Application Effect and Accuracy Analysis of Electrochemiluminescence Immunoassay and Enzyme-Linked Immunosorbent Assay in the Serological Test of Hepatitis B Virus.

Objective: To explore the validity and accuracy of electrochemiluminescence immunoassay (ECLIA) and enzyme-linked immunosorbent assay (ELISA) in the serological detection of the hepatitis B virus.
Methods: From 6 February 2019 to 1 March 2020, 96 patients diagnosed with hepatitis B virus infection in our hospital were recruited and assigned at a ratio of 1 : 1 to experimental groups A (GA) and B (GB), with 48 cases in each group, and the five major serological indicators of hepatitis B were tested and analyzed using ECLIA and ELISA. In addition, 50 suspected patients were selected for two tests, respectively, to compare the accuracy of the two test methods.
Results: ECLIA was associated with significantly higher expression levels and higher detection rates of HBeAg, HBeAb, HBsAg, and HBsAb versus ELISA (P < 0.05), and the difference in the expression and detection rates of HBcAb levels between the two groups did not come up to the statistical standard (P > 0.05). ECLIA yielded significantly higher sensitivity and specificity than ELISA (P < 0.05), while the two methods showed comparable detection accuracy (P > 0.05).
Conclusion: Despite the inconsistent results of the latest studies on the serological detection of hepatitis B by the two techniques, ECLIA is consistently superior to ELISA and provides better diagnostic benefits and merits promotion.

1. Introduction

Hepatitis B is a hepatic disease caused by the hepatitis B virus (HBV) [1]. According to the World Health Organization (WHO), there are about 257 million chronic HBV infections worldwide, with 68% in Africa and the Western Pacific, and about 887,000 deaths from HBV infection each year [2]. The hepatitis B virus enters the body, invades the liver, and replicates in large numbers, causing abnormalities in immune function [3]. Immune cells attack hepatocytes infected with the hepatitis B virus, causing degeneration, edema, and necrosis, resulting in a decrease in liver function and liver disease and digestive symptoms such as jaundice, fatigue, nausea, loss of appetite, and abdominal distension [4]. The disease deteriorates after serious damage to the liver causing liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Some cases may develop serious complications, such as upper gastrointestinal bleeding and hepatic encephalopathy, which endanger patients' lives [5, 6]. It has been shown that entecavir is an antifibrotic and antiviral agent that postpones and reverses the progression of liver disease, but long-term use of entecavir is associated with adverse effects and compromises the therapeutic effect [7]. According to traditional Chinese medicine (TCM), the basic pathogenesis of hepatitis B is “dampness, heat, stasis, and toxicity.” Hypersplenism induces congestion and enlargement of the spleen, which can be classified as “accumulation” and subdivided into the blood or Qi accumulation. Clinical TCM treatment mostly adopts Qi and Blood enhancement therapy. However, the efficiency leaves much to be desired. Therefore, early diagnosis with reliable diagnostic tools is of major significance for the treatment and prognosis of the disease.

Currently, the clinical diagnosis of hepatitis B mostly uses hepatitis B virus serum markers, anti-HBc, anti-HBe, HBeAg, anti-HBs, and HBsAg. HBV status and infectivity are determined based on the diagnostic results. There are two serological assays commonly used in clinical practice: electrochemiluminescence immunoassay (ECLIA) and enzyme-linked immunosorbent assay (ELISA). During ECLIA, the concentration of a test substance in a chemistry detection system under certain conditions is linearly quantified with the chemiluminescence intensity of the system, and the amount of the test substance is determined by using the instrument to measure the chemiluminescence intensity of the system [8]. During ELISA, the test specimen is reacted with an enzyme-labeled antigen or antibody; the antigen or antibody is added to the surface of the solid phase carrier with an enzymatic reaction substrate, which turns into a colored product under the catalysis of enzymes, the amount of which is directly related to the amount of the test substance in the specimen. Qualitative or quantitative analysis is performed according to the shade of the color reaction [9]. ELISA is widely used in the primary care system with extensive applicability and low cost, However, the lack of quantitative analysis, especially when serum specimens are too mobile and highly susceptible to false negatives, has led to a general consensus in recent years on the feasibility of ECLIA superseding ELISA as the recommended diagnostic method [10]. Currently, there is a paucity of clinical studies related to the detection of serological markers of hepatitis B virus infection by two different immunoassays.

To this end, 96 patients diagnosed with hepatitis B virus infection in our hospital from February 6, 2019, to March 1, 2020, were recruited to retrospectively analyze the effectiveness and accuracy of ECLIA and ELISA in hepatitis B virus serological tests, so as to explore a more efficient diagnostic modality for clinical purposes.

2. Materials and Methods

2.1. Baseline Data

From 6 February 2019 to 1 March 2020, 96 patients diagnosed with hepatitis B virus infection in our hospital were recruited and assigned at a ratio of 1 : 1 to experimental groups A (GA) and B (GB), with 48 cases in each group, and the five major serological indicators of hepatitis B were tested and analyzed using ECLIA and ELISA. The randomization was carried out using an online web-based randomization tool (freely available at http://www.randomizer.org/). For concealment of allocation, the randomization procedure and assignment were managed by an independent research assistant who was not involved in the screening or evaluation of the participants. In addition, 50 suspected patients were selected as experimental group C (GC) for two tests, respectively, to compare the accuracy of the two test methods. Informed consent was obtained from patients and signed prior to enrollment in this study. The study protocol was approved by the hospital ethics committee. Ethics number: SH-YUX20190206. All processes were in accordance with the Declaration of Helsinki ethical guidelines for clinical research.

2.2. Inclusion and Exclusion Criteria

2.2.1. Inclusion Criteria

For GA and GB:

(1) Patients with a pathologically confirmed diagnosis of hepatitis B [11].

(2) Patients without other viral infections.

(3) Patients without other liver diseases (e.g., liver injury, autoimmune hepatitis, etc.).

(4) Patients with complete clinical data.

For GC: Suspected of having hepatitis B.

2.2.2. Exclusion Criteria

Patients with poor adherence or psychiatric disorders.

Patients with concurrent malignancies.

Patients with a long history of alcohol abuse.

Pregnant or lactating women.

2.3. Assay Method

For 96 confirmed patients, 5 ml of morning fasting venous blood was collected from all patients in the experimental groups was centrifuged at maximum speed for 10 minutes and the supernatant was retained and stored at −20°C for processing.

Experimental group A: Patient sera were tested using ECLIA. The sera were placed in polystyrene tubes with hepatitis B virus antigen and then labeled with diluted 30% hydrogen peroxide (H2O2). The tubes were incubated at 37°C for 120 minutes and then immunocoated, followed by rinsing off the plate five times with buffer. After adding 100 μl of H2O2 and an equal amount of luminescence reagent (luminol) again, the samples were assayed by chemiluminescence.

Experiment Group B: Patient sera were tested using ELISA. The reagents were incubated with the microtiter plate at room temperature (approximately 20°C) for 30 minutes and then the serum samples were loaded into the microtiter wells and set up as blank control, positive control, and negative control. The plates were covered with sealing film and incubated in a water bath at 37°C for 60 minutes and then washed 5 times. The procedure was repeated once and the termination solution was loaded onto the plate, followed by an assessment of assay results.

After the ECLIA and ELISA serological tests, fluorescence quantitative polymerase chain reaction (PCR) analysis and liver histopathological examination were used to confirm the diagnosis of the 50 suspected participants, so as to compare the accuracy of the two test methods and the detection rate of serological indicators.

2.4. Observations

The serum test markers of both groups were recorded, and the positive detection rate was calculated to compare the two methods.

The reference standards for the five indicators were as follows (Table 1) [12, 13]:

Table 1

The reference value range of five serological indexes of hepatitis B.

Serological indicators	Reference value (more than this value is positive)
HBeAg	≥ 0.05 NCU/Ml
HBeAb	≥ 2.00 NCU/mL
HBsAg	≥ 10.00 mU/mL
HBsAb	≥ 0.20 ng/mL
HBcAb	≥ 1.50 NCU/mL

Hepatitis B e antigen (HBeAg): HBeAg is considered positive with a concentration of ≥ 0.05 NCU/mL. The presence of HBeAg indicates active HBV replication and high infectivity.

Hepatitis B e antibody (HBeAb): HBeAb ≥ 2.0 NCU/mL is considered positive. The disappearance of HBeAg and the production of HBeAb is called “serological conversion” when HBV is mostly in a low replication state and the infectiousness is reduced.

Hepatitis B surface antigen (HBsAg): HBsAg is considered positive with a concentration of ≥ 10.0 mU/mL. HBsAg only turns positive two weeks after HBV infection, and a positive reaction indicates current HBV infection.

Hepatitis B surface antibody (HBsAb): HBsAb is positive with a concentration of ≥ 0.2 n/mL. Positive HBsAb indicates that the body is immune to HBV, while some patients do not always produce anti-HBs.

A positive HBsAg and HBsAb may simultaneously occur in variant strains of infection, where the HBsAb produced by the original HBV does not remove the HBsAg of the mutant strain.

Hepatitis B core antibody (HBcAb): HBcAb is considered positive with a concentration of ≥ 1.5 NCU/mL. HBcAb is a total antibody, including HBcAbIgM and HBcAbIgG, but it is mainly an HBcAbIgG antibody. Antibodies to HBcAbIgM are observed in both acute hepatitis and acute attacks of chronic hepatitis. If both HBcAbIgM and HBcAbIgG are positive, it is indicative of an acute attack of chronic hepatitis B.

2.5. Statistical Analysis

The experimental data were processed using SPSS 22.0 statistical software. The Shapiro–Wilk line normal distribution test was used for measurement data. Normally distributed measures were expressed as mean plus or minus standard deviation (n=2(μ+μ)2p(1 − p)/δ2). Comparisons of means between two groups were first performed with the chi-squared F-test. The variance chi-square was tested by independent samples t-test, the variance nonchi-square was tested by independent samples t' test, and the within-group prepost comparison was tested by paired samples t-test. The count data were expressed as (n (%)), and the chi-square test was used to compare the differences between groups. Differences were considered statistically significant when P < 0.05.

3. Results

3.1. Patient Characteristics

There were 26 males and 22 females in the GA group, aged 47.87 ± 6.26 years, with a BMI of 24.74 ± 1.66. There were 20 males and 28 females in the GB group, aged 44.22 ± 8.68 years, with a BMI of 24.93 ± 1.57. The patient characteristics between the two groups were comparable (P > 0.05). (Table 2).

Table 2

Comparison of key baseline data between the two groups ( ± s).

Groups	n	Gender		Age (years-old)	BMI
		Male	Female
EA	48	26	22	47.87 ± 6.26	24.74 ± 1.66
EB	48	20	28	44.22 ± 8.68	24.93 ± 1.57
Χ2/t		0.032		0.824	0.525
p		0.846		0.126	0.557

3.2. Comparison of the Five Serum Indicators Tested by ECLIA and ELISA

In GA patients, the HBeAg level was 0.11 ± 0.04 NCU/mL, HBeAb level was 2.84 ± 0.52 NCU/mL, HBsAg level was 10.78 ± 1.67 mU/mL, HBsAb level was 0.34 ± 0.08 ng/mL, and HBcAb level was 1.71 ± 0.13 NCU/mL by chemiluminescence/mL. In GB patients, the HBeAg level was 0.09 ± 0.02 NCU/mL, HBeAb level was 2.19 ± 0.49 NCU/mL, HBsAg level was 9.86 ± 1.58 mU/mL, HBsAb level was 0.33 ± 0.07 ng/mL, and HBcAb level was 1.69 ± 0.11 NCU/mL by chemiluminescence/mL. HBeAg, HBeAb, HBsAg, and HBsAb levels discovered by the two tests were therefore statistically significant (P < 0.05); however, the difference in HBcAb levels between the two groups was not (P > 0.05) Table 3.

Table 3

Comparison of the five serum indicators tested by ECLIA and ELISA ( ± s).

Methods	Serological indicators
	HBeAg (NCU/mL)	HBeAb (NCU/mL)	HBsAg (mU/mL)	HBsAb (ng/mL)	HBcAb (NCU/mL)
GA (ECLIA)	0.12 ± 0.04	2.84 ± 0.52	10.78 ± 1.67	0.36 ± 0.08	1.71 ± 0.13
GB (ELISA)	0.09 ± 0.02	2.19 ± 0.49	9.86 ± 1.58	0.30 ± 0.05	1.69 ± 0.11
T	6.254	14.967	6.331	11.151	1.471
p	< 0.05	< 0.05	< 0.05	< 0.05	0.084

3.3. Comparison of the Positive Detection Rates of the Five Serum Indicators Tested by ECLIA and ELISA [n(%)]

The positive detection rate of HBeAg in GA patients was 100%, of HBeAb was 91.67%, of HBsAg was 96.33%, of HBsAb was 89.58% and of HBcAb was 93.75%. The positive detection rate of HBeAg in GB patients was 83.33%, of HBeAb was 77.08%, of HBsAg was 81.25%, of HBsAb was 75.00%, and of HBcAb was 87.50%. ECLIA was associated with significantly higher positive detection rates for HBeAg, HBeAb, HBsAg, and HBsAb versus ELISA (P < 0.05), while the difference in positive detection rates for HBcAb was not statistically significant (P > 0.05). (Table 4).

Table 4

Comparison of the positive detection rates of the five serum indicators tested by ECLIA and ELISA [n (%)].

Methods	n	Serological indicators
		HBeAg	HBeAb	HBsAg	HBsAb	HBcAb
GA (ECLIA)	48	48 (100.00)	44 (91.67)	46 (96.33)	43 (89.58)	45 (93.75)
GB (ELISA)	48	40 (83.33)	37 (77.08)	39 (81.25)	36 (75.00)	42 (87.50)
Χ2		5.912	5.887	6.024	5.545	1.283
p		0.011	0.014	0.009	0.021	0.256

3.4. Comparison of Diagnostic Efficacy between ECLIA and ELISA

After histopathological examination of the liver and quantitative PCR analysis, 28 of the 50 suspected hepatitis B patients were diagnosed. Their sera were tested by ECLIA and ELISA and the results showed the accuracy, sensitivity, and specificity were 96.00%, 100.00%, and 95.45% for ECLIA, and 90.00%, 89.29%, and 81.82% for ELISA. There was no significant difference in accuracy between these two procedures (P > 0.05); however, ECLIA had better sensitivity and specificity than ELISA (P < 0.05). (Table 5).

Table 5

Comparison of diagnostic efficacy between ECLIA and ELISA [n (%)].

Methods	n	Accuracy (n = 50)	Sensitivity (n = 28)	Specificity (n = 22)
GA (ECLIA)	50	48 (96.00)	28 (100.00)	21 (95.45)
GB (ELISA)	50	45 (90.00)	25 (89.29)	18 (81.82)
Χ2		0.457	1.582	1.338
p		0.365	0.047	0.31

4. Discussion

HBV can be transmitted through mother-to-child, blood and blood products, broken skin mucous membranes, and sexual contact [14]. Due to viral, host, and environmental variables, HBV infection is associated with various clinical presentations [15]. With prolonged disease, chronic hepatitis B will develop intrahepatic complications such as cirrhosis and even hepatocellular carcinoma [16]. Furthermore, individuals with substantially reduced liver function are predisposed to major extrahepatic consequences including infections, upper gastrointestinal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome [17]. In addition, hepatitis B may also be misdiagnosed with other liver diseases, such as Wilson disease and alcoholic liver disease [18, 19]. Currently, drug injections are mostly used to treat hepatitis B with the aim of enhancing the therapeutic effect in terms of leukocytes, antivirals, and antiplatelets [20]. Although surgical intervention is available for the progression of hepatitis B to cirrhosis, patients are highly susceptible to complications and immune function abnormalities [21]. The insights gained from the introduction of TCM treatment for hepatitis B have been widely accepted in terms of clinical efficacy [22]. According to TCM theory, hepatitis B is attributed to the long-term effects of exertion, diet, and emotions, resulting in internal dampness, liver qi stagnation, and prolonged depression that damages the spleen [23, 24]. The pathological factors of this disease include epidemic toxicity, qi stagnation, fatigue, and blood and water dampness. The overall pathogenesis is qi deficiency and blood stasis, which requires treatment to strengthen the spleen, invigorate qi, resolve blood stasis, activate blood circulation, and soften hardness and disperse nodules [25].

Currently, enzyme-linked immunosorbent assay and electrochemiluminescence immunoassay techniques are effective and widely used clinically in hepatitis B virological testing. ECLIA and ELISA are both biological tests, differing only in the index system used. ECLIA is quantitative by photon counting, while ELISA is different by color shades [26] Enzyme-linked immunosorbent assay features low cost, easy operation, low sensitivity and is suitable for preliminary qualitative diagnosis of diseases; therefore, it is widely used for large volume sample detection, but the quantitative analysis is insufficient and only qualitative analysis is available [27]. Additionally, the antibody and antigen reagents produced by different manufacturers vary largely, and the operation is susceptible to the impact of relevant factors (e.g., repeated washing of plates), which increases the contamination of the sample and results in a predisposition to inaccurate results such as false positives or false negatives, compromising the sensitivity of the test and preventing a dynamic diagnosis of hepatitis B virus infection [28]. ECLIA is a sensitive method for the detection of antigens and antibodies to viruses and is considered an alternative to radioimmunoassay and ELISA for the detection of antibodies and antigens in vivo due to its high sensitivity, simplicity and speed of analysis, short detection time, and wide diagnostic range [29]. It is based on the principle that under certain conditions, the concentration of antibodies and antigens in the specimen is linearly quantified with the luminescence intensity of the chemical formula. The luminescence intensity of the system is measured using the instrument to directly determine the content of antigens and antibodies [30]. Herein, the five major serum indicators of hepatitis B patients were tested separately using these two methods. The results showed that the levels of HBeAg, HBeAb, HBsAg, and HBsAb detected by the two tests were statistically significant (P < 0.05), but the difference in HBcAb levels between the two groups was not significant (P > 0.05).

Hepatitis B surface antigen is a marker of hepatitis B virus infection [31]. Positive results are detectable in serum at 2–6 months of infection with hepatitis B virus and 2 to 8 weeks before the elevation of alanine aminotransferase, which is indicative of acute hepatitis B, chronic hepatitis B patients, or pathogens carriers [32]. Most patients with acute hepatitis B may show negative results in the early course of the disease, and patients with chronic hepatitis B or virus carriers persist with positive surface antigen [33]. Positive hepatitis B surface antibodies are immunological and protective antibodies against the hepatitis B virus that suggest either a past viral infection that was eradicated or immunization against hepatitis B that created protective antibodies [34]. The higher the titer of hepatitis B surface antibody in the serum, the more protective it is. However, a small number of people who are positive for hepatitis B surface antibody develop hepatitis B, which may be ascribed to infection with a different hepatitis B virus subtype or a mutation of the hepatitis B virus [35]. A positive e antigen test results in a highly infectious hepatitis B that is actively multiplying in the body. A positive e antibody suggests that the patient is less infectious or in remission. Although there are some cases with e antibody positive results yet their disease persists, most typically owing to infection with a mutant hepatitis B virus [36, 37]. HBcAg is generally not detectable in the serum, while HBcAb can be detected. High titers of core antibodies indicate that the hepatitis B virus is replicating and infectious and persists for years to decades, while low titers of core antibodies indicate the previous infection with the hepatitis B virus [38].

When the hepatitis B virus enters the body, the body produces corresponding antibodies to the hepatitis B virus antigens, namely hepatitis B surface antibodies, hepatitis B e antibodies, and hepatitis B core antibodies, which bind to the corresponding antigens of the hepatitis B virus in the blood [39]. The body's immune system is then activated, particularly the T cells, which attack the hepatitis B virus. The hepatitis B core antigens made by the hepatitis B virus bind to cell membrane lipoproteins as hepatitis B virus-sensitized human T cells, and while these antigens and hepatocyte endotoxins are eliminated, the hepatocytes are similarly damaged and the corresponding clinical manifestations occur [40]. In the present study, the HBeAg, HBeAb, HBsAg, and HBsAb positive detection rates were significantly higher in ECLIA than in ELISA (P < 0.05), but the difference in HBcAb positive detection rates between the two groups was not statistically significant (P > 0.05). The study by Hang showed that the ECLIA group had a higher rate of HBeAb, HBeAg, and HBsAg positivity than the ELISA group (P < 0.05), while there was no statistically significant difference in the rate of HBsAb and HBcAb positivity between the two groups (P > 0.05) [41]. At this stage, the results of these two methods for serological detection of hepatitis B are incompatible, as in the study by Yao, there was no statistical difference in the hepatitis B virus serum HBsAg, HBsAb (P > 0.05), HBeAg, and HBcAb detected by the two assays, while there was a statistical difference in the HBeAb results (P < 0.05) [42]. Overall, the higher ECLIA detection rate is most probably attributable to the rapid disappearance of electromagnets from the fully automated ECLIA instrument, which facilitates the separation of free and compound antibodies, reduces false-negative results caused by free antibodies, and improves the specificity of the ECLIA assay [43]. The results herein showed that the sensitivity and specificity of ECLIA were higher than ELISA (P < 0.05), but the accuracy of the two methods was not significantly different (P > 0.05). ELISA is a relatively new clinical test that effectively combines the merits of both electrochemiluminescence and immunoassay methods. It allows the labeling of antibodies with the aid of electrochemiluminescent agents and the accurate separation of antigens and antibodies, thereby considerably improving the disease detection rate of hepatitis B. The electrochemiluminescence immunoassay technique has unique repeatability, and its effective use of electromagnets automates the test operation, thus avoiding possible human errors and greatly improving the accuracy of the test operation, as well as averting false-positive detection due to hemolysis and contamination that may be caused by improper specimen handling, thereby further enhancing the accuracy of the detection of serologically relevant indicators [44, 45].

The current experiment, however, has certain drawbacks. Firstly, the small sample size of this study may result in bias. Second, the devices with different versions made by different manufacturers differ, which may also be attributed to potential errors. In addition, this did not expand over time to examine the influence of various diagnostic methods on prognosis and quality of life.

5. Conclusion

Despite the inconsistent results of the latest studies on the serological detection of hepatitis B by the two techniques, ECLIA is consistently superior to ELISA and provides better diagnostic benefits and merits promotion.