Abstract
Background
To establish a chemiluminescence method for detecting anti‐E1 and anti‐E2 antibodies in the serum of patients with hepatitis C virus (HCV) infection.
Methods
The microplate was coated with recombinant envelope proteins E1 and E2 by indirect method, respectively, and the kits for detecting anti‐E1 and anti‐E2 antibodies were prepared. The methodological indexes were evaluated.
Results
The methodological indexes of the kits were as follows: precision test (the variation coefficient of anti‐E1 antibody 6.71%–8.95% for within run and 9.91%–12.16% for between run, the variation coefficient of anti‐E2 antibody 6.06%–8.44% for within run and 10.77%–13.98% for between run, respectively). The blank limit and detection limit were 1.18 RLIR and 3.16 RLIR for the anti‐E1 antibody, and 1.26 RLIR and 3.32 RLIR for the anti‐E2 antibody, respectively. The correlation coefficients (r) of anti‐E1 and anti‐E2 were 0.9963 and 0.9828, the analysis and measurement ranges (AMR) were 1.66–41.28 RLIR and 1.55–19.46 RLIR, and the average recovery was 96.4% and 93.7%, respectively. The rheumatoid factor and other positive serum samples had no interference or cross‐reaction to the test, and the kits were stable within 15 months. The positive rates of anti‐E1 and anti‐E2 antibodies in 45 patients with HCV infection were 35.6% (16/45) and 44.4% (20/45), respectively.
Conclusions
The kits for detecting anti‐E1 and anti‐E2 meet the requirements of methodology, and can be used in screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism, and epidemiological studies of HCV infection. The HCV envelope proteins E1 and E2 have an immune response in HCV‐infected patients.
Keywords: antibody, detection, envelope protein, evaluation, hepatitis C virus
To establish a chemiluminescence method for detecting anti‐E1 and anti‐E2 antibodies in the serum of patients with hepatitis C virus (HCV) infection. It can be used in screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological study of HCV infection.
1. INTRODUCTION
Viral hepatitis is a group of infectious diseases secondary to hepatitis virus infection (commonly hepatitis B and C virus infection) that seriously threatens the public health and safety of society and has a high rate of chronicity and mortality. 1 Hepatitis C virus (HCV) has a history of more than 30 years since its discovery, 2 and most infected individuals (55%–85%) become chronic, and long‐term chronic hepatitis C can lead to cirrhosis and even liver cancer. 3 Although medicine has made great progress in recent years with the advent of direct‐acting antiviral agents (DAAs), making HCV infection a curable disease, because HCV is a rapidly evolving RNA virus, long‐term exposure to DAAs triggers strong viral drug selection, causing the virus itself to develop partially drug‐resistant mutants to DAAs. 4 The efficacy of anti‐HCV drugs is not satisfactory, and therefore prevention and control of infection are the current priorities. In addition, there is no vaccine to prevent hepatitis C infection, and there are still some problems in HCV pathogenesis, immune response, disease recurrence, and timely diagnostic monitoring, which makes it difficult to completely eradicate HCV globally. 5 The course of chronic hepatitis C development and related treatment is a long process, and most chronic hepatitis C mostly evolves from acute hepatitis C. Due to the enormous compensatory capacity of the liver, most people infected with the hepatitis C virus do not have any symptoms and are often found incidentally during physical examinations or many years later when they develop uncomfortable symptoms. Chronic hepatitis C is caused by infection with the hepatitis C virus (HCV), a small RNA virus classified as a genus of hepatovirus in the family Flaviviridae, 6 which consists of three structural and six nonstructural proteins, of which the core coat protein (Core), envelope proteins (E1, E2), and transmembrane protein (P7) work together as the shell protein skeleton to assemble the complete viral particle, while the envelope glycoproteins E1 as a fusion subunit and envelope glycoprotein E2 as a receptor binding subunit play important roles in the process of HCV invasion into host cells, 7 respectively. E1 and E2 envelope proteins form heterodimers on the surface of the virus, which are important for the maturation and infectivity of viral particles and have potential glycosylation sites in the outer membrane domain. These glycosylation sites may be important for the stability and antigenicity of HCV particles, 8 and E1 and E2 also play important roles in virus infection and immune escape, and hence important roles in the process of HCV chronicity, and some studies have shown that E1 and E2 are highly glycosylated proteins, 9 in which it is generally believed that E2 envelope protein contains important neutralizing epitopes and E2 antibodies have potential protective neutralizing antibodies. This protein is the main protein that mediates the sequential interaction of HCV with multiple receptors on the surface of hepatocytes and triggers endocytosis of the cell membrane. In addition, in recent years, some achievements have been made in HCV envelope protein vaccines. E1 protein has a neutralizing epitope and CTL recognition site; E2 protein is an important immune antigen; and stimulating the body with E1 protein alone can cause a weak immune response. Purified E1 and E2 protein complexes can produce high‐titer anti‐E1 and anti‐E2 antibodies. These results indicate that envelope proteins E1 and E2 may be necessary components of HCV vaccine. 10 Cross‐neutralizing antibodies against each genotype can be detected in the sera of HCV‐infected patients, and the main target of these antibodies is the E2 protein. E1 does not interact directly with the host receptor, and its viral entry function is mainly through direct binding to the receptor by interacting with E2. 9 Neutralizing antibodies against conformational epitopes of the envelope protein on the surface of E1 and E2 viral particles also play an important role in providing protection against chronic HCV infection and promoting viral clearance. Most scholars consider the detection of anti‐E1 antibodies and anti‐E2 antibodies as a new indicator for the diagnosis of HCV. The detection of E1 and E2 antigen components on the basis of HCV antibody detection kits is expected to improve their detection rate. Therefore, it is important to detect the antibodies produced by HCV envelope glycoproteins E1 and E2 to evaluate the infectiousness, disease progression, and drug efficacy of HCV‐infected patients. However, the study of envelope glycoproteins E1 and E2 in the serum of HCV‐infected patients is subject to some limitations, and the main difficulties of the study are as follows: there are no available kits for the detection of antibodies to E1 and E2 proteins; the existing commercial kits are mostly for the combined detection of antibodies to multiple proteins (in order to improve the detection rate of HCV infection); the inability to complete the detection of anti‐E1 and anti‐E2 individual antibodies and the small fragments of E1 and E2 proteins, which are difficult to express and purify, 11 ; and the difficulty of preparing detection kits. Therefore, in order to understand the humoral immune response of HCV envelope glycoproteins (E1, E2) in the host, this paper developed a chemiluminescence detection kit for anti‐E1 and anti‐E2 antibodies in HCV‐infected sera based on the development of kits for detecting anti‐P7 and anti‐NS2 antibodies, 12 , 13 and 45 cases of HCV‐infected sera were tested and 16 cases of anti‐E1 and 20 cases of anti‐E2 antibodies were detected, respectively. The results indicated that a certain percentage of anti‐E1 and anti‐E2 antibodies existed in HCV‐infected patients, suggesting the existence of an immune response to the HCV envelope protein. The results of these preliminary studies provide technical means to further reveal whether there are differences in the production of antibodies and possible circulating immune complexes (anti‐E1‐IC, anti‐E2‐IC) in HCV‐infected patients with different genotypes, different clinical stages (chronic, cirrhotic, and hepatocellular carcinoma), before and after antiviral therapy, and provide technical guidelines for clinical assessment of infectivity, disease progression, and drug efficacy in HCV‐infected patients.
2. MATERIALS AND METHODS
2.1. Reagents and apparatus
96 microplate Nunc (Thermo Fisher, USA), recombinant envelope protein E1 (100 μg/tube, LOT: LCO8AU1802, Sino Biological Inc., Beijing), recombinant envelope protein E2 (20 μg/stem, lot 715PHCVE2, ProSpec‐Tany, Israel), U Tube protein ultrafiltration concentration tube (Novagen, USA), horseradish peroxidase‐labeled sheep anti‐human IgG marker (1 mg/mL, Hangzhou Longji Biotechnology Co., Ltd.), and anti‐HCV total antibody screening kit (Xiamen Suntrust Co., China) were used. Separate preparation: (1) coating solution (0.02 M phosphate buffer solution at pH 7.4), (2) closure solution (containing 5% bovine serum albumin, 1% sucrose, 4% gelatin, 0.2% Proclin 300 preservative, 0.01 M phosphate buffer salt solution at pH 7.4), (3) wash solution (containing 0.5% Tween‐20, 0.2% Proclin 300 preservative, pH 7.4 in 0.01 M phosphate buffer solution), (4) sample dilution solution (containing 20% bovine serum, pH 7.4 in 0.01 M phosphate buffer solution), (5) substrate solution A (containing 0.8 g/L luminol, 0.008 g/L o‐phenylphenol, 0.025 g/L 4‐imidazole phenol, pH 9.0 in 0.1 M carbonate buffer solution), stored at 2–8°C and protected from light, (6) substrate solution B (containing 0.3 g/L peroxiredoxin, 0.2 M phosphate buffer pH 7.4), and (7) enzyme‐labeled antibody diluent (containing 5% bovine serum albumin, 0.5% enzyme stabilizer, 0.1% Tween‐20, 5 μg/mL anti‐mouse antibody blocker, 1 μg/L red pigment, 0.2% Proclin 300 preservative, pH 7.4 in 0.01 M phosphate buffered salt solution). The washing solution can be stored at 2–30°C, and all other prepared reagents need to be stored at 2–8°C (substrate solution A needs to be stored away from light), with a validity of 12 months. All the above chemical reagents and drugs were purchased from Shanghai Aladdin Biochemical Technology Co. Chemiluminescence immunoassay analyzer (TZD‐CL‐200S, Xiamen Tianzhongda Biotechnology Co., Ltd.).
2.2. Specimen collection
The serum or plasma (anticoagulation of disodium ethylenediamine tetraacetic acid or potassium salt [EDTA]) of 50 healthy patients (anti‐HCV antibody negative, control group) and 45 patients (anti‐HCV total antibody positive, study group) infected with HCV were randomly collected from the 903th Hospital of the Joint Logistic Support Force from August 2019 to October 2020. Within 1 week, it can be stored at 2–8°C, and >1 week, it can be stored at −20°C and avoid repeated freeze–thaw. In addition, the mixed serum or plasma of non‐HCV‐infected persons and HCV‐infected persons (anti‐E1 or E2 antibody‐positive) with negative human immunodeficiency virus antibodies, treponema pallidum antibodies, and hepatitis B virus surface antigens were collected. After heat inactivation, 0.2% ProClin 300 preservative was added. Negative control serum, positive control serum for anti‐E1 antibody, and positive control serum for anti‐E2 antibody were prepared and stored at 2–8°C. All samples were approved by the Medical Ethics Committee of the 903 Hospital of the PLA Joint Logistic Support Force (approval No. 20200608/06/02/204).
2.3. Experimental methods
2.3.1. Recombinant envelope protein E1‐, E2‐coated microtiter plate
The microtiter plate was coated by the indirect method principle, with specific steps: (1) select the optimal envelope concentration of recombinant envelope protein E1, E2 by square (checkerboard) titration method; (2) dilute recombinant envelope protein E1, E2 to the optimal working concentration by using coating solution; (3) add 100 μL of diluted envelope protein E1, E2 to each well; (4) add 300 μL of washing solution to each well to wash away the unadsorbed proteins, repeat washing 3 times and pat dry; (5) add 200 μL of closing solution to each well and place at 2–8°C for 20–24 h; (6) pat dry and attach the sealing film, place in a sealed bag (containing desiccant) and store at 2–8°C for backup.
2.3.2. Preparation of enzyme marker working solution
Dilute 1 mg/mL of horseradish peroxidase‐labeled sheep anti‐human IgG marker with enzyme‐labeled antibody diluent and select the most suitable enzyme marker working solution for detection of anti‐E1 antibody and anti‐E2 antibody in a microtiter plate by square (checkerboard) titration method, respectively.
2.3.3. Detection of anti‐E1 antibody and anti‐E2 antibody in serum
(1) Sample addition: set up 2 wells of negative control and 1 well of positive control in a 96‐microtiter plate that has been coated with envelope protein E1 or E2, add 100 μL of negative and positive control quality control sera, set up 1 well of blank control without adding any sample, add 100 μL of sample dilution to the remaining wells, then add 10 μL of the sample to be tested, cover with sealing film, and mix well at 37°C for 30 min, discard the liquid in the hole and wash for 3 times; (2) Add enzyme marker working solution: except for the blank control wells, add 100 μL of enzyme marker working solution to the remaining wells; (3) Incubate: cover with a sealing membrane and mix well, place at 37°C for 30 min; (4) Wash: discard the liquid in the wells, fill each well with washing solution, leave for 30 s, discard the washing solution in the wells, and repeat 5 times to pat dry; (5) Color development: add 50 μL each of substrate solution B and substrate solution A to each well in turn, shake and mix for 15 min at room temperature and avoid light; (6) Detection: measure the relative luminescence intensity value (RLIV) of each well with the TZD‐CL‐200S chemiluminescence immunoassay analyzer and calculate the relative luminescence intensity ratio of each well (RLIR = sample RLIV/negative control RLIV mean value).
2.3.4. Judgment of results
(1) When the ratio of sample RLIV to the mean value of negative control RLIV is ≥2.1, it is positive; otherwise, it is negative. (2) Quality control: the blank value should be <30,000 RLIV; each negative control value should be E1 ≤ 115,000 RLIV, E2 ≤ 300,000 RLIV; and the difference between the readings of the two negative control wells and the mean value of the negative control should be ≤10%; each positive control value should be E1 > 241,500 RLIV, E2 > 630,000 RLIV; otherwise, the experiment is invalid and the test needs to be repeated.
2.3.5. Evaluation of methodological indexes of anti‐HCV envelope protein E1 and E2 antibody detection kit 14 , 15
(1) Precision test, intra‐batch imprecision: take anti‐E1 antibody and anti‐E2 antibody obtained by U‐Tube protein ultrafiltration concentration to prepare one sample each of high‐ and low‐concentration anti‐E1 and anti‐E2 antibodies, and repeat the test 20 times for different concentrations of sample anti‐E1 and anti‐E2 antibodies, respectively (well), calculate the mean and SD of RLIR of anti‐E1 and anti‐E2 antibodies, and calculate the intra‐batch CV% (<10% is qualified); inter‐batch imprecision: take one sample of each of the above high and low concentrations of anti‐E1 and anti‐E2 antibodies, and test the samples of different concentrations of anti‐E1 and anti‐E2 antibodies once a day in the morning and afternoon, and carry out 20 batches for 10 days, calculate the mean and SD of RLIR of anti‐E1 and anti‐E2 antibodies. SD: calculate the inter‐batch CV% (<15% is qualified); (2) blank limit and detection limit, blank limit: select 5 sera of uninfected HCV subjects (anti‐E1, anti‐E2 antibody negative), test anti‐E1 and anti‐E2 antibodies once a day in the morning and afternoon, respectively, for a total of 6 days, record RLIR values, and obtain a total of 60 results for each anti‐E1 and anti‐E2 antibodies; according to the class I error (α = 0.05), LoB has a 5% probability of containing the substance to be tested; a non‐parametric test was used to rank the results of blank samples from smallest to largest, and the 95th percentile value was the anti‐E1 and anti‐E2 antibody blank limit LoB; detection limit: 5 anti‐E1 and anti‐E2 antibody serum specimens with expected concentrations of 1–5 times LoB were selected as low concentration samples, and each was tested twice a day for 6 consecutive days. The results of 12 consecutive determinations of 5 low‐concentration samples were non‐normally distributed, and the LOD of anti‐E1 and anti‐E2 antibodies was estimated using a non‐parametric procedure, respectively, i.e., LOD = LOB + Ds.β, where Ds.β is the difference between the median (M) and the 5th percentile value of the measured values of low‐concentration samples of anti‐E1 and anti‐E2 antibodies; (3) Linearity evaluation and analytical measurement range (AMR): according to the EP6‐A2 document requirements, 16 serum from uninfected HCV subjects (low value L: anti‐E1 antibody 1.66 RLIR; anti‐E2 antibody 1.55 RLIR) and serum from HCV‐infected subjects with anti‐E1 and anti‐E2 antibodies (high value H: anti‐E1 antibody 41.28 RLIR; anti‐E2 antibody 19.46 RLIR) were taken, according to 5H, 4H + 1L, 3H + 2L (2H + 3L, 1H + 4L, 5L), 6 samples were prepared according to the ratio of 5H, 4H + 1L, 3H + 2L, 2H + 3L, 1H + 4L, 5L, and each specimen was tested twice to take the mean value (actual value), and the correlation coefficient was calculated using the theoretical and actual values as vertical and horizontal coordinates, respectively (r 2 > 0.95 was qualified); (4) Accuracy evaluation 17 : anti‐E1 antibody (41.2 RLIR) and anti‐E2 antibody (19.5 RLIR) obtained by ultrafiltration of U‐Tube protein were taken. 19.5 RLIR samples were added to anti‐E1 and anti‐E2 antibody‐negative sera at 5% and 10%, respectively, and tested three times in parallel, and the average recovery was calculated (90%–110% was considered as qualified); (5) interference test: hepatitis B virus surface antibody, hepatitis B virus e antibody, treponema pallidum antibody, rheumatoid factor, human immunodeficiency virus type I and/or type II antibody positive serum specimens were mixed with anti‐E1 (20.64 RLIR), anti‐E2 (9.73 RLIR) in equal amounts, and the sample diluents were used as controls. The results were observed for interference or cross‐reaction; (6) stability observation: Store the same batch of anti‐E1, E2 antibody test kit (containing coated microplate, sample diluent, negative control, positive control, enzyme marker working solution, substrate solution A, substrate solution B, and wash solution) at 2–8°C, test the positive quality control serum of the same batch of the same number at 0, 3, 6, 9, 12, 15, and 18 months, respectively, and take the mean value of 3 repetitions each time and compare with the initial, observe the decrease in RLIR of the positive quality control serum anti‐E1, anti‐E2 antibody (<10% is qualified). Compare with the first time, observe the decrease in RLIR of anti‐E1 and anti‐E2 antibodies of positive quality control serum (<10% is qualified); (7) stability observation: the same batch of anti‐E1, E2 antibody test kits (containing coated microplates, sample dilutions, negative control, positive control, enzyme markers) will be mixed with the same amount of anti‐E1 and E2 antibody test kits (containing coated microplates, sample dilutions, negative control, positive control, and enzyme markers). Working solution, substrate solution A, substrate solution B, and washing solution were stored at 2–8°C, and the positive control sera of the same batch were tested at 0, 3, 6, 9, 12, 15, and 18 months, respectively, and the mean values were obtained by repeating 3 times each time, and the decrease in RLIR of positive control sera anti‐E1 and anti‐E2 antibodies (<10% was qualified) was observed when compared with the first time; (8) example application: the prepared anti‐E1 and anti‐E2 antibodies were used to detect the anti‐E1 and anti‐E2 antibodies. E1 and anti‐E2 antibody test kits were used to detect anti‐anti‐E1 and anti‐E2 antibodies in the sera of 45 cases of HCV‐infected patients and 50 cases of healthy physical examiners, respectively, and the positive rates of anti‐E1 and anti‐E2 antibodies in the two groups were counted and compared.
2.3.6. Statistical treatment and methodological evaluation indexes
Precision was expressed as the coefficient of variation of imprecision (CV%); sensitivity was expressed as the blank limit and detection limit and calculated by the non‐parametric method; linearity and measurement range were expressed as r 2; accuracy was expressed as the mean recovery; specificity was evaluated by the interference test; stability was evaluated as relative error (%) and two groups of anti‐E1 and anti‐E2 antibody detection. The results were compared by chi‐square test, and the differences were statistically significant at p < 0.05.
3. RESULTS
3.1. The optimal concentration of recombinant HCV envelope proteins E1 and E2 and the selection of enzyme marker working fluid
The square matrix (checkerboard) titration method was used to select the optimal coating concentration of recombinant HCV envelope proteins E1 and E2 and the concentration of enzyme marker working liquid. All the above used sample volumes were operated at 100‐μL spiked volume. The optimal coating concentration of envelope proteins E1 and E2 was 0.5 μg/mL (Table 1), and the optimal concentration of enzyme marker working liquid was 0.05 μg/mL (Table 2).
TABLE 1.
Coating concentration selection of glycoprotein E1 and E2 (RLIV).
| Coating concentration | Negative control anti‐E1 antibody | Negative control anti‐E1 antibody | Negative control anti‐E2 antibody | Positive control anti‐E2 antibody |
|---|---|---|---|---|
| 2 μg/mL | 41,815 | 139,633 | 189,653 | 1,486,881 |
| 1 μg/mL | 40,575 | 128,462 | 183,347 | 1,495,735 |
| 0.5 μg/mL | 34,196 | 112,711 | 152,901 | 1,382,944 |
| 0.25 μg/mL | 18,725 | 55,684 | 81,445 | 521,740 |
TABLE 2.
Selection of workload of enzyme‐labeled antibody (RLIV).
| Enzyme‐labeled antibody addition (μL) | Negative control anti‐E1 antibody | Positive control anti‐E1 antibody | Negative control anti‐E2 antibody | Positive control anti‐E2 antibody |
|---|---|---|---|---|
| 50 | 25,013 | 74,616 | 73,482 | 181,609 |
| 100 | 33,005 | 122,102 | 172,953 | 1,043,589 |
| 150 | 41,434 | 141,588 | 293,656 | 1,288,747 |
3.2. Evaluation of methodological indicators of the kit
(1) Precision test: intra‐batch coefficient of variation CV% < 10%, inter‐batch coefficient of variation CV% < 15% (Table 3); (2) Non‐parametric method was used to estimate the 95th percentile of blank sample measurement results; the 95th percentile anti‐E1 antibody and anti‐E2 antibody results were 1.18 RLIR and 1.26 RLIR, respectively (Table 4). The median value of anti‐E1 antibody series diluted samples tested was 3.64 RLIR and 1.98 RLIR, so the LoD of anti‐E1 antibody was 3.16 RLIR; the median value of anti‐E2 antibody series diluted samples tested was 3.68 RLIR and 2.06 RLIR, so the LoD of anti‐E2 antibody was 3.32 RLIR (Table 5); (3) The linear regression equations of anti‐E1 antibody and anti‐E2 antibody were y = 0.955X + 0.4782 and y = 0.9763X‐0.2601, respectively (Figures 1 and 2), and the regression coefficients b were between 0.95 and 1.05, and the correlation coefficients were r ≥ 0.975 (r 2 ≥ 0.95), so the analytical measurement ranges of anti‐E1 antibody and anti‐E2 antibody were 1.66–41.28 RLIR and 1.55–19.46 RLIR; (4) Accuracy evaluation: the average recoveries of anti‐E1 antibody and anti‐E2 antibody were 96.4% and 93.7%, respectively; (5) Interference test: serum specimens positive for hepatitis B virus surface antibody, hepatitis B virus e antibody, syphilis spirochete antibody, rheumatoid factor, and human immunodeficiency virus type I and/or II antibody did not interfere with this test or cross‐reactivity; (6) Stability results: the results of the kit were stable within 15 months, and the decrease of anti‐E1 antibody and anti‐E2 antibody in the 15th month of detection was <10%; (7) Example application: the positive rates of anti‐envelope protein E1 antibody and anti‐E2 antibody were 35.6% (16/45), 44.4% (20/45), and 0% and 0%, respectively, and the results are shown in Table 6.
TABLE 3.
Statistical results and performance judgment of precision experiment.
| Items | Statistical parameters | Low level (n = 20) | High level (n = 20) | ||
|---|---|---|---|---|---|
| In batch test | Inter batch test | In batch test | Inter batch test | ||
| Anti‐E1 antibody | Mean value () | 3.28 | 3.33 | 41.12 | 41.28 |
| Standard deviation (s) | 0.22 | 0.33 | 3.68 | 5.02 | |
| Coefficient of variation (CV, %) | 6.71 | 9.91 | 8.95 | 12.16 | |
| Judgment | Meet the requirements | Meet the requirements | |||
| Anti‐E2 antibody | Mean value () | 2.97 | 2.97 | 19.90 | 19.46 |
| Standard deviation (s) | 0.18 | 0.32 | 1.68 | 2. 72 | |
| Coefficient of variation (CV, %) | 6.06 | 10.77 | 8.44 | 13.98 | |
| Judgment | Meet the requirements | Meet the requirements | |||
TABLE 4.
Determination results of blank limit (RLIR).
| Items | Original data | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| First batch | Second batch | ||||||||||
| E1 | Day 1 | 0.88 | 1.01 | 0.87 | 1.11 | 0.81 | 0.94 | 1.14 | 1.16 | 1.07 | 1.1 |
| Day 2 | 0.89 | 0.95 | 0.84 | 0.89 | 1.19 | 0.92 | 0.88 | 1.05 | 0.84 | 0.98 | |
| Day 3 | 0.86 | 1.18 | 0.93 | 1.03 | 0.92 | 0.94 | 1.18 | 0.84 | 0.86 | 0.89 | |
| Day 4 | 0.98 | 1.04 | 0.98 | 1.03 | 0.83 | 1.08 | 0.94 | 0.86 | 1.16 | 1.16 | |
| Day 5 | 0.89 | 0.89 | 1.2 | 1.12 | 1.08 | 0.91 | 0.97 | 1.06 | 0.86 | 1.18 | |
| Day 6 | 1.1 | 0.85 | 0.81 | 1.2 | 1.12 | 0.86 | 1.03 | 0.84 | 0.9 | 1.17 | |
| E2 | Day 1 | 0.89 | 1.09 | 0.87 | 1.16 | 1.06 | 1.03 | 0.92 | 1.08 | 0.96 | 1.26 |
| Day 2 | 0.86 | 0.97 | 1.26 | 0.97 | 0.94 | 1.3 | 1.02 | 1.01 | 1.06 | 0.88 | |
| Day 3 | 0.94 | 0.86 | 1.02 | 1.26 | 0.86 | 0.94 | 0.93 | 1.11 | 1.12 | 1.2 | |
| Day 4 | 1.07 | 1.29 | 0.87 | 1.03 | 1.23 | 0.86 | 1.22 | 1.06 | 0.92 | 1.08 | |
| Day 5 | 1.05 | 0.97 | 0.93 | 0.94 | 1.02 | 1.25 | 1.12 | 1.08 | 1.24 | 1.1 | |
| Day 6 | 1.17 | 1.28 | 1.16 | 0.87 | 1.07 | 0.99 | 1.01 | 0.93 | 0.89 | 0.92 | |
TABLE 5.
Detection limit test results (RLIR).
| Items | Original data | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| First batch | Second batch | ||||||||||
| E1 | Day 1 | 2.05 | 2.52 | 3.79 | 4.06 | 5.05 | 1.96 | 2.56 | 3.73 | 4.31 | 5 |
| Day 2 | 2.03 | 2.56 | 3.74 | 4.2 | 5.21 | 2.17 | 2.77 | 3.38 | 4.39 | 5.22 | |
| Day 3 | 1.99 | 2.71 | 3.65 | 4.34 | 5.01 | 1.8 | 2.47 | 3.71 | 4.28 | 5.42 | |
| Day 4 | 1.55 | 2.79 | 3.77 | 4.02 | 5.21 | 2.14 | 2.42 | 3.45 | 4.38 | 5.1 | |
| Day 5 | 1.56 | 2.87 | 3.53 | 4.13 | 5.29 | 2.09 | 2.74 | 3.54 | 4.28 | 5.01 | |
| Day 6 | 1.66 | 2.5 | 3.49 | 4.41 | 5.31 | 2.13 | 2.41 | 3.62 | 4.42 | 5.16 | |
| E2 | Day 1 | 1.76 | 2.66 | 3.71 | 4.41 | 5.21 | 1.79 | 2.68 | 3.62 | 4.37 | 5.22 |
| Day 2 | 1.72 | 2.66 | 3.79 | 4.14 | 5.38 | 1.74 | 2.82 | 3.37 | 4.49 | 5.44 | |
| Day 3 | 2.16 | 2.43 | 3.38 | 4.48 | 5.11 | 2.13 | 2.75 | 3.4 | 4.19 | 5.29 | |
| Day 4 | 2.02 | 2.66 | 3.31 | 4.49 | 5.37 | 1.54 | 2.69 | 3.35 | 4.36 | 5.48 | |
| Day 5 | 1.94 | 2.64 | 3.48 | 4.33 | 5.46 | 2.06 | 2.7 | 3.61 | 4.46 | 5.18 | |
| Day 6 | 1.55 | 2.63 | 3.55 | 4.18 | 5.07 | 1.56 | 2.53 | 3.35 | 4.3 | 5.37 | |
FIGURE 1.
Determination of anti‐E1 antibody linear relationship by chemiluminescence.
FIGURE 2.
Determination of anti‐E2 antibody linear relationship by chemiluminescence.
TABLE 6.
Detection results of anti‐E1 and anti‐E2 antibody in two groups (n).
| Group | n | Anti HCV antibody a | Anti‐E1 antibody b | Anti‐E2 antibody b |
|---|---|---|---|---|
| Research group | 45 | 45 | 16 | 20 |
| Control group | 50 | 0 | 0 | 0 |
Note: The study group was compared with the control group (anti‐E1 antibody: χ 2 = 21.38, p < 0.05; anti‐E2 antibody: χ 2 = 28.15, p < 0.05).
Commercial kits were used to detect anti HCV mixed antibodies (four recombinant proteins of nucleocapsid, NS3, NS4, and NS5).
The recombinant envelope proteins E1 and E2 were used to detect anti‐E1 and anti‐E2 antibodies, respectively.
4. DISCUSSION
HCV infection can cause both acute and chronic hepatitis, with chronic infection occurring in about 55%–85% of people and leading to progressive liver fibrosis and cirrhosis, with the risk of cirrhosis ranging from 15% to 30%. HCV is highly insidious, with no obvious symptoms in the early stages of the disease, and most patients are unaware of their disease, which can easily develop into liver cancer. The cure rate for hepatitis C is more than 80% for patients with the disease for less than 5 years, 60% for patients with the disease for about 8 years, and even less for patients with the disease for more than 10 years. Although the risk of cirrhosis and hepatocellular carcinoma associated with HCV infection varies significantly among patients, approximately 1%–3% of patients with cirrhosis associated with HCV infection progress to hepatocellular carcinoma each year. 3 The World Health Organization estimates that there are approximately 1.75 million new cases of hepatitis C virus infection each year, mainly due to injecting drug use and poor health care conditions. Thus, HCV infection has gradually shifted from blood transfusion as the main route of infection to injecting drug use, people serving prison sentences, and gay men, thus posing new challenges to the prevalence and prevention and control of HCV, which has become a more serious social and public health problem. 3 , 18 The information in “National epidemic situation of statutory infectious diseases” published by the National Health and Wellness Commission shows that the incidence of viral hepatitis has been the first in China over the years, and hepatitis C is the second highest in the incidence of viral hepatitis, and has remained high since 2012 when the incidence rose to 200,000. The incidence of hepatitis C in China is 3% to 5%, and it has become a viral hepatitis that cannot be ignored. The hepatitis C virus is the causative agent of hepatitis C. Its envelope glycoproteins E1 and E2 play fusion and binding roles in the process of HCV invasion into host cells, 7 , 19 respectively. And most scholars believe that the detection of antibodies to HCV envelope proteins E1 and E2 is a new indicator for the diagnosis of HCV. The detection of E1 and E2 antigen components was designed on the basis of HCV antibody detection kits, and the detection of positive patient sera with good antigenicity by the HCVE1E2 fusion protein is expected to improve its detection rate. It has been reported that CD81 is the receptor for HCV, and HCV E2 is an important protein that recognizes CD81 and is involved in the recognition and attachment of HCV to host cells. 20 The HVR1 region of the HCV E2 protein is the most variable region in the whole viral gene, which is the strongest immunogenic determinant and the main cause of immune heterogeneity among individuals infected with the virus. It is mainly composed of neutralizing epitopes, which are considered to be the main factor leading to escape from the host immune response. 21 Therefore, antibodies produced by HCV envelope glycoproteins E1 and E2 were detected to understand the immune response of E1 and E2 proteins in the host and to further reveal the correlation between changes in anti‐E1 and E2 protein antibody content and different genotypes and types of HCV‐infected persons, clinical infection course progression, and before and after DAA treatment to provide technical support. It is of great significance to evaluate the infectivity, disease progression, and drug efficacy of HCV‐infected individuals, and the research in this field has received extensive attention from experts in molecular biology, infectious diseases, and epidemiology. More recently, the structure of the mbE1E2 complex bound to the antibodies AR4A, AT1209, and IGH505 was determined by cryoelectron microscopy (cryo‐EM), 22 revealing details of the interface between E1 and E2 and AR4A and the complex, which will greatly assist in the design of anti‐HCV vaccines. In addition, the recently developed soluble secreted E1E2 complex (sE1E2) 23 , 24 and the determination of its structure 25 further enable E1E2‐based NPs as a vaccine candidate. Therefore, in order to understand whether vaccines containing E1 and E2 proteins as targets will stimulate the immune response to produce antibodies against E1 and E2 proteins, as well as their changes in the different courses of virus infection before and after medication, it is necessary to first develop a kit capable of detecting antibodies against E1 and E2 proteins. 26 The current kits for routine clinical diagnosis of HCV infection are mainly fourth‐generation reagents encapsulated with mixed antigens (recombinant protein C‐22 in the nucleocapsid region, recombinant protein C‐200 in the NS3 region, and recombinant protein C‐200 and NS5 in the NS4 region). 27 It detects mixed anti‐HCV antibodies without anti‐E1 and anti‐E2 antibodies, cannot distinguish which protein component produces the antibodies, and cannot understand the immune response of different protein components of HCV in the host, nor can it reveal the clinical significance of different protein components of HCV in the immune response of HCV‐infected patients, disease monitoring, prognosis assessment, and epidemiological studies. Domestic kits and related studies on the detection of anti‐HCV envelope proteins E1 and anti‐E2 have not been reported, and Sillanpää M et al. 28 established an immunoblotting method to detect antibodies to the corresponding proteins in the sera of HCV‐infected patients using recombinant Core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins, and found that the antibody positivity rate of different proteins in the sera of HCV‐infected patients varied greatly, and the analysis may be related to the different disease progression, different genotypes, before and after antiviral treatment, and the presence of circulating immune complexes in the sera of HCV‐infected patients.
The main methods used to detect antibodies are enzyme‐linked immunosorbent assay (ELISA), colloidal gold method, immunoblotting method, chemiluminescence method, etc. The ELISA method is technically mature, but the operation is complicated, the detection cycle is long and needs to be completed manually, and the false positive rate and leakage rate are high; the colloidal gold method has low sensitivity and is prone to false negatives; the immunoblotting method has low sensitivity and qualitative results; and the chemiluminescence immunoassay uses acridine ester to combine chemiluminescence technology and magnetic particle separation technology for determination, which has the advantages of high sensitivity, high specificity, rapidity, and stability compared with some technologies such as ELISA. Therefore, in this paper, we developed a chemiluminescence kit for the detection of anti‐E1 and anti‐E2 antibodies in HCV‐infected sera and used two chemiluminescence enhancers (o‐phenylphenol and 4‐imidazolidinol) to play a synergistic role to significantly reduce the background signal and make the luminescence signal stable for a long time, which indirectly amplifies the immunoreactivity results with high sensitivity and stability of the assay. The performance verification of the precision, precision near the critical value, minimum detection limit, inter‐method comparison, and analytical measurement range of the chemiluminescence assay in this study can meet clinical needs. We tested the sera of 45 HCV‐infected patients and detected 16 anti‐E1 antibodies and 20 anti‐E2 antibodies, with a positive detection rate of 36% and 44%, respectively, and an overall positive detection rate of 80%. The above results indicated that there was a certain proportion of anti‐envelope protein antibodies in HCV‐infected patients (Table 2), suggesting that there was an immune response to HCV envelope protein. These preliminary findings provide further insight into the stimulation of antibody production and possible circulating immune complexes (anti‐E1‐IC, anti‐E2‐IC) in HCV‐infected patients with different genotypes and clinical stages (chronic, cirrhosis, and hepatocellular carcinoma) and whether differences exist before and after antiviral therapy provide technical means.
In this paper, the methodological evaluation of the developed kit for the detection of anti‐E1 and anti‐E2 antibodies by chemiluminescence was carried out, and the results showed that the indicators meet the methodological requirements and that the kit can be applied to the screening and diagnosis, disease monitoring, prognosis assessment, disease mechanism, and epidemiological study of HCV‐infected patients.
FUNDING INFORMATION
This work was supported by Medical Science and Technology Innovation Foundation of Nanjing Military Region (15DX025), Social Development Research Project of Hangzhou Science and Technology Bureau of Zhejiang Province (20160533B75), The Medical and Health Research Project of Zhejiang Province (2016KYB250, 2021433791), and Zhejiang Provincial Natural Science Foundation, LGF21H200003. The product of this project has been applied for national invention patent (202010946271.1).
CONFLICT OF INTEREST STATEMENT
No conflict of interest.
HUMAN OR ANIMAL EXPERIMENTATION
All samples were approved by the Medical Ethics Committee of the 903 Hospital of the PLA Joint Logistic Support Force (approval no. 20200608/06/02/204).
ACKNOWLEDGEMENTS
Not applicable.
Wang, Ningning , Liu Qingqing, Che Feihu, Sun Qingyang, Wang Yue, Yang Chunli, Dai Yuzhu and Cheng Jun. 2024. “Establishment and methodological evaluation of a chemiluminescence assay for detection of anti‐envelope protein (E1, E2) antibodies in the serum of hepatitis C virus‐infected patients.” Journal of Clinical Laboratory Analysis 38 (4): e25011. 10.1002/jcla.25011
Contributor Information
Yuzhu Dai, Email: dyz5895@qq.com.
Jun Cheng, Email: cj1171967@163.com.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
REFERENCES
- 1. Pisano MB, Giadans CG, Flichman DM, Ré VE, Preciado MV, Valva P. Viral hepatitis update: Progress and perspectives. World Journal of Gastroenterology. 2021;27(26):4018‐4044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Weiner AJ, Kuo G, Bradley DW, et al. Detection of hepatitis C viral sequences in non‐a, non‐B hepatitis. Lancet. 1990;335(8680):1‐3. [DOI] [PubMed] [Google Scholar]
- 3. Guidelines for the Care and Treatment of Persons Diagnosed with Chronic Hepatitis C Virus Infection. World Health Organization; 2018. [PubMed] [Google Scholar]
- 4. Tsukiyama‐Kohara K, Kohara M. Hepatitis C virus: viral quasispecies and genotypes. International Journal of Molecular Sciences. 2017;19(1):23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Lombardi A, Mondelli MU. ESCMID study group for viral hepatitis (ESGVH). Hepatitis C: is eradication possible. Liver International. 2019;39(3):416‐426. [DOI] [PubMed] [Google Scholar]
- 6. Martinez MA, Franco S. Therapy implications of hepatitis C virus genetic diversity. Viruses. 2020;13(1):41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Kumar A, Hossain RA, Yost SA, et al. Structural insights into hepatitis C virus receptor binding and entry. Nature. 2021;598(7881):521‐525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Cao L, Yu B, Kong D, et al. Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus. PLoS Pathog. 2019;15(5):e1007759. doi: 10.1371/journal.ppat.1007759 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Tzarum N, Wilson IA, Law M. The neutralizing face of hepatitis C virus E2 envelope glycoprotein. Frontiers in Immunology. 2018;9:1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Xiaodong Z, Li W, Guocai C, et al. Wuhan region of hepatitis c virus envelope protein E1 bioinformatics research. J Central China Normal Univ (Nat Sci Ed). 2018;52(3):366‐372. doi: 10.19603/j.carolcarrollnki [DOI] [Google Scholar]
- 11. Cocquerel L, Meunier JC, Pillez A, Wychowski C, Dubuisson J. A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. Journal of Virology. 1998;72(3):2183‐2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Yu Y, Cheng J, Dai Y, Mei C. Detection of serum anti‐NS2 antibody and recombinant hepatitis C virus nonstructural protein 2 (NS2): its development and evaluation. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022;38(4):361‐367. [PubMed] [Google Scholar]
- 13. Zhou H, Wu J, Yu Y, et al. Establishment and evaluation of recombinant expression of HCV transmembrane protein (p7) and detection of anti‐p7 antibody in serum of HCV‐infected patients by chemiluminescence. Laboratoriums Medizin. 2022;27:lmac113. [DOI] [PubMed] [Google Scholar]
- 14. Clinical and Laboratory Standards Institute . User Demonstration of Performance for Precision and Accuracy. CLSI EP15‐A2; 2004. [Google Scholar]
- 15. China National Accreditation Service for Conformity Assessment . CNAS‐CL02:2012 Accereditation Criteria for the Quality and Competence of Medical Laboratories. Standards Press of China; 2012. [Google Scholar]
- 16. Clinical and Laboratory Standards Institute . Evaluation of the Linearity of Quantitative Measurement Procedures. CLSI EP6‐A2; 2003. [Google Scholar]
- 17. State Food and Drug Administration . Guidelines for technical review of analytical performance evaluation (Accuracy‐recovery test) of in vitro diagnostic reagents. Letter from the office of the State Food and Drug Administration on medical devices. No. 116 [2011]. 2011.
- 18. Jin F, Dore GJ, Matthews G, et al. Prevalence and incidence of hepatitis C virus infection in men who have sex with men: a systematic review and meta‐analysis. The Lancet Gastroenterology & Hepatology. 2021;6(1):39‐56. [DOI] [PubMed] [Google Scholar]
- 19. Chigbu DI, Loonawat R, Sehgal M, Patel D, Jain P. Hepatitis C virus infection: host‐virus interaction and mechanisms of viral persistence. Cell. 2019;8(4):376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Owsianka AM, Timms JM, Tarr AW, et al. Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. Journal of Virology. 2006;80(17):8695‐8704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Prentoe J, Bukh J. Hypervariable region 1 in envelope protein 2 of hepatitis C virus: a linchpin in neutralizing antibody evasion and viral entry. Frontiers in Immunology. 2018;9:2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Torrents de la Peña A, Sliepen K, Eshun‐Wilson L, et al. Structure of the hepatitis C virus E1E2 glycoprotein complex. Science. 2022;378(6617):263‐269. doi: 10.1126/science.abn9884 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Guest JD, Wang R, Elkholy KH, et al. Design of a native‐like secreted form of the hepatitis C virus E1E2 heterodimer. Proc Natl Acad Sci USA. 2021;118(3):e2015149118. doi: 10.1073/pnas.2015149118 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Wang R, Suzuki S, Guest JD, et al. Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate. Proc Natl Acad Sci USA. 2022;119(11):e2112008119. doi: 10.1073/pnas.2112008119 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Metcalf MC, Janus BM, Yin R, et al. Structure of engineered hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies. Nat Commun. 2023;14(1):3980. doi: 10.1038/s41467-023-39659-z [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Toth EA, Andrianov AK, Fuerst TR. Prospects for developing an hepatitis C virus E1E2‐based nanoparticle vaccine. Reviews in Medical Virology. 2023;33(5):e2474. doi: 10.1002/rmv.2474 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Warkad SD, Song KS, Pal D, Nimse SB. Developments in the HCV screening technologies based on the detection of antigens and antibodies. Sensors (Basel). 2019;19(19):4257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Sillanpää M, Melén K, Porkka P, et al. Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection. Virology Journal. 2009;6:84. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.