settings and for testing of multiple biomarkers using ELISA. We believe this will help us to develop and validate high-throughput assays and to enable us to conduct large nationwide clinical trials in future in an emergency situation. In addition, few other groups had also reported not only antibody measurement but also measured naïve T cell population from DBS [29]. Earlier, different serological assays performance has been evaluated using dried blood spots as a surrogate specimen and found similar observation to those of serum and/or plasma specimens [16, 20].

The objective of the study was to evaluate and confirm a commercially available ELISA assay for determination of HEV IgG using dried blood spot that can be comparable to that seen in matched plasma samples. Through a long procedure, we developed this assay by minor modification of a commercially available ELISA kit that provides acceptable measurement of HEV IgG antibodies against the viral antigen in blood spots that were prepared directly from venous blood. In our study, DBS sample absorbance were found higher than that of matched plasma samples in the initial dilution (1: 11) that is not consistent with the study done by Eick et al., [20] where plasma antibody titers were higher than that of DBS. DBS has not yet been suggested as biological sample to be analyzed in the employed ELISA diagnostic reagent kit, there remains every possibility of interference (matrix-induced effect and hematocrit effect) that might be the possible explanation for the higher value in DBS in our study. Again, 3.2 mm punch from a 50 μl dried blood spot is assumed to contains ~ 1.5μl of serum at a hematocrit of 55 percent [16], the exact volume cannot be ascertained from DBS sample. However, a strong positive correlation has been found between sample absorbance derived from DBS and plasma HEV IgG antibody assay that explored good concordance with DBS elutes when the samples were diluted 200 times in kit diluents for samples (R2=0.975).

Since limited data are available for comparative assay performance using DBS with plasma samples for detection of HEV IgG antibody, the present study offers a highly sensitive and specific sampling strategy to detect antibody and monitoring the level of antibodies in remote settings, thus provides a great opportunity for screening mass population. Evaluation of immune responses from DBS in other infections and vaccination has recently been accomplished [23]. It showed very good agreement between conventional collected blood specimens and DBS. Large clinical trial can easily be designed by following such kind of collection in remote and less facilitated area. Though some other technical aspects (eg: volume of blood in DBS, temperature and duration of storage of DBS etc) should be properly handled to get comparable and robust results from the field settings.

Therefore, the commercially-available ELISA assay that detected HEV IgG antibody in this study was validated for use with DBS. Hopefully, in future it will facilitate continued investigation of HEV IgG antibody titers in DBS, though the use of DBS is not a new idea and has already been applied for diagnosis of many infectious diseases. However, for successful implementation of this easy and more comfortable procedure for measuring HEV antibodies in low- and middle-income country, a standardized approach for sampling, storage, and processing DBS samples can be considered essential since this is not always feasible to draw blood from every location of Bangladesh especially from children. All the subjects analyzed in the present study were female that will justify the necessity of HEV IgG detection before and after mass vaccination of the women of child bearing age, though initially seemed to be a limitation of the study. In summary, dried blood spot platform can have extreme 324 potential for massive future use and to add more parameters that is applicable to global health concerns in an endemic setting.

Acknowledgement

This research was supported by the Global Emerging Leader Award (K43TW010362 [T.R.B]), R01 AI130378 [T.R.B.] of the National Institutes of Health (NIH) and icddr,b. icddr,b is grateful to NIH for its research support. icddr,b is also grateful to the Governments of Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support.

Footnotes

Potential conflicts of interest

All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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