Elisa ARS

Procedia in Vaccinology 2 (2010) 311
1877-282X/$see front matter 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.provac.2010.03.001
3
rd
Vaccine Global Congress, Singapore 2009
Validation of indirect ELISA for quantitative testing of rabies antibodies
during production of Antirabies serum using equines
N. C. Salvi
a *
, R. L. Deopurkar
b *
, A. B. Waghmare
a
, M. V. Khadilkar
a
,
M. Y. Kalolikar
a
, S. K. Gade
a
, L. S. Mohite
c
a
Antitoxins & Sera Dept., Haffkine Biopharmaceutical Corporation Limited, Pune, India
b
Dept. of Microbiology, University of Pune, Pune, India
c
Research & Development Dept., Haffkine Biopharmaceutical Corporation Limited, Mumbai, India
Abstract
Rabies is still a cause of global concern, particularly in the developing countries. The treatment for post exposure prophylaxis is a combination of
administration of vaccine and specific antiserum. Although equine antirabies immunoglobulin is available, the only approved method by World
Health Organization for its testing is the Virus Neutralization Test (VNT), performed using mice. Since a large number of
in-process samples are generated during antirabies immunoglobulin F(ab)
2
production., VNT is time consuming, utilizes a large number of mice,
results are prone to errors; therefore cumbersome and costly for routine testing. Hence, there is an urgent need to develop an alternative test for
screening large number of in-process samples.
In the present study an attempt has been made to evaluate the possibility of applying Enzyme Linked Immunosorbent Assay (ELISA) for
quantifying the samples, viz: Equine sera, Plasma, Purified bulk sera and Batch. A total of 4,946 samples of sixty equines were monitored by indirect
ELISA at an interval of two weeks for a period of five years. When seven equines of the above sixty were compared for VNT and indirect ELISA
during their primary phase of immunization, a good correlation was observed (r = 1.0). Based on this, the equines could be segregated into Low
responders (<50 IU/ml), Medium responders (between 50-100 IU/ml) and High responders (>100 IU/ml). This segregation of equines based on
their antibody titres proved to be very useful for initiating corrective remedial measures. On comparison of 14 Plasma samples, 31 Purified bulk sera
samples and 17 Batch samples for VNT and indirect ELISA, a good correlation (r = 0.82, 0.923 & 0.874 respectively) was observed. The sensitivity
values observed were 100 %, 100%, 90.7 % & 90.9% and specificity values were 100%, 100%, 94.7% & 100% for Equine sera, Plasma, Purified
bulk sera and Batch samples respectively. The Cohens Kappa index reflected a good agreement between the indirect ELISA test and the VNT test
for Equine sera samples (1.0), Plasma samples (1.0), Purified bulk sera samples (0.931) and Batch samples (0.874).
Thus, we conclude that indirect ELISA, which eliminates the use of a large number of mice, gives fast, yet reliable and reproducible results with
less labor than the VNT test could be used as an optimum and ethical method for screening of antibody titers of all the in-process samples during
manufacturing of antirabies serum.
2009 Elsevier B.V. All rights reserved
Keywords: Rabies antibodies, Virus Neutralization test, Indirect ELISA, Validation.
1. Introduction
Although rabies, a fatal zoonotic infection of the Central nervous System transmitted by the bite of rabid animal and capable of
infecting all mammalian species (17), the advent of molecular biology and advancements in research related to rabies, mankind has
been able to understand and control, if not eliminate it (24). Rabies invariably remains a serious, re-emerging public health problem in
many developing countries affecting over 55,000 people world wide, especially Asia where over 30,000 people succumb annually,
40% of whom are children less than 15 years of age (19,23,26). The situation seems all the more aggravated due to the reports that
rabies has been spreading from neighboring infected areas to rabies free areas like Japan, Singapore and Taiwan (23); and also
paradigm of rabies, presumed predominantly in canine, being proliferated in bats (4), raccoons, coyotes (12) and other wild animals;
thereby remaining an undeniable itch to the supposedly rabies free developed countries (24). Despite gravity of the disease, with the
help of strategic management plans (20), information sharing workshops on rabies between countries (19), Epidemiological
*Corresponding authors.
E-mail addresses: nitinvibha@yahoo.com (N. C. Salvi), writetodeopurkar@gmail.com (R. L. Deopurkar)
Available online at www.sciencedirect.com
2010 Published by Elsevier Ltd.
4 N.C. Salvi et al. / Procedia in Vaccinology 2 (2010) 311
surveillance, organized mass scale pre-exposure vaccination programs, dog population management, community participation
(25, 30), and WHO guidelines (27) and proper post exposure prophylaxis comprising of rabies vaccine and rabies immunoglobulins
(RIG), it can not only be effectively controlled but gradually eliminated.
Considering the magnitude of the disease, and the indispensability of RIG in the post exposure regimen for WHO category 3
exposures which constitute about 60% of all cases, the requirement of RIG at the global level annually is estimated to be over nine
million vials per year and of the two RIGs currently available, the burden to fulfill this demand falls on Equine Rabies
Immunoglobulin (ERIG), rather than Human Rabies Immunoglobulins (HRIG), (9, 13, 28). Whether HRIG or ERIG, there are only
two WHO approved methods for testing the potency of rabies antibodies, Virus Neutralization Test using mice (VNT) and Rapid
Fluorescent Focus-Inhibition Test (RFFIT) in cell cultures; but VNT is more widely used although both require Challenge Virus
Strain (CVS) in their testing methodologies (29). Due to the potential hazards involved in working with CVS and the fact that VNT
using mice is extremely laborious, time consuming, results obtained are not perfectly reproducible because of biological variations in
strains, and not of least concern is the utilization of large number of animals. During production of Antirabies serum (ARS) using
equines, a large number of in-process samples are generated to be tested for knowing the potency of rabies antibodies, a need for an
alternative test like ELISA has always been felt. Moreover this need for an alternative test complies with the three R concepts of
Reduction, Refinement and Replacement in relation to animal experimentation which is the principle aim of the European Centre for
the Validation of Alternative Methods (ECVAM), (3, 6, 11). This communication focuses on the Replacement aspect of the three
Rs.
In the present study an attempt was made to standardize indirect ELISA for quantification of antirabies antibodies in equines used
for antirabies serum production and secondly, explore the possibility of using indirect ELISA for routine use in place of Virus
Neutralization Test (VNT) as an alternative test.
2. Materials and methods
2.1. Animal selection and immunization
Under the Equideae family, Horses, Mules and Ponies are generally used for immunization during production of antiserum. In
the current experiments, all the above mentioned three categories, viz: Horses, Mules and Ponies were selected for immunization
during production of antirabies serum. For the sake of convienience the words Equines or Horses are used alternatively and
signify Horses, Mules or Ponies.

Table 1: Protocol used for raising of antirabies immunoglobulins and monitoring of titre during primary immunization in equines.

Time(weeks) Total dose
a
(ml) SC
b
Mean ELISA OD
c
Mean VNT Titre
c

0 .0 ml antigen + 1.0 ml CFA adjuvant --
3 1.0 ml antigen + 1.0 ml ISA 206 adjuvant 0.176 + 0.015 18.71 + 1.15
6 2.0 ml antigen + 2.0 ml ISA 206 adjuvant 0.240 + 0.032
12 1.0 ml antigen (Plain Dose) 0.429 + 0.027
14 1.0 ml antigen (Plain Dose) 0.529 + 0.026 51.5 7+ 0.65
19 2.5 ml antigen + 2.5 ml ISA 206 adjuvant 0.697 + 0.026 66 + 0.63
25 1.0 ml antigen (Plain Dose) 0.890 + 0.037 85.71 + 1.13
27 1.0 ml antigen (Plain Dose)

0.952 + 0.040

1.064 + 0.048 100.71 + 1.60

a
Total dose (ml). Antigen used: commercially available vaccine like Rabipur
(R)
. Dose includes equal quantity of antigen and adjuvant,
administered at 3 week intervals. Plain dose was without adjuvant, administered at 2 week intervals.
b
SC, Subcutaneous injections.
c
OD of indirect ELISA of seven horses were monitored in duplicate, with samples withdrawn after 10 days after the dose administration and expressed as
Mean OD + SEM (n = 14 ). Virus Neutralization Titres of the same seven horses were monitored at 3
rd
, 9
th
, 14
th
, 19
th
, 25
th
, 29
th
weeks and mean neutralization
are presented in IU / ml of serum + SEM (n = 14 ).
Corelation considered is VNT titre: 100 IU / ml = ELISA O.D. 1.0. All the values were found to be statistically significant (p< 0.05).

Time(weeks) Total dose
a
(ml) SC
b
Mean ELISA OD
c
Mean VNT Titre
c

0 .0 ml antigen + 1.0 ml CFA adjuvant --
19 2.5 ml antigen + 2.5 ml ISA 206 adjuvant 0.697 + 0.026 66 + 0.63

0.952 + 0.040

1.064 + 0.048
0.429 + 0.027
0.529 + 0.026
0.890 + 0.037
0.952 + 0.040
1.064 + 0.048
--
Mean ELISA OD
c
Mean VNT Titre
c

51.5 7+ 0.65
85.71 + 1.13

100.71 + 1.60
2.0 ml antigen + 2.0 ml ISA 206 adjuvant
N.C. Salvi et al. / Procedia in Vaccinology 2 (2010) 311 5
A total of 60 equines under antirabies serum production were included in the study. Of these 27 equines were already in the
secondary phase of immunization when the study commenced. Subsequently 33 new equines inducted for antirabies serum production
were also included in the study. All these horses were fit and healthy and did not posses any external wounds and signs of contagious/
infectious diseases or any permanent physical deformity. The newly received equines were kept in designated and isolated stables
under quarantine for observation for a period of minimum twenty one days before experimentation. During the quarantine period
equines were checked for physical examination temperature, weight as well as for hematological and biochemical values estimations.
Prophylactic vaccination was carried out with the Tetanus toxoid (5.0 ml of 25 lf / ml) on eighth day of quarantine and on 21
st
day
(10 ml). The absence of Glanders in horses was also confirmed by Mallein Antigen inoculation during quarantine period. The
average body weight of equines ranged between 250 500 Kg. Montanide group of adjuvants were obtained from Seppic, France;
while Complete Freunds adjuvant (CFA) was purchased from Difco, USA.
The proportion of Rabipur
(Chiron Behring Vaccines Pvt. Ltd.) and adjuvant (CFA / Montanide ISA 206) was 1:1 (v/v). The
equines were immunized with rabipur vaccine with or without adjuvants, subcutaneously (s.c.) in the cervical region. Immunization
was carried out at an interval of three weeks for vaccine-adjuvant doses and at an interval of two weeks for plain vaccine doses. The
booster doses were administered with similar procedure only slight increments in vaccine quantity. In primary immunization phase the
equines were repeatedly administered booster doses with vaccine adjuvants until the equines blood serum exhibit required minimum
antibody level (100 IU/ml) and these were termed as responder equines. The primary phase of immunization continued for 29 weeks
as depicted in Table 1.
In the secondary immunization phase, the responder equines were untaken for bleeding cycle to obtain plasma as raw material for
making antirabies serum. Once a month, a 3.0 ml plain intramuscular dose or a 6.0 ml intramuscular dose consisting of 3.0 ml of
vaccine plus 3.0 ml of adjuvant (Montanide ISA 206) was administered and after two weeks blood was withdrawn from the animal at
the proportion of 1.5% v/w of its body weight. This sequence of immunization and bleeding constituted the secondary immunization
phase. The alternation from vaccine adjuvant doses to plain doses was done every third month to get an enhanced titre of antirabies
antibodies. The cycle was continued till horses showed satisfactory level of antibody, physical condition and biochemical values
remained within acceptable limits.
In the current study four types of samples were withdrawn during different stages of antirabies serum production, viz: Horse Sera,
Plasma, Purified Bulk Sera, and Batch Sera containing the Finished Product. The horse sera samples were withdrawn at an
interval of every 15 days corresponding to immunization and the bleeding activities which occurred usually once a month. Plasma
samples were collected when a lot of pooled plasma was undertaken for purification of sera. During purification of serum from
plasma, after the ultrafiltration step, purified bulk sera samples were withdrawn. A total of 4946 equine sera samples were collected
over a span of five years and tested by indirect ELISA test. Sera of seven equines during their primary phase of immunization were
tested for VNT as well as indirect ELISA tests. Similarly, 14, 31 and 17 samples of Plasma, Purified bulk sera and batch sera
respectively were collected throughout the study and tested for VNT as well as indirect ELISA tests.
2.2. Virus Neutralization Test
The Virus Neutralization Test (VNT), was carried out as per the testing procedure laid down in the WHO Manual "Laboratory
Techniques in rabies" (29). Interpretation was done using statistical method of Spearman-Karber. Samples were assayed along with
National Reference Standard Antirabies Sera of 90 IU/ml, supplied by Central Drugs Laboratory (CDL), Himachal Pradesh, India.
Based on the neutralizing dose 50 (ND
50
) values of Reference Standard & the sera samples, the potency or the amount of neutralizing
antibodies were estimated.
The sera to be tested were inactivated at 56
0
C for 30 mins. Two-fold serial dilutions of each serum sample to be tested and the
reference standard serum were prepared using sterile Normal saline(NSS). A challenge virus dose (CVD) using CVS of known titre
was prepared. Equal quantity (0.5 ml) of CVD was mixed with each dilution of the serum. The serum virus mixture was incubated at
37
0
C for one hour. The actual quantity of virus used in the test was determined by mixing equal quantity (0.5 ml) of CVD and three
subsequent 10-fold dilutions with the diluent. The reaction mixture containing 0.03 ml dose was inoculated intracerebrally in 10 mice
(each weighing 11-14 gms.) and the results were monitored until the 14
th
day. Neutralization Dose (ND
50
) for sera samples and Lethal
dose (LD
50
) for virus control, were calculated using Reed & Meunch Method (21). IU /ml of sera was calculated as follows:
Potency (IU/ ml) = ND
50
of Sample x Potency of Ref. serum
ND
50
of Ref. serum
2.3. Indirect ELISA Test
The indirect ELISA was performed as per the method described in Antibodies Manual (1).Before the samples could be tested by
indirect ELISA; checker board standardization (5) was performed for equine serum, plasma, purified bulk serum and batch serum
using a known positive control whose VNT value was predetermined. The negative controls were the corresponding equivalents of
Antisnake Venom Serum differing only in the specific neutralizing antibodies. Based on the Checker Board Titrations performed,
for all the types of samples to be tested, the antigen dilution and the primary antibody (sera samples) dilution was 1:1000, the
secondary antibody dilution was 1:20,000 and 100 IU / ml, 100 IU / ml, 800 IU / ml, & 300 IU / ml, corresponded with 1.0 O.D., 1.2
O.D. 1.0 O.D., 1.0 O.D. for equine sera, plasma, purified bulk sera and batch samples respectively.
Briefly, 96 well microplates (Nunc-ImmunoplateMaxisorp USA) were coated with 100 l of rabies antigen (Rabipur vaccine,
manufactured by Chiron B , USA) diluted in phosphate buffered saline (pH 7.4) and kept overnight at 4
0
C. After four washings with
100 l /well phosphate buffered saline containing 0.1% Tween 80 (PBST), the plates were treated with 100 l / well of blocking
solution (1%Bovine Serum Albumin) for 1 hr at 37 C. After four washings with PBST, 100 l of sera to be tested (1:500) were
added to duplicate wells and incubated for 30min at room temperature. The plates were again washed with PBST and incubated with
100 l / well of a 1:20,000 dilution of anti-horse IgG peroxidase conjugate (Sigma USA.) for 30min at 37 C. After four washings
with PBST, 100 l of substrate (3, 3, 5, 5-Tetramethyl-benzidine Sigma 10 mg; 0.003% H
2
O
2
) in citrate buffer, 0.5 M, pH 5.0 was
added to each well. Plates were then incubated for 15min at 37 C, when the reaction was terminated by adding 100 l / well of 1N
HCL. The absorbance was measured in an ELISA plate reader (Multiskan, Titertek) at 450 nm.
(A) High Responding Horses
0
0.5
1
1.5
2
2.5
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126
O
.
D
.

4
5
0

n
m
(B) Medium Responding Horses
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127
O
.
D
.

4
5
0

n
m
(C) Low Ressponding Horses
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126
Period (Weeks)
O
.
D
.

4
5
0

n
m
Fig. 1: Based on the comparison of the mean ELISA O.D. values and mean VNT titre during
immunization of horses as given in Table 1, they could be segregated into High Responders
(O.D. > 1.0), Medium Responders (O.D. between 0.5 1.0) and Low Responders (O.D. < 0.5)
signifying their antibody conversion ability.
2.4. Statistical analysis
All the statistical analysis was performed using Graphpad Prism 5 software and Medcalc version10 software.
3. Results and discussion
During the production of antirabies serum using equines, one of the main important aspects is testing of the numerous in-
process samples generated right from immunization of equines till the release of finished product. A need for a simple, fast,
sensitive, reproducible and a reliable test like ELISA has been envisaged despite the fact that only VNT and RFFIT confirm the
presence of neutralizing antibodies. But before using any test routinely it has to be standardized and validated and hence, in the
present study, an attempt was made to explore this possibility.
The VNT widely used by the manufacturers of antirabies serum utilizes a large number of mice. About 144 mice are required
to test one sample including 60 mice for reference serum and 24 mice for virus control and not more than three samples are
advisable per test. Utilization of these many mice is unethical and also adds to a sizeable cost factor thereby having an effect on
the productivity. In case of ELISA, considerable work has been done to quantify antibodies by ELISA (14, 15, 22) and more
specifically rabies antibodies (2, 7, 10, 16).
HORSE SERA
1.4 1.6 1.8 2.0 2.2
-0.5
0.0
0.5
1.0
1.5
Log. Potency
O
.
D
.

(
5
4
0

n
m
)
PLASMA
1.8 1.9 2.0 2.1 2.2 2.3 2.4
1.0
1.2
1.4
1.6
1.8
Log. Potency
O
.
D
.

(
5
4
0

n
m
)
PURIFIED SERA
2.4 2.6 2.8 3.0 3.2 3.4
0.0
0.5
1.0
1.5
2.0
Log. Potency
O
.
D
.

(
5
4
0

n
m
)
FINAL SERA
1.5 2.0 2.5 3.0
-0.5
0.0
0.5
1.0
1.5
Log. Potency
O
.
D
.

(
5
4
0

n
m
)
Fig. 2. Regression analysis was performed on the above four types of samples and based on this regression equation as given below was based
on the ELISA O.D. (X value), corresponding VNT Titre (Y value) can be estimated.
1) Horse Sera : Y = -1.4382 + 1.1961 X
2) Plasma : Y = -0.6839 + 0.9612 X
3) Purified Sera : Y = -5.1721 + 2.1443 X
4) Final batch : Y = -1.3250 + 0.9029 X
During primary immunization, seven horses were selected and monitored for their antibody response against rabipur vaccine.
The samples were tested by indirect ELISA method as well VNT. Although indirect ELISA was performed on these samples of
seven equines in duplicate, every time an antigen dose was administered, VNT was performed on the samples in duplicate, only
on those withdrawn at 3
rd
, 9
th
, 14
th
, 19
th
, 25
th
and 29
th
weeks. The mean ELISA optical density (O.D.) and the mean VNT titers as
depicted in Table 1, reflect a perfect correlation. (r=1). Based on the above correlation and depending on the ELISA readings of
the 4,946 equine sera samples, a profile of immune response reflected by the ability produce antibodies by each individual horse
could be adjudged and subsequently it was possible to segregated them in to three categories, viz: Low Responders, Medium
Responders and High Responders; the ELISA O. D. being < 0.5, between 0.5 1.0 and > 1.0 respectively (Fig. 1) and 0.5 O D.
was correlated with 50 IU / ml.. This proved to be a very important tool because now corrective actions like regrouping the
equines, reorganizing their immunization schedule and improvising the antigenic dose could be thought of for deserving horses.
Of the 60 equines studied, 34, 16 and 10 equines reflected a consistent titer >100 IU / ml, between 50-100 IU / ml and <50 IU / ml
respectively. Based on our experience, during immunization, generally 10- 15 % of the equines dont exhibit expected response in
terms of antibody titer. Horse nos. 246 & 611 were Late Responders, but by 86
th
week and 98
th
week respectively, they were in
the category of Medium Responders. Similarly, Horse no. 339 which was a Medium Responder achieved High Responder
status by 88
th
week and horse nos. 616 and 620 also Medium Responders became High Responders by 66
th
week. In antirabies
serum production, it is not advisable to maintain a fixed immunization schedule, because antibody response being a function of
many factors, a lot of individual variations are observed and during purification of plasma to obtain purified F(ab)
2
antibodies
plasma of various horses are pooled together and hence group dynamics come into play, so therefore the antibody titer of horses
have to be monitored on a continuous basis to ensure that maximum number of equines remain under the High Responder
category.
The coefficients of correlation between the indirect ELISA values and the VNT titers for horse sera samples reached unity
(r = 1.0, p < 0.005) and were satisfactory incase of plasma samples (r = 0.82, p < 0.005), purified sera (r = 0.923, p < 0.005) as
well as for final batch sera (r = 0.874, p < 0.005) (Fig. 2 & Table 2). These results supported the earlier work related to
quantification of antibodies to rabies virus for which VNT using indirect immunoperoxidase technique was validated and
standardized (18).
Table 2: Summary of statistical parameters assessed during validation of Indirect ELISA
__________________________________________________________________
Sample Sensitivity Specificity Inter-rater Correlation (r)
Reliability index
_________________________________________________________________
Horse Sera 100 100 1.0 1.0
Plasma 100 100 1.0 0.82
Purified Sera 90.7 94.7 0.931 0.923
Final Batch 90.9 100 0.933 0.874
__________________________________________________________________
Horse Sera Samples showed excellent results for Sensitivity, Specificity, Inter-rater
reliability index (Cohens Kappa value), whereas all the other three types of samples,
viz.: Plasma, Purified Sera and Final Batch Sera showed satisfactory results
The receiver-operator Characteristic (ROC) curve analysis is an important part while performing validation and standardization
of ELISA test (8). It is a graphical plot of sensitivity versus 1-specificity which ascertains the sensitivity and specificity of
indirect ELISA in relation to VNT for all the different types of samples tested. The sensitivity and specificity values were 100%
for equine sera, plasma, whereas for purified sera sensitivity was 90.7 % and specificity was 94.7 % and for batch samples
sensitivity was 90.9 % and specificity was 100 % (Fig. 3 and Table 2).
The samples of plasma, purified sera and batch sera were analyzed repeatedly to determine the consistency of indirect ELISA
test. The mean + SEM value for all the plasma samples was 1.284 +0.022 (for n = 14) and each sample was analyzed at least 10
times. Similarly, the mean + SEM value for all the purified sera samples was 0.967 + 0.027 (for n = 31) and each sample was
analyzed at least 8 times. Lastly, the mean + SEM value for all the batch sera samples was 0.853 + 0.029 (for n = 17) and each
sample was analyzed at least 11 times (Fig. 4). The mean values for plasma correlated satisfactorily with the initial value
considered for standardization of indirect ELISA, where 100 IU / ml of VNT titer was considered equivalent to 1.2 O. D. by
indirect ELISA (r = 0.82, p < 0.005). Similarly, the mean values for purified sera correlated satisfactorily with the initial values
considered for standardization of indirect ELISA, where 800 IU / ml of VNT titer was considered equivalent to 1.0 O. D. by
indirect ELISA (r = 0.923, p < 0.005). Lastly, for batch sera correlation observed was also satisfactory, where 300 IU / ml of VNT
titer was considered equivalent to 1.0 O. D. by indirect ELISA (r = 0.874, p < 0.005). Incase of batch sera along with the specified
potency (300 IU / ml) of the finished product, some finished product samples of different manufacturer with an altered potency
(200 IU / ml) were also considered in the study.
The comparative study between indirect ELISA and VNT showed a very good agreement and the index for inter-rater
reliability as calculated by Cohens Kappa were 1.0, 1.0, 0.931 and 0.933 for equine sera, plasma, purified sera and batch sera
respectively; where a value >0.7 is considered satisfactory (Table 2).
In conclusion, indirect ELISA proves to be an excellent tool to replace VNT during antirabies production. Considering the
ethical issues involved in utilizing a large number of mice for the VNT and the related productivity challenges faced by the
manufacturers, initiatives can be taken by WHO to conduct further trails of this test with manufacturers of antirabies serum
globally to think of possible replacement of VNT by indirect ELISA.
HORSE SERA
0 20 40 60 80 100
0
50
100
150
Sensitivity%
Identity%
100% - Specificity%
S
e
n
s
i
t
i
v
i
t
y
PLASMA
0 20 40 60 80 100
0
50
100
150
Sensitivity%
Identity%
100% - Specificity%
S
e
n
s
i
t
i
v
i
t
y
PURIFIED SERA
0 50 100 150
0
50
100
150
Sensitivity%
Identity%
100% - Specificity%
S
e
n
s
i
t
i
v
i
t
y
BATCH SERA
0 20 40 60 80 100
0
50
100
150
Sensitivity%
Identity%
100% - Specificity%
S
e
n
s
i
t
i
v
i
t
y
Fig. 3 Sensitivity and Specificity the two very important aspects to validate the Indirect ELISA
samples, viz: Horse Sera, Plasma, Purified Sera and Batch Sera was one at p < 0.005.
test were ascertained using the ROC curve analysis. The area under the curve for all the types of
PLASMA
0
0.5
1
1.5
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
No. of ELISA Readings
O
D

(
4
5
0

n
m
)
PURIFIED SERA
0
0.5
1
1.5
2
1 2 3 4 5 6 7 8 9 10 11 12
o
.
d

(
4
5
0

n
m
)
FINAL BATCH
0
0.5
1
1.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
O
.
D

(
4
5
0

n
m
)
Fig. 4: Plasma samples, Purified sera samples and Final Batch samples were tested repeatedly
for Indirect ELISA test to confirm the repeatability of the test. (n = 14, 31 and 17 for plasma,
purified bulk sera and batch sera respectively).
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Procedia in Vaccinology 2 (2010) 311

1877-282X/$see front matter 2010 Elsevier B.V. All rights reserved.

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