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Sensitive ELISA tests for detection of anti-hepatitis A virus antibodies
Serodiagnosis & Imnlunotherapy IN I N F E C T I O U S
ELSEVIER
DISEAaE
Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65
Letter to the Editor
Sensitive ELISA tests for detection of anti-hepatitis A virus antibodies S.D. Chitambar, M.S. Joshi, V.A. Arankalle, K. Banerjee National Institute of Virology, 20.A, Dr. Ambedkar Road, P.O. Box No, 11, P~me-411001, India
Received 8 September 1995; accepted 30 October 1995
Serodiagnosis of hepatitis A infection involves detection of hepatitis A virus-specific IgM and IgG antibodies. For the accurate detection and measurement of these antibodies, sensitive immunoassays are appropriate methods. We describe here indigenously developed enzyme-linked immunosorbant assay (ELISA) tests for the detection of total anti-HAV antibodies and evaluate their performance in comparison with HAVAB RIA or EIA test of Abbott Laboratories, USA. HAV and anti-HAV IgGs were derived by procedures described earlier [1]. Test sera were collected from healthy subjects, hepatitis A patients and from a rhesus monkey prior to infection with HAV and thereafter on alternate days for determination of serostatus. The blocking assay procedure involved reactivity of test serum alone with HAV captured on anti-HAV antibody bound to the solid phase, followed by probing with antiHAV IgG-horseradish peroxidase (HRP) conjugate. A competitive inhibition assay was based on the competitive binding of specific antibody in the test sample and enzyme-labeled antibody in a ratio of 1:1 to the antigen captured on anti-HAV antibody bound to the solid phase [2]. The optimum concentrations of capture and conjugated antibodies and antigen in blocking and competitive inhibition assays were determined in the checkerboard assay. HAVAB RIA/EIA was performed for comparison according to the protocol furnished in the kit [2,3]. The cut off value was determined as (mean optical density (OD) of negative controls + mean OD of positive controls)/2 and was thus equivalent to 50% neutralization. The percentage of blocking or neutralization was calculated according to the formula described by Safford et al. [3]. For titration of sera, specimens were serially diluted in 0.05 M phosphate buffered saline (PBS), pH 7.2. Titration of anti-HAV IgM antibodies was performed by anti-HAV IgM capture ELISA [1]. 0888-0786/96/$15.00 © 1996 ElsevierScience B,V. All rights reserved SSD! 0888-0786(95)01053-B
Pre- and post-infection sera of a rhesus monkey infected with HAV were tested for seroconversion to antiHAV antibodies. By HAVAB EIA, blocking and competitive inhibition assays, seroconversion was demonstrated on post-inoculation day (PID) 31. Thus, the specificity of blocking and competitive inhibition assays was comparable to that of HAVAB EIA. The performance of blocking and competitive inhibition assays was compared with HAVAB RIA by employing three sets of sera which included RIA strong positives (N/S ratio > I0), weak positives (N/S ratio < 10) and anti-HAV negatives (N/S ratio <2). The resuits are summarized in Fig. 1. The blocking and competitive inhibition assays could identify all the HAVAB RIA strong (22) and weak (13) positives. In addition, 2/19 and 6/19 samples negative in the HAVAB RIA were scored positive by competitive and blocking assay, respectively. The higher sensitivity of the blocking assay seems to be due to elimination of the dilution factor (1:20) of HAVAB tests. Retesting of six blocking ELISA positive/HAVAB RIA negative samples at the dilution of 1:20 gave negative results. For the evaluation of recently developed hepatitis A vaccines, immunoassays have been modified or developed to yield more sensitive test systems [4,5]. In view of this, the blocking assay may be a useful choice for such studies and needs to be evaluated for the same. Anti-HAV IgG antibody titres in commercial preparations of human immune serum globulins (2) and sera from seropositive healthy blood donors (5) were determined by the blocking assay. The titres were 2 to 4 times higher than those of HAVAB EIA. In the blocking assay, anti-HAV titres of 44 sera from hepatitis A patients collected between day 1 and 195 after the onset of the disease were found to be between 1:40 and 1:128 000. To determine the end point dilution, selected sera were titrated in blocking ELISA, HAVAB EIA and
64
S.D. Chitambar et aL / Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65 9013
22
SO-
Test sample with H A V A B R I A N / S ratio ;~ 1 0 :
•
Test sample with H A V A B R I A N I S ratio < 1 0 : Test sample with H A V A B R I A N / S ratio < 2 :
o q,
22 70N/S:Menn
negative control O D / S a m p l e
OD
22
60-
5013
40-
"..
, 30-
..
..
•
•
-.
: o
•
•
20-.
• •
13
.
10,
19
""
o o
.
q,
--
19
-- o
*
4.%'kq
•k,t ,~,t ,t .~~ .t .~ ,, .k.k4.
.HAVAB
RIA
Blocking ELISA
*
."
~
• •
~e
19 _ ,b
-~'
Cut off
I Competitive inhibition E L I S A
Fig. I. Comparison of N/S ratio values of anti-HAV negative and positive sere in HAVAB RIA and blocking and competitive inhibition ELISAs.
anti-HAV IgM capture ELISA. The reactivity of each assay was then analysed. A rise in anti-HAV antibody titres with respect to time after infection was significantly higher in the blocking assay than in the HAVAB EIA. Table 1 Anti-HAV antibody titres in sera from HAV-infeeted rhesus monkey and hepatitis A patients PostAnti-HAV antibody titre in inoculation/ onset day Blocking HAVAB Anti-HAV ELISA EIA IgM capture ELISA
Rhesus monkey 31 75 Patient I 7 42 2 8 29 3 !1 18 4
5
7 42 3 34 172
biT: not tested.
!:16 1:800
1:64 1:200
< I:100 1:8000 1:400 1:3200 1:!600 1:3200 1:200 1:1600 < 1:200 1:1600 1:3200
1:4000 1:1600 1:4000 1:3200 i:!000 1:6400 1:200 1:800 bit !:3200 1:3200
1:400 < 1:25 1:16000 I:1000 1:64000 1:4000 1:64000 1:256000 1:64000 hlO00 1:64000 !:16000 1:1000
Interestingly, HAVAB EIA titres were distinctly higher than blocking assay titres in acute phase/early convalescent phase sere. However, in convalescent phase sampies, HAVAB EIA titres were equivalent to or lower than the blocking assay titres. A decrease in HAVspecific IgM was shown in convalescent phase sara by anti-HAV IgM capture ELISA (Table 1). Anti-HAV IgM ELISA showed highest titres of antibodies of the IgM type. However, total anti-HAV antibody titres noted in HAVAB EIA were considerably low. The blocking assay showed affinity for IgG antibodies similar to the IAHA test described earlier by Miller et al. [6]. The selective binding of antibodies displayed in these assays could be attributed to different protocols employed in the tests, differences in the solid phase coated antigen, and/or steric hinderance in binding of IgM molecules. In conclusion, the ELISAs reported here are highly specific and sensitive and can provide useful tools for the study of the epidemiology of hepatitis A, for preand post-vaccination screening of individuals, as well as for evaluation of different hepatitis A vaccines. Acknowledgements This work was supported by the Department of Biotechnology, New Delhi. The authors are grateful to Dr. J.R. Navarange and his staff at Ajinkya Hospital
S.D. Chitambar et al./ Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65
and Dr. A n a n d Pandit a n d his staff at K E M Hospital, Pune for collection of sera from hospitalized children and Dr. Shubhada Bopegamage and Ms. Swati Aiyer for the d o n a t i o n of normal h u m a n serum (negative for anti-HAV antibodies). The technical assistance of Ms. Rekha Nair is acknowledged.
References [I] Chitambar SD, GrewalSM, Bokil M, ArankalleVA, Gore MM, Banerjee K. Indigenous anti-hepatitis A virus IgM capture ELISA for the diagnosisof hepatitis A. Indian J Med Res 1994; 99: 243-251. [2] Safford SES, Ncedleman SB, Decker RH. Radioimmunoassay for detectionof antibody to hepatitis A virus. Results of clinical evaluation. Am J Clin Path 1980; 74: 25-31.
65
[3] Decker RH, Overby LR, Ling CM, Frosner C.rG,Deinhardt F, Boggs J. Serologic studies of transmission of hepatitis A in humans. J Infect Dis 1979; 139: 74-82. [4] Miller WJ, Clark W, Humi W, Kuter B, SchofieldT, Nalin D. Sensitiveassays for hepatitis A antibodies. J Med Virol 1993;41: 201-204. [5] Hess G, Faatz E, Melchoir W, Bayer H. Analysis of immunoassaysto detect antibodies to hepatitis A virus (anti-HAV) and anti-HAV immunoglobulinM. J. Virol. Methods 1995; 51: 221-228. [6] MillerWJ, Provost PJ, McAleer WJ, Ittensohn OL, ViUarejos VM, Hilleman MR. Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologicinvestigations.Proc Soc Exp Biol Med 1975; 149: 245-261.
ELSEVIER
DISEAaE
Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65
Letter to the Editor
Sensitive ELISA tests for detection of anti-hepatitis A virus antibodies S.D. Chitambar, M.S. Joshi, V.A. Arankalle, K. Banerjee National Institute of Virology, 20.A, Dr. Ambedkar Road, P.O. Box No, 11, P~me-411001, India
Received 8 September 1995; accepted 30 October 1995
Serodiagnosis of hepatitis A infection involves detection of hepatitis A virus-specific IgM and IgG antibodies. For the accurate detection and measurement of these antibodies, sensitive immunoassays are appropriate methods. We describe here indigenously developed enzyme-linked immunosorbant assay (ELISA) tests for the detection of total anti-HAV antibodies and evaluate their performance in comparison with HAVAB RIA or EIA test of Abbott Laboratories, USA. HAV and anti-HAV IgGs were derived by procedures described earlier [1]. Test sera were collected from healthy subjects, hepatitis A patients and from a rhesus monkey prior to infection with HAV and thereafter on alternate days for determination of serostatus. The blocking assay procedure involved reactivity of test serum alone with HAV captured on anti-HAV antibody bound to the solid phase, followed by probing with antiHAV IgG-horseradish peroxidase (HRP) conjugate. A competitive inhibition assay was based on the competitive binding of specific antibody in the test sample and enzyme-labeled antibody in a ratio of 1:1 to the antigen captured on anti-HAV antibody bound to the solid phase [2]. The optimum concentrations of capture and conjugated antibodies and antigen in blocking and competitive inhibition assays were determined in the checkerboard assay. HAVAB RIA/EIA was performed for comparison according to the protocol furnished in the kit [2,3]. The cut off value was determined as (mean optical density (OD) of negative controls + mean OD of positive controls)/2 and was thus equivalent to 50% neutralization. The percentage of blocking or neutralization was calculated according to the formula described by Safford et al. [3]. For titration of sera, specimens were serially diluted in 0.05 M phosphate buffered saline (PBS), pH 7.2. Titration of anti-HAV IgM antibodies was performed by anti-HAV IgM capture ELISA [1]. 0888-0786/96/$15.00 © 1996 ElsevierScience B,V. All rights reserved SSD! 0888-0786(95)01053-B
Pre- and post-infection sera of a rhesus monkey infected with HAV were tested for seroconversion to antiHAV antibodies. By HAVAB EIA, blocking and competitive inhibition assays, seroconversion was demonstrated on post-inoculation day (PID) 31. Thus, the specificity of blocking and competitive inhibition assays was comparable to that of HAVAB EIA. The performance of blocking and competitive inhibition assays was compared with HAVAB RIA by employing three sets of sera which included RIA strong positives (N/S ratio > I0), weak positives (N/S ratio < 10) and anti-HAV negatives (N/S ratio <2). The resuits are summarized in Fig. 1. The blocking and competitive inhibition assays could identify all the HAVAB RIA strong (22) and weak (13) positives. In addition, 2/19 and 6/19 samples negative in the HAVAB RIA were scored positive by competitive and blocking assay, respectively. The higher sensitivity of the blocking assay seems to be due to elimination of the dilution factor (1:20) of HAVAB tests. Retesting of six blocking ELISA positive/HAVAB RIA negative samples at the dilution of 1:20 gave negative results. For the evaluation of recently developed hepatitis A vaccines, immunoassays have been modified or developed to yield more sensitive test systems [4,5]. In view of this, the blocking assay may be a useful choice for such studies and needs to be evaluated for the same. Anti-HAV IgG antibody titres in commercial preparations of human immune serum globulins (2) and sera from seropositive healthy blood donors (5) were determined by the blocking assay. The titres were 2 to 4 times higher than those of HAVAB EIA. In the blocking assay, anti-HAV titres of 44 sera from hepatitis A patients collected between day 1 and 195 after the onset of the disease were found to be between 1:40 and 1:128 000. To determine the end point dilution, selected sera were titrated in blocking ELISA, HAVAB EIA and
64
S.D. Chitambar et aL / Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65 9013
22
SO-
Test sample with H A V A B R I A N / S ratio ;~ 1 0 :
•
Test sample with H A V A B R I A N I S ratio < 1 0 : Test sample with H A V A B R I A N / S ratio < 2 :
o q,
22 70N/S:Menn
negative control O D / S a m p l e
OD
22
60-
5013
40-
"..
, 30-
..
..
•
•
-.
: o
•
•
20-.
• •
13
.
10,
19
""
o o
.
q,
--
19
-- o
*
4.%'kq
•k,t ,~,t ,t .~~ .t .~ ,, .k.k4.
.HAVAB
RIA
Blocking ELISA
*
."
~
• •
~e
19 _ ,b
-~'
Cut off
I Competitive inhibition E L I S A
Fig. I. Comparison of N/S ratio values of anti-HAV negative and positive sere in HAVAB RIA and blocking and competitive inhibition ELISAs.
anti-HAV IgM capture ELISA. The reactivity of each assay was then analysed. A rise in anti-HAV antibody titres with respect to time after infection was significantly higher in the blocking assay than in the HAVAB EIA. Table 1 Anti-HAV antibody titres in sera from HAV-infeeted rhesus monkey and hepatitis A patients PostAnti-HAV antibody titre in inoculation/ onset day Blocking HAVAB Anti-HAV ELISA EIA IgM capture ELISA
Rhesus monkey 31 75 Patient I 7 42 2 8 29 3 !1 18 4
5
7 42 3 34 172
biT: not tested.
!:16 1:800
1:64 1:200
< I:100 1:8000 1:400 1:3200 1:!600 1:3200 1:200 1:1600 < 1:200 1:1600 1:3200
1:4000 1:1600 1:4000 1:3200 i:!000 1:6400 1:200 1:800 bit !:3200 1:3200
1:400 < 1:25 1:16000 I:1000 1:64000 1:4000 1:64000 1:256000 1:64000 hlO00 1:64000 !:16000 1:1000
Interestingly, HAVAB EIA titres were distinctly higher than blocking assay titres in acute phase/early convalescent phase sere. However, in convalescent phase sampies, HAVAB EIA titres were equivalent to or lower than the blocking assay titres. A decrease in HAVspecific IgM was shown in convalescent phase sara by anti-HAV IgM capture ELISA (Table 1). Anti-HAV IgM ELISA showed highest titres of antibodies of the IgM type. However, total anti-HAV antibody titres noted in HAVAB EIA were considerably low. The blocking assay showed affinity for IgG antibodies similar to the IAHA test described earlier by Miller et al. [6]. The selective binding of antibodies displayed in these assays could be attributed to different protocols employed in the tests, differences in the solid phase coated antigen, and/or steric hinderance in binding of IgM molecules. In conclusion, the ELISAs reported here are highly specific and sensitive and can provide useful tools for the study of the epidemiology of hepatitis A, for preand post-vaccination screening of individuals, as well as for evaluation of different hepatitis A vaccines. Acknowledgements This work was supported by the Department of Biotechnology, New Delhi. The authors are grateful to Dr. J.R. Navarange and his staff at Ajinkya Hospital
S.D. Chitambar et al./ Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 63-65
and Dr. A n a n d Pandit a n d his staff at K E M Hospital, Pune for collection of sera from hospitalized children and Dr. Shubhada Bopegamage and Ms. Swati Aiyer for the d o n a t i o n of normal h u m a n serum (negative for anti-HAV antibodies). The technical assistance of Ms. Rekha Nair is acknowledged.
References [I] Chitambar SD, GrewalSM, Bokil M, ArankalleVA, Gore MM, Banerjee K. Indigenous anti-hepatitis A virus IgM capture ELISA for the diagnosisof hepatitis A. Indian J Med Res 1994; 99: 243-251. [2] Safford SES, Ncedleman SB, Decker RH. Radioimmunoassay for detectionof antibody to hepatitis A virus. Results of clinical evaluation. Am J Clin Path 1980; 74: 25-31.
65
[3] Decker RH, Overby LR, Ling CM, Frosner C.rG,Deinhardt F, Boggs J. Serologic studies of transmission of hepatitis A in humans. J Infect Dis 1979; 139: 74-82. [4] Miller WJ, Clark W, Humi W, Kuter B, SchofieldT, Nalin D. Sensitiveassays for hepatitis A antibodies. J Med Virol 1993;41: 201-204. [5] Hess G, Faatz E, Melchoir W, Bayer H. Analysis of immunoassaysto detect antibodies to hepatitis A virus (anti-HAV) and anti-HAV immunoglobulinM. J. Virol. Methods 1995; 51: 221-228. [6] MillerWJ, Provost PJ, McAleer WJ, Ittensohn OL, ViUarejos VM, Hilleman MR. Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologicinvestigations.Proc Soc Exp Biol Med 1975; 149: 245-261.