The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (K D rel) of 2.9±0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described.
Hepatitis B virus (HBV) poses a major public health problem worldwide. The virus is estimated to infect more than one-third of the world's population and there are about 350 million carriers of HBV worldwide (Jung and Pape, 2002). High prevalence areas have been identified in South-East Asia, China and Africa (reviewed by Lee, 1997). Prolonged carriage of HBV may progress to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the presence of effective vaccines, about 1–2 million people die of HBV infection each year (Jung and Pape, 2002). In Malaysia, about 5% of healthy blood donors are chronic carriers of HBV (Merican et al., 2000).
Three morphological distinct forms of particle are found in sera of infected individuals (Bayer et al., 1968). They exist as spherical and filamentous particles of 20 nm diameter as well as 42 nm double-shelled Dane particles (Dane et al., 1970). The latter is the infectious form and it consists of an outer envelope derived from the host cell membrane. Embedded in the envelope are three distinct but related forms of the surface antigen (HBsAg): L-HBsAg (large) , M-HBsAg (middle) and S-HBsAg (small). Internal to the envelope is the viral nucleocapsid which is made of many copies of the core antigen (HBcAg). Within the nucleocapsid is the polymerase protein (P) which is covalently attached to a partially double-stranded circular DNA of about 3.2 kb (Ganem, 1991).
The three forms of surface antigens, L-HBsAg, M-HBsAg and S-HBsAg are encoded by one single open reading frame of the viral genome by using three different in-frame start codons and a common stop codon. Hence, the proteins differ at their N-termini, but have a large sequence in common within their C-terminal ends. The longest of the three, L-HBsAg, has the PreS1 region of 108 or 119 amino acid residues (depending on serotype), followed by the PreS2 region of 55 residues and the S region which comprises 226 amino acid residues (Heermann et al., 1984). The second largest protein, the M-HBsAg, contains the PreS2 and S regions. The smallest of these polypeptides, S-HBsAg, contains 226 residues of the S region. HBsAg can be expressed in a variety of heterologous systems, including yeasts (Valenzuela et al., 1982, Miyanohara et al., 1983, Hitzeman et al., 1983, Murray et al., 1984), animal cells (Dubois et al., 1980, Hirschman et al., 1980, Moriarty et al., 1981, Christman et al., 1982, Gough and Murray, 1982, Siddiqui, 1983, Shih et al., 1984) and plants (Mason et al., 1992). In these systems, the recombinant HBsAg assembles into 20 nm particles which are morphologically and antigenically similar to the spherical particles derived from human serum.
Serologically, the HBsAg contains dominant neutralizing epitopes, denoted as the ‘a’ determinant epitopes. They are located within a double-looped structure, the first loop lies between amino acid residues 107–137, and the other between residues 138–149 (Zuckerman and Zuckerman, 2003). These sets of epitopes which are known as the immunodominant region are highly conformational (Ashton-Rickardt and Murray, 1989) and its three-dimensional structure at atomic resolution has yet to be determined by X-ray crystallography or nuclear magnetic resonance. In this paper, we describe the isolation of ligands that interact with this region from a disulfide-constrained heptapeptide library and the application of the fusion phage harbouring the ETGAKPH sequence for detecting HBsAg in serum samples.
HBsAg purified from human plasma [subtype ad (Biodesign); 3 μg/ml in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl); 100 μl] was coated onto a microtiter plate well overnight at 4°C and then blocked with blocking buffer (0.1 M NaHCO 3, pH 8.6, 5 mg/ml BSA, 0.02% NaN 3; 200 μl) for 2 h at room temperature (∼27°C). The well was then added with a disulfide-constrained heptapeptide phage display library (New England Biolabs, USA) that had been diluted to 1×10 11 plaque forming unit (pfu) in TBS (50 mM Tris–HCl, pH
The peptide sequences selected from three rounds of biopanning against HBsAg are shown in Table 1. In the first round, 20% of the phages carried the peptide sequence ETGAKPH. This dominant sequence increased to 50% and 75% in the second and third rounds, respectively. This suggests that there was an enrichment of peptide sequences that interact with HBsAg. About 17% of the phages in the third round harbored peptide sequences ETG E KP Q and Q TG E KP Q, which are highly related to this dominant
HBsAg is one of the immunologic markers of HBV infection, and therefore, many immunological methods, especially ELISA, have been developed to detect the antigen in blood samples. Most of the presently available methods depend on polyclonal or monoclonal antibodies which are isolated from sera of immunized animals or culture media of hybridomas, as recognition reagents. However, the production and purification of these antibodies are difficult and time-consuming, which dramatically increase
This study was supported by the grant no. 09-02-04-0355-EA001 from the Ministry of Science, Technology and Innovation of Malaysia (MOSTI). G.H. Tan is supported by the National Science Fellowship of Malaysia.
