Use of Epitope Mapping To Identify a PCR Template for Protein Amplification and Detection by Enzyme-Linked Immunosorbent Assay of Bovine Herpesvirus Type 1 Glycoprotein D

Virus strains and cell culture.The Los Angeles (LA) strain of BHV-1 was obtained from the American Type Culture Collection (Manassas, Va.). Madin-Darby bovine kidney (MDBK) cells were grown in minimum essential medium (GIBCO Life Technologies) containing 100 U of penicillin/ml, 100 μg of streptomycin/ml, 25 μg of fungizone (Invitrogen Life Technologies)/ml, and 10% fetal bovine serum and maintained in 75-cm² flasks in minimum essential medium with Earle’s salts with the same concentration of antibiotics and the fetal bovine serum reduced to 2%. The flasks were incubated at 37°C with a 5% CO₂ air atmosphere, and the cell monolayers were infected the next day with virus diluted 1:10. The virus was harvested after 3 to 4 days and purified on a Ficoll 400 (Amersham Pharmacia Biotech) step gradient (25:10) after precipitation with ammonium sulfate. The protein concentration in the purified virus was estimated by spectrophotometry at 280 nm prior to storage at −80°C.

BHV-1-infected cell lysate.MDBK cells infected with the LA strain of BHV-1 were used 16 h postinfection. They were lysed with 0.5% Nonidet P-40 (NP-40) (Sigma Chemicals, St. Louis, Mo.) and clarified. Uninfected cell lysate was similarly prepared, and the protein concentration was estimated and samples were stored as for the purified virus.

In vitro spiking of semen with BHV-1.Semen samples collected from a bull and stored at −80°C were diluted 1:20 to a final volume of 90 μl using PCR buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], 2.5 mM MgCl₂, 0.1 mg of gelatin/ml, 0.45% NP-40, and 0.45% Tween 20). Tenfold serial dilutions of BHV-1 strain LA stock culture containing 10^5.5 50% tissue culture infective doses (TCID₅₀)/100 μl were made in the PCR buffer, and 10 μl of each dilution from 10⁻¹ to 10⁻⁶ was added to the diluted semen.

Construction of pGEX expression plasmids and preparation of fusion proteins.The BL21(DE3)(pLysS) strain of E. coli transformed with pGEX plasmids containing the entire open reading frame (ORF) of the gD gene (14) was used to express the full-length glutathione S-transferase (GST)-gD fusion protein. In order to obtain overlapping fragments of the gD ORF, three forward primers designated A, C, and E and two reverse primers designated B and D, based on the published nucleotide sequence of the gD gene of the P8-2 strain of BHV-1 from nucleotide positions 77 to 1341 (20), were used in the PCR. The forward primers were designed to contain a BamHI restriction site (italic) with primer A located from nucleotide positions 84 to 98 (5′ GGA TCC ATG CAA GGG CCG AC 3′), primer C located from nucleotide positions 730 to 744 (5′ GGA TCC GCC CGG GAT TAC GA 3′), and primer E located from nucleotide positions 412 to 425 (5′ GGA TCC ATC GAG AGC CGG TG 3′). The reverse primers were designed to contain the Streptag sequence (lowercase) (19) and XhoI restriction site (italic) with primer B located from nucleotide positions 997 to 1013 (5′ CTC GAG tca acc gaa ctg cgg gtg acg cca agc gct CC GTC GCC TTC GGG TCC 3′) and primer D located from nucleotide positions 1315 to 1335 (5′ CTC GAG tca acc gaa ctg cgg gtg acg cca agc gct CCC GGG CAG CGC GCT GTA GTT 3′). For the in vitro transcription and translation reactions, forward primer C was designed to contain T7 RNA polymerase promoter sequence with an ATG codon (5′ GTA AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AG GG ATG GCC CGG GAT TAC GA 3′) and the reverse primer D was designed without the Streptag sequence and XhoI restriction enzyme site (5′ TCA CCC GGG CAG CGC GCT GTA GTT 3′) (29). The BHV-1 DNA preparation and PCR procedure used to obtain specific amplification of the gD gene DNA were described previously (29). The PCR products were purified from low-melting-point agarose gels (5) and then cloned into the TA cloning vector pCRII (Invitrogen Life Technologies). The inserts were subcloned into the pGEX-5X-3 expression vector, which was then used to transform competent E. coli strain BL21(DE3)(pLysS). The transformed E. coli cells were grown overnight in 2× YT (yeast and tryptone) medium (Becton Dickinson and Co., Paramus, N.J.) supplemented with 0.1 ml of 5-mg/ml ampicillin, 5 μl of 35-μg/ml chloramphenicol (Roche Molecular Biochemicals), and 0.25 ml of 40% glucose. Protein expression was induced by adding IPTG (isopropyl-β-d-thiogalactoside) (Amersahm Pharmacia Biotech) to a final concentration of 0.1 mM. The GST-gD fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The purified GST-gD fusion proteins were concentrated using centricon (Millipore Corp., Bedford, Mass.). The concentrations of the purified proteins were estimated by spectrophotometry at 280 nm, and their purity and integrity were analyzed by resolving them on discontinuous sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12.5% PAGE) (11) and visualizing them with Coomassie blue R-250 (Bio-Rad) and, if necessary, by staining them with a silver staining kit (Bio-Rad). The BL21 strain of E. coli without a gD gene was lysed with 0.5% NP-40 (Sigma) and then clarified at 700 × g to obtain the bacterial lysate without gD.

For the amplification of the CD fragment of the gD ORF from semen samples infected with BHV-1, the semen samples were digested with proteinase K for 60 min at 55°C and then heated for 10 min to denature the proteinase K and clarified at 13,000 × g for 30 min, and 2.5 μl of the supernatant was directly used for PCR (29). PCR with a DNA template prepared from the purified BHV-1 was used as a positive control.

GST-gD-specific MAbs.The GST-gD-specific hybridoma cells of clones 26, 12, 4F7, 5D4, 2D7, 2C4, and 10B5, developed and characterized previously (14), were used to raise mouse ascites fluid. For each clone, this was done by intraperitoneal inoculation of 10⁷ hybridoma cells into BALB/c mice that had been primed 2 to 3 weeks previously with 0.5 ml of pristane (Sigma). The clarified ascites fluid containing immunoglobulin M (IgM) MAbs were used as is, and IgG MAbs were purified by affinity chromatography using protein G columns (17) and were then concentrated using polyvinyl pyrrolidone (Sigma).

Western blotting.Purified GST-gD and BHV-1 proteins were transferred from the SDS-PAGE gel to the nitrocellulose membrane and then blocked with 3% skim milk. The membrane was then reacted with a 1:100 dilution of the MAb. Subsequent steps were performed with protein A protein L-horseradish peroxidase conjugate (Clontech Laboratories) and a peroxidase substrate kit (Bio-Rad) according to the directions of the manufacturers.

ELISA.The full-length GST-gD fusion protein and the MAbs were used to develop the ELISA, in which purified BHV-1 and infected cell lysate were used as positive controls and uninfected cell lysate and bacterial lysate without gD served as negative controls. To determine the optimum volumes and concentrations of the reagents, preliminary checkerboard titration experiments were conducted with all the antigens and MAbs. The best results were obtained when 10 μg of GST-gD fusion protein and 20 μg of purified BHV-1 or infected cell lysate were used to coat each well of the plate and were reacted with 1:100 dilution of the MAbs. For the indirect ELISA, the antigens were diluted in 0.2 M bicarbonate buffer, pH 9.6, and 100 μl was used to coat each well of a 96-well Immulon II microtiter immunoassay plate (Dynatech Laboratories, Chantilly, Va.). The plates were incubated overnight at 4°C. The antigens were removed, and the plates were blocked with 100 μl of 5% horse serum diluted in 1× Dulbecco's phosphate-buffered saline (Invitrogen Life Technologies) containing 0.05% Tween 20 and incubated at room temperature for 1 h. The plates were then treated with 100 μl of a 1:100 dilution of MAbs/well and incubated at room temperature for 2 h. This was followed by incubation with 100 μl of a 1:30,000 dilution of alkaline phosphatase-labeled goat anti-mouse IgM-IgG (Sigma)/well for 1 h at room temperature. The plates were reacted with the substrate p-nitrophenyl phosphate (Bio-Rad) until the color development was complete. The optical density was measured at 405 to 490 nm (OD_405-490) using a Spectra MAX 340 (Molecular Devices Corp., Sunnyvale, Calif.) ELISA reader. The steps were separated by extensive washing with 1× Dulbecco's phosphate-buffered saline containing 0.05% Tween 20. All the experiments were done in triplicate, and the mean OD was calculated. The ODs of the positive samples were expressed after subtracting the OD of the corresponding negative control. An OD_405-490 cutoff value for the ELISA to differentiate positive from negative samples was set at 2 standard deviations above the average OD_405-490 of the negative antigens. Values above this cutoff were scored positive (3). For the detection of CD polypeptide in the in vitro transcription-translation reaction, 96-well microtiter plates were coated with 50 μl of the TNT reaction mixture diluted to 100 μl in 0.2 M bicarbonate coating buffer, and ELISA was carried out as described above using MAb 26. In vitro transcription and translation reactions without PCR products served as negative controls, and the E. coli-expressed CD fragment of gD served as the positive control.

Epitope mapping.MAbs 2D7 (IgG), 26, 12, 4F7, and 5D4 (IgM) were reacted individually with full-length GST-gD and the overlapping protein fragments AB, CD, and ED in indirect ELISA to identify their epitopes on recombinant proteins. Bacterial lysate without gD was used as the negative control. Epitopes of the MAbs were located based on the reactivities of the MAbs with individual protein fragments and the full-length GST-gD. OD readings equal to or above the OD readings of full-length GST-gD were considered positive. OD readings below the OD readings of full-length GST-gD but above the cutoff value were considered partial reactions. The percentages of reactions of various fragments with different MAbs in indirect ELISA were also calculated by considering the reaction of the full-length GST-gD as 100%.

Preparation of CD protein fragment of gD from PCR products.CD peptides from PCR products were prepared as described previously (29) using the TNT T7-coupled reticulocyte lysate system (TNT-RLS; Promega). Briefly, 2.5 μl of the PCR products of the CD fragment from purified BHV-1 DNA with the forward primer containing the T7 promoter sequence (29) were used directly in an in vitro transcription-translation mix containing 40 μCi of [³⁵S]methionine (Amersham Pharmacia Biotech) in 50-μl reaction volumes, using the TNT-RLS according to the manufacturer's specifications. A luciferase DNA (supplied by Promega) was used as a positive control. The reaction products were analyzed for size by SDS-PAGE and autoradiography. Nonradioactive TNT-RLS reactions with cold methionine were set up with the PCR products of the CD fragment of the gD ORF from purified BHV-1 DNA and infected semen samples. The CD peptide was then detected by indirect ELISA.

Analysis of various BHV-1 gD antigens by MAbs.ELISA was used to determine the antigenic characteristics of GST-gD, purified BHV-1, and infected cell lysate and to confirm the presence of specific epitopes. All the MAbs reacted specifically with the purified GST-gD, purified BHV-1, and infected cell lysate. The results are summarized in Table 1. The reactions of MAbs 26 and 5D4 with the infected cell lysate were weaker than the reactions of the rest of the MAbs, and MAb 2C4 did not react at all with the infected cell lysate. The purified IgG MAb 2D7 gave a positive reaction with a concentration of GST-gD as low as 0.312 μg/well, whereas the IgM MAbs were not able to detect concentrations below 1.25 μg/well. However, all MAbs failed to react with GST-gD and purified BHV-1 in Western blotting except MAb 26, which showed a weak but specific reaction with purified BHV-1.

DNA amplification of gD gene.PCR using primers A and B gave a PCR product of 971 bp, primers C and D gave a PCR product of 648 bp, primers E and D gave a PCR product of 966 bp, and primers A and D gave the full-length gD gene PCR product of 1,293 bp shown in Fig. 1.

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FIG. 1.

PCR amplification of gD gene DNA and overlapping fragments of gD ORF. Lane 1, 1-kbp-plus DNA ladder; lane 2, gD gene DNA; lanes 3, 4, and 5, overlapping fragments AB, ED, and CD, respectively.

Protein expression and SDS-PAGE analysis.Expression of BHV-1 gD and the overlapping fragments AB, CD, and ED (designated according to the primers used to amplify their sequences) with GST tags allowed the purification of the fusion proteins by affinity chromatography on glutathione-Sepharose 4B columns. Figure 2 shows the E. coli-expressed GST-gD fusion protein and overlapping protein fragments analyzed by SDS-PAGE. Separation of GST from the fusion proteins occurred during SDS-PAGE. The amino acid sequences expressed by the various protein fragments are represented schematically in Fig. 3.

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FIG. 2.

SDS-PAGE analysis of affinity-purified GST-gD and various overlapping protein fragments. Lane 1, protein standards (Bio-Rad), with values indicated on the left; lanes 2, 3, and 4, overlapping protein fragments CD, ED, and AB, respectively; lane 5, full-length gD. In each case, the GST fragment has been cleaved away (as discussed in the text) and is indicated at the low-molecular-weight end of the gel. The positions of gD, CD, ED, AB, and GST are indicated on the right.

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FIG. 3.

Schematic representation of BH-1 gD and its segments that were expressed by E. coli as GST fusion proteins. Each protein is fused with a GST tag at the amino end and Streptag at the carboxyl end.

Epitope mapping.In the epitope-mapping experiments, all the MAbs reacted specifically with full-length GST-gD and protein fragments CD and ED, suggesting that their epitope lies between amino acid positions 216 and 417, i.e., on the protein fragment CD. The results are summarized in Table 2. A partial reaction ranging from 25 to 66% of the reaction to the full-length protein was observed with protein fragment AB.

Detection of BHV-1 in virus-infected semen by protein amplification-ELISA.In vitro transcription-translation of the CD fragment PCR product yielded only one protein band with approximately the same size as that of the CD fragment expressed in E. coli (Fig. 4). In ELISA, MAb 26 reacted specifically with the CD polypeptide in the reactions where PCR products were amplified from purified BHV-1 and from semen infected with virus dilutions of 10⁻¹ to 10⁻⁴. The comparison of PCR and protein amplification-ELISA for the detection of BHV-1 in bovine semen is summarized in Table 3. PCR could detect the virus only in semen infected with 10⁻¹ virus sample having a virus titer of 790 TCID₅₀/50 μl (Fig. 5), whereas protein amplification-ELISA detected BHV-1 in semen seeded with 10⁻⁴ virus sample with a virus titer of 0.79 TCID₅₀/50 μl. Thus, even without considering the different amounts of samples used in the two methods, there is a direct observation of a 1,000-fold increase in sensitivity by protein amplification-ELISA over traditional PCR.

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FIG. 4.

Detection of the CD peptide of gD by autoradiography after in vitro transcription and translation. Lane 1, luciferase control reaction product at 61 kDa full length; lane 2, CD peptide; lane 3, in vitro transcription and translation reaction product without the template DNA.

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FIG. 5.

PCR amplification of CD fragment of gD gene using purified BHV-1 DNA and infected semen samples. Lane 1, 1-kbp-plus DNA ladder (Invitrogen Life Technologies); lane 2, PCR product from purified BHV-1 DNA; lanes 3 to 8, PCR products from semen samples infected with virus dilutions from 10⁻¹ to 10⁻⁶, respectively.