Indirect-blocking ELISA for detecting antibodies against glycoprotein B (gB) of porcine cytomegalovirus (PCMV)

Journal of Virological Methods 186 (2012) 30–35

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Indirect-blocking ELISA for detecting antibodies against glycoprotein B (gB) of porcine cytomegalovirus (PCMV) Xiao Liu a , Ling Zhu a , Xiaohong Shi a , Zhiwen Xu a,b,∗ , Miao Mei a , Weiwei Xu a , Yuancheng Zhou a , Wanzhu Guo a , Xiaoyu Wang a a b

Animal Biotechnology Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya’an 625014, China Key Laboratory of Animal Disease and Human Health, College of Veterinary Medicine of Sichuan Agricultural University, Ya’an 625014, China

a b s t r a c t Article history: Received 27 January 2012 Received in revised form 22 August 2012 Accepted 30 August 2012 Available online 5 September 2012 Keywords: Porcine cytomegalovirus (PCMV) Glycoprotein B (gB) Major epitope region of gB Western blot analysis Indirect-blocking ELISA

The major epitope region of the glycoprotein B (gB) gene of the porcine cytomegalovirus (PCMV), with a length of 270 bp, was cloned and expressed in Escherichia coli Rosetta (DE3). The major gB epitope was detected using an agar gel precipitation and Western blot analysis with the polyclonal antibodies specific for the major epitope. An indirect-blocking enzyme-linked immunosorbent assay (ELISA) was developed using the expressed major gB epitope as a coating antigen for the detection of PCMV antibodies. The results of the tests show that the indirect-blocking ELISA has 98% specificity and 97.8% sensitivity. No cross-reactions were observed between the major gB epitope and the antibodies against other virus, which indicates that the gB epitope is specific for PCMV antibodies. The indirect-blocking ELISA is a highly specific, sensitive method for detecting anti-PCMV gB antibodies. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Porcine cytomegalovirus (PCMV) belongs to the genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesvirus (Roizmann et al., 1992). In 1955, the herpes virus-like particles of PCMV were isolated (Plowright et al., 1976). PCMV grows slowly in cell cultures, and produces intranuclear inclusions in giant cells (Booth et al., 1967). Nowadays, PCMV is distributed widely around the world, with reported cases in Britain, Japan, Germany and the United States (Corner et al., 1964). PCMV is an opportunistic virus that spreads mainly through the upper respiratory tract, but it can also spread by horizontal transmission, or by vertical transmission (Edington et al., 1988). PCMV often induces fatal systemic infections in young animals, and causes the death of piglets and embryonic death in susceptible pigs. Infected pigs present with clinical symptoms of pneumonia, rhinitis, and dysplasia, whereas the pigs that recover develop lifelong latent infection. Although a large number of neutralizing antibodies are present in the serum of recovered pigs, their low levels of immunity cannot eradicate the virus completely (Edington et al., 1976; Staczek, 1990).

Considering the fact that pigs are often infected latently, and the serologic diagnosis is necessary for confirming PCMV infections, an immunofluorescent serologic method for detecting PCMV antibodies was developed (Assaf et al., 1982; Tajima et al., 1993), but commercial enzyme-linked immunosorbent assay (ELISA) kits for PCMV detection are lacking. This study aimed to develop a reliable, sensitive, and specific indirect-blocking ELISA for large-scale detection of PCMV infections. PCMV glycoprotein B (gB) is an important transmembrane glycoprotein that plays a major role in the fusion and adhesion of the virus onto the cell membrane of the host cells when the virus enters cells or transfers between cells (McGregor et al., 2004). The gB epitope has a good immunogenicity (Mitchell et al., 2002; Mocarski and Kemble, 1996), well-conserved as well as highly specific (Widen et al., 2001). An indirect-blocking ELISA for detecting antibodies against the PCMV gB was developed using the expressed fragment of the major gB epitope as the coating antigen. This method detects antibodies in clinical serum samples to confirm PCMV infections with good specificity and sensitivity. 2. Materials and methods

∗ Corresponding author at: Key Laboratory of Animal Disease and Human Health of Sichuan Province and Animal Biotechnology Center, College of Veterinary Medicine of Sichuan Agricultural University, 46# Xinkang Road, Yucheng District, Ya’an 625014, Sichuan Province, China. Tel.: +86 835 2885846; fax: +86 835 2885302. E-mail address: [email protected] (Z. Xu). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.08.024

2.1. PCMV DNA and serum samples Serum samples were collected from farms in Sichuan Province, China. The PCMV strain SC DNA was provided by the Animal Biotechnology Center of Sichuan Agricultural University.

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2.2. Construction, expression, and purification of the major gB epitope The characteristics of gB were analyzed by using Protean (DNASTAR, Madison, WI, USA), and the 2305–2574 bp major epitope region of the PCMV gB gene was selected as the target fragment. Primers were designed by using Oligo 6.0 (Molecular Biology Insights, Cascade, USA) based on the PCMV nucleotide sequence reported in GenBank (FJ595497.1). EcoRI and HandIII restriction sites, an ATG initiation codon, and the TAG stop codon were also added to the primers. The forward primer was gBF: 5 GAATTCATGAGTTCTTCGGGAACTG-3 , and the reverse primer was gBR: 5 -AAGCTTCTACACGTCCTCGGTGGAT-3 . The target fragment of the major gB epitope was amplified via polymerase chain reaction (PCR) by using the primers gBF and gBR, and the amplified product was purified by using a DNA gel extraction kit (Tiangen Biotech, Beijing, China). The 270 bp PCR product was cloned into the EcoRI and HandIII sites of the pET32a(+) vector (Novagen, Madison, WI, USA), and the recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) (Invitrogen, CA, USA). The positive clones were confirmed through nucleotide sequencing and designated as pET32-major epitope of gB. The clones were selected for large-scale production and purification of the target fragment. The pET32-major epitope of gB was induced with isopropyl-␤d-1-thiogalactopyranoside (IPTG), and the expressed major gB epitope was purified using a HisTrap affinity column (GE Healthcare, Burlington, USA). The protein concentration in the purified product was determined using a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, USA).

2.3. Preparation of polyclonal antibodies and agar gel precipitation test The purified major gB epitope region was used to immunize one healthy male rabbit to produce specific polyclonal antibodies (Penrose et al., 1995). The rabbit was immunized initially with 1 mL of the purified product (containing 0.62 mg protein) mixed with 1 mL of complete Freund’s adjuvant. Two weeks later, the rabbit was immunized with 2 mL of the purified product (containing 1.24 mg protein) mixed with 2 mL of incomplete Freund’s adjuvant once a week for the second and third injection. A week later, 0.5 mL of the purified product (containing 0.31 mg protein) was injected into the ear vein of the rabbit as the fourth injection. The specific antibody titers were detected using an agar gel precipitation test, and the polyclonal antibodies were purified from the blood of the immune rabbit for later use as blocking antibodies.

2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis SDS-PAGE loading buffer was mixed with the recombinant protein and separated using 12% SDS-PAGE. The protein was transferred onto a nitrocellulose (NC) membrane from the gel using a Western Blot System Automatic apparatus (Bio-Rad, Hercules, USA), and then post-coated with 5% skimmed milk in phosphatebuffered saline (PBS) (pH 7.4) for 12 h at 4 ◦ C. After 2 h of incubation with polyclonal antibodies (diluted by 1:50) at 37 ◦ C, the NC membrane was washed with PBS containing 0.05% Tween-20 (PBS-T, pH 7.4), and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:50 dilution; Jackson ImmunoResearch, Baltimore, USA) for 2 h at 37 ◦ C. The target protein formed bands on the NC membrane after the color-developing agent was added.

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2.5. Indirect-blocking ELISA To establish the indirect-blocking ELISA for the detection of antibodies against PCMV gB, the purified fragment containing the major epitope region of gB was used as the antigen to detect the anti-gB antibodies. The coating buffer and blocking solution were selected as the following listed: 0.05 M carbonate buffer (pH 9.6), 0.01 M phosphate buffer (pH 7.4), 0.05 M citrate buffer (pH 4.6), and 0.01 M NaOH (pH 12.0). The blocking solution was selected as the following listed: 50 g/L liquid gluten, 5 g/L PVA, 3% BSA, and 5% skimmed milk. The reaction conditions between the antigen and the blocking antibody were optimized by using the matrix titration method. The dilution of HRP-conjugated goat anti-rabbit IgG was optimized via indirect ELISA reactions with serial dilutions ranging from 1:8000 to 1:18,000. The optimal reaction condition for the test serum was also determined by indirect-blocking ELISA. The sensitivity, specificity, and reproducibility of the indirect-blocking ELISA were tested as follows. The ELISA plate (Thermo Fisher Scientific, Rockville, USA) was coated with 100 ␮L of 0.62 mg/mL purified target protein in 0.05 M carbonate buffer (pH 9.6), which had been previously incubated for 12 h at 4 ◦ C. The antigen-coated plate was washed three times with PBS-T, and blocked with PBS containing 3% BSA for 1.5 h at 37 ◦ C. After washing thrice with PBS-T, the test serum (with final dilution of 1:4 in dilution buffer) and the blocking antibodies were added to the wells, and then incubated for 1.5 h at 37 ◦ C. After washing thrice with PBS-T, 100 ␮L of the diluted blocking antibodies was added into the wells, and then the ELISA plate was incubated for 1 h at 37 ◦ C. After washing, the plate was incubated for 40 min at 37 ◦ C with HRP-conjugated goat anti-rabbit IgG at 100 ␮L/well, which was diluted by dilution buffer. Following another washing step, 50 ␮L of tetramethyl benzidine dihydrochloride (TMB) was added into the wells, and the substrate–chromogen reaction was terminated with the addition of 50 ␮L of 2 M H2 SO4 per well. The optical density (OD) of each well was determined by using a Model 680 Microplate reader (Bio-Rad Laboratories, USA) at 450 nm. The OD values of 20 negative control samples were also detected by using the optimized indirect-blocking ELISA, and the percent inhibition (PI) values were obtained by using the following formula:

PI (%) =

ODblocking antibody − ODtest sample ODblocking antibody

× 100

The critical value was determined according to the following formula (Morenkov et al., 1997):critical value = average PI of the negative control samples + 3(or + 2) × standard deviation. 2.6. Western blot analysis of sera A total of 184 serum samples were tested by Western blot analysis and indirect-blocking ELISA. The compliance rate between the Western blot analysis and the indirect-blocking ELISA was derived from the results of these tests. 3. Results 3.1. Cloning, expression, and purification of the major gB epitope The gB gene was analyzed using molecular biology software, and the 2305–2574 bp major epitope of the PCMV gB gene was selected as the target fragment. The amplified PCR product was purified and sequenced, with an expected size of 270 bp (Fig. 1A). The fragment containing the gB epitope was cloned into the pET32a(+) vector, and the recombinant plasmid pET32-major epitope of gB was identified by sequencing. After induction with isopropyl-␤-d-1-thiogalactopyranoside (IPTG), the 28.3 kDa major

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X. Liu et al. / Journal of Virological Methods 186 (2012) 30–35

Fig. 1. Construction of the pET32-major epitope of gB plasmid and identification of the major gB epitope from the pET32-major epitope of gB plasmid using SDSPAGE. (A) Lane 1, DNA marker; lane 2, pET32-major epitope of gB plasmid; and lane 3, pET32-major epitope of gB plasmid after EcoRI and HandIII enzyme digestion, with the 270 bp band of the gB epitope (indicated by an arrow). (B) Lane 1, lowrange protein molecular weight marker; lane 2, E. coli Rosetta (DE3) expressing the pET32a(+) vector; lane 3, E. coli Rosetta (DE3) expressing the pET32-major epitope of gB after IPTG induction; lane 4, the supernate of pET32-major epitope of gB lysate after IPTG induction; lane 5, precipitate of pET32-major epitope of gB lysate after IPTG induction; and lane 6, the purified 28.3 kDa fragment of the major gB epitope (indicated by an arrow).

gB epitope (including the 18.3 kDa His tag), was expressed in E. coli Rosetta (DE3), and purified successfully as revealed by SDS-PAGE (Fig. 1B). 3.2. Polyclonal antibody, agar gel precipitation test, and Western blot analysis

Fig. 2. Serologic responses to the major gB epitope and the polyclonal antibody, as showed by agar gel precipitation test and Western blot. (A) Well 1, the major gB epitope; well 2, 1:1 dilution of the polyclonal antibodies; well 3, 1:2 dilution of the polyclonal antibodies; well 4, 1:4 dilution of the polyclonal antibodies; well 5, 1:8 dilution of the polyclonal antibodies; well 6, 1:16 dilution of the polyclonal antibodies; well 7, antibody-negative control serum. (B) Lane 1, prestained molecular weight marker; and lane 2, E. coli Rosetta (DE3) expressing the pET32-major epitope of gB; and lane 3, E. coli Rosetta (DE3) expressing the pET32a(+) vector.

selected as the coating buffer (Fig. 3), and PBS containing 3% BSA, with the highest P/N value, was selected as blocking solution of indirect-blocking ELISA. The optimal dilutions of the antigen, blocking antibody, and HRP-conjugated goat anti-rabbit IgG were determined via the matrix titration method in indirect-ELISA. Likewise, the optimal reaction conditions for the test serum were determined by using the same matrix titration method. The optimal concentration for the purified gB epitope, used as the coating antigen, was 1.94 ␮g/mL, and the optimal dilution of the blocking antibodies was 1:1600, with a P/N of 16.4 (Table 1). The optimal dilution for the IgG conjugate was 1:10,000, with a P/N of 5.7 (Fig. 4), whereas the test serum was 1:5.

A rabbit was immunized with the purified major gB epitope combined with Freund’s adjuvant. Polyclonal antibodies were extracted from the blood of the immunized rabbit. The polyclonal antibodies were used as blocking antibodies in the indirectblocking ELISA. The results reveal that the purified protein fragment was antigenic and induced a specific immune response in the immunized rabbit. The specific immune response between the purified gB epitope and the polyclonal antibody was confirmed by agar gel precipitation (Fig. 2A). The resulting 28.3 kDa band was identified by Western blot analysis (Fig. 2B). 3.3. Establishment of an indirect-blocking ELISA By using the major gB epitope, an indirect-blocking ELISA was established for the detection of antibodies against PCMV gB. The 0.05 M carbonate buffer (pH 9.6) with the highest P/N ratio was

Fig. 3. Optimization of coating buffer. The carbonate buffer (0.05 M, pH 9.6) was chosen as the coating buffer for the indirect-blocking ELISA.

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Table 1 Determination of the coating concentration of the major epitope of gB (antigen) and the dilution of the blocking antibody. Concentration of antigen (␮g/mL)

Serum OD450

Dilution of the blocking antibody

1:100

1:200

1:400

1:800

1:1600

1:3200

31

P N

1.478 0.441

1.403 0.338

1.456 0.217

1.347 0.142

1.285 0.11

1.156 0.096

15.5

P N

1.51 0.42

1.432 0.304

1.54 0.195

1.403 0.151

1.385 0.126

1.182 0.088

7.75

P N

1.535 0.378

1.465 0.242

1.357 0.182

1.344 0.132

1.272 0.098

1.139 0.103

3.88

P N

1.454 0.356

1.465 0.234

1.39 0.211

1.34 0.124

1.206 0.087

1.151 0.085

1.94

P N

1.412 0.22

1.321 0.212

1.235 0.152

1.312 0.112

1.2 0.073

1.081 0.085

0.97

P N

1.285 0.207

1.396 0.198

1.315 0.176

1.291 0.101

1.127 0.086

1.026 0.08

0.48

P N

1.053 0.199

1.522 0.169

1.239 0.131

1.107 0.082

1.215 0.075

0.962 0.081

Fig. 4. Determination of the optimal concentration of the HRP-conjugated goat anti-rabbit IgG. The P/N was at maximum when the dilution of the anti-antibody was 1:10,000.

The indirect-blocking ELISA results from the 20 antibodynegative control serum samples had an average PI of 10.2%, with a standard deviation of 0.0478%. Based on the formula for computing the critical value, the PCMV gB was antibody-positive when the PI of the serum sample was greater than 24.5%, and it was antibodynegative when the PI of serum sample was less than 19.8%. The indirect-blocking ELISA showed a specificity of 98.0% (241 out of 246) (Fig. 5A), and a sensitivity of 97.8% (135 out of 138) (Fig. 5B) when tested with the PCMV antibody-negative serum and the PCMV-infected porcine serum samples, respectively. The negative results from the indirect-blocking ELISA showed no crossreactions between the major gB epitope and the sera positive for antibodies against the pseudorabies virus (PRV), the porcine reproductive and respiratory syndrome virus (PRRSV), and the classical swine fever virus (CSFV). An antibody-positive serum sample was tested in each well of a coated microtiter plate using indirect-blocking ELISA, and

the coefficient of variation of PI was 2.4%. One antibody-positive serum sample was also tested in each well of five coated plates at different times of one week, and the coefficient of variation of the PI values was 6.2%. These data show the good reproducibility of indirect-blocking ELISA results.

Table 2 Comparison of the Western blot analysis and the indirect-blocking ELISA.

4. Discussion

Indirect-blocking ELISA

Positive Negative

Western blot Positive

Negative

95 0

33 56

3.4. Comparison of Western blot analysis and indirect-blocking ELISA The results of the Western blot analysis and the indirectblocking ELISA coincided in 151 of the 184 serum samples, and the compliance rate between the Western blot analysis and indirectblocking ELISA was 82.1%. Based on the test results, 33 serum samples that were antibody-negative under Western blot were antibody-positive under indirect-blocking ELISA. No serum samples negative under indirect-blocking ELISA were positive by Western blot analysis (Table 2).

PCMV suppresses the immune function and defense mechanisms of the host, especially by suppressing T cell function. The low immunity caused by immune suppression causes superinfections in pigs, which are usually infected with PCMV, PRRSV, porcine circovirus Type 2, PRV, CSFV, and Mycoplasma pneumoniae in the

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Fig. 5. Frequency distribution of PI values according to the results from the PCMV antibody-negative sera (n = 246) and PCMV antibody-positive sera (n = 138) tested using indirect-blocking ELISA. PCMV gB was antibody-positive when PI was ≥24.5% in the serum sample, and antibody-negative when PI was ≤19.8%. The sera were tested at an optimal dilution of 1:5 in the indirect-blocking ELISA.

form of mixed infections. Haemophilus parasuis, Streptococcus, Actinobacillus, and Pasteurella suis infections often occur in pigs after PCMV infection, thus making prevention and treatment more difficult. Currently, the mortality of pigs infected with PCMV is high. The growth of infected pigs can be retarded, and the latently infected pigs cause unforeseen problems in the breeding industry. PCMV has caused huge financial losses, but the disease has not received due attention because of the lack of studies on PCMV and the similarity of the symptoms of PCMV infections to other epidemic diseases. In recent years, with in-depth research on the pathogenesis of mixed infections, the damage caused by PCMV to the porcine immune system has been receiving increasing attention. At present, many methods are used for detecting members of the Herpesvirus family. Immunofluorescence assay, ELISA, nucleic acid hybridization technology, flow cytometry, and gene chip technology have been used for the laboratory detection of human cytomegalovirus (HCMV), whereas ELISA and PCR have also been applied widely for the detection of PRV (Essa et al., 2000; Janssen et al., 1987; Ogawa-Goto et al., 2003; Song and Stinski, 2002; Tiran et al., 1999). However, studies on serological PCMV detection methods are lacking; therefore, the development of a practical, efficient, and economical ELISA for the detection of PCMV antibodies is important. gB, one of the major structural proteins of PCMV, has good immunogenicity, and induces production specific antibodies (Speckner et al., 1999), but whether it is a protective antibody has not been confirmed. The homology between the gB nucleotide sequences of the PCMV isolated from China and the nucleotide sequences published in GenBank was higher than 97.6%, whereas

the amino acid sequence homology was more than 97%. However, the difference between the gB nucleotide sequences of PCMV and the HCMV is large (Widen et al., 2001). This indicates that the gB gene is well-conserved and highly specific, making it ideal for research on PCMV serologic testing methods. The study of the structure and function of gB will also help to clarify the pathogenesis of PCMV, and provide a new way of treating and preventing the disease. Considering the hydrophobic region of gB may affect the specificity of the ELISA, a molecular analysis was carried out on the PCMV gB gene. The rare codon, signal peptide, and transmembrane sequences of the protein were excluded, and the major epitope region was chosen for prokaryotic expression and polyclonal antibody production; the expressed major gB epitope was used as the blocking antibody in the indirect-blocking ELISA (Gathmann et al., 2006; Jolivet-Reynaud et al., 1998; Morenkov et al., 1997). The application of the major gB epitope in this study reduced the difficulty of antigen production, and increased the specificity and sensitivity of the ELISA. Given the lack of reliable cell cultures for PCMV, this study used indirect-blocking ELISA to avoid cultivation of PCMV, with its demanding technical requirements, and effectively reduced the cost of the study. The strength of this study is the use of a large number of clinical samples selected at random. These samples were collected from 12 districts of Sichuan, China. A total of 246 PCMV antibody-negative porcine serum samples were tested by indirect-blocking ELISA. The results showed 98% specificity, with only five serum samples having PI values higher than the critical value. The negative results with indirect-blocking ELISA showed there was no cross-reaction between the major gB epitope and the antibody-positive serum

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of other infectious diseases, which indicates the high specificity of the indirect-blocking ELISA. The indirect-blocking ELISA showed 97.8% sensitivity when it was used to test 138 PCMV-infected serum samples. At present, no commercial ELISA kit is available for PCMV antibody detection; therefore, Western blot analysis was used for comparison. According to the results, Western blot analysis was not consistent with the indirect-blocking ELISA, and indirect-blocking ELISA had higher reliability and sensitivity for detecting PCMV gB antibodies. The results of the experiments confirmed that the major gB epitope can be used as a coating antigen in indirect-blocking ELISA for detecting anti-PCMV gB antibodies. It also showed that the indirectblocking ELISA is a rapid, specific, sensitive, and simple detection method for PCMV. Funding This study was supported by Fund Project for Youth Science and Technology in Sichuan Province (FPYSTSP; Project No. 200930421). Competing interests No conflicts of interest. Ethical approval Not required. Acknowledgment We would like to acknowledge all members of Animal Biotechnological Center for their contribution to this study. References Assaf, R., Bouillant, A., Di Franco, E., 1982. Enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to porcine cytomegalovirus. Canadian Journal of Comparative Medicine 46, 183. Booth, J.C., Goodwin, R.F., Whittlestone, P., 1967. Inclusion-body rhinitis of pigs: attempts to grow the causal agent in tissue cultures. Research in Veterinary Science 8, 338–345. Corner, A.H., Mitchell, D., Julian, R.J., Meads, E.B., 1964. A generalized disease in piglets associated with the presence of cytomegalic inclusions. Journal of Comparative Pathology 74, 192–199. Edington, N., Plowright, W., Watt, R.G., 1976. Generalized porcine cytomegalic inclusion disease: distribution of cytomegalic cells and virus. Journal of Comparative Pathology 86, 191–202. Edington, N., Wrathall, A., Done, J., 1988. Porcine cytomegalovirus (PCMV) in early gestation. Veterinary Microbiology 17, 117–128.

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