Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

In the test,three sets of sera were included: 1). 60 sera collected from naive cattle before vaccination or infection. No serum neutralization antibody to FMDV was detected for these sera.2).249 "post-vaccination" sera were collected from cattle 3 weeks after the third vaccination with inactivated commercial FMD vaccine. 3). 66 "post-infection" sera,collected from 5 cattle experimentally infected with FMDV China/99 and sequentially bled at regular intervals for up to 12 months post-infection. Two of them were collected for up to 15 months post-infection.

NsPs antibody were examined using three ELISA kits: the commercially available Ceditest^® kit (Netherlands),the UBI^® kit (USA) and the FMDV 3ABC-Ⅰ-ELISA kit developed in the Lanzhou Veterinary Research Institute,China.

ELISA plates were coated with purified 3ABC fusion proteins in carbonate / bicarbonate buffer at pH 9.6. Plates were washed five times between stages with phosphate buffered saline (PBS) containing 0.05% Tween-20. After blocking with 5% sucrose,10% equine sera in PBST,test or reference sera were diluted 1:100 in blocking buffer (PBST,5% non-fat dry milk,10% equine sera,1% E.coli lysate) and added to the plates in duplicate. Plates were incubated for 1h at 37℃ and washed 5 times with PBST,and then incubated with 1:1 500 dilution of anti-bovine IgG peroxidase conjugate (Sigma chromogen.) for 1h at 37℃. After the final wash,chromogen mixture was added (OPD plus 0.004% H2O2 in phosphate-citrate buffer,pH 5.5).Color develop-ment was stopped after 15 min by the addition of 1.25 mol/L H2SO4. The optical density (OD) of each well was read at 492 nm for background correction.Results were calculated as follows using the means of the pairs of samples and the median of the 4 positive and negative controls on each plate.

Value=(ODsample-ODneg)/(ODpos-ODneg)

We consider values below 0.2 as negative,values between 0.2 and 0.3 as ambiguous and values above 0.3 as positive.

The Ceditest^® kit was performed according to the manufacturer′s instructions. Briefly,ELISA plates coated with 3ABC specific monoclonal antibody were incubated with the 3ABC protein. After washing plates,sera samples were dispensed to the wells and incubated. After wash,conjugate was diluted accord-ing to the recommendation of the manufacturer and was incubated for 1h at room temperature. After the final wash,the chromo-gen/substrate was allowed to react for 20 min at room temperature before addition of stop solution. Optical density (OD) was read at a wavelength of 450 nm.

The percentage inhibition (PI) of the reference sera and the test sera were calculated according to the formula below.

PI=100-(OD₄₅₀ test serum/OD₄₅₀maxium×100)

The manufacturer′s recommended interpreta-tion is PI less than 50 is negative and greater than or equal to 50 is positive.

According to the manufacturer′s instructions,the serum was diluted 1:21 and added to 96 wells microtitre plate pre-coated with the purified,synthetic FMDV 3B NS peptide. Antibodies to FMDV 3B were bound to the antigen forming an antigen/antibody complex on the plate surface. Unbound antibody was washed away. A conjugate was added which bound to any antibody/antigen complexes. Unbound conjugate was removed by washing. After adding the TMB substrate,blue color developed in proportioned to the amount of FMDV NS-specific antibodies present,if any,in the serum samples tested. This enzyme-substrate reaction was terminated by the addition of a solution of sulfuric acid to give a yellow color.

The color changes that had occur-red in each well were then measured spectrophotometrically at a wavelength of 450 nm,the Cutoff valuf was calculated according to the formula below.

Cutoff value=0.23×ODpos

The manufacturer′s recommended interpreta-tion is absorbance values of specimens less than the Cutoffvalue are negative,greater than or equal to the Cutoff value are positive.

The specificity (Sp) of the FMDV 3ABC-Ⅰ-ELISA kit was evaluated with sera collected from non-vaccinated and vaccinated animals. A total of 60 sera from non-vaccinated cattle were tested for antibodies to 3ABC,observing that 1 out of 60 sera gave a positive reaction (Table 1). In addition,249 sera,collected from vaccinated cattle were also tested,indicating that 8 out of 249 gave a positive reaction (Table 1).

Table 1.  Specificity of the FMDV 3ABC-Ⅰ-ELISA kit

Thus,the specificity of the FMDV 3ABC-Ⅰ-ELISA kit was over 96% when used to examine sera from naive and vaccinated cattle (Table 1). For the calculation of specificity,results in the "doubtful" range were counted as positive.

The sensitivity (Se) of the assay was evaluated by examining 66 serum samples from experimen-tally infected cattle. All sera gave a positive reac-tion,thus the sensitivity of the assay was 100%.

The specificity and the sensitivity of the Ceditest^® kit and UBI^® kit were evaluated using the same sera. Sixty-seven out of 249 vaccinated sera were not tested because of limited Ceditest^® kit (Table 2).

Table 2.  Specificity and sensitivity of the Ceditest^® kit and UBI^® kit

The sensitivity of Ceditest^® kit and UBI^® kit was evaluated by examining 66 sera from experimentally infected cattle (Table 2). As the table showed,the specifi-city of the Ceditest kit exceeded 96% when used to examine vaccinated or non-vaccinated sera. By comparison,the specifi-city of the UBI^® kit was extremely high though the sensitivity was low.

The results showed that the UBI^® kit had a low sensitivity,the sensitivity was only 81.8%,54 out of 66 sera gave positive results,12 out of 66 sera gave negative results,most of these sera were bled after the 12th month post-infection. In contrast,the Ceditest^® kit had a high degree of sensitivity.

The results obtained from the FMDV 3ABC-Ⅰ-ELISA kit was compared with those of two foreign ELISA kits (Table 3 and Table 4). 302(70+232) out of 308 showed the same results as shown in Table 3,corresponding to a coincidence rate of 98.05%. In contrast,only 354(55+299) out of 375 showed the same results as that indicated in Table 4,corresponding to a coincidence rate of 94.4%. One possible reason is that the UBI^® kit detects the anti-3B antibody which exists in sera of cattle not more than 364 days,so some experimentally infected sera gave negative results (Table 4). However,anti-3ABC antibodies could be detected in experimentally infected cattle up to 742 days post-infection (1).

Table 3.  Comparison of FMDV 3ABC-Ⅰ-ELISA kit with ceditest^® kit

Table 4.  Comparison of FMDV 3ABC-Ⅰ-ELISA kit with UBI^® kit

The results indicate that both the Ceditest^® kit and the FMDV 3ABC-Ⅰ-ELISA kit perform particularly well as compared with the UBI^® kit,the sensitivity of the UBI^® kit is low though the specificity is very high. It is worthy to note that the sensitivity and the specificity are not fixed values and will vary between sub-population and between populations conditional on the distribution of influential covariates. Although the UBI^® kit is less than perfect,it still may be useful depending on the circumstances in which it is used.

The 3ABC protein was the most antigenic of the NS proteins and it had been argued that antibody specific for this protein could be the most useful marker of viral replication (2). The results described here support these hypothesis,as all sera from infected cattle gave positive results in both FMDV 3ABC-Ⅰ-ELISA kit and Ceditest^® kit. In contrast,12 out of 66 sera from infected cattle gave negative results in the UBI^® kit. A possible reason is that the UBI^® kit use a synthetic peptide 3B as the antigen for detection of antibodies to FMDV NSP. The use of this synthetic peptide minimizes the incidence of non-specific reactions originating from antibody reactivity in the specimen towards host cell antigens or expression vector antigens which are copurified with virus-derived or recombinant-derived antigens (7, 9, 12). However,antibody to 3B falls below detectable levels before antibody to 3ABC of FMDV (which may be detectable 742 days post-infection).

In short,by comparing with two foreign ELISA kits,we can draw the following conclusions: the FMDV 3ABC-Ⅰ-ELISA kit developed in China can be used in laboratories with limited facilities for FMD diagnosis. It is well suited for use in large-scale serological survey. In addition,the high sensitivity and specificity of the test make the FMDV 3ABC-Ⅰ-ELISA kit a potentially valuable diagnostic tool for FMD eradication campaigns and control programs. The study reported here demon-strates that animals that have been infected with FMDV can be differentiated from those that have been vaccinated on the basis of the detection of antibody to 3ABC.