A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions, A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.Facultad de Ciencias Veterinaria

Echeverría, María Gabriela

Etcheverrigaray, María Elisa

Nosetto, Edgardo Omar

Journal of Veterinary Diagnostic Investigation

English

SEDICI - Repositorio de la UNLP

J Vet Diagn Invest 12:266-268 (2000)
Evaluation of a blocking ELISA using a urease conjugate for the detection of 
antibodies to pseudorabies virus
Maria Gabriela Echeverria, Edgardo Omar Nosetto, Maria Elisa Etcheverrigaray
Abstract. A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) 
was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 
serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and 
U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, 
respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing 
visual assessment of results.
Pseudorabies (Aujeszky’s disease) virus (PRV) is a very 
important pathogen for the swine industry worldwide, and 
eradication programs for PRV are in progress in many coun­
tries. Most if not all of PRV eradication programs are based 
on the use of differential vaccines and appropriate sérodi­
agnostic techniques. In Argentina, the National Animal 
Health Service established in 1996 a PRV control program 
based on serologic detection of infected animals with inter­
diction of vaccine usage. In accordance with the National 
PRV control program, the seropositive animals are segre­
gated and/or sent to slaughter. Two serologic techniques are 
accepted as official by Argentina’s animal health authorities: 
the latex agglutination test4 and the nondifferential enzyme- 
linked immunosorbent assay (ELISA).8 The kits for these 2 
different tests are imported from other countries, which re­
sults in a significant additional surcharge for the perfor­
mance of PRV serology by diagnostic laboratories in Argen­
tina. Likewise, there are few laboratories in the country that 
can run the commercially available PRV ELISAs because 
ELISA plate readers are not readily available. Therefore, the 
development of sérodiagnostic techniques that are reliable, 
easy to perform, and require minimal equipment are of fun­
damental importance for the control and eradication of PRV 
in developing countries such as Argentina. The ELISA is 
probably the most widely used test for screening of PRV 
exposure in pig populations. In 1977, indirect ELISAs (I- 
ELISAs) for detection of antibodies to PRV were developed 
in the USA.8 Since then, a number of reports on the appli­
cation of ELISA as a sérodiagnostic test for PRV have been 
published.1’2’10 Most of the ELISA methods use alkaline 
phosphatase (AP) or horseradish peroxidase (HRPO) anti­
body conjugates. The evaluation of the results of these tests 
usually requires the use of an ELISA plate reader. If a urease 
conjugate is used instead, results can be obtained visually 
without using specialized equipment. The urea-containing 
substrate causes a pH shift, which in turn is visualized by 
the use of bromocresol purple as a pH indicator. In the pres­
ent study, a blocking ELISA using a urease conjugate (U- 
B-ELISA) was evaluated for screening sera for antibodies to 
PRV under field conditions. This test combines the sensitiv­
ity and specificity of the blocking ELISA format while al­
lowing visual assessment of results.
From the Area of Virology, Faculty of Veterinary Sciences, Na­
tional University of La Plata, 60 and 118, La Plata 1900, Argentina.
Received for publication March 16, 1999.
Two groups of serum samples were used throughout this 
study. Five hundred porcine serum samples were obtained 
from an epizootic that occurred in the province of Buenos 
Aires in 1990 and subsequently were stored at —20 C until 
use.5 These samples were used for the HRPO blocking 
ELISA (HRPO-B-ELISA) and the U-B-ELISA but were not 
suitable for use in the virus neutralization (VN) test. Two 
hundred sixty-four serum samples were obtained from dif­
ferent breeding farms at various locations in Santa Fe and 
Buenos Aires provinces between 1995 and 1997 and were 
analyzed by VN test and U-B-ELISA. Reference negative 
and positive anti-PRV sera were used in each case.
Rabbit anti-PRV hyperimmune gamma globulin was pre­
pared by immunizing rabbits with potassium tartrate-purified 
PRV (106 5 TCIDjo) which had been inactivated by ultraviolet 
light and mixed with Freund’s complete adjuvant. The im­
munization schedule consisted of 4 subcutaneous applica­
tions at intervals of 2-3 weeks. Six days after the last im­
munization, the rabbits were euthanized and serum was col­
lected and precipitated with saturated ammonium sulfate. 
The precipitate was resuspended in distilled water and ex­
tensively dialyzed against phosphate-buffered saline.
For the VN test, all serum samples were heat treated at 
56 C for 30 minutes before titration. Antibodies to PRV were 
detected by adding 100 TCID50/0.025 ml of virus to 2-fold 
dilutions of serum in microtiter plates. Each sample was test­
ed in duplicate. Following incubation for 1 hour at 37 C, 
RK13 cells were added to each well. The neutralizing titer 
was expressed as the reciprocal value of the highest serum 
dilution that neutralized 100 TCID50 of virus.2 An endpoint 
titer of >1:4 was considered positive.
PK15 cells were grown in Eagle’s minimum essential 
medium3 supplemented with 10% fetal calf serum, 0.3 mg/ 
ml glutamine, 200 IU/ml penicillin, and 0.5 mg/ml strepto­
mycin. The local PRV CL 15 strain5 was propagated in the 
PK15 cell cultures. The virus culture was harvested when 
90% of the cells exhibited cytopathic effect, and the culture 
was clarified by centrifugation. Nonidet P-40b at a concen­
tration of 1% in Tris ethylenediaminetetraacetic acid 
(EDTA)-saline buffer was used to lyse and solubilize the 
pellet of PRV-infected cells. These lysates were used as the 
source of antigen for the U-B-ELISA.6
To standardize the U-B-ELISA, 0.1 ml of the predeter­
mined antigen dilution (1:1,000) in carbonate/bicarbonate 
buffer (0.05 M, pH 9.6) was used in all microtiter wells. 
After incubation at 4 C overnight in a humid chamber, 4- 
266
Brief Communications 267
fold dilutions of pig sera to be tested were added to the 
antigen-coated wells in duplicate and incubated overnight at 
room temperature. Subsequently, rabbit anti-PRV serum di­
luted 1:1,000 was employed as secondary antibody to detect 
antigen sites that had not been blocked by the primary sera 
through incubation for 30 minutes at 37 C. Anti-rabbit ure­
ase conjugateb diluted 1:1,000 was used as a third antibody 
and incubated for 60 minutes at 37 C. In all cases, phosphate 
buffer containing 0.05% Tween 20 and 0.1% bovine serum 
albumin (BSA) was used as the diluent. The washing solu­
tion (WS) was similar to the diluent but did not include BSA. 
After each step of incubation, the plates were washed 3 times 
with WS and 3 additional times with distilled water before 
the addition of substrate. The substrate containing 0.1% urea, 
0.008% bromocresol purple,b and 0.2 mM EDTA was added 
to each well. The plates were sealed and incubated at room 
temperature for 30-60 minutes until the negative control se­
rum developed a purple color without noticeable change in 
the positive control, which remained yellow.
As expected for the blocking test format, absence of an­
tibodies in the pig serum samples resulted in the binding of 
the secondary antibody (rabbit anti-PRV serum). The absor­
bance values were measured in a microplate spectrophotom­
eter/' Optimal dilutions of the components of the system 
were determined by checkerboard titration, to identify the 
concentration of each reagent that would provide the best 
discrimination between the positive and negative reference 
sera. Additionally, various blocking substances (to adsorb 
nonspecific antibodies from the rabbit anti-PRV serum), i.e., 
skimmed milk, BSA, and egg albumin, were tested as dilu­
tion buffers. In each plate, 2 wells (to which only anti-rabbit 
conjugate was added) were used for control antigen, 2 wells 
were positive controls, 2 wells were negative controls, and 
2 wells were controls for the overall detection system (an­
tigen + rabbit anti-PRV + anti-rabbit conjugate). The repro­
ducibility of the results obtained by this test was determined 
by using 2 negative and 2 positive sera and by agreement 
of the positive and negative control sera in 10 different rep­
etitions. To perform the HRPO-B-ELISA, an ELISA kit was 
used.d The steps are similar to those described for U-B- 
ELISA except for the use of peroxidase as the enzyme with 
H2O2 as substrate and 3'3'5'5'-tetramethyl benzidine as 
chromogen. The reaction was stopped by adding 1 M H2SO4, 
and the results were read on a microplate spectrophotometer/'
A total of 764 serum samples were analyzed by U-B- 
ELISA (Table 1). Of these, 264 sera were studied by both 
VN and U-B-ELISA, and the results of the 2 tests were 
compared. Using the chi-square method, the agreement be­
tween the serologic techniques was significant: y2 = 249, P 
< 0.001. U-B-ELISA showed 98.5% sensitivity and 98.9% 
specificity (Table 1). Absorbance values for the urease re­
action were determined by spectrophotometer using a 450- 
nm filter. Samples were considered positive if the OD value 
was at least 3 SD above the mean of 70 negative serum 
samples for both VN and U-B-ELISA (0.593). The choice 
of the optimal concentration of antigen used for coating onto 
the wells was based on obtaining maximal absorbance read­
ings for PRV-positive control samples and minimal back­
ground for PRV-negative control samples. Rabbit anti-PRV 
antibodies are added to bind to the specific sites on the an-
Table 1. Comparison of results of urease blocking ELISA (U- 
B-ELISA), virus neutralization (VN), and peroxidase blocking 
ELISA (HRPO-B-ELISA) for pseudorabies virus obtained with 764 
field serum samples.
VN HRPO-B-ELISA
Test + - + -
U-B-ELISA + 68 2 112 13*
U-B-ELISA - 1 193 0 375
Total 69 195 112 388
Relative sensitivity (°o) 98.5 100
Relative specificity (°o) 98.9 96
* Sera tested by ELISAs but unfit for VN testing.
tigen free from pig’s antibodies. Weak positive sera develop 
a greenish color. Development of a purple color indicates 
unequivocally negative sera. Using repeated measures anal­
ysis of variance, the reproducibility of the test was accept­
able (F = 2.38) for the sera analyzed on 10 occasions.7
This study demonstrates the efficacy of an alternative ure­
ase conjugate that permits the visual evaluation of the end­
point of the PRV blocking ELISA. The use of the regular 
peroxidase enzyme for ELISAs has some disadvantages, 
e.g., HRPO conjugates can lose activity if they become con­
taminated with microorganisms. This problem cannot be 
overcome by the use of bacteriostatic agents because HRPO 
activity is very sensitive to antibacterial agents such as meth­
anol and sodium azide.11 In addition, the substances used as 
substrates are frequently unstable. The use of an ELISA 
plate reader is always necessary. Further, both HRPO and 
AP are present in many mammalian tissues, which limits 
their application in assays in which whole cells are used as 
substrate.3 These problems can be overcome by using urease 
as the enzyme label. The urease substrate is stable in aque­
ous solution at ambient temperature, and the enzymatic re­
action can be stopped by the addition of a small quantity of 
thiomersal. Incubation at room temperature for 24 hours was 
preferred because evaporation at 37 C causes drying, espe­
cially at the wells along the edge of the plate. A visual for­
mat using urease as the enzyme marker was adapted for this 
blocking-ELISA: the nonreactive sera developed a definite 
purple color. When the U-B-ELISA was compared with the 
HRPO-B-ELISA, there was no evidence that 1 enzyme was 
superior to the other for increasing the sensitivity or the 
speed of the assay.6 The endpoint as determined by the ure­
ase conjugates may appear more clear-cut than that obtained 
with other enzyme conjugates.3 Experience does indicate that 
the urease conjugates provides more clear-cut endpoints than 
do other enzymes. HRPO and AP conjugates give a gradual 
color development, which makes accurate assessment of ti­
tration endpoint difficult. This difficulty does not occur with 
the urease conjugate.
The use of urease conjugates in ELISAs offers the advan­
tages of convenience and safety. However, use of urease 
conjugates may result in false-negative reactions upon pro­
longed incubation when wells containing substrate solution 
absorb ammonia liberated from an adjacent reactive well. 
Also, the urease test system is extremely sensitive to pH 
changes and thus demands special care during the perfor­
268 Brief Communications
mance of the test. This system was highly sensitive when 
the substrate solution was used at pH 4-4.2. A close cor­
relation between the results obtained with the blocking 
ELISA and with the VN test has been reported.9 In this com­
parison, close overall correlations between VN and the U- 
B-ELISA (261/264 samples = 98.1%) and between the 
HRPO-B-ELISA and the U-B-ELISA (487/500 samples = 
97.4%) were obtained.
The HRPO-B-ELISA has been used in this laboratory for 
several years with satisfactory results. However, because of 
the implementation of a national plan for control of pseu­
dorabies in Argentina, it has become necessary to transfer 
the testing to less well-equipped laboratories located 
throughout the swine-producing region of the country. The 
overall unavailability of ELISA plate readers in those small- 
scale laboratories precludes the use of the conventional 
HRPO-B-ELISA. The use of the U-B-ELISA offers a solu­
tion to this problem. The U-B-ELISA was sensitive, specific, 
precise, rapid, and easily automated. The test can be easily 
transferred for use in routine serodiagnosis in those condi­
tions where access to an ELISA plate reader is limited, as it 
often occurs in developing countries.
Acknowledgements. We are grateful to M. Mondragon and 
D. D’Andrea for their technical assistance. We also S. G. 
Corva and F. Osorio for their useful suggestions and correc­
tions on the manuscript. This work was supported in part by 
the Japan International Cooperation Agency.
Sources and manufacturers
a. Nissui, Tokyo, Japan.
b. Sigma Chemical Co., St. Louis, MO.
c. Titertek Multiskan Plus, EFLAB, Finland.
d. Blocking ELISA for detection of antibodies against pseudorabies 
virus, the Joint FAO/International Atomic Energy Agency Divi­
sion, Seibersdorf, Austria.
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Evaluation of a blocking ELISA using a urease conjugate for the detection of antibodies to pseudorabies virus

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Evaluation of a blocking ELISA using a urease conjugate for the detection of antibodies to pseudorabies virus

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