An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein sigma B as the coating antigen

An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.     

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.     

Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody

For the rapid and accurate evaluation of the IgA antibody level of porcine epidemic diarrhea virus (PEDV) in pig serum and milk,a specific PEDV IgG monoclonal antibody (MAb) 8A3 was screened from four strains of PEDV MAb,which could capture all virus particles (inactivated virus cell culture medium) of PEDV efficiently.In this method,the coating concentration of 6.0 μg/mL showed the optimal performance of MAb 8A3,the cut-off value (D_{450 nm}) was settled as 0.34,it had no cross-reactivity with the positive serums of common porcine viruses.Compared with immune-peroxidase monolayer assay (IPMA),the concordance rates of established ELISA for positive and negative serum detection were 98.7% (152/154) and 98.0% (145/148),respectively.For positive and negative samples of colostrum and milk,the concordance rates of the established ELISA compared with IPMA were 100% (60/60) and 95.8% (23/24),respectively.IgA levels in colostrum and milk samples during lactation detected by established ELISA were highly correlated with trends in neutralizing titers (kappa=0.835).Collectively,the indirect ELISA in this study had high sensitivity and specificity,it was a rapid and objective method suitable for large-scale detection of PEDV IgA in clinical samples.     

Comparison of immunogenic properties of native and inactivated Mannheimia haemolytica Lkt in calves

The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorbance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MTT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.     

Protection of laying hens against infectious bronchitis with inactivated emulsion vaccines

Commercially-reared laying chickens were challenged at 31 weeks of age with a virulent infectious bronchitis (IB) virus. They showed a sharp drop in egg production, despite having been vaccinated at four and eight weeks old with live attenuated IB vaccines to a recommended schedule. In contrast, similar birds that had been further immunised at point-of-lay with inactivated oil emulsion IB vaccine, or with a combined IB/Newcastle disease (ND) emulsion vaccine, showed no detectable fall in egg production after the same challenge. Unvaccinated susceptible specific pathogen-free birds challenged at the same time stopped laying almost completely. In the birds revaccinated with emulsion vaccine, measurement of haemagglutination inhibition antibody levels to IB showed their geometric mean titres to be raised from less than 5 log2 at the time of vaccination to over 10 log2 four weeks later. Their antibody levels did not rise further followining the IB challenge whereas in the birds that had not been revaccinated antibody rises to nearly 10 log2 were detected after the same challenge. For pullets vaccinated earlier with live IB vaccine, revaccination with inactivated IB or IB/ND oil emulsion vaccine at point-of-lay provides a safe and effective way of protecting their egg production against IB infection.     

Functional analysis of antibody responses of feedlot cattle to bovine respiratory syncytial virus following vaccination with mixed vaccines.

The antibody response of cattle to bovine respiratory syncytial virus (BRSV) immunization was investigated using 4 different commercially available mixed vaccines. Forty, 5-6 month old, beef calves, randomly assigned to groups of 10, were vaccinated on day 0 and 21 with 1 of 3 inactivated vaccines, (3 groups), or a modified live virus (MLV) vaccine. BRSV-specific antibody responses were measured prior to vaccination and on day 35 by using an enzyme linked immunosorbent assay (ELISA), virus neutralization assay (VN), a fusion inhibition assay (FI); and responses were also measured for their ability to facilitate antibody dependent, complement mediated cytotoxicity (ADCMC) of BRSV infected cells. Sera from day 35 were, in addition, analyzed by use of an IgG1, IgG2 isotype specific ELISA. All vaccines induced significant increases in BRSV specific IgG antibody as measured by ELISA, but only one inactivated and the MLV vaccine induced significant increases in VN titers. Fusion inhibiting antibody titers were low or undetected in calves vaccinated with the inactivated vaccines. Vaccination with modified live virus induced significantly higher titers of fusion inhibiting antibodies, which are considered to be most highly correlated with protection. The VN to ELISA and FI to ELISA ratio of the calves that received MLV vaccine were significantly greater than the calves receiving the 3 inactivated vaccines. Vaccination with MLV induced the highest IgG2/IgG1 ratio. This difference was small, and only significant relative to 2 of the inactivated vaccine groups, which were not significantly different from each other. The higher proportion of IgG2 isotype in the MLV sera was not associated with lower ADCMC, a function not attributed to this isotype. The VN and FI titers, but not the ELISA value of the sera, were most predictive of ADCMC. The inactivation processes apparently alter epitopes and affect the induction of functional antibodies.     

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

Antibody responses to inactivated vaccines and natural infection in cattle using bovine viral diarrhoea virus ELISA kits: Assessment of potential to differentiate infected and vaccinated animals

Bovine viral diarrhoea virus (BVDV) is one of the most common and economically important viral infections of cattle. As vaccination is common in most European countries, differentiation between infected and vaccinated animals is one of the key challenges facing BVDV eradication campaigns. This study was designed to compare the ability of commercial ELISA kits to differentiate antibodies generated following vaccination with four different commercial inactivated BVDV vaccines from antibodies generated following challenge with virulent BVDV. Although none of the tested vaccine–ELISA combinations was able to differentiate an infected from a vaccinated animal (DIVA) at the individual animal level, p80 blocking ELISAs, in combination with inactivated BVDV vaccines, may have some value under certain circumstances at herd level. In most cases, antibody responses to BVDV vaccines cannot be clearly distinguished from responses seen in the early phase of natural infection. No commercial BVD vaccine showed true marker qualities for DIVA using p80 blocking ELISAs.     

Evaluation of the results of ELISA in the quantitative determination of antibodies against infectious bursitis in poultry]

Specific antibodies were investigated in serums of chicks vaccinated with live vaccine and revaccinated with inactivated vaccine against the infectious bursal disease virus, using three methods. An ELISA technique was used to determine antibody titres at a fourfold serum dilution; the reaction was evaluated visually. The results were compared with the titres of neutralizing antibodies and percentage of samples with precipitin activity (Fig. 1). The average values of neutralizing antibody titres and ELISA titres were found to have an analogical pattern; a decrease in maternal antibodies on the first days of chick life and an increase in the antibodies after vaccination, accompanied by an increase in precipitin activity, were typical in these tests. Using the different ELISA technique, 138 samples of fowl serum were examined (Fig. 2). The high correlation, r = 0.85, was found between the ELISA titres determined by visual reading of the reaction within the fourfold serum dilutions and the absorbance values determined at single serum dilution. Applying a spectrophotometer programme, a scale of antibody quantification was made up pursuant to the intensity of immunoenzymatic reaction. For the purposes of method reproducibility, the evaluation was made within the average values of absorbance of positive serum on the one hand and of negative serum on the other. The span of these values was divided into ten equal parts, designated by degrees 0 to 9. Corresponding degrees of positivity were assigned to the examined samples in dependence on the determined value of absorbance (Fig. 3). The correlation r = 0.61 was found between the ELISA values and the titres of neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody responses of naive cattle to two inactivated bovine viral diarrhoea virus vaccines,measured by indirect and blocking ELISAS and virus neutralisation

Two groups of naive heifers were given primary courses of two inactivated bovine viral diarrhoea (BVD) virus vaccines licensed for use in the UK. Their humoral responses in serum and milk were assayed by means of an indirect ELISA detecting antibodies to structural viral glycoproteins, a blocking ELISA specific for antibodies to the non-structural protein NS2-3 and the virus neutralisation test (VNT). For each assay, the numbers of serum or milk samples testing positive at each sample point and the mean values were determined. In both vaccine groups, serum antibody responses were detected by the indirect ELISA and the VNT, with both the numbers of seropositive animals and mean values peaking five weeks after the second vaccination. In the 23 heifers vaccinated with Bovilis BVD, the mean NS2-3-specific ELISA values remained low throughout the trial, with no serum or milk samples testing positive. In the 24 heifers vaccinated with Bovidec, the mean NS2-3 responses peaked below the level of positivity five weeks after the second vaccination, before declining again; NS2-3-specific antibodies were detected in one serum sample and one milk sample from two heifers in this group. A pooled milk sample from each vaccine group tested negative by both ELISAS 12 weeks after the second vaccination.     

Cell mediated and humoral immune response of chickens to inactivated oil-emulsion infectious bronchitis vaccine

A lymphocyte transformation microassay was used to study cell mediated immunity (CMI) in chickens following primary and secondary vaccination with inactivated oil emulsion infectious bronchitis (IB) vaccine and subsequent challenge with Massachusetts-41 (M-41). Humoral immunity was monitored for comparison, using the haemagglutination inhibition (HI) microassay. Positive stimulation indices (2 to 2.7 after primary and 2 to 4.8 after secondary vaccination) were lower and HI titres were higher than those previously reported following primary and secondary vaccination with live IB vaccines. The highest HI titres appeared in birds which had received the inactivated vaccine as a secondary vaccination. Challenge of vaccinated and revaccinated birds resulted in strong HI and weak CMI secondary responses. There was no correlation between CMI and HI antibody production. Monitoring egg production and clinical signs showed that a high level of protection against challenge resulted from revaccination with an inactivated oil adjuvant vaccine.     

Adjuvanted Outer Membrane Protein Vaccine Protects Poultry against Infection with Salmonella enteritidis

The immunogenicity of a sonicated extract (SE) and of outer membrane proteins (OMP) of Salmonella enteritidis was tested in birds of about 8 weeks of age. The dose, route of vaccination and the adjuvant used varied in different groups of birds. Two vaccine doses with or without adjuvant were given parenterally or orally 3 weeks apart. OMP vaccines gave significantly higher antibody titres than SE vaccines, as indicated by ELISA. The vaccines adjuvanted with oil produced higher antibody titres than those without any adjuvant. A dose of 1 mg of vaccine produced higher antibody titres than 0.5 mg of vaccine. Adjuvanted vaccine given subcutaneously elicited higher antibody responses than oral vaccines given without adjuvant. The birds were challenged with virulent S. enteritidis organisms at the end of the second week after a booster dose. None of the birds given 1 mg of OMP vaccine subcutaneously shed the organisms when tested by culturing cloacal swabs, although a few birds vaccinated with 0.5 mg of OMP vaccine did so. In general, adjuvanted OMP vaccines gave better protection than SE vaccines.     

Mucosal vaccination with formalin-inactivated avian metapneumovirus subtype C does not protect turkeys following intranasal challenge

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.     

通过检测非结构蛋白抗体评价口蹄疫疫苗纯度的研究

为探索通过检测非结构蛋白(NSPs)抗体的方法评价口蹄疫疫苗纯度的可行性,用不同纯化工艺制备口蹄疫O型、亚洲1型、A型三价灭活疫苗,重复免疫牛至少3次后,定期检测NSPs的ELISA抗体并统计分析抗体情况。结果显示,高度纯化疫苗组的NSPs抗体阴性率远高于普通纯化疫苗组,表明通过检测NSPs抗体评价口蹄疫疫苗纯度可以作为质量控制的一种方法。     

检测鸡球虫四价弱毒疫苗抗体应答的ELISA方法的建立及初步应用

本研究旨在建立检测鸡球虫四价弱毒疫苗(柔嫩艾美耳球虫、毒害艾美耳球虫、堆型艾美耳球虫、巨型艾美耳球虫)抗体应答的酶联免疫吸附试验(ELISA),并初步阐明鸡免疫四价弱毒疫苗及攻虫后的抗体应答。将300羽岭南黄鸡分为A(免疫攻虫组)、B(不免疫不攻虫组)、C(不免疫攻虫组)共3个组。A组雏鸡在4日龄时进行首免,11日龄时鸡开始二免,二免完成后(17日龄时)对A组及C组的鸡进行攻虫(28万个4种球虫的混合卵囊/羽),7 d后检查A、C两组鸡的存活率。在鸡二免和三免完成后,分别对A、B组进行采血,通过优化最佳抗原包被浓度、酶结合物工作浓度及最佳血清稀释度,建立了ELISA方法来检测鸡体对球虫四价弱毒疫苗的抗体应答情况。A组鸡血清抗体的平均D值为0.870和0.904,明显高于B组鸡的平均D值0.261和0.270,证明了鸡体免疫疫苗后可以刺激产生相应抗体。免疫鸡攻虫后的存活率和抗体应答的检测结果,都充分说明了此鸡球虫弱毒四价疫苗具有良好的免疫原性,所建立的ELISA方法为今后评价鸡球虫弱毒四价疫苗的免疫效力提供了有效手段。     

Measurement of isotype-specific antibody responses to Aujeszky's disease virus in sera and mucosal secretions of pigs.

Enzyme-linked immunosorbent assays (ELISAs) for the detection of porcine IgM, IgA, IgG1 and IgG2 antibodies directed against Aujeszky's disease virus (ADV) are described. ADV-specific IgA and IgM were detected in an antibody capture assay, and ADV-specific IgG1 and IgG2 were detected in an indirect double antibody sandwich assay. A selected set of samples was tested in the four ELISAs and in a 24 h virus neutralization assay. Comparison of the results showed that the ELISAs were isotype-specific, sensitive, and reproducible. Samples with ADV antibody of one isotype showed that ADV-specific IgG1, IgG2 and IgM were able to neutralize the virus in vitro. In vitro neutralization of virus can be enhanced by complement. ADV-specific IgA neutralized virus only weakly. ADV-infected cells activated complement in the absence of antibody. Specific IgG2 and IgM enhanced complement activation. Analysis of the time course of antibody responses after infection or vaccination revealed that the isotype-specific ELISAs are suitable to study the humoral antibody response of pigs to the virus in mucosal secretions. Wild-type virus (strain NIA-3) and an attenuated vaccine strain (Bartha) administered intranasally induced mucosal IgM and IgA responses to the virus. In contrast, a killed vaccine (Nobivac) administered intramuscularly induced only weak mucosal IgM responses. The attenuated vaccine strain primed for a mucosal IgA memory response evoked upon challenge infection with wild-type virus.     

Vaccination of day-old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines

Day-old broilers were administered live and/or inactivated vaccines to assess vaccine efficacy against challenge with Newcastle disease (ND) and infectious bursal disease (IBD). Chicks were from commercial breeder pullets vaccinated against ND and IBD using several live vaccine primers followed by an inactivated ND-IBD vaccine at 18 weeks. The most efficacious initial ND-IBD vaccination program was live ND virus by eye drop and live IBD vaccine injected subcutaneously (SQ) followed 2 hours later with inactivated ND-IBD vaccine SQ. The next two most efficacious programs were live vaccine alone and the inactivated vaccine only. Inactivated vaccine given SQ had no adverse effect on live IBD vaccine given 2 hours earlier in a similar site. Administration of inactivated vaccine by vent was not as efficacious as administration SQ. A booster of a second live ND-IBD vaccine drinking water at 18 days significantly increased levels of circulating antibody, regardless of the initial vaccination program.     

Calcium phosphate coupled Newcastle disease vaccine elicits humoral and cell mediated immune responses in chickens

Calcium phosphate (CaP) particles were coupled with inactivated Newcastle disease virus (NDV) vaccine. The surface morphology of CaP particles coupled to NDV was found to be spherical, smooth and with a tendency to agglomerate. The mean (± SE) size of CaP particles was found 557.44 ± 18.62 nm. The mean percent encapsulation efficiency of CaP particles coupled to NDV assessed based on total protein content and haemagglutination (HA) activity in eluate was found to be 10.72 ± 0.89 and 12.50 ± 2.09, respectively. The humoral and cell mediated immune responses induced by CaP coupled NDV vaccine were assessed in comparison to a commercial live vaccine (RDV ‘F’). CaP coupled NDV vaccine elicited prolonged haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titres in the serum even at fourth and fifth week post-vaccination (PV), unlike RDV ‘F’ inoculated chickens whose titres declined to insignificant levels by this time. CaP coupled NDV vaccine could stimulate HI antibodies in tracheal washings and tears from second and first week PV, respectively. IgA ELISA antibodies were also seen in tracheal washings of these birds from third week PV and in tears from second week PV. CaP coupled NDV vaccine elicited cell mediated immune responses (CMI) from two to four weeks PV. The stimulation indices obtained after stimulation with specific antigen was not significantly different between CaP coupled antigen and live NDV virus except on first week PV. However, CaP coupled antigen did not cause suppression of lympo proliferation as indicated by statistically similar responses to mitogen, concanavalin A between the two groups. Overall, CaP coupled NDV vaccine elicited stronger and prolonged immune responses in comparison to the commercial live vaccine. No increase in the serum calcium and phosphorous levels were seen in CaP coupled NDV vaccine inoculated chickens.     

Protection against bovine respiratory syncytial virus challenge following a single dose of vaccine in young calves with maternal antibody

Twenty-one young calves with maternally derived antibody to bovine respiratory syncytial virus (BRSV) were divided into three groups of seven, each group balanced for BRSV antibody titre. The calves had no evidence of previous exposure to BRSV. The calves in one group were given a single dose of a monovalent modified live BRSV vaccine; the calves in the second group were given a single dose of an inactivated combined BRSV, parainfluenza virus type 3, Mannheimia haemolytica vaccine and the calves in the third group were left as unvaccinated controls. Three weeks after the single doses of vaccine, all the calves were challenged with BRSV. The clinical signs of disease were mild, and virus excretion was limited to two calves in the group given the inactivated vaccine, compared with six in the negative controls (P = 0.05) and five in the group given the live vaccine. The mean virus excretion titres after the challenge were not significantly different between the groups. There was little seroconversion before the challenge, but six of the seven calves in the group given the inactivated vaccine showed significant seroconversion within two weeks after the challenge, compared with only one calf in each of the other two groups (P = 0.015).     

A double-antibody enzyme-linked immunosorbent assay for the detection of turkey hemorrhagic enteritis virus antibody and antigen

A highly sensitive and specific double-antibody enzyme-linked immunosorbent assay (ELISA) is described for the detection of antigen and antibody of turkey hemorrhagic enteritis virus (HEV). The assay utilizes a virus-neutralizing monoclonal antibody (MAb) to capture the antigen and turkey antiserum against HEV as the second antibody. Microtiter plates were first coated with a dilution of 1:3000 of the MAb (300 ng immunoglobulin/well) and are used for detection of both antigen and antibody. For antibody detection, MAb-coated plates were treated with an appropriate dilution of a cell-culture-propagated HEV antigen and then reacted with the test turkey serum. For detection of HEV antigen, MAb-coated plates were treated with appropriate dilutions of test antigens and then reacted with purified anti-HEV turkey immunoglobulins. The assay for HEV antibody detection was more sensitive and specific than previously described single-antibody ELISAs. Using the double-antibody ELISA, it was found that the spleen of HEV-infected turkeys harbors very high levels of antigen. Traces of HEV antigen are present in some other organs. Infectivity assay for HEV is found to be about two orders of magnitude more sensitive than the ELISA for detection of virus.