Characterization and Application of Monoclonal Antibody Against Hepatitis C Virus Nonstructual Protein Three

Abstract

Developing assays for detecting HCV antigens could be beneficial because viral proteins appear earlier than antibodies and are more stable than RNA in the serum. Monoclonal antibody was prepared by immunization and cell fusion. Subclass, specificity, and efficiency of monoclonal antibodies were determined by ELISA. Epitope specificity of monoclonal antibodies was analyzed by ELISA additivity test. HCV antigen in serum of hepatitis patients was examined by double monoclonal antibody sandwich ELISA. Five hybridoma cell lines were screened and named HCV₁, HCV₂, HCV₃, HCV₄, and HCV₅. These five monoclonal antibodies had high specificity and efficiency. The additivity test showed that HCV₂, HCV₄, and HCV₅ recognized different epitopes, which can be matched in ELISA. Of 173 anti-HCV positive patients, 37 (21.4%) were positive for HCV antigen. Of 1498 anti-HCV negative patients, 10 (0.67%) were positive for HCV antigen. Fifty normal controls were negative for HCV antigen. HCV antigen detection had moderate agreement and correlation with HCV RNA detection (kappa=0.577, p<0.01; r=0.59, p<0.01). This result indicates that the monoclonal antibody against HCV NS₃ may be a potential diagnostic reagent, which would provide a foundation for developing a sandwich ELISA of HCV antigen detection.

Introduction

Hepatitis C virus (HCV) is the major etiologic agent of post-transfusion and sporadic non-A, non-B hepatitis.⁽¹⁾ The majority of individuals with acute HCV infection subsequently develop chronic infection and liver cirrhosis as well as hepatocellular carcinoma (HCC).^(2–4) Diagnosis of HCV infection is based primarily on the detection of antibody by recombinant viral protein or on detection of viral RNA by reverse transcription and polymerase chain reaction (RT-PCR).⁽⁵⁾ Developing assays for detecting HCV antigens could be beneficial in that viral proteins appear earlier than antibodies and are more stable than RNA in the serum.^(6,7)

The antibody is very important as a reagent detecting HCV antigen. Good antibody is usually sufficient to establish the experimental procedure. In this case, monoclonal antibodies are the first choice because of higher specificity, efficiency, and yield than polyclonal antibodies. It is also necessary to use a series of monoclonal antibodies directed against the same antigenic molecule, but with different epitope specificities.⁽⁸⁾

The purpose of this study was to develop monoclonal antibodies against the HCV antigen as an early diagnostic reagent. In this study, analysis of epitope specificity was a very crucial step. Based on this step, a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of HCV antigen was developed.

Materials and Methods

Antigen

Highly purified HCV antigen and HBV recombinant surface antigen were donated by the Viral Institute of Chinese Academy of Science (Beijing, China). HCV antigen contains a nonstructural protein three region (nucleotide [nt], 3890∼4716, amino acids [a], 1008∼1616], with a molecular weight of 68 kDa. HDV recombinant antigen was prepared at the Beijing Municipal Liver Diseases Institute (Beijing, China).

Reagents

1640 medium and fetal bovine serum was obtained from Gibco (Carlsbad, CA). SP2/0 myeloma cells and BALB/c mice were obtained from the Military Medical Science Institute (Beijing, China). The immunoglobulin subclass kit and goat anti-mouse IgG peroxidase conjugate were obtained from Sigma (St. Louis, MO). HCV-RNA and anti-HCV assay kits were purchased from Sino-American Biotechnology Co (Luoyang, China).

Patients

The patients were from the outpatient and inpatient units in Beijing Youan Hospital, who were diagnosed in terms of the standards set at the 10th National Infection and Verminosis Symposium at Xi'an in 2000.⁽⁹⁾ Fifty normal controls (NC) were healthy volunteers, negative for anti-HCV antibody and hepatitis B virus surface antigen. The study protocol was approved by the Ethics and Research Committee of Beijing Youan Hospital of Capital Medical University. All the participants signed written informed consents.

Immunization and cell fusion

BALB/c mice were injected subcutaneously with 100 μg HCV recombinant antigen in complete Freund's adjuvant. After 14 days, BALB/c mice were injected subcutaneously with a similar dose in incomplete Freund's adjuvant. After another 14 days, an intraperitoneal injection with a similar dose in phosphate buffer saline was used as the booster immunization 3 days before a hybridoma fusion. The fusion procedure followed the technique described by Chen and associates⁽¹⁰⁾ with PEG-1500 as fusion reagent. The spleen cells of the immunized mice were harvested and fused with SP2/0 myeloma cells. The ratio of spleen cells to myeloma cells was 5:1. The cells were plated at 2×10⁵ cells per well in 96-well plates in RPMI 1640 medium containing HAT supplement. The positive population was cloned by limiting dilution. The clones retained were cultured and injected intraperitoneally into pristine-primed BALB/c mice to induce ascites. Subclass analysis was determined by an ELISA kit.

ELISA for anti-HCV NS3

Microplates were coated with 0.25 μg HCV NS3 antigen in 100 μL coating buffer (0.05 mol/L sodium bicarbonate buffer [pH 9.6]) per well and incubated at 4°C overnight. The plates were then blocked with blocking buffer (2% bovine serum albumin and 0.05% Tween-20 in phosphate buffer saline) and incubated at 4°C overnight. After washing, a 200 μL sample (e.g., ascitic fluids or supernatant) diluted in washing buffer (0.05% Tween-20 in phosphate buffer saline) was added to each well and incubated at 37°C for 2 h. The serum of normal BALB/c mouse was used as a negative control. After washing, 100 μL of goat anti-mouse IgG peroxidase conjugate (1:50,000) were added to each well and incubated at 37°C for 2 h. Finally 100 μL of substrate solution (3,3′,5,5′-tetramethylbenzidine) was added and incubated at room temperature for 10–15 min. The reaction was stopped with 2 mol/L sulfate. Plates were read with a microplate reader (Bio-Rad, Hercules, CA). Samples were confirmed to be positive with P/N≥2.1 (P, optical density value of the sample; N, optical density value of the negative control).

To analyze the specificity of monoclonal antibodies and HCV NS3 recombinant antigen, we determined whether there was a cross-reaction between ascitic fluid and HBV recombinant surface antigen, HDV recombinant antigen, and Escherichia coli BL-21, respectively. The experimental procedures are described above.

ELISA additivity test

The sandwich ELISA we tried to develop was based on an estimation of the number of antigenic sites simultaneously available to a pair of antibodies on the antigen. Estimating this number by an ELISA additivity test required that the antigen be saturated with each antibody. The saturation curve of the antigen by each antibody was determined by ELISA. The ascitic fluid was diluted to a series of titers from 1:10 to 1:10⁷. The experimental procedures are described above. The optical density (OD) value did not change markedly when increasing the titer of monoclonal antibodies, which suggested that the coated antigen had reached saturation. The titer of each ascitic fluid corresponded to the titer at which the saturation curve turned to a plateau. After the titer of each ascitic fluid was determined, the ELISA additivity test was used to analyze the epitope specificity of the monoclonal antibodies. Only one step was different from the ELISA described above. After coating and blocking, two ascitic fluids were added to each well simultaneously, each volume was half of the total volume, and each titer was sufficient to saturate the coated antigen.

Additivity index

To quantify the experimental results of the additivity test, an additivity index (AI) has been defined for a pair of monoclonal antibodies as follows:⁽⁸⁾

\documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*} {\rm Al} = \left \{ \frac {2 {\rm A}_{1 + 2}} {{\rm A} _1 + {\rm A} _2} \right\} - 1 \times 100 \% \end{align*} \end{document}

A₁, A₂, and A₁₊₂ are the absorption values reached in ELISA with the first antibody alone, the second antibody alone, and the two antibodies together, respectively. If the two antibodies bind randomly at the same site, A₁₊₂ should be equal to the mean value of A₁ and A₂, and AI will be equal to zero. On the contrary, if the two antibodies bind independently at a distinct site, A₁₊₂ should be the sum of A₁ and A₂, and AI will be equal to 100%. The additivity index at 50% is thought as a cut-off value.⁽¹¹⁾

Double monoclonal antibody sandwich ELISA for HCV antigen

Microplates were coated with monoclonal antibody (HCV₄, 1:1000) and incubated at 4°C overnight. The plates were then blocked with blocking buffer (2% bovine serum albumin and 0.05% Tween-20 in phosphate buffer saline) and incubated at 4°C overnight. After washing, 200 μL of the sera diluted in washing buffer (0.05% Tween-20 in phosphate buffer saline) were added to each well and incubated at 37°C for 2 h. The serum of a healthy volunteer was used as a negative control. After washing, 100 μL of monoclonal antibody (HCV₅, 1:8000) was added to each well and incubated at 37°C for 2 h. After washing, 100 μL of goat anti-mouse IgG peroxidase conjugate (1:50 000) was added to each well and incubated at 37°C for 2 h. Finally 100 μL of substrate solution (3,3′,5,5′-tetramethylbenzidine) was added and incubated at room temperature for 10–15 min. The reaction was stopped with 2 mol/L sulfate. Plates were read with a microplate reader (Bio-Rad). Samples were confirmed to be positive with P/N≥2.1 (P, OD value of the sample; N, OD value of the negative control).

HCV RNA assay and anti-HCV assay

HCV RNA was tested with reverse transcription and nested polymerase chain reaction (RT-PCR) according to the manual. Anti-HCV was tested by ELISA according to the manual.

Statistical analysis

Paired four-fold tables were analyzed by the McNemar test, kappa test, and correlation. A value of p<0.05 was considered as statistically significant. All data were analyzed with SPSS11.5 software.

Results

Establishment of monoclonal antibodies

Five hybridoma cell lines were screened, which can secrete stably specific monoclonal antibodies against HCV nonstructural protein three after being resuscitated and regenerated two times. They were named HCV₁, HCV₂, HCV₃, HCV₄, and HCV₅.

Identification of monoclonal antibodies

In the five monoclonal antibodies, HCV₃ was found with IgA subclass and kappa chain, while the others were found with IgG₁ subclass and kappa chain. All monoclonal antibodies reacted with HCV recombinant antigen, and did not cross-react with HDV recombinant antigen and Escherichia coli BL-21 (data not shown). Data showed that these five monoclonal antibodies had high efficiency (Table 1).

Table 1.

Efficiency of Five Monoclonal Antibodies

Hybridoma	Titer of supernatant	Titer of ascitic fluid
HCV₁	1×10³	1×10⁴
HCV₂	1×10³	1×10⁵
HCV₃	1×10³	1×10³
HCV₄	2×10³	1×10⁴
HCV₅	2×10³	5×10⁵

Epitope specificity analysis of monoclonal antibodies

It is well known that IgG is more than 80% of total immunoglobulin in serum. So we chose four monoclonal antibodies with IgG₁ subclass to develop an early diagnostic reagent.

First, we determined the saturation curve of the antigen for each monoclonal antibody. The results are shown in Figure 1. It can be seen that for the four ascitic fluids, a plateau is particularly reached in certain dilutions of HCV₁, HCV₂, HCV₄, and HCV₅. We found the dilutions of HCV₁, HCV₂, HCV₄, and HCV₅ to be 1:1000, 1:1000, 1:1000, and 1:8000, respectively, in which the coated antigen had reached saturation.

Fig. 1.

Saturation curve of coated antigen and monoclonal antibodies.

The epitope specificity of these four monoclonal antibodies was then analyzed by an ELISA additivity test. The four monoclonal antibodies were divided into six pairs. The results are shown in Table 2. The AI of HCV₂ and HCV₄, HCV₂ and HCV₅, and HCV₄ and HCV₅ were more than 50%, so we thought the pair recognized different epitopes. The AI of HCV₁ and HCV₂, HCV₁ and HCV₄, and HCV₁ and HCV₅ was less than 50%, so we thought the pair recognized the same epitope.

Table 2.

ELISA Additivity Test of Four Monoclonal Antibodies

Hybridoma and titer	A₁₊₂	A₁+A₂	AI (%)
HCV₁ (1:1000) and HCV₂ (1:1000)	1.321	1.958 (0.653+1.305)	34.9
HCV₁ (1:1000) and HCV₄ (1:1000)	1.044	1.463 (0.653+0.81)	42.7
HCV₁ (1:1000) and HCV₅ (1:8000)	1.55	2.153 (0.653+1.5)	44
HCV₂ (1:1000) and HCV₄ (1:1000)	1.69	2.115 (1.305+0.81)	59.8
HCV₂ (1:1000) and HCV₅ (1:8000)	2.259	2.805 (1.305+1.5)	61.7
HCV₄ (1:1000) and HCV₅ (1:8000)	2.001	2.31 (0.81+1.5)	73.2

HCV antigen detection with double monoclonal antibody sandwich ELISA

Of 173 anti-HCV positive patients, 37 (21.4%) were positive for HCV antigen. Of 1498 anti-HCV negative patients, 10 (0.67%) were positive for HCV antigen. Fifty normal controls were negative for HCV antigen. Data showed the HCV antigen detection with double monoclonal antibodies sandwich ELISA had high specificity and better sensitivity (Table 3).

Table 3.

HCV Antigen Detection in Sera of Hepatitis Patients and Normal Controls

		Total no.	Number of HCV antigen positive	HCV antigen positive ratio
Anti-HCV positive samples		173	37	21.4%
Anti-HCV	HAV positive	81	0	0
Negative	HBV positive	1350	10	0.74%
Samples	HEV positive	67	0	0
Normal controls		50	0	0

Comparison between HCV antigen and HCV RNA detection

A total of 173 anti-HCV positive samples was detected by HCV antigen and HCV RNA simultaneously. This result showed that the agreement between two methods was moderate (kappa=0.577, p<0.01), and there were statistical differences between the two methods (p=0.013<0.05). There was also correlation between the two methods (r=0.59, p<0.01; Table 4).

Table 4.

Crosstab of HCV Antigen and HCV RNA Detection

		HCV RNA
		Positive	Negative	Total
HCV	Positive	30	7	37
Antigen	Negative	21	115	136
Total		51	122	173

Discussion

In this study, we found that the monoclonal antibodies against HCV NS₃ had high specificity and efficiency, and that HCV₂, HCV₄, and HCV₅ recognized different epitopes. We also found that the HCV antigen detection with double monoclonal antibody sandwich ELISA developed by HCV₄ and HCV₅ had high specificity and better sensitivity, which had moderate agreement and correlation with HCV RNA detection (kappa=0.577, p<0.01; r=0.59, p<0.01). These results indicate that the monoclonal antibody against HCV NS₃ may be a potential diagnostic reagent, which would provide a foundation for developing a sandwich ELISA of HCV antigen detection.

Similar to our study, Xie and colleagues⁽¹²⁾ found that the HCV antigen detection showed perfect specificity, high sensitivity, and good stabilization, which can be used for the early diagnosis of HCV. But different from our study, in the double antibody sandwich ELISA it was established that one antibody was polyclonal antibody and another was monoclonal antibody. We had the same sensitivity as in their study, but higher specificity than in their study. Ergünay and colleagues⁽¹³⁾ had consistent results in the agreement between HCV antigen and HCV RNA (kappa=0.554) with us. We also found that the subclass in most of the monoclonal antibodies were IgG₁. Sallberg and colleagues⁽¹⁴⁾ demonstrated that HCV NS₃ immunization in mice resulted in predominantly IgG₁ antibody. In addition, Pu and colleagues⁽¹⁵⁾ found that the NS_3-e protein beared the antigenic determinant recognized by both human sera (93%) and murine monoclonal antibody (100%). These studies provide a foundation for our study.

It has been well documented that the HCV NS₃ region contains multiple conservative antigenic determinants for B cells that induce antibodies in HCV infection,⁽¹⁶⁾ which is the main target antigen for the study of HCV diagnosis and vaccine.^(17–19) Therefore, it is very possible to find two monoclonal antibodies against NS₃ protein with different epitopes, which is very important to establish the HCV detection assay. The assay that we want to develop is an ELISA for antigen detection sandwiched between two monoclonal antibodies. The basic mechanism of the experiment is antigen-antibody reaction. The two monoclonal antibodies bind simultaneously to one antigen molecule, so the two monoclonal antibodies matched in the experiment must recognize different antigenic determinants. If the two monoclonal antibodies recognize the same antigenic site, the sandwich ELISA would not be established. Furthermore, because the proximity of antigenic sites creates steric hindrance between antibody molecules, the antigenic determinants that the monoclonal antibodies recognized had better distribution in different places in the antigen, and the one antigenic determinant should be far away from the other. The further the distance between the two antigenic determinants, the better the result of the sandwich ELISA. The combination between one monoclonal antibody and antigen should not interfere with the other or decrease the interference effect to a minimum extent. Thus, the sandwich ELISA has high sensitivity and specificity.

To screen epitope specificity of the monoclonal antibody, we used the ELISA additivity test and determined the additivity index for each pair of monoclonal antibodies. The method we used turned out to be very valuable in screening epitope specificity of the monoclonal antibody. It is very easy to use for routine screening since it requires a simple, stable, and commercially available reagent.⁽⁸⁾ In addition, it can distinguish whether two monoclonal antibodies recognize the same antigenic site or if they are proximate in the antigen. However, this method has one disadvantage. If the antigen oscillates between two conformations, and if the antibody binds exclusively to one conformation while the other binds exclusively to the other, it will be impossible for the two antibodies to bind simultaneously to the antigen even if they are different epitopes. Indeed “allosteric” behavior⁽²⁰⁾ of a protein for the binding of a monoclonal antibody has been reported.⁽²¹⁾ But fortunately, the HCV antigen we used is a recombinant antigen, and it is a linear epitope. We found that there was some difference of epitope specificity between monoclonal antibodies, and the AI of HCV₂ and HCV₄, HCV₂ and HCV₅, and HCV₄ and HCV₅ was more than 50%. So we believe these pairs recognized different epitopes and they can be matched in ELISA. These results indicate that a sandwich ELISA for detecting the HCV antigen through these monoclonal antibodies can be established.

As is already known, patients infected with HCV have a long preseroconversion window phase of about 70 days, from HCV infection to the appearance of HCV antibody in serum.^(22,23) HCV RNA detection as an early diagnostic means has some disadvantages including RNA instability and contamination problems.^(24–26) However, the HCV antigen appears in the serum earlier than the HCV antibody by about 40 days, almost at the same time as the HCV RNA.⁽²⁷⁾ In addition, there is a good correlation between HCV antigen and HCV RNA.^(28–30) Therefore, HCV antigen detection may be substituted for RNA detection as an early diagnosis of HCV infection. It will improve the detection of HCV infection in the preseroconversion window phase, and help to limit HCV propagation by monitoring HCV infection in donor blood. In the future, a test that combines both HCV antigen and HCV antibody immunoassay will be required for HCV diagnosis.^(31–33)

Footnotes

Acknowledgments

This study was supported by grants from the National High Technology Research and Development Program of China (863) (no. 2007AA03Z315) and the Capital Medical Developing Scientific and Research Fund (no. 2007-3080).

Author Disclosure Statement

The authors have no financial interests to report.

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