Veterinary Research Communications, 29 (2005) 487^497 # 2005 Springer. Printed in the Netherlands
Development of a Latex Agglutination Test Using The Major Epitope Domain of Glycoprotein E of Pseudorabies Virus Expressed in E. coli to Di¡erentiate Between Immune Responses in Pigs Naturally Infected or Vaccinated with Pseudorabies Virus Tang Yong1,2, Chen Huan-chun1*, Xiao Shao-bo1, Qin Ya-li1, He Qi-gai1 and Ren Yu-qi1 1 Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China 2 Present address: Department of Bioengineering, JiNan University, Guangzhou 510632, People's Republic of China *Correspondence: E-mail:
[email protected] Tang Yong, Chen Huan-chun, Xiao Shao-bo, Qin Ya-li, He Qi-gai and Ren Yu-qi, 2005. Development of a latex agglutination test using the major epitope domain of glycoprotein E of pseudorabies virus expressed in E. coli to di¡erentiate between immune responses in pigs naturally infected or vaccinated with pseudorabies virus. Veterinary Research Communications, 29(6), 487^497 ABSTRACT A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-b-d -thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to di¡erentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic speci¢city and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic speci¢city and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/ 260). No signi¢cant di¡erence was found between the two methods (p40.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-speci¢c antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the di¡erential diagnosis of PRV infection. Keywords: pseudorabies virus, glycoprotein E, latex agglutination test Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FMDV, foot and mouth disease virus; gE, glycoprotein E; HCV, hog cholera virus; IPTG, isopropyl-b-d -thiogalactopyranoside; JEV, Japanese encephalitis virus; LAT, latex agglutination test; LB, Luria broth; PBS, phosophate-bu¡ered saline; PCV, porcine circovirus; PEG, polyethylene glycol; PPV, porcine parvovirus; PRRSV, porcine reproductive and respiratory syndrome virus; PRV, pseudorabies virus; SDS, sodium dodecyl sulphate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis 487
488
INTRODUCTION Aujeszky disease is one of the most signi¢cant infectious diseases in the pig industry (Kluge et al., 1992) and is caused by pseudorabies virus (PRV). Most eradication campaigns are based on the use of marker vaccines that lack the nonessential glycoprotein E (gE). It enables the detection of wild-type PRV-infected pigs and vaccinated pigs by the use of serological assays that detect antibodies to gE (Fujitu, 1994; Mettenleiter, 1994). One object of this study was to develop a sensitive, speci¢c, e¤cient and safe test for the detection of antibodies to vaccinated as well as wild-type virus-infected pigs. In recent years, Gut and colleagues (1999) and Kimman and colleagues (1996) have developed several commercial enzyme-linked immunosorbent assays (ELISAs). Many countries have eradicated or controlled pseudorabies by using marker vaccines and ELISAs of this kind (Medveczky, 1999). However, in some areas, such as Asia and Latin America, that have many medium and small-sized piggeries, there are di¤culties in using ELISAs as diagnostic assays because of the high cost and the shortage of necessary laboratory equipment. Thus, the latex agglutination test (LAT) is an economic and low-technology alternative to ELISAs. Several PRV-LATs for the detection of antibodies to PRV in pigs have been developed and evaluated (Schoenbaum et al., 1990; Rodgers et al., 1996), but no report has been published describing a LAT for the detection of antibodies to the glycoprotein E of PRV in pigs. This report describes a gE latex agglutination test (gE-LAT) that uses the major epitope domain of gE of PRV as antigen bound to latex beads to detect antibodies to gE in wild-type but not in vaccinated pigs. MATERIALS AND METHODS Bacteria, plasmids and vaccines E. coli BL21(DE3) and vector pET-28b were obtained from Invitrogen corporation and EMD Inc., respectively. Plasmid pSBE804, containing the major epitope domain (52^ 238 amino acids) (Jacobs and Kimman, 1994) of the gE gene of PRV, was supplied by Dr Xiao Shao-bo. Killed PRV vaccine (containing gE) was obtained from Ke Qian Biological Goods Co. Ltd, Wuhan, China. The gE-deleted vaccine (Bartha) was obtained from Harbin Veterinary Research Institute, Harbin, China. Pig sera for development and evaluation of gE-LAT Porcine anti-PRV antisera were obtained from pigs that had been vaccinated with killed PRV vaccine (containing gE); PRV-negative sera were obtained from pigs that were not inoculated. Serum samples for validating whether gE-LAT can be used to di¡erentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs were obtained from 8 pigs experimentally infected with wild PRV, and from 7 pigs immunized with gE-deletion vaccines. For the evaluation of gE-LAT, the 534 serum samples (62 serum samples from pigs that were neither vaccinated with any PRV vaccines nor infected by PRV; 212 serum samples from pigs that had been vaccinated
489
with PRV killed vaccines (containing gE); and 260 serum samples from pigs in a herd with a natural PRV outbreak that had been vaccinated with gE-deleted vaccine (Bartha)) were obtained from Guandong Provincial Station of Animal Science and Veterinary Medicine. Ten serum samples for quality control (which were tested by ¢ve lots of gE-LAT) were obtained from pigs from a herd with a natural PRV outbreak. Serum samples for analytic speci¢city testing from pigs that were previously infected with heterologous viruses (porcine parvovirus (PPV), hog cholera virus (HCV), foot and mouth disease virus (FMDV), Japanese encephalitis virus (JEV), porcine circovirus (PCV), and porcine reproductive and respiratory syndrome virus (PRRSV)) but were neither infected by PRV nor vaccinated with any PRV vaccines were obtained from Lanzhou Veterinary Research Institute, Lanzhou, China. For the comparison of the ability of gE-LAT and blocking gE-ELISA to detect sera with low titrer of gEspec¢c antibody, the 2263 experimental sera were obtained from 22 pigs vaccinated with PRV (containing gE) killed vaccine from the Laboratories of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China. Each one of these 22 pigs was bled on days 0, 7 and 14 post vaccination. Kit and reagent Latex solution (average diameter, 0.8 nm) was from the Medical Examination Institute of Shanghai, China; n-lauroyl sarcosine sodium salt and PEG 4000 were from SigmaAldrich Co. Ltd, Poole, UK. PRV-LAT kit wa from Ke Qian Bioproducts Co. Ltd, Wuhan, China and gE-ELISA kit was from IDEXX Laboratories, Inc., Westbrook, ME, USA. Recombinant gE antigen preparation For the construction of the E. coli gE expression vector, pET-28b was digested with BamHI and EcoRI; plasmid DNA was recovered by glassmilk (Sambrook and Russell, 2001); a fragment (804 bp) containing the major epitope domain of the gE gene was excised from vector pSB804 with BamHI and EcoRI; and this fragment was ligated to the BamHI^EcoRI site of pET-28b under the transcriptional control of the T7 promoter (fused with six histidine). The resultant construct of pETgE804 was used to transform the BL21(DE3) E. coli strain (named pETgE804). This transformed E. coli was cultured overnight and then diluted 1:100 in Luria broth (LB) containing kanamycin sulphate and ampli¢ed at 378C on a shaker (250 rpm) until the culture optical density (OD600nm) reached 0.6. The culture was induced by adding isopropy1-bd -thiogalactopyranoside (IPTG) to a ¢nal concentration of 1 mmol/L for 3 h at 378C and harvested by centrifugation at 10 000g for 10 min. The cell pellet samples of pETgE804 were suspended in SDS running bu¡er (50 mmol/L Tris-HCl (pH 6.8) 100 mmol/L DTT, 2% SDS, 10% glycerol and 0.1% bromophenol blue). The localization of the pETgE804 insert expressing product was determined by SDS-PAGE. The ability of the expressed product of pETgE804 to bind with antibody to PRV was con¢rmed by western immunoblotting analysis. Proteins from SDS-PAGE were transferred electro-
490
phoretically onto a nitrocellulose membrane. Membranes were blocked with 2% (w/v) BSA in PBS and incubated with a PRV-positive pig serum (1:100 dilution) in PBS containing 0.05% Tween 20 and 1% (w/v) BSA (PBS/BSA/Tween). Goat anti-pig IgG horseradish peroxidase conjugate (1:4000 diluted) in PBS/BSA/Tween was used as the secondary antibody. To harvest the expressed gE protein for preparation of the antigencoated latex particles, the bacterial cell pellet was solubilized, denatured and renatured. To do this, the cell pellet (0.2 g) was resuspended in 20 ml of bu¡er A (50 mmol/L TrisHCl (pH 7.9), 0.5 mmol/L EDTA, 50 mmol/L NaCl, and 5% glycerol) and was then ultrasonicated (20 000 Hz) for 6 min. After these treatments, the cell lysate was centrifuged at 13 000g for 10 min. The pellet was resuspended in 20 ml of bu¡er A containing 0.3% n-lauroyl sarcosine sodium salt, 1 mmol/L glutathione (oxidized), 2 mmol/L glutathione (reduced) and 0.2% PEG 4000; the suspension was incubated at room temperature for 30 min, and the lysate was dialysed with 0.9% NaCl for 3 days at 48C (Marshak et al., 1996). This product was then be used as antigen to coat the latex beads. Optimum of gE-LAT In order to develop the gE-LAT, optimum dilution of gE antigen, temperature and time for sensitization were established by systematic checkerboard titration. To standardize the procedure for antigen-coating the latex beads, the antigen solution was diluted in a 1:2 series with PBS (pH 7.4). Then an equal volume of 2% (v/v) latex that had been pretreated according to Xuenan Xuan and colleagues (2001) was mixed with each of the di¡erent dilutions of the antigen. The binding was carried out at of 378C, 458C or 568C and for 15, 30, 45 or 60 min. The combination of conditions was chosen that detected the highest titer of the positive serum. For the gE-LAT procedure, serum samples were diluted 1:2 to 1:128 with PBS. The diluted serum samples (15 ml) were mixed with equal volumes of latex antigen on a clean microscope slide. The slide was rotated by hand for 3 min, and the agglutination was determined visually against black paper. The test was considered positive when the latex agglutination was observed at a dilution of 1:2 and above. The highest titre of serum that agglutinated the latex antigen was considered to be the antibody titre. Evaluation of gE-LAT and comparison with gE-ELISA Ten serum samples from pig herds with a natural PRV outbreak were tested by ¢ve lots gE-LAT for quality control testing. The analytic speci¢city of gE-LAT for PRV gE antibody was determined with gE latex antigen by testing PRV-negative sera, sera from pigs immunized with gE-deletion vaccine and sera from pigs previously infected with PPV, HCV, FMDV, JEV, PCV or PRRSV but neither PRV infected nor vaccinated with any PRV vaccines. For the diagnostic speci¢city test, 62 serum samples from pigs that were neither vaccinated with any PRV vaccines nor infected by PRV were analysed with gE-LAT and PRV-LAT. For the diagnostic sensitivity test, 212 serum samples from pigs that had been vaccinated with PRV killed vaccines (containing gE) were tested with gE-LAT and PRV-LAT. The comparison between gE-LAT and gE-
491
ELISA was performed by testing 260 serum samples from pigs in a herd with a natural PRV outbreak that were vaccinated with gE-deleted vaccine (Bartha). For the comparison between the ability of gE-LAT and gE-ELISA to detect sera with low titres of gE-speci¢c antibody, 66 sera from 22 pigs were tested. RESULTS The expression of recombinant gE antigen The expressed products were determined by SDS-PAGE. Results indicated that the product expressed from construct pETgE804 was 38 kDa (as shown by the arrow) and was localized in the inclusion body (Figure 1a). The ability of the expressed product of pETgE804 to bind with antibody to PRV was con¢rmed by western immunoblotting analysis. A speci¢c band (as shown by the arrow in Figure 1b) was identi¢ed by the antibody (anti-PRV), indicating that the expressed gE was recognized by the antibody. The optimal gE-LAT Optimum dilution of gE antigen, temperature and time for sensitization were established by systematic checkerboard titration. The optimal antigen concentration for coating was 1.05 mg/ml, and the optimal time and temperature were 30 min and 378C, respectively.
Figure. 1. Expression of pETgE804 in E. coli and the identi¢cation of the ability of the expressed product to bind with antibody to PRV. (a) SDS-PAGE was performed as described under Materials and Methods. The speci¢c band is approximately 38 kDa (arrow) in lane 2. M, protein marker; lane 1, pET-28b in BL21(DE3). (b) The multi-clone antibody (anti-PRV) of swine was reactive to the expressed product of pETgE804. A speci¢c band (arrow) was identi¢ed by the antibody (anti-PRV) in lane 2. Lane 1, pET-28b in BL21(DE3); lane 2, pETgE804 in BL21(DE3)
492
Evaluation of gE-LAT and comparison with gE-ELISA To validate whether gE-LAT can be used to di¡erentiate the sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs, serum samples from pigs experimentally infected with wild PRV, and from pigs immunized with gE-deletion vaccines were tested by gE-LAT. All serum samples from 8 pigs experimentally infected with wild PRV were positive, whereas serum samples from 7 pigs immunized with gEdeletion were negative. This indicates that gE-LAT was able to di¡erentiate very clearly sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. Ten serum samples for quality control were tested by ¢ve lots of gE-LAT. The results with ¢ve independently produced lots of gE-LAT antigen exihibited nearly perfect reproducibility and agreement in lot-to-lot testing. The analytic speci¢city of gE-LAT for PRV gE antibody was determined with gE latex antigen by testing PRV-negative sera, sera from pigs immunized with gE-deletion vaccine and sera from pigs previously infected with PPV, HCV, FMDV, JEV, PCV, or PRRSV but neither PRV-infected nor vaccinated with any PRV vaccines. The results indicated that the gE latex antigen did not agglutinate with the PRV-negative sera, gE-deletion vaccine-immunized porcine sera, or the sera positive to any of the six heterologous viruses. For the diagnostic speci¢city test, 62 serum samples from pigs that were neither vaccinated with any PRV vaccines nor infected by PRV were tested with gE-LAT (2 positives; 60 negatives) and PRV-LAT(Ke Qian Bioproducts Co. Ltd., Wuhan, China) (3 positives; 59 negatives). The diagnostic speci¢city of gE-LAT was 96.77% (60/62) (Table I). For the diagnostic sensitivity test, 212 serum samples from pigs that had been vaccinated with PRV killed vaccines (containing gE) were tested with gE-LAT (203 positives; 9 negatives) and PRV-LAT (206 positives; 6 negatives). The diagnostic sensitivity of gE-LAT was 95.76% (203/212) (Table II). Both the diagnostic speci¢city and the sensitivity of gE-LAT (96.77%, 95.76%) are very similar to those of PRV-LAT (95.16%, 97.17%). The comparison between gE-LAT and gE-ELISA(IDEXX Laboratories, Inc., Westbrook, ME, USA.) was performed by testing 260 serum samples from pigs in a herd with a natural PRV outbreak that had been vaccinated with gE-deleted vaccine. Only 57.77% (150/260) positive serum samples and 110 negative serum samples were determined by gE-LAT. For gE-ELISA, 58.46% (152/260) positive serum samples and 108 negative serum samples were detected. The number of positive serum samples determined by gE-LAT is very similar to that of gE-ELISA. The total coincidence was 96.94% (252/260). There was no obvious di¡erence between the two assays (p40.05) (Table III). The comparison between gE-LAT and gE-ELISA to detect sera with low titres of gE-speci¢c antibody was performed by testing 66 sera from 22 pigs. Neither gE-LAT nor gE-ELISA detected gE-speci¢c antibodies from the sera sampled before 7 days post vaccination. At 14 days post vaccination, 21 positive sera were determined by gE-ELISA and 20 positive sera were determined by gE-LAT. The positive rate (90.9%, 21/22) of gE-LAT is little lower than that of gE-ELISA (94.5%, 20/22) (Table IV).
493
TABLE I Diagnostic speci¢city test of gE-LAT gE-LAT öööööööööööööööööööööööö Positive Negative Total PRV-LAT Positive Negative Total
1 1 2
2 58 60
3 59 62a
a
62 serum samples from pigs that were neither vaccinated with any PRV vaccines nor infected with PRV were obtained from Guandong Provincial Station of Animal Science and Veterinary Medicine
TABLE II Diagnostic sensitivity test of gE-LAT gE-LAT öööööööööööööööööööööööö Positive Negative Total PRV-LAT Positive Negative Total
201 2 203
5 4 9
206 6 212a
a
212 serum samples from pigs that had been vaccinated with PRV killed vaccines (containing gE) were obtained from Guandong Provincial Station of Animal Science and Veterinary Medicine
TABLE III Comparison between gE-ELISA and gE-LAT gE-LAT öööööööööööööööööööööööö Positive Negative Total gE-ELISA Positive Negative Total a
147 3 150
5 105 110
152 108 260a
260 serum samples from pigs in a herd with a natural PRV outbreak that had been vaccinated with gEdeleted vaccine (Bartha) were obtained from Guandong Provincial Station of Animal Science and Veterinary Medicine
494
TABLE IV Comparison of the abilities gE-LAT and blocking gE-ELISA to detect sera with low titres of gE-spec¢c antibodiesa Pig no.
Day post vaccination
PRV-LAT
gE-ELISA
gE-LAT
1
0 7 14
^e 1:2 1:8
^ ^ +c
^ ^ 1:2
2
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
3
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:4
4
0 7 14
^ ^ 1:4
^ ^ +
^ ^ 1:2
5
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
6
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
7
0 7 14
^ 1:4 1:8
^ ^ +
^ ^ 1:2
8
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
9
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
10
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
11
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
495
TABLE IV (continued) Pig no.
Day post vaccination
PRV-LAT
gE-ELISA
gE-LAT
12
0 7 14
^ 1:2 1:8
^ ^ +
^ ^ 1:2
13
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
14
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
15
0 7 14
^ 1:4 1:8
^ ^ +
^ ^ 1:2
16
0 7 14
^ ^ 1:4
^ ^ +
^ ^ 1:2
17
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ ^
18
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
19
0 7 14
^ ^ 1:2
^ ^ ^
^ ^ ^
20
0 7 14
^ 1:4 1:4
^ ^ +
^ ^ 1:2
21
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
22
0 7 14
^ 1:2 1:4
^ ^ +
^ ^ 1:2
a The 2263 experimental sera obtained from 22 pigs vaccinated with PRV (containing gE ) killed vaccine from Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, China b Negative result c Positive result
496
DISCUSSION Many vectors were used in this study in the attempt to express the full-length gE gene in E. coli, but none was successful. However, the major epitope domain of gE was expressed at high e¤ciency in E. coli BL21(DE3) using vector pET-28b. Failure to express the full-length gE gene in E. coli may be related to an accumulation of fulllength gE, including the transmembrane region, in the bacterial membrane, resulting in inhibition of gE protein synthesis and low levels of production of the expressed protein (Hulst et al., 1993; Makrides, 1996). The gE gene was inserted downsream of the codons of six histidines; the gE protein was fused with six histidines when it was expressed. The six histidines can be used in a¤nity chromatography for puri¢cation, but will not in£uence the antigenicity of gE protein because of the size of the six histidines. In the comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-speci¢c antibody, the sensitivity of gE-LAT is lower than that of PRVLAT because the PRV virion has many more epitopes (many other proteins) than gE. The reason for the positive rate of gE-LAT being a little lower than that of the blocking gE-ELISA may be related to the fact that blocking ELISA can detect all types of immunoglobulin whereas LAT mainly detects the agglutinative immunoglobulins such as IgM. However, there is no obvious di¡erence between these two assays and gE-LAT is economic, convenient and rapid. The assay can be accomplished in 3 min without the use of any other apparatus. Thus, the gE-LAT is very useful in situations in which the time, equipment and technology required for ELISA are not available or are inappropriate (Xuenan Xuan et al., 2001). Aujeszky disease has been eradicated in some countries but virtually none of the countries in Asia and Latin America have eradicated this disease. The disease and its virus not only do harm to animal husbandry in these countries, but can also become the source of PRV for other countries. It is therefore urgently necessary to eradicate Aujeszky disease. A suitable di¡erential diagnostic assay is essential for eradication campaigns. A gE-LAT based on gE antigen expressed by E. coli was developed in this study and the assay's reproducibility, speci¢city and sensitivity were evaluated. The results showed that it possesses good reproducibility and high speci¢city and sensitivity. The comparison of gE-LAT to gEELISA indicated that gE-LAT is a good alternative to the di¡erential diagnostic assay in an eradication programme. REFERENCES Fujitu, T., 1994. Aujesky's disease control program in Japan. OIE Symposium on Aujeszky's Disease, Thailand, 85^97 Gut, M., Jacobs, L., Tyborowska, J., Szewczyk, B. and Bienkowska-Szewczyk, K., 1999. A highly speci¢c and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed pseudorabies virus glycoprotein gE and gI complex. Veterinary Microbiology, 69, 239^249 Hulst, M.M., Westra, D.F., Wensvoort, G. and Mrormann, R.J.M., 1993. Glycoprotein E1 of hog cholera virus virus expressed in insect cell prot2ects swine from hog cholera. Journal of Virology, 67, 5435^5442 Jacobs, L. and Kimman, T.G., 1994. Epitope-speci¢c antibody response against glycoprotein E of pseudorabies virus. Clinical Diagnostic Laboratory Immunology, 1, 500^505
497 Kluge, J.P., Beran, G.W., Hill, H.T. and Platt, K.B., 1992. Pseudorabies. In: A.D. Leman, B.E. Straw, W.L. Mengeling, S. D'Allaire and D.J. Taylor (eds), Disease of Swine 7th edn, (Iowa State University Press, Ames, IA), 312^323 Kimman, T.G., De Leeuw, O., Kochan, G., Szewczyk, B., van Rooij, E., Jacobs, L., Kramps, J.A. and Peeters, B., 1996. An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibody to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs. Clinical and Diagnostic Laboratory Immunology, 3, 167^174 Marshak, D.R., Kadonaga, J.T., Burgess, R.R., Knurth, M. and Brennan, W.A., 1996. Strategies for Protein Puri¢cation and Characterization: A Laboratory Course Manual, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Makrides Savvas, C., 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews, (Sept.), 512^538 Medveczky, I., 1999. Current epizootiological status of the Eastern European countries for Aujeszky's disease. Proceedings of PRRS and Aujeszky's Disease, France, 397^398 Mettenleiter, T.C., 1994. Pseudorabies (Aujesky's disease) virus: State of the art. Acta Veterinaria Hungarica, 42, 153^177 Rodgers, S.J., Karges, S.L. and Saliki, J.T., 1996. Evaluation of a semiautomated latex agglutination test for the detection of pseudorabies antibody in swine sera. Journal of Veterinary Diagnostic Investigation, 8, 168^171 Schoenbaum, M.A., Beran, G.W. and Murphy, D.P., 1990. A study comparing the immunologic responses of swine to pseudorabies viral antigens based on the ELISA, serum virus neutralization, and latex agglutination tests. Journal of Veterinary Diagnostic Investigation, 2, 29^34 Sambrook, J. and Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd edn, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Xuenan Xuan, Ikuo Igarashi, Tetsuya Tanaka, Shinya Fukumoto, Hideyuki Nagasawa, Kozo Fujisaki and Takeshi Mikami, 2001. Detection of antibodies to Babesia equi in horses by a latex agglutination test using recombinant EMA-1. Clinical and Diagnostic Laboratory Immunology, 8, 645^646 (Accepted: 28 May 2004)
