Volume 38, Issue 1, Pages 49-56 (January 2007)

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ABSTRACT

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A phage-displayed single chain variable fragment that interacts with hepatitis B core antigen: Library construction, selection and diagnosis

Received 13 April 2006; received in revised form 11 August 2006; accepted 19 September 2006. published online 03 November 2006.

Abstract

Background

Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen.

Objectives

To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen.

Study design

Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (VH) and light (VL) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples.

Results

A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10ng of HBcAg can be detected by the assay.

Conclusion

The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.

Keywords: Phage display, Combinatorial antibody libraries, Hepatitis B core antigen, Diagnosis, Phage-ELISA

Article Outline

• Abstract

• 1. Introduction

• 2. Materials and methods

• 2.1. Construction of ScFv

• 2.2. Biopanning

• 2.3. Inhibition study

• 2.4. Development of phage-ELISA using C4 phage

• 3. Results

• 3.1. Construction and biopanning of ScFv library

• 3.2. Phage-peptide inhibition assay

• 3.3. Phage-ELISA for detecting HBcAg in serum samples

• 4. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

Hepatitis B virus (HBV) is a human pathogen that poses a major health problem worldwide despite the existence of effective recombinant vaccines. More than 350 million people are known to be carriers of this virus and they have an increased risk of developing primary liver cancer and cirrhosis (Jung and Pape, 2002).

HBV is enveloped by a lipid bilayer membrane containing three forms of surface antigens: L (large), M (middle) and S (short)-HBsAg (Ganem, 1991). Within the envelope is an icosaheral nucleocapsid composed of 180 or 240 subunits (Crowther et al., 1994) of core protein or known as core antigen (HBcAg). Each subunit contains 183 or 185 amino acid residues and it is able to self assemble into large and small capsids when expressed in Escherichia coli in the absence of other viral proteins (Cohen and Richmond, 1982, Crowther et al., 1994). Electron cryomicroscopy and image reconstruction revealed that the three-dimensional structure of the capsids produced in E. coli are similar to those isolated from infectious virions (Roseman et al., 2005).

HBsAg and anti-HBcAg are the primary markers for the identification of acute HBV infection in routine diagnosis. Serological tests for HBeAg and anti-HBeAg are performed for patients with chronic hepatitis B to elucidate their level of infectivity and seroconversion status (Hatzakis et al., 2006). Quantification of HBV DNA has been employed to monitor HBV replication, disease progression and assessing the efficacy of drug treatment (Hendricks et al., 1995). HBxAg and anti-HBxAg have been used as prognostic markers for the development of hepatocellular carcinoma (Hwang et al., 2003). HBcAg could be a marker for virus load but it is not routinely tested. Assays for the detection of HBcAg have been reported (Bredehorst et al., 1985a, Bredehorst et al., 1985b, Usuda et al., 1998), but due to low sensitivity and complexity in the procedures, their application has been limited (Kimura et al., 2003). HBV has been extensively studied ever since the emergence of the disease and enzyme immunoassays have been developed for the detection of the virus as early as the 1980s. Radioimmunoassay (Rizzetto et al., 1981) and counter-immunoelectrophoresis (Freeman and Hambling, 1978) were developed to detect HBcAg and its antibody, respectively. Through the years, significant progress has been made in research on detection and diagnosis of HBV. Nevertheless, there are always setbacks and problems with respect to the capabilities of the assays developed.

HBV capsid is highly immnunogenic, naturally it functions as both a T-cell independent and a T-cell dependent antigen (Milich and McLachlan, 1986), and preferentially primes T-helper-1 (Th1) cells in mice and human (Cao et al., 2001). It is thought that the unique three-dimensional structure of the capsid favors this strong immune response (Milich et al., 1997). The X-ray structure of HBV capsid at 3.3Å resolution clearly shows that residues 78–82 which constitute the major epitope are located at the tip of the capsid spike (Wynne et al., 1999).

Phage display technology has been widely used to display antibody libraries on the surface of filamentous bacteriophages. The libraries allow the selection of antibodies with high specificity and affinity for any antigens. In this study, we describe (i) the construction of a single chain variable fragment (ScFv) library towards HBcAg, (ii) affinity selection of the library against HBcAg, (iii) characterization of a ScFv phage clone that interacts tightly with HBcAg, and (iv) the application of the phage clone as a diagnostic reagent for detecting HBcAg via a phage-ELISA.

2. Materials and methods

2.1. Construction of ScFv

Two female Balb/C mice were immunized with truncated HBcAg (residues 3–148, subtype adyw) purified from E. coli (Dyson and Murray, 1995). Pre-immune sera were collected from the tails and used as negative control. HBcAg (1mg/ml; 50μg) emulsified in Freund's complete adjuvant (Sigma, USA) was injected subcutaneously and four boosters with the same dose were given at 1 week interval. Their spleens were harvested 3 days after the fourth booster and homogenized with Tri Reagent (Invitrogen, USA). cDNA synthesis was carried out using total RNA (5μg), Oligo (dT)12–18 (0.5μg/μl), dNTPs (200μM), SuperScript II reverse transcriptase (5U; Invitrogen, USA), RNase inhibitor (4U), MgCl2 (25mM), 10× RT buffer [200mM Tris–HCl (pH 8.4), 500mM KCl] and DTT (0.1M). The mixture was incubated at 42°C for 1h and heated at 70°C for 15min.

Primers used to amplify the light (VL) and heavy (VH) chain genes are listed in Table 1. PCR reaction for the VL was carried out in a reaction containing MgCl2 (25mM), dNTPs (200μM), 10× buffer [100mM Tris–HCl (pH 9.0), 500mM KCl, 1% Triton X-100], MSCVL-1 forward primer (2μM), MSCJL-B reverse primer (2μM), cDNA (0.5μg) and High Fidelity Platinum Taq Polymerase (5U; Invitrogen, USA). For VH region amplification, the forward MSCVH [(MSCVH1–MSCVH19), 2μM] and reverse MSC primers mix [(MSCGlab-B, MSCG3-B and MSCM-B), 2μM] were utilized. A total of 24 tubes of VL and VH PCR reactions were heated at 94°C for 2min followed by a 35-cycle reaction (94°C/15s; 56°C/30s; 72°C/90s) and at 72°C for 10min.

Table 1.

List of primers for amplification of variable heavy and light chains


	Primers	Sequences
	(a) Primers for light chain (VL)
	MSCVL-1	5′GGG CCC AGG CGG CCG AGC TCG ATG CTG TTG TGA CTC AGG AAT C3′
	MSCJL-B	5′GGA AGA TCT AGA GGA ACC ACC GCC TAG GAC AGT CAG TTT GG3′

	(b) Primers for heavy chain (VH)
	MSCVH-1	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTR MAG CTT CAG GAG TC3′
	MSCVH-2	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTB CAG CTB CAG CAG TC3′
	MSCVH-3	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAG CTG AAG SAS TC3′
	MSCVH-4	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAR CTG CAA CAR TC3′
	MSCVH-5	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTY CAG CTB CAG CAR TC3′
	MSCVH-6	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTY CAR CTG CAG CAG TC3′
	MSCVH-7	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAC GTG AAG CAG TC3′
	MSCVH-8	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAS STG GTG GAA TC3′
	MSCVH-9	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AWG YTG GTG GAG TC3′
	MSCVH-10	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAG SKG GTG GAG TC3′
	MSCVH-11	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAM CTG GTG GAG TC3′
	MSCVH-12	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAG CTG ATG GAR TC3′
	MSCVH-13	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAR CTT GTT GAG TC3′
	MSCVH-14	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTR AAG CTT CTC GAG TC3′
	MSCVH-15	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAR STT GAG GAG TC3′
	MSCVH-16	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTT ACT CTR AAA GWG TST G3′
	MSCVH-17	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAA CTV CAG CAR CC3′
	MSCVH-18	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAG TTG GAA GTG TC3′
	MSCVH-19	5′GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAG GTG ATC GAG TC3′
	MSCGlab-B	5′CCT GGC CGG CCT GGC CAC TAG TGA CAG ATG GGG STG TYG TTT TGG3′
	MSCG3-B	5′CCT GGC CGG CCT GGC CAC TAG TGA CAG ATG GGG CTG TTG TTG T3′
	MSCM-B	5′CCT GGC CGG CCT GGC CAC TAG TGA CAT TTG GGA AGG ACT GAC TCT C3′

	(c) Primers for overlapping PCR
	RSC-F	5′GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC3′
	RSC-B	5′GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT GGC CAC TAG TG3′

Purified VL and VH DNAs were fused by overlapping PCR in a reaction containing MgCl2 (25mM), dNTPs (200μM), 10× buffer [100mM Tris–HCl (pH 9.0), 500mM KCl, 1% Triton X-100], RSC-F (2μM), RSC-B (2μM), cDNA (0.5μg) and High Fidelity Platinum Taq Polymerase (5U; Invitrogen, USA)]. The mixture was heated at 94°C for 5min, followed by a 35-cycle reaction (94°C/15s; 56°C/30s; 72°C/2min) and at 72°C for 10min. Purified ScFv was subsequently digested with SfiI and ligated to the pComb3x vector (Barbas III et al., 1991). Competent ER2537 cells were transformed with the ligation mixture by electroporation.

2.2. Biopanning

Purified HBcAg (300ng, 100μl) in TBS (50mM Tris–HCl, pH 7.5, 150mM NaCl) was coated onto a microtiter plate well overnight at 4°C and then blocked with 10% milk diluent (KPL, USA). Phage library (1.0×1011pfu/ml; 100μl) was added and incubated for 2h at room temperature. The well was washed with TBST [TBS containing 0.05% Tween 20], bound phage was eluted with glycine–HCl (0.1M, pH 2.2; 120μl) and neutralized with Tris–HCl (1M, pH 9.0; 15μl). The eluted phage was then amplified by infecting E. coli XL1-Blue and the panning process was repeated for another three rounds. Selected phages were amplified and their titers were determined (Sambrook et al., 1989).

Randomly selected phage clones were screened by ELISA by adding the phages (1.0×1010pfu/ml) into wells coated with HBcAg. After 2h of incubation, the wells were washed with TBST and anti-M13 mAb conjugated to horseradish peroxidase (1:5000; Amersham Pharmacia Biotech; 200μl) was added for another hour. ABTS was added and A405 determined. The nucleotide sequence of the insert was determined as described in Tan et al. (2005)

2.3. Inhibition study

A constant concentration of a phage displaying ScFv, namely C4 (1.0×1012pfu/ml; 50μl) was mixed with series dilutions of the cyclic peptide WSFFSNI or ETGAKPH (0.0001–1mM; 50μl). The mixtures (100μl) were then added into wells coated with HBcAg, incubated at 4°C for 2h and washed six times with TBST. The bound phages were eluted with glycine–HCl (0.1M, pH 2.2; 120μl) and the amount was determined by titration (Tan et al., 2005) and expressed as plaque forming unit (pfu).

2.4. Development of phage-ELISA using C4 phage

HBcAg (10ng to 1μg; 100μl) was coated onto microtiter plate as described above and purified C4 phage (1.0×1010–1.0×1012pfu/ml; 100μl) was added. After 2h of incubation at 4°C, the wells were washed and bound phage was detected as described above. Specificity of the assay was studied by replacing the HBcAg with HBsAg and HBeAg (0.3μg; Biodesign, USA). HBcAg was also detected using the anti-HBcAg mAb [Clone C1–5; Chemicon, USA], followed by anti-mouse AP conjugated (1:5000, Chemicon) and PNPP was used as substrate. HBV positive and negative sera were tested using the established phage-ELISA. These samples were first treated with SDS (15%) and CHAPS (5%) for 30min at 70°C to release the HBcAg from the virion. For each well, the treated serum (1:10 dilution; 120μl) was coated overnight at 4°C and 1.0×1012pfu/ml of phage was used.

3. Results

3.1. Construction and biopanning of ScFv library

The titer of anti-HBcAg polyclonal antibody raised in Balb/C mice was determined by ELISA before the spleens were harvested for RNA extraction. The titer was estimated to be 1 in 100,000 after fourth booster. The coding regions of VL and VH chains were amplified by PCR, analyzed on agarose gel and gave the expected DNA fragments of approximately 350bp [Fig. 1(a)]. A short linker (GGSSRSS) was incorporated to link the VL and VH chains into a single chain in the assembly PCR to produce ScFv DNA fragments of about 750bp [Fig. 1(b)]. The ScFv was then cloned into pComb3x vector.

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Fig. 1. Amplification of light and heavy chain fragments of antibody with PCR. (a) First PCR products of VL (lane 1) and VH (lane 2) with a size of approximately 350bp. (b) Assembly of PCR products to form ScFv of anti-HBcAg antibodies [(lane 1, 2μl) and (lane 2, 4μl)] with a size of approximately 750bp. Lane M: GeneRuler DNA Ladder Mix in bp. The PCR products were analyzed on 1.5% (w/v) agarose gel.

A total of 31 clones were selected from the fourth round of panning against HBcAg and their ability to interact with HBcAg is shown in Fig. 2. Only one phage clone, namely C4, showed the highest absorbance of about 0.4. Its phagemid was extracted and the nucleotide sequence of the insert was determined. The deduced amino acid residues of the VH and VL are given in Fig. 3. Amino acid sequence comparison revealed 94% similarity to the mouse IgG lamda-2 chain (Pennell, 1988).

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Fig. 2. Phage-ELISA of phage clones selected randomly from fourth round of biopanning. Wells were coated with HBcAg and blocked with 10% milk diluent. Phage clones were added into each well and incubated at room temperature for 2h. The wells were washed six times with TBST and anti-M13 monoclonal antibody conjugated to HRP was added followed by ABTS substrate. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

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Fig. 3. Nucleotide and deduced amino acid sequences for the variable domain genes of C4 phage. The amino acid residues and CDR limits are according to Kabat et al. (1991). (a) VL domain of C4 phage. The residues before and after CDR-L1 are always Cys and Trp, the length is always 10–17 residues. The residues after CDR-L2 are always Ile-Tyr, but also Val-Tyr, Ile-Lys, Ile-Phe. The residues before and after CDR-L3 are always Cys, and Phe-Gly-X-Gly. (b) VH domain of C4 phage. The residues before CDR-H1 are always Cys-XXX, and the residue after is always Trp, typically Trp-Val. For CDR-H2, the residues before are always Leu-Glu-Trp-Ile-Gly, but a number of variations; and after CDR-H2 are Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. The residues before CDR-H3 are always Cys-X-X (typically Cys-Ala-Arg/Leu), and after are Trp.

3.2. Phage-peptide inhibition assay

To locate the binding site of the C4 phage on the HBV capsid, an inhibition assay was carried out by incubating the phage with different concentrations of peptide WSFFSNI (Ho et al., 2003). This peptide has been shown to compete with the anti-HBcAg mAb C1–5 for a binding site on the immunodominant epitope (residues 78–82) of HBcAg (Ho et al., 2003). In this study, the amount of C4 phage bound to HBcAg decreased when the concentration of peptide increased (Fig. 4). This suggests that C4 phage is most likely interacted with the immunodominant region of HBcAg. The negative control using an unrelated peptide with the sequence ETGAKPH that binds tightly to HBsAg (ad subtype) (Tan et al., 2005) did not inhibit the binding of the C4 phage to HBcAg.

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Fig. 4. Peptide-phage competition assay. C4 phage clone was incubated with different concentrations of cyclic peptide WSFFSNI. The mixtures were then added into wells that had been coated with truncated HBcAg. The phage that interacted with the immobilised HBcAg was determined by titration. Cyclic peptide ETGAKPH in the presence and absence of C4 phage were included as negative controls. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

3.3. Phage-ELISA for detecting HBcAg in serum samples

The C4 phage carrying the ScFv molecule was employed as a diagnostic reagent for detecting HBcAg via a phage-ELISA. Fig. 5a shows that the phage could detect purified HBcAg down to 10ng or lower when 1012pfu/ml of phage was used. The phage-ELISA is as sensitive as the ELISA whereby the phage was replaced by an anti-HBcAg mAb (Fig. 5b). The newly established phage-ELISA was then applied to detect HBcAg in HBV positive sera. Fig. 6a shows that all the 15 HBV positive sera produced significant readings compared to those of the negative sera. A similar profile was observed for all the sera when the phage was substituted with the anti-HBcAg mAb (Fig. 6b). Cross reactivity assay revealed that the phage reacted specifically to HBcAg with the highest absorbance value of about 0.7, but it did not cross react with HBsAg and HBeAg (Fig. 7).

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Fig. 5. Sensitivity test of the phage-ELISA and ELISA. (a) Detection of purified HBcAg with C4 phage ScFv via phage-ELISA. HBcAg (0–1000ng) was coated on microtiter plate wells and detected with C4 phage (1.0×1010–1.0×1012pfu/ml). Anti-M13 conjugated to HRP was added and followed by its substrate. M13 wild-type phage and milk diluent was used as negative controls. (b) Detection of purified HBcAg with anti-HBcAg monoclonal antibody C1–5. HBcAg coated on wells were detected using anti-HBcAg mAb C1–5 instead of the C4 phage. Anti-mouse conjugated to alkaline phosphatase (AP) was added followed by its substrate. Wells coated with milk diluent served as negative controls. The assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

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Fig. 6. Comparison of C4 phage with anti-HBcAg mAb C1–5 for detection of HBcAg in the treated sera. (a) Microtiter plate wells were coated with treated HBV positive (labeled as 1–15) and negative sera (labeled as N1–N7) overnight and blocked with 10% milk diluent. The C4 phage was added and detected with anti-M13 conjugated to HRP and its substrate. (b) Treated sera were coated on the wells and the anti-HBcAg mAb C1–5 (1:5000) was added and detected with anti-mouse conjugated to AP. The straight line represents cut-off value, calculated as the mean value of the negative control plus 3 standard deviations. Assays were performed in triplicates and the error bars represent the standard deviation.

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Fig. 7. Specificity study of C4 phage ScFv towards HBcAg, HBsAg and HBeAg. Microtiter plate wells were coated with HBcAg, HBsAg, HBeAg (0.3μg) or human plasma and detected using 1.0×1012pfu/ml of C4 phage. The assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

4. Discussion

In the life cycle of HBV, HBcAg associates with the viral nucleic acid and assembles into nucleocapsid before being enveloped to form an infectious virion and subsequently released in the blood circulation. Therefore, the concentration of HBcAg in serum is correlated with the viral load and also proportional to the level of HBV genome (Bredehorst et al., 1985a, Bredehorst et al., 1985b, Kimura et al., 2003). Thus, HBcAg is one of the markers for HBV infection.

In this study, a phage library of ScFv against HBcAg was constructed and a phage clone displaying a good affinity towards the antigen was isolated by biopanning. Restriction enzyme analysis and sequencing confirmed that the C4 phage carried a ScFv gene. The arrangement of the VL and VH domains in the ScFv gene fulfills the criteria of the Kabat numbering scheme (Kabat et al., 1991). The two variable regions are linked by a short linker (GGSSRSS) which enhances the stability of the Fv-fragment and thus extending its application as a diagnostic reagent (Glockshuber et al., 1990).

The three-dimensional structure of HBV capsid determined by X-ray crystallography revealed that the immunodominant loop in the region of residues 78–82 in HBcAg (Salfeld et al., 1989) is maximally exposed at the spikes on the surface of the capsid (Wynne et al., 1999). It is likely that the ScFv displayed on the filamentous phage interacts with the immunodominant loop because the peptide WSFFSNI (Ho et al., 2003) that inhibits the binding of mAb C1–5 (recognizes a linear epitope located between residues 78–83; Pushko et al., 1994) also inhibited the binding of the C4 phage to the capsid.

In this study, the C4 phage was employed as a diagnostic reagent for detecting HBcAg. The newly established phage-ELISA is capable to detect a minimum amount of 10ng of purified HBcAg using 1012pfu/ml of C4 phage. The phage-ELISA was applied to detect HBcAg in pretreated sera to release the antigen from the virion. Positive readings were obtained for the HBV positive serum samples and the negative controls showed negligible readings. Specificity study showed that the C4 phage only reacted with HBcAg but not with HBsAg and HBeAg.

Currently there is no standardized commercial kit available for detecting HBcAg in the routine diagnostic laboratory. However, a number of anti-HBcAg mAbs have been produced as research reagents for applications in immunoblotting and ELISA. Recombinant phages may potentially replace the monoclonal antibodies that are available in the market. The advantages of this type of phage-based diagnostic reagent over monoclonal antibodies are: (i) lower production cost; (ii) the recombinant M13 can be propagated rapidly within hours and; (iii) the purification of phage particles involves lesser steps. In other words, the phage-based diagnostic reagent can save time, cost and energy compared to monoclonal antibodies. Thus, the C4 phage provides a better option and can be potentially used as a diagnostic reagent.

Acknowledgement

This study was supported by the Grant No. 09-02-04-0355-EA001 from the Ministry of Science, Technology and Innovation of Malaysia (MOSTI).

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a Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

b Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

c Department of Clinical Laboratory Science, Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Corresponding author. Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +60 3 89466715; fax: +60 3 89430913.

PII: S1386-6532(06)00346-5

doi:10.1016/j.jcv.2006.09.010

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