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. 2004 Nov;42(11):5087-93.
doi: 10.1128/JCM.42.11.5087-5093.2004.

Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses

Eiji Konishi  1 , Mizue Shoda, Naoko Ajiro, Takashi Kondo
Affiliations

Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses

Eiji Konishi et al. J Clin Microbiol. 2004 Nov.

Abstract

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.

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Figures

FIG. 1.
FIG. 1.
Immunoprecipitation of culture fluids from Vero cells infected with JEV and CHO cells transfected with pcJENS1NS2A (3G8 cells) or pcJENS1 (MF6 cells) with a monoclonal antibody to NS1 (2D5). Samples heated or unheated under nonreduced conditions were run on an 8.5% polyacrylamide gel and detected by silver staining.
FIG. 2.
FIG. 2.
Comparison of ELISA reactions obtained with 3 dilutions of capture antibody (rabbit anti-NS1 hyperimmune serum) and 5 dilutions of 3G8 cell culture fluid containing 0.01 to 100 ng of NS1 antigen per ml (corresponding to 0.001 to 10 ng/well) and with highly positive (solid squares), moderately positive (solid circles), low-positive (open diamonds), and negative (open triangles) sera.
FIG. 3.
FIG. 3.
Dose-response absorbance curves of highly positive (solid circles), moderately positive (open circles), low-positive (solid triangles), and negative (open triangles) sera. Microplates were sensitized with a 1:10,000 dilution of rabbit anti-NS1 hyperimmune serum and 3 ng of NS1 antigen.
FIG. 4.
FIG. 4.
Comparison of the immunostaining and ELISA methods with horse sera positive (48 samples) or negative (47 samples) by immunostaining. The ordinate indicates ELISA values, and the abscissa indicates NS1 antibody titers obtained by the immunostaining method. A dotted line indicates the cutoff value for ELISA (0.122). NS1 antibody titers of less than 1:80 by immunostaining were regarded as negative.
FIG. 5.
FIG. 5.
Comparison of the one-dilution method and the endpoint method in ELISA for quantifying NS1 antibodies, using 30 horse serum samples.
FIG. 6.
FIG. 6.
Comparison of results obtained in duplicated ELISA tests, using 20 sera positive and 25 sera negative for NS1 antibodies.
FIG. 7.
FIG. 7.
Time course of NS1 antibody levels in sera serially collected from four subclinically infected horses during a JE epizootic in Japan.
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References

    1. Andersen, M. M., and T. Ronne. 1991. Side-effects with Japanese encephalitis vaccine. Lancet 337:1044. - PubMed
    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
    1. Berg, S. W., B. S. Mitchell, R. K. Hanson, R. P. Olafson, R. P. Williams, J. E. Tueller, R. J. Burton, D. M. Novak, T. F. Tsai, and F. S. Wignall. 1997. Systemic reactions in U.S. Marine Corps personnel who received Japanese encephalitis vaccine. Clin. Infect. Dis. 24:265-266. - PubMed
    1. Burke, D. S., and T. P. Monath. 2001. Flavivirus, p. 1043-1125. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
    1. Halstead, S. B., and J. Jacobson. 2003. Japanese encephalitis. Adv. Virus Res. 61:103-138. - PubMed

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