Vol. 9, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1979, p. 459-465 0095-1 137/79/04-0459/07$02.00/0
Solid-Phase Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A-Specific Immunoglobulin M STEPHEN A. LOCARNINI,* ANTHONY G. COULEPIS, ANN M. STRATTON, JAKOV KALDOR, AND IAN D. GUST Virus Laboratory, Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield, Victoria, 3078, Australia Received for publication 5 December 1978
A solid-phase enzyme-linked immunosorbent assay was developed for the detection of immunoglobulin M antibody to hepatitis A virus. The system was capable of detecting hepatitis A-specific immunoglobulin M in a single dilution of serum and appears to be a reliable and rapid means of establishing a diagnosis of hepatitis A infection. Specific immunoglobulin M was only detected in patients with serologically confirmed hepatitis A and not in patients with other forms of hepatitis, chronic liver disease, or autoimmune disease. In patients with hepatitis A, specific immunoglobulin M was usually detectable for 6 weeks after the onset of dark urine, and the longest period for which it was present in any patient was 115 days. This enzyme-linked immunosorbent assay is rapid, simple to perform, and does not require complicated equipment. Provided adequate supplies of purified reagents can be obtained, this enzyme-linked immunosorbent assay procedure is likely to simplify hepatitis A serology, because the same antibodycoated plates can be utilized to detect hepatitis A virus, anti-hepatitis A virus, and hepatitis A-specific immunoglobulin M.
The visualization of the hepatitis A virus (HAV) by Feinstone et al. (5) in 1973 has been followed by the rapid development of a number of methods for detecting both the virus and antibody developed to it (anti-HAV). The methods described to date, immune electron microscopy (IEM) (3, 5, 11, 13), complement fixation (20), immune adherence hemagglutination (16, 17), solid-phase radioimmunoassay (SPRIA) (7, 22), and enzyme-linked immunosorbent assay (ELISA) (12, 15), have been utilized to study both naturally and experimentally acquired hepatitis A. Acute infections with HAV can be confirmed either by detecting virus in fecal specimens collected during the late incubation period and acute phase of the illness, or by demonstrating a significant rise in anti-HAV in paired serum specimens collected early in the illness and during the convalescent phase. Because many patients have ceased shedding virus before admission to a hospital (4, 12, 13), and several weeks may be required before a significant rise in antiHAV can be detected between paired sera (3, 10, 15-17, 20, 22, 23), laboratory confirmation of hepatitis A infection is often delayed. Recently, we reported (10, 11) the detection of hepatitis A-specific immunoglobulin M (IgM) by IEM and SPRIA in serum specimens from patients with acute hepatitis A and established
its value for the confirmation of infection with HAV. The method used involved fractionation of serum specimens by sucrose density gradient ultracentrifugation and was hence of limited value to most clinical laboratories. This communication reports the successful adaptation of the widely available ELISA technique for the detection of hepatitis A-specific IgM. SUBJECTS AND METHODS Patients. The five groups of subjects studied were: (group I) 18 patients with acute hepatitis A; (group II) 10 patients with acute hepatitis B; (group III) 5 patients with non-A, non-B hepatitis; (group IV) 10 patients with infectious mononucleosis; and (group V) 28 patients with autoimmune disease (with and without accompanying liver disease). Groups I to IV were patients admitted to Fairfield Hospital for Communicable Diseases, Melbourne, Australia, between October 1973 and December 1977. Several serum specimens, including paired acute and convalescent sera, were collected from each patient and stored at -20°C until tested. Group V were patients admitted to the Alfred Hospital, Melbourne, between October 1977 and January 1978. A single serum specimen was collected from each patient. In all cases, the diagnoses were made by specialist physicians on the basis of clinical assessment aided by laboratory tests. A patient was regarded as having hepatitis A if HAV was detected in fecal specimens collected early in the illness and/or a rising titer of anti-HAV was 459
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demonstrated in paired sera (13). Tests for detecting a human IgG immunoadsorbent. The immunoadsorbHAV and anti-HAV were performed by SPRIA as ent polymer was prepared by cross-linking purified previously described (8, 10). These paired sera were human IgG (Commonwealth Serum Laboratories; also tested for hepatitis B surface antigen and antibody Cohn fraction V further purified by ion-exchange chroby SPRIA, using commercially available reagents matography as described above) with glutaraldehyde (Ausria II and Ausab respectively, Abbott Laborato- (Taab, Reading, England), using the method of Avrameas and Ternyck (1). The specificity of the absorbed ries, North Chicago, Ill.). All sera were negative. A diagnosis of hepatitis B was made if hepatitis B antisera was checked by double immunodiffusion in surface antigen was present in acute phase sera but gel and by immunoelectrophoresis against purified absent from sera collected during the convalescent human IgG, IgM, and IgA, as well as against human phase of the illness. Hepatitis B surface antigen was whole serum (Behringwerke Laboratories, Marburg, detected by SPRIA as described above, and all positive Germany). After absorption, the IgG component of each serum results were confirmed by specific neutralization tests. The paired sera from these ten patients were also was purified by ammonium sulfate precipitation and tested for anti-HAV by SPRIA as described above. A ion-exchange chromatography as described above, and 3.0, stationary level was detected in four patients, whereas conjugated with horseradish peroxidase (RZ the remaining six were negative for anti-HAV anti- Sigma Type VI; Sigma Chemical Co., St. Louis, Mo.) by the method of Nakane and Kawaoi (19). body. ELISA assay. The method used was modified from Patients with acute hepatitis in whom there was no serological evidence of infection with HAV, hepatitis that of Wolters et al. (28). The optimal dilution of B virus, Epstein-Barr virus, cytomegalovirus, and reagents used was determined by checkerboard titraherpes simplex virus were classified as non-A, non-B tion. The substrate was prepared by dissolving 80 mg hepatitis. Tests for infection with Epstein-Barr virus, of 5 amino-salicylic acid (Merck, Munich, Germany) cytomegalovirus, and herpes simplex virus were per- in 100 ml of hot distilled water, the pH was adjusted to 6.0 with 1 N NaOH, and then nine parts were added formed by standard techniques. Infectious mononucleosis was diagnosed when the to one part of 0.05% (vol/vol) H202 and used immedipatient's acute-phase serum contained heterophile an- ately. The wells of polyvinyl microtiter plates (Cooke tibody which could not be removed by prior adsorption with guinea pig kidney cells and/or a rising titer of Engineering Inc., Alexandria, Va.) were coated with a anti-Epstein-Barr virus was demonstrable by indirect 1:1,000 dilution of the human convalescent hepatitis A immunofluorescence (14). IgG in 0.85% (wt/vol) saline and incubated for 4 h at Tests for rheumatoid factor (RF) were performed room temperature. After washing three times with by latex agglutination (Commonwealth Serum Labo- phosphate-buffered saline (pH 7.4) containing 0.05% ratories, Melbourne, Australia) and hemagglutination (vol/vol) Tween 20 (PBS-T), the wells were filled with of sensitized sheep red blood cells (Denver Laborato- 1% (wt/vol) bovine serum albumin (Commonwealth ries, Cranbury, N.J.). Antibodies to nuclear factor, Serum Laboratories) in saline and allowed to stand at smooth muscle, and mitochondria were detected by 4°C overnight. After washing three times with PBSindirect immunofluorescence on a composite block of T, 50 ,ll of purified HAV suspended in PBS was added to each well, and the plates were incubated at 4°C for rat kidney, mouse stomach, and rabbit liver (18). Reagents. (i) Coating antibody. The antibody 16 h. After washing three times with PBS-T, 50 ,ld of used to coat the microtiter plates was obtained from the test sera, diluted 1:1,000 in PBS (pH 7.4), were an adult male 4 months after his admission to hospital added to each well and incubated at 37°C for 1 h. The with serologically confirmed hepatitis A. IgG was pu- samples were then aspirated, each well was washed six rified from 10.0 ml of the convalescent serum specimen times with PBS-T and 50 IA of peroxidase conjugate by precipitation with ammonium sulfate followed by (diluted 1:400 in PBS-T) was added, and the plates ion-exchange chromatography on diethylaminoethyl- were incubated at 37°C for 1 h. Finally, after aspirating cellulose (DE-52, Whatman, England) as described by the conjugate and washing each well six times with Purcell et al. (21). Purified IgG was stored at -20°C. PBS-T, 50 Au of substrate was added, and the plates (ii) Hepatitis A antigen. A fecal specimen and were incubated in the dark at room temperature for acute and convalescent sera were collected from two 20 min. The enzyme-substrate reaction was stopped patients with serologically confirmed hepatitis A. HAV by the addition of 50 ,ll of 1% (wt/vol) sodium azide. was detected in each crude fecal suspension by SPRIA The color produced in each well was recorded visually, and IEM as described elsewhere (9, 10, 13). No other using a 0 to 3+ scale: 0, no color reaction; 1+, a light virus-like particles were visualized by IEM. HAV was to medium color; 2+, a medium to strong color; 3+, a purified from each fecal specimen as described previ- very strong color reaction. A 1+ difference in reading ously (2, 9). Briefly, this procedure involved extraction was considered significant (12). In addition, the optical of the feces, clarification by low-speed centrifugation, density of the substrate solution was measured by pelleting of the virus, extraction with chloroform and, diluting the samples to 300 ,ul in distilled water (final finally, agarose gel filtration on Sepharose CL-2B dilution, 1:6) and reading the results in a spectrophotometer (Centrifichem System 400, Union Carbide (Pharmacia Fine Chemicals, Uppsala, Sweden). (iii) Conjugates. Anti-human IgM (mu chain spe- Corp.) at 550 nm. To measure any nonspecific binding to the IgGcific) prepared in goats was obtained commercially (Hyland, Travenol Laboratories, Melbourne) and nor- coated plates, each serum was also tested on a control mal goat serum was from Commonwealth Serum Lab- plate to which PBS-T had been added in place of the oratories. Both sera were absorbed several times with purified HAV. Substrate and conjugate controls were
also included on each microtiter plate. All testing of sera was performed under code, and each specimen was tested in duplicate.
0.5
Specific conditions. (i) Coating antibody. In preliminary tests, the purity of the IgG coating antibody was found to be critical, because traces of IgM produced unacceptably high background levels of color when reacted against the goat anti-humAn IgM conjugate. This difficulty was overcome by using IgG purified by ammonium sulfate precipitation and ionexchange chromatography. Log dilutions of purified IgG were tested to determine the optimal dilution for use. A dilution of i0' was found to provide maximum use of the material without compromising the sensitivity of the test. (ii) Hepatitis A antigen. In preliminary tests using crude or chloroform-extracted fecal specimens, nonspecific binding was found to occur which produced unacceptably high background levels. To reduce all nonspecific binding, HAV was purified until only minimal background debris was evident by direct electron microscopic examination (2, 9). Agarose gel filtration, using Sepharose CL-2B, usually yielded purified HAV suitable for the ELISA assay. (iii) Conjugates. Since commercially available anti-human IgM antisera often contain traces of antihuman IgG reactivity, immunoadsorption with the human IgG polymer before conjugation was found necessary to obtain monospecific activity. After immunoadsorption, the conjugate control wells showed no reactivity on the IgG-coated wells. The peroxidase-labeled normal goat IgG conjugate demonstrated no reactivity with any of the test sera or reagents used in the ELISA assay. (iv) Determination of cut-off point. The results of the ELISA assay were expressed as a positive/ negative (P/N) ratio, where N was the mean optical density of six negative control sera (three preinfection and three late convalescent hepatitis A sera) tested at
OD,,. 0.3-
the same dilution as the test sera (1:1,000). Using this N value, the individual P/N ratio for each control serum was calculated. The mean (1.10) plus 3 standard deviations of these six P/N values was 1.6. This value was used to signify the lowest level at which hepatitis A-specific IgM was considered present (29).
RESULTS (i) Effect of serum dilution on reactivity. Preinfection, early-acute-phase and late-convalescent sera were available from three patients with serologically confirmed hepatitis A, and decimal dilutions of each were tested for hepatitis A-specific IgM. All sera showed some nonspecific reactivity, when tested undiluted or at 1:10, which was entirely abolished at a dilution of 1:1,000 (Fig. 1). At this dilution, the acute-phase serum samples (collected 1 to 2 weeks after the onset of dark urine) had an optical density greater than 0.4, whereas the preinfection and late-convalescent-phase sera yielded values of less than 0.2. These results provided the basis for screening
461
SOLID-PHASE ELISA
VOL. 9, 1979
0.4 I
I
20min
I
I
, I
0.2
,
&
I
-
-
0.1
-.C-
:*
__r_
tb
-
-
:;:;-
I --.ri
1,000 10,000 100000 1,000,000 100 RECIPROCAL OF DILUTION
FIG. 1. Titrations of preinfection, acute, and convalescent sera from apatient with hepatitis A (patient 1, Table 1) for hepatitis A-specific IgM determinations. Symbols: A, A, preinfection serum tested against control and HAV test wells, respectively; 0, 0, acute-phase serum tested against control and test wells, respectively; O, U, convalescent serum tested against control and test wells, respectively.
all subsequent sera at 1:1,000. (ii) Detection of hepatitis A-specific IgM in serum from patients with hepatitis A. At least two serum specimens from each of 18 patients with serologically confirmed hepatitis A infection were tested for hepatitis A-specific IgM. The results of the 70 sera tested are shown in Table 1 and Fig. 2, in relation to time from the onset of illness, measured from the first day on which dark urine was observed. In three patients (patients 1 to 3, Table 1), one or more specimens had been collected before the onset of illness, and none of these were found to contain hepatitis A-specific IgM. In 10 of 11 patients, hepatitis A-specific IgM was detected within 7 days of the onset of dark urine. All 18 patients showed maximum levels of hepatitis A-specific IgM within the first 3 weeks from the onset of dark urine. Specific IgM was detected in 39 of 41 sera collected within 6 weeks of the onset of dark urine (Fig. 2). Beyond this period, its frequency of detection declined abruptly, with 2 of 5 sera collected between 43 and 63 days, 3 of 9 collected between 64 and 119 days, and 0 of 10 collected between 120 and 416 days, being positive. The longest period for which specific IgM was detected in any patient (patient 12, Table 1) was 115 days (measured to the last positive test). None of the 70 sera showed any nonspecific binding to control wells in which HAV was replaced by PBS-T. (iii) Specificity of the test. Acute and convalescent sera from 25 patients with hepatitis B, non-A, non-B hepatitis, and infectious mononucleosis (Groups II to IV) and single sera from 28 patients with liver and autoimmune disease (Group V) were also tested for hepatitis A-spe-
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LOCARNINI ET AL.
TABLE 1. Levels of hepatitis A-specific IgM in 18 patients with hepatitis A (group I) Days after onset of Hepatitis A-speDays after onset of HpttsAse Patient no. dark urine at sePatient no. dark urne at scTc IgM P/Na cic IgM P/Na 1rum colection rum-collf 10
5 67 181
P 2.30 1.37 1.06
11
8 26 43
2.06 2.33 1.60
12
2 9 115 169
3.10 2.26 1.77 1.16
13
13 20 27 31 46 66
2.73 1.90 2.03 2.03 1.06 1.09
1.00 1.73 1.81
14
3 119 178
3.33 1.00 1.10
3 8 22
2.53 2.36 2.43
15
13 17 43
2.50 1.60 1.00
6 12 64
3.11 2.37 0.94
16
6
3 102
2.63 0.88
9 16 19 37
2.76 2.60 2.83 2.53
17 7
5 8 185
3.43 3.58 1.00
7 15 29
2.53 2.06 1.00
8
18 7 86 142
2.26 1.63 1.13
10 16 38 64
3.33 3.46 2.45 1.90
9
4 47 68
3.26 1.57 1.19
1
2
3
4
5
-158 -92 6 10 17 24 34 61 305 416
1.25 1.15 1.43 2.87 3.27 2.33 2.33 1.66 0.78 0.78
-184 -81 11 22 85 156 396
1.00 0.97 1.63 1.63 1.53 0.75 0.72
-210 25 39
II
c
a P/N ratio calculated as described in the text.
cific IgM as described above (Table 2). Of the 78 tested, only one had a P/N ratio l1.6 (patient 71, Table 2). The serum specimen also reacted in the control wells (identical P/N ratio), indicating that the binding was nonspecific. The specimen had been obtained from a patient with autoimmune disease and contained RF. In the remaining 77 sera, the P/N ratios of the test and control wells were similar. (iv) Comparison between optical density estimation and visual reading. Before the sera
enzyme-substrate reaction mixture was read
spectrophotometrically, visual readings were taken. In general, there was very good correlation between the two methods, except borderline samples (1.6 P/N < 1.8) were often scored as negative when read visually. Readings of 1+, 2+, and 3+ corresponded to P/N ratios in the range of approximately 1.8 to 2.2, 2.3 to 2.8, and 2.9 to 3.58, respectively. All of the 18 patients with hepatitis A were scored positive visually on at least one serum sample. -
VOL. 9, 1979
SOLID-PHASE ELISA
.0o
30
I.
i
P 2.0
N
if
-
C PRFE
t
onset
1-7
8-14
INTERVAL
15-21
22-28 29-35 36-42
4363
64-119 120-416
FROM ONSET OF DARK URINE (DAYS)
FIG. 2. Hepatitis A-specific IgM levels in sera collected from the 18 patients with serologically confirmed hepatitis A (Table 1). Individual P/N ratios are given for each group of sera collected at intervals, including preinfection, from the onset of dark urine.
DISCUSSION ELISAs have been used for the detection of class-specific antibody to other human viruses (24, 29), and the data presented above demonstrate that this system can also be successfully adapted for the detection of hepatitis A-specific IgM. When sera are diluted 1:1,000, to minimize nonspecific binding to the immunoglobulincoated wells, IgM was detected only in the acutephase sera of all patients with hepatitis A studied. It was never detected late (greater than 120 days after the onset of dark urine) in the convalescent phase of the illness, nor was it found in patients with other forms of liver or autoimmune disease. Hepatitis A-specific IgM becomes detectable within 1 week from the onset of dark urine and tends to persist for about 7 weeks. The longest period for which it was detected in any patient was 115 days. A major problem in hepatitis A serology is specificity. The lack of availability of monospecific nonprimate hyperimmune antisera to HAV has necessitated the use of human or chimpanzee convalescent anti-HAV sera in most serological tests and, since these sera contain antibodies to other antigens commonly found in primate feces (15), false-positive results are not uncommon. Therefore, the quality of reagents used in this ELISA assay is of prime importance, i.e., the purity of the HAV preparation and the convalescent human hepatitis A IgG, and a monospecific enzyme-linked goat anti-human IgM conjugate. High background optical density and "nonspecific" reactions with control or negative sera were abolished -only when such reagents used. One pitfall with antiviral IgM tests is that they may detect an RF IgM, which will react
were
with IgG bound to the solid phase, giving a falsepositive reaction (25). These RF-positive sera can be detected readily in this assay by the incorporation of controls in which purified HAV is replaced by buffer. Since all test sera were diluted 1:1,000, it was not surprising that only one of the RF-positive sera reacted significantly (P/N 1.6) against the human IgG coating antibody control plates. In the other 17 RFpositive cases, the control wells had identical P/ N values (0.10 P/N < 1.6) to the test wells (Table 2). All these 18 sera were therefore considered negative for hepatitis A-specific IgM. Several studies of serum immunoglobulin levels in patients after acute hepatitis A infection have demonstrated a characteristic polyclonal increase of IgM or IgM hypergammaglobulinemia (6, 27). It is probable that not all of the raised serum IgM observed during acute hepatitis A is virus specific. In light of the specificity problems associated with hepatitis A serology, the role of antigens that are carried, for example, by intestinal bacteria and dietary antigens (26) in the nonspecific stimulation of the immune system after hepatitis A could warrant investigation. Until recently, laboratory confirmation of hepatitis A was largely dependent upon demonstration of rising titers of anti-HAV in adequately spaced serum samples. Detection of HAV in the feces was of less value, because many patients have stopped shedding virus by the time they are admitted to the hospital (4, 12, 13). Usually, a period of 3 to 4 weeks between paired sera is required to detect a significant rise in anti-HAV by IEM (3, 10, 13, 23), SPRIA (7, 22), ELISA (15), or immune adherence hemagglutination (16, 17, 23). There is usually a delay of 2 to 6 weeks after the onset of acute illness before anti-HAV can be detected by immune adherence hemagglutination, whereas IEM can detect anti-HAV in the acute phase of illness, but levels rise slowly before a seroconversion can be established (10, 13, 23). SPRIA and ELISA can also detect early antibody and subsequent seroconversion, but high titers of anti-HAV are usually found in acute-phase sera on admission of patients to the hospital (8, 10, 12). Recently it has been proposed that a diagnosis of hepatitis A could be made on a single acute-phase serum if IEM, SPRIA, or ELISA were positive for antiHAV and immune adherence hemagglutination was negative (15, 23). This "window" result does not appear to be useful for reliable diagnosis, since immune adherence hemagglutination responses are variable and not many laboratories would run two serological tests for anti-HAV routinely. Thus, detection of hepatitis A-specific ,
.
10
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LOCARNINI ET AL:
TABLE 2. Levels of hepatitis A-specific IgM in the control patients (groups II to V) P/Na
Group
Patient
Acute serum
Convalescent serum
II: hepatitis B
19 20 21 22 23 24 25 26 27 28
1.14 0.45 1.16 0.69 1.03 0.50 0.10 0.10 1.28 0.40
0.89 0.38 0.55 0.65 0.51 0.10 0.10 0.10 0.40 0.35
III: non-A, non-B hepatitis
29 30 31 32 33
0.10 0.10 0.40 0.75 0.20
0.10 0.40 0.40 0.30 0.40
IV: infectious mononucleosis
34 35 36 37 38 39 40 41 42 43
0.56 0.47 0.47 0.78 0.35 0.47 0.60 0.70 1.10 0.30
0.34 0.71 0.50 0.60 0.57 0.50 0.90 0.50 0.90 0.10
Patient
P/N: single seruMa
Antibody'
V(a) primary biliary cirrhosis
44 45 46 47
0.85 1.09 1.28 0.40
AMA 2+ AMA 2+ AMA 2+ AMA 2+
V(b):
48
0.38
49 50 51 52 53
1.37 0.32 0.53 0.10 0.30
SMA 2+ ANF 2+ SMA 2+ SMA 2+ SMA 2+ SMA 2+ SMA 2+
Group
chronic active hepatitis
54 0.30 0.10 55 56 0.10 0.10 57 0.10 58 0.10 59 0.10 60 61 0.63 0.46 62 63 0.46 64 0.46 65 1.32 0.25 66 67 0.25 1.19 68 0.10 69 70 1.06 1.80 71 a P/N ratios calculated as described in the text. The very low P/N ratios observed with some of the control sera reflect the fact that only six control sera from the hepatitis A group were used for the determination of the N value. b ANA, SMA, and ANF denote antibody to mitochondrial antigen, smooth muscle antigen, and nuclear factor, respectively, as detected by indirect immunofluorescence (18).
IgM in sera collected early in the illness provides reliable means of establishing a diagnosis within a few days of the patient being seen by a physician (10). To date, detection of hepatitis A-specific IgM has required prior fractionation of the serum sample on sucrose density gradients (10), which has severely limited its value as a laboratory a
test. The ELISA test described in this report
has a number of advantages over this previously described system. Antibody-coated plates can be prepared in batches and stored until they are required and large collections of sera can be tested. A diagnosis of hepatitis A infection can now be reliably established on a single acute-
V(c):
positive RF
phase serum specimen, and results could be obtained 1 day after specimen collection. In addition, because sera are tested at a dilution of 1: 1,000, as little as 1 ,ul of serum is sufficient to allow the ELISA test to be performed. Provided adequate conjugates are available, the same antibody-coated plates can be used for the detection of HAV (12), total anti-HAV (15), and hepatitis A-specific IgM, which will greatly simplify hepatitis A serology. The only limiting factor at present is an adequate supply of puri-
fied reagents. ACKNOWLEDGMENTS This work
was
supported by
grants
from the National
Health and Medical Research Council of Australia.
SOLID-PHASE ELISA
VOL. 9, 1979
We are most grateful to the medical, nursing, and laboratory staff of Fairfield Hospital for their cooperation, anid in particular to Noreen Lehmann for radioimmunoassay testing of sera for anti-HAV. We thank Jennifer Rolland, Department of Pathology and Immunology, Monash Medical School, Melbourne, for supplying the sera from patients with autoimmune and chronic liver disease. LITERATURE CITED 1. Avrameas, S., and T. Ternyck. 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6:53-66. 2. Coulepis, A. G., S. A. Locarnini, A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1978. The polypeptides of hepatitis A virus. Intervirology 10:24-31. 3. Dienstag, J. L., D. W. Ailing, and R. H. Purcell. 1976. Quantitation of antibody to hepatitis A antigen by immune electron microscopy. Infect. Immun. 13:12091213. 4. Dienstag, J. L., S. M. Feinstone, A. Z. Kapikian, R. H. Purcell, J. D. Boggs, and M. E. Conrad. 1975. Faecal shedding of hepatitis A antigen. Lancet i:765767. 5. Feinstone, S. M., A. Z. Kapikian, and R. H. Purcell. 1973. Hepatitis A: detection by immune electron microscopy of a viruslike antigen associated with acute illness. Science 182:1026-1028. 6. Giles, J. P., and S. Krugman. 1969. Viral hepatitis: immunoglobulin response during the course of the disease. J. Am. Med. Assoc. 208:497-503. 7. Hollinger, F. B., D. W. Bradley, J. E. Maynard, G. R. Dreesman, and J. L. Melnick. 1975. Detection of hepatitis A viral antigen by radioimmunoassay. J. Immunol. 115:1464-1466. 8. Lehmann, N. I., and I. D. Gust. 1977. The prevalence of antibody to hepatitis A virus in two populations in Victoria. Med. J. Aust. 2:731-732. 9. Locarnini, S. A., A. G. Coulepis, A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1978. Purification of hepatitis A virus from human feces. Intervirology 10:300308. 10. Locarnini, S. A., A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1977. The antibody response following hepatitis A infection. Intervirology 8:309-318. 11. Locarnini, S. A., A. A. Ferris, A. C. Stott, and I. D. Gust. 1974. The relationship between a 27 nm viruslike particle and hepatitis A as demonstrated by immune electron microscopy. Intervirology 4:110-118. 12. Locarnini, S. A., S. M. Garland, N. I. Lehmann, R. C. Pringle, and I. D. Gust. 1978. Solid-phase enzymelinked immunosorbent assay for detection of hepatitis A virus. J. Clin. Microbiol. 8:277-282. 13. Locarnini, S. A., L. D. Gust, A. A. Ferris, A. C. Stott, and M. L. Wong. 1976. A prospective study of acute viral hepatitis with particular reference to hepatitis type A. Bull. W.H.O. 54:199-206. 14. McKinnon, G. T., and R. C. Pringle. 1974. A serological study of antibody to Epstein-Barr virus in an Australian population. Med. J. Aust. 2:243-246. 15. Mathiesen, L. R., S. M. Feinstone, D. C. Wong, P. Skinhoej, and R. H. Purcell. 1978. Enzyme-linked
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immunosorbent assay for detection of hepatitis A antiin stool and antibody to hepatitis A antigen in sera: comparison with solid-phase radioimmunoassay, immune electron microscopy, and immune adherence hemagglutination assay. J. Clin. Microbiol. 7:184-193. 16. Miller, W. J., P. J. Provost, W. J. McAleer, 0. L. Ittensohn, V. M. Villarejos, and M. R. Hilleman. 1975. Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologic investigations. Proc. Soc. Exp. Biol. Med. 149: gen
254-261. 17. Moritsugu, Y., J. L. Dienstag, J. Valdesuso, D. C. Wong, and R. H. Purcell. 1976. Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination. Infect. Immun. 13:898-908. 18. Nairn, R. C. 1976. Fluorescent protein tracing, 4th ed. Churchill Livingstone, Edinburgh. 19. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. 20. Provost, P. J., 0. L. Ittensohn, V. M. Villarejos, and M. R. Hilleman. 1975. Specific complement-fixation test for human hepatitis A employing CR326 virus antigen. Diagnosis and epidemiology. Proc. Soc. Exp. Biol. Med. 148:962-969. 21. Purcell, R. H., D. C. Wong, H. J. Alter, and P. V. Holland. 1973. Microtiter solid-phase radioimmunoassay for hepatitis B antigen. Appl. Microbiol. 26:478484. 22. Purcell, R. H., D. C. Wong, Y. Moritsugu, J. L. Dienstag, J. A. Routenberg, and J. D. Boggs. 1976. A micro-titer solid-phase radioimmunoassay for hepatitis A antigen and antibody. J. Immunol. 116:349-356. 23. Rakela, J., D. Stevenson, V. M. Edwards, I. Gordon, and J. W. Mosley. 1977. Antibodies to hepatitis A virus: patterns by two procedures. J. Clin. Microbiol. 5: 110-111.
24. Schmitz, H., H.-W. Doerr, D. Kampa, and A. Vogt. 1977. Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus. J. Clin. Microbiol. 5:629-634. 25. Shirodaria, P. V., K. B. Fraser, and F. Stanford. 1973. Secondary fluorescent staining of virus antigen by rheumatoid factor and fluorescein-conjugate anti-IgM. Ann. Rheum. Dis. 32:53-57. 26. Triger, D. R., M. H. Alp, and R. Wright. 1972. Bacterial and dietary antibodies in liver disease. Lancet i:60-63. 27. Wollheim, F. A. 1968. Immunoglobulins in the course of viral hepatitis and in cholestatic and obstructive jaundice. Acta Med. Scand. 183:473-479. 28. Wolters, G., L. P. C. Kuijpers, J. Kacaki, and A. H. W. M. Schuurs. 1977. Enzyme-linked immunosorbent assay for hepatitis B surface antigen. J. Infect. Dis. 136: S311-S317. 29. Yolken, R. H., R. G. Wyatt, H. W. Kim, A. Z. Kapikian, and R. M. Chanock. 1978. Immunological response to infection with human reovirus-like agent: measurement of anti-human reovirus-like agent immunoglobulin G and M levels by the method of enzymelinked immunosorbent assay. Infect. Immun. 19:540546.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1979, p. 459-465 0095-1 137/79/04-0459/07$02.00/0
Solid-Phase Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A-Specific Immunoglobulin M STEPHEN A. LOCARNINI,* ANTHONY G. COULEPIS, ANN M. STRATTON, JAKOV KALDOR, AND IAN D. GUST Virus Laboratory, Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield, Victoria, 3078, Australia Received for publication 5 December 1978
A solid-phase enzyme-linked immunosorbent assay was developed for the detection of immunoglobulin M antibody to hepatitis A virus. The system was capable of detecting hepatitis A-specific immunoglobulin M in a single dilution of serum and appears to be a reliable and rapid means of establishing a diagnosis of hepatitis A infection. Specific immunoglobulin M was only detected in patients with serologically confirmed hepatitis A and not in patients with other forms of hepatitis, chronic liver disease, or autoimmune disease. In patients with hepatitis A, specific immunoglobulin M was usually detectable for 6 weeks after the onset of dark urine, and the longest period for which it was present in any patient was 115 days. This enzyme-linked immunosorbent assay is rapid, simple to perform, and does not require complicated equipment. Provided adequate supplies of purified reagents can be obtained, this enzyme-linked immunosorbent assay procedure is likely to simplify hepatitis A serology, because the same antibodycoated plates can be utilized to detect hepatitis A virus, anti-hepatitis A virus, and hepatitis A-specific immunoglobulin M.
The visualization of the hepatitis A virus (HAV) by Feinstone et al. (5) in 1973 has been followed by the rapid development of a number of methods for detecting both the virus and antibody developed to it (anti-HAV). The methods described to date, immune electron microscopy (IEM) (3, 5, 11, 13), complement fixation (20), immune adherence hemagglutination (16, 17), solid-phase radioimmunoassay (SPRIA) (7, 22), and enzyme-linked immunosorbent assay (ELISA) (12, 15), have been utilized to study both naturally and experimentally acquired hepatitis A. Acute infections with HAV can be confirmed either by detecting virus in fecal specimens collected during the late incubation period and acute phase of the illness, or by demonstrating a significant rise in anti-HAV in paired serum specimens collected early in the illness and during the convalescent phase. Because many patients have ceased shedding virus before admission to a hospital (4, 12, 13), and several weeks may be required before a significant rise in antiHAV can be detected between paired sera (3, 10, 15-17, 20, 22, 23), laboratory confirmation of hepatitis A infection is often delayed. Recently, we reported (10, 11) the detection of hepatitis A-specific immunoglobulin M (IgM) by IEM and SPRIA in serum specimens from patients with acute hepatitis A and established
its value for the confirmation of infection with HAV. The method used involved fractionation of serum specimens by sucrose density gradient ultracentrifugation and was hence of limited value to most clinical laboratories. This communication reports the successful adaptation of the widely available ELISA technique for the detection of hepatitis A-specific IgM. SUBJECTS AND METHODS Patients. The five groups of subjects studied were: (group I) 18 patients with acute hepatitis A; (group II) 10 patients with acute hepatitis B; (group III) 5 patients with non-A, non-B hepatitis; (group IV) 10 patients with infectious mononucleosis; and (group V) 28 patients with autoimmune disease (with and without accompanying liver disease). Groups I to IV were patients admitted to Fairfield Hospital for Communicable Diseases, Melbourne, Australia, between October 1973 and December 1977. Several serum specimens, including paired acute and convalescent sera, were collected from each patient and stored at -20°C until tested. Group V were patients admitted to the Alfred Hospital, Melbourne, between October 1977 and January 1978. A single serum specimen was collected from each patient. In all cases, the diagnoses were made by specialist physicians on the basis of clinical assessment aided by laboratory tests. A patient was regarded as having hepatitis A if HAV was detected in fecal specimens collected early in the illness and/or a rising titer of anti-HAV was 459
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J. CLIN. MICROBIOL.
demonstrated in paired sera (13). Tests for detecting a human IgG immunoadsorbent. The immunoadsorbHAV and anti-HAV were performed by SPRIA as ent polymer was prepared by cross-linking purified previously described (8, 10). These paired sera were human IgG (Commonwealth Serum Laboratories; also tested for hepatitis B surface antigen and antibody Cohn fraction V further purified by ion-exchange chroby SPRIA, using commercially available reagents matography as described above) with glutaraldehyde (Ausria II and Ausab respectively, Abbott Laborato- (Taab, Reading, England), using the method of Avrameas and Ternyck (1). The specificity of the absorbed ries, North Chicago, Ill.). All sera were negative. A diagnosis of hepatitis B was made if hepatitis B antisera was checked by double immunodiffusion in surface antigen was present in acute phase sera but gel and by immunoelectrophoresis against purified absent from sera collected during the convalescent human IgG, IgM, and IgA, as well as against human phase of the illness. Hepatitis B surface antigen was whole serum (Behringwerke Laboratories, Marburg, detected by SPRIA as described above, and all positive Germany). After absorption, the IgG component of each serum results were confirmed by specific neutralization tests. The paired sera from these ten patients were also was purified by ammonium sulfate precipitation and tested for anti-HAV by SPRIA as described above. A ion-exchange chromatography as described above, and 3.0, stationary level was detected in four patients, whereas conjugated with horseradish peroxidase (RZ the remaining six were negative for anti-HAV anti- Sigma Type VI; Sigma Chemical Co., St. Louis, Mo.) by the method of Nakane and Kawaoi (19). body. ELISA assay. The method used was modified from Patients with acute hepatitis in whom there was no serological evidence of infection with HAV, hepatitis that of Wolters et al. (28). The optimal dilution of B virus, Epstein-Barr virus, cytomegalovirus, and reagents used was determined by checkerboard titraherpes simplex virus were classified as non-A, non-B tion. The substrate was prepared by dissolving 80 mg hepatitis. Tests for infection with Epstein-Barr virus, of 5 amino-salicylic acid (Merck, Munich, Germany) cytomegalovirus, and herpes simplex virus were per- in 100 ml of hot distilled water, the pH was adjusted to 6.0 with 1 N NaOH, and then nine parts were added formed by standard techniques. Infectious mononucleosis was diagnosed when the to one part of 0.05% (vol/vol) H202 and used immedipatient's acute-phase serum contained heterophile an- ately. The wells of polyvinyl microtiter plates (Cooke tibody which could not be removed by prior adsorption with guinea pig kidney cells and/or a rising titer of Engineering Inc., Alexandria, Va.) were coated with a anti-Epstein-Barr virus was demonstrable by indirect 1:1,000 dilution of the human convalescent hepatitis A immunofluorescence (14). IgG in 0.85% (wt/vol) saline and incubated for 4 h at Tests for rheumatoid factor (RF) were performed room temperature. After washing three times with by latex agglutination (Commonwealth Serum Labo- phosphate-buffered saline (pH 7.4) containing 0.05% ratories, Melbourne, Australia) and hemagglutination (vol/vol) Tween 20 (PBS-T), the wells were filled with of sensitized sheep red blood cells (Denver Laborato- 1% (wt/vol) bovine serum albumin (Commonwealth ries, Cranbury, N.J.). Antibodies to nuclear factor, Serum Laboratories) in saline and allowed to stand at smooth muscle, and mitochondria were detected by 4°C overnight. After washing three times with PBSindirect immunofluorescence on a composite block of T, 50 ,ll of purified HAV suspended in PBS was added to each well, and the plates were incubated at 4°C for rat kidney, mouse stomach, and rabbit liver (18). Reagents. (i) Coating antibody. The antibody 16 h. After washing three times with PBS-T, 50 ,ld of used to coat the microtiter plates was obtained from the test sera, diluted 1:1,000 in PBS (pH 7.4), were an adult male 4 months after his admission to hospital added to each well and incubated at 37°C for 1 h. The with serologically confirmed hepatitis A. IgG was pu- samples were then aspirated, each well was washed six rified from 10.0 ml of the convalescent serum specimen times with PBS-T and 50 IA of peroxidase conjugate by precipitation with ammonium sulfate followed by (diluted 1:400 in PBS-T) was added, and the plates ion-exchange chromatography on diethylaminoethyl- were incubated at 37°C for 1 h. Finally, after aspirating cellulose (DE-52, Whatman, England) as described by the conjugate and washing each well six times with Purcell et al. (21). Purified IgG was stored at -20°C. PBS-T, 50 Au of substrate was added, and the plates (ii) Hepatitis A antigen. A fecal specimen and were incubated in the dark at room temperature for acute and convalescent sera were collected from two 20 min. The enzyme-substrate reaction was stopped patients with serologically confirmed hepatitis A. HAV by the addition of 50 ,ll of 1% (wt/vol) sodium azide. was detected in each crude fecal suspension by SPRIA The color produced in each well was recorded visually, and IEM as described elsewhere (9, 10, 13). No other using a 0 to 3+ scale: 0, no color reaction; 1+, a light virus-like particles were visualized by IEM. HAV was to medium color; 2+, a medium to strong color; 3+, a purified from each fecal specimen as described previ- very strong color reaction. A 1+ difference in reading ously (2, 9). Briefly, this procedure involved extraction was considered significant (12). In addition, the optical of the feces, clarification by low-speed centrifugation, density of the substrate solution was measured by pelleting of the virus, extraction with chloroform and, diluting the samples to 300 ,ul in distilled water (final finally, agarose gel filtration on Sepharose CL-2B dilution, 1:6) and reading the results in a spectrophotometer (Centrifichem System 400, Union Carbide (Pharmacia Fine Chemicals, Uppsala, Sweden). (iii) Conjugates. Anti-human IgM (mu chain spe- Corp.) at 550 nm. To measure any nonspecific binding to the IgGcific) prepared in goats was obtained commercially (Hyland, Travenol Laboratories, Melbourne) and nor- coated plates, each serum was also tested on a control mal goat serum was from Commonwealth Serum Lab- plate to which PBS-T had been added in place of the oratories. Both sera were absorbed several times with purified HAV. Substrate and conjugate controls were
also included on each microtiter plate. All testing of sera was performed under code, and each specimen was tested in duplicate.
0.5
Specific conditions. (i) Coating antibody. In preliminary tests, the purity of the IgG coating antibody was found to be critical, because traces of IgM produced unacceptably high background levels of color when reacted against the goat anti-humAn IgM conjugate. This difficulty was overcome by using IgG purified by ammonium sulfate precipitation and ionexchange chromatography. Log dilutions of purified IgG were tested to determine the optimal dilution for use. A dilution of i0' was found to provide maximum use of the material without compromising the sensitivity of the test. (ii) Hepatitis A antigen. In preliminary tests using crude or chloroform-extracted fecal specimens, nonspecific binding was found to occur which produced unacceptably high background levels. To reduce all nonspecific binding, HAV was purified until only minimal background debris was evident by direct electron microscopic examination (2, 9). Agarose gel filtration, using Sepharose CL-2B, usually yielded purified HAV suitable for the ELISA assay. (iii) Conjugates. Since commercially available anti-human IgM antisera often contain traces of antihuman IgG reactivity, immunoadsorption with the human IgG polymer before conjugation was found necessary to obtain monospecific activity. After immunoadsorption, the conjugate control wells showed no reactivity on the IgG-coated wells. The peroxidase-labeled normal goat IgG conjugate demonstrated no reactivity with any of the test sera or reagents used in the ELISA assay. (iv) Determination of cut-off point. The results of the ELISA assay were expressed as a positive/ negative (P/N) ratio, where N was the mean optical density of six negative control sera (three preinfection and three late convalescent hepatitis A sera) tested at
OD,,. 0.3-
the same dilution as the test sera (1:1,000). Using this N value, the individual P/N ratio for each control serum was calculated. The mean (1.10) plus 3 standard deviations of these six P/N values was 1.6. This value was used to signify the lowest level at which hepatitis A-specific IgM was considered present (29).
RESULTS (i) Effect of serum dilution on reactivity. Preinfection, early-acute-phase and late-convalescent sera were available from three patients with serologically confirmed hepatitis A, and decimal dilutions of each were tested for hepatitis A-specific IgM. All sera showed some nonspecific reactivity, when tested undiluted or at 1:10, which was entirely abolished at a dilution of 1:1,000 (Fig. 1). At this dilution, the acute-phase serum samples (collected 1 to 2 weeks after the onset of dark urine) had an optical density greater than 0.4, whereas the preinfection and late-convalescent-phase sera yielded values of less than 0.2. These results provided the basis for screening
461
SOLID-PHASE ELISA
VOL. 9, 1979
0.4 I
I
20min
I
I
, I
0.2
,
&
I
-
-
0.1
-.C-
:*
__r_
tb
-
-
:;:;-
I --.ri
1,000 10,000 100000 1,000,000 100 RECIPROCAL OF DILUTION
FIG. 1. Titrations of preinfection, acute, and convalescent sera from apatient with hepatitis A (patient 1, Table 1) for hepatitis A-specific IgM determinations. Symbols: A, A, preinfection serum tested against control and HAV test wells, respectively; 0, 0, acute-phase serum tested against control and test wells, respectively; O, U, convalescent serum tested against control and test wells, respectively.
all subsequent sera at 1:1,000. (ii) Detection of hepatitis A-specific IgM in serum from patients with hepatitis A. At least two serum specimens from each of 18 patients with serologically confirmed hepatitis A infection were tested for hepatitis A-specific IgM. The results of the 70 sera tested are shown in Table 1 and Fig. 2, in relation to time from the onset of illness, measured from the first day on which dark urine was observed. In three patients (patients 1 to 3, Table 1), one or more specimens had been collected before the onset of illness, and none of these were found to contain hepatitis A-specific IgM. In 10 of 11 patients, hepatitis A-specific IgM was detected within 7 days of the onset of dark urine. All 18 patients showed maximum levels of hepatitis A-specific IgM within the first 3 weeks from the onset of dark urine. Specific IgM was detected in 39 of 41 sera collected within 6 weeks of the onset of dark urine (Fig. 2). Beyond this period, its frequency of detection declined abruptly, with 2 of 5 sera collected between 43 and 63 days, 3 of 9 collected between 64 and 119 days, and 0 of 10 collected between 120 and 416 days, being positive. The longest period for which specific IgM was detected in any patient (patient 12, Table 1) was 115 days (measured to the last positive test). None of the 70 sera showed any nonspecific binding to control wells in which HAV was replaced by PBS-T. (iii) Specificity of the test. Acute and convalescent sera from 25 patients with hepatitis B, non-A, non-B hepatitis, and infectious mononucleosis (Groups II to IV) and single sera from 28 patients with liver and autoimmune disease (Group V) were also tested for hepatitis A-spe-
462
J. CLIN. MICROBIOL.
LOCARNINI ET AL.
TABLE 1. Levels of hepatitis A-specific IgM in 18 patients with hepatitis A (group I) Days after onset of Hepatitis A-speDays after onset of HpttsAse Patient no. dark urine at sePatient no. dark urne at scTc IgM P/Na cic IgM P/Na 1rum colection rum-collf 10
5 67 181
P 2.30 1.37 1.06
11
8 26 43
2.06 2.33 1.60
12
2 9 115 169
3.10 2.26 1.77 1.16
13
13 20 27 31 46 66
2.73 1.90 2.03 2.03 1.06 1.09
1.00 1.73 1.81
14
3 119 178
3.33 1.00 1.10
3 8 22
2.53 2.36 2.43
15
13 17 43
2.50 1.60 1.00
6 12 64
3.11 2.37 0.94
16
6
3 102
2.63 0.88
9 16 19 37
2.76 2.60 2.83 2.53
17 7
5 8 185
3.43 3.58 1.00
7 15 29
2.53 2.06 1.00
8
18 7 86 142
2.26 1.63 1.13
10 16 38 64
3.33 3.46 2.45 1.90
9
4 47 68
3.26 1.57 1.19
1
2
3
4
5
-158 -92 6 10 17 24 34 61 305 416
1.25 1.15 1.43 2.87 3.27 2.33 2.33 1.66 0.78 0.78
-184 -81 11 22 85 156 396
1.00 0.97 1.63 1.63 1.53 0.75 0.72
-210 25 39
II
c
a P/N ratio calculated as described in the text.
cific IgM as described above (Table 2). Of the 78 tested, only one had a P/N ratio l1.6 (patient 71, Table 2). The serum specimen also reacted in the control wells (identical P/N ratio), indicating that the binding was nonspecific. The specimen had been obtained from a patient with autoimmune disease and contained RF. In the remaining 77 sera, the P/N ratios of the test and control wells were similar. (iv) Comparison between optical density estimation and visual reading. Before the sera
enzyme-substrate reaction mixture was read
spectrophotometrically, visual readings were taken. In general, there was very good correlation between the two methods, except borderline samples (1.6 P/N < 1.8) were often scored as negative when read visually. Readings of 1+, 2+, and 3+ corresponded to P/N ratios in the range of approximately 1.8 to 2.2, 2.3 to 2.8, and 2.9 to 3.58, respectively. All of the 18 patients with hepatitis A were scored positive visually on at least one serum sample. -
VOL. 9, 1979
SOLID-PHASE ELISA
.0o
30
I.
i
P 2.0
N
if
-
C PRFE
t
onset
1-7
8-14
INTERVAL
15-21
22-28 29-35 36-42
4363
64-119 120-416
FROM ONSET OF DARK URINE (DAYS)
FIG. 2. Hepatitis A-specific IgM levels in sera collected from the 18 patients with serologically confirmed hepatitis A (Table 1). Individual P/N ratios are given for each group of sera collected at intervals, including preinfection, from the onset of dark urine.
DISCUSSION ELISAs have been used for the detection of class-specific antibody to other human viruses (24, 29), and the data presented above demonstrate that this system can also be successfully adapted for the detection of hepatitis A-specific IgM. When sera are diluted 1:1,000, to minimize nonspecific binding to the immunoglobulincoated wells, IgM was detected only in the acutephase sera of all patients with hepatitis A studied. It was never detected late (greater than 120 days after the onset of dark urine) in the convalescent phase of the illness, nor was it found in patients with other forms of liver or autoimmune disease. Hepatitis A-specific IgM becomes detectable within 1 week from the onset of dark urine and tends to persist for about 7 weeks. The longest period for which it was detected in any patient was 115 days. A major problem in hepatitis A serology is specificity. The lack of availability of monospecific nonprimate hyperimmune antisera to HAV has necessitated the use of human or chimpanzee convalescent anti-HAV sera in most serological tests and, since these sera contain antibodies to other antigens commonly found in primate feces (15), false-positive results are not uncommon. Therefore, the quality of reagents used in this ELISA assay is of prime importance, i.e., the purity of the HAV preparation and the convalescent human hepatitis A IgG, and a monospecific enzyme-linked goat anti-human IgM conjugate. High background optical density and "nonspecific" reactions with control or negative sera were abolished -only when such reagents used. One pitfall with antiviral IgM tests is that they may detect an RF IgM, which will react
were
with IgG bound to the solid phase, giving a falsepositive reaction (25). These RF-positive sera can be detected readily in this assay by the incorporation of controls in which purified HAV is replaced by buffer. Since all test sera were diluted 1:1,000, it was not surprising that only one of the RF-positive sera reacted significantly (P/N 1.6) against the human IgG coating antibody control plates. In the other 17 RFpositive cases, the control wells had identical P/ N values (0.10 P/N < 1.6) to the test wells (Table 2). All these 18 sera were therefore considered negative for hepatitis A-specific IgM. Several studies of serum immunoglobulin levels in patients after acute hepatitis A infection have demonstrated a characteristic polyclonal increase of IgM or IgM hypergammaglobulinemia (6, 27). It is probable that not all of the raised serum IgM observed during acute hepatitis A is virus specific. In light of the specificity problems associated with hepatitis A serology, the role of antigens that are carried, for example, by intestinal bacteria and dietary antigens (26) in the nonspecific stimulation of the immune system after hepatitis A could warrant investigation. Until recently, laboratory confirmation of hepatitis A was largely dependent upon demonstration of rising titers of anti-HAV in adequately spaced serum samples. Detection of HAV in the feces was of less value, because many patients have stopped shedding virus by the time they are admitted to the hospital (4, 12, 13). Usually, a period of 3 to 4 weeks between paired sera is required to detect a significant rise in anti-HAV by IEM (3, 10, 13, 23), SPRIA (7, 22), ELISA (15), or immune adherence hemagglutination (16, 17, 23). There is usually a delay of 2 to 6 weeks after the onset of acute illness before anti-HAV can be detected by immune adherence hemagglutination, whereas IEM can detect anti-HAV in the acute phase of illness, but levels rise slowly before a seroconversion can be established (10, 13, 23). SPRIA and ELISA can also detect early antibody and subsequent seroconversion, but high titers of anti-HAV are usually found in acute-phase sera on admission of patients to the hospital (8, 10, 12). Recently it has been proposed that a diagnosis of hepatitis A could be made on a single acute-phase serum if IEM, SPRIA, or ELISA were positive for antiHAV and immune adherence hemagglutination was negative (15, 23). This "window" result does not appear to be useful for reliable diagnosis, since immune adherence hemagglutination responses are variable and not many laboratories would run two serological tests for anti-HAV routinely. Thus, detection of hepatitis A-specific ,
.
10
463
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J. CLIN. MICROBIOL.
LOCARNINI ET AL:
TABLE 2. Levels of hepatitis A-specific IgM in the control patients (groups II to V) P/Na
Group
Patient
Acute serum
Convalescent serum
II: hepatitis B
19 20 21 22 23 24 25 26 27 28
1.14 0.45 1.16 0.69 1.03 0.50 0.10 0.10 1.28 0.40
0.89 0.38 0.55 0.65 0.51 0.10 0.10 0.10 0.40 0.35
III: non-A, non-B hepatitis
29 30 31 32 33
0.10 0.10 0.40 0.75 0.20
0.10 0.40 0.40 0.30 0.40
IV: infectious mononucleosis
34 35 36 37 38 39 40 41 42 43
0.56 0.47 0.47 0.78 0.35 0.47 0.60 0.70 1.10 0.30
0.34 0.71 0.50 0.60 0.57 0.50 0.90 0.50 0.90 0.10
Patient
P/N: single seruMa
Antibody'
V(a) primary biliary cirrhosis
44 45 46 47
0.85 1.09 1.28 0.40
AMA 2+ AMA 2+ AMA 2+ AMA 2+
V(b):
48
0.38
49 50 51 52 53
1.37 0.32 0.53 0.10 0.30
SMA 2+ ANF 2+ SMA 2+ SMA 2+ SMA 2+ SMA 2+ SMA 2+
Group
chronic active hepatitis
54 0.30 0.10 55 56 0.10 0.10 57 0.10 58 0.10 59 0.10 60 61 0.63 0.46 62 63 0.46 64 0.46 65 1.32 0.25 66 67 0.25 1.19 68 0.10 69 70 1.06 1.80 71 a P/N ratios calculated as described in the text. The very low P/N ratios observed with some of the control sera reflect the fact that only six control sera from the hepatitis A group were used for the determination of the N value. b ANA, SMA, and ANF denote antibody to mitochondrial antigen, smooth muscle antigen, and nuclear factor, respectively, as detected by indirect immunofluorescence (18).
IgM in sera collected early in the illness provides reliable means of establishing a diagnosis within a few days of the patient being seen by a physician (10). To date, detection of hepatitis A-specific IgM has required prior fractionation of the serum sample on sucrose density gradients (10), which has severely limited its value as a laboratory a
test. The ELISA test described in this report
has a number of advantages over this previously described system. Antibody-coated plates can be prepared in batches and stored until they are required and large collections of sera can be tested. A diagnosis of hepatitis A infection can now be reliably established on a single acute-
V(c):
positive RF
phase serum specimen, and results could be obtained 1 day after specimen collection. In addition, because sera are tested at a dilution of 1: 1,000, as little as 1 ,ul of serum is sufficient to allow the ELISA test to be performed. Provided adequate conjugates are available, the same antibody-coated plates can be used for the detection of HAV (12), total anti-HAV (15), and hepatitis A-specific IgM, which will greatly simplify hepatitis A serology. The only limiting factor at present is an adequate supply of puri-
fied reagents. ACKNOWLEDGMENTS This work
was
supported by
grants
from the National
Health and Medical Research Council of Australia.
SOLID-PHASE ELISA
VOL. 9, 1979
We are most grateful to the medical, nursing, and laboratory staff of Fairfield Hospital for their cooperation, anid in particular to Noreen Lehmann for radioimmunoassay testing of sera for anti-HAV. We thank Jennifer Rolland, Department of Pathology and Immunology, Monash Medical School, Melbourne, for supplying the sera from patients with autoimmune and chronic liver disease. LITERATURE CITED 1. Avrameas, S., and T. Ternyck. 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6:53-66. 2. Coulepis, A. G., S. A. Locarnini, A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1978. The polypeptides of hepatitis A virus. Intervirology 10:24-31. 3. Dienstag, J. L., D. W. Ailing, and R. H. Purcell. 1976. Quantitation of antibody to hepatitis A antigen by immune electron microscopy. Infect. Immun. 13:12091213. 4. Dienstag, J. L., S. M. Feinstone, A. Z. Kapikian, R. H. Purcell, J. D. Boggs, and M. E. Conrad. 1975. Faecal shedding of hepatitis A antigen. Lancet i:765767. 5. Feinstone, S. M., A. Z. Kapikian, and R. H. Purcell. 1973. Hepatitis A: detection by immune electron microscopy of a viruslike antigen associated with acute illness. Science 182:1026-1028. 6. Giles, J. P., and S. Krugman. 1969. Viral hepatitis: immunoglobulin response during the course of the disease. J. Am. Med. Assoc. 208:497-503. 7. Hollinger, F. B., D. W. Bradley, J. E. Maynard, G. R. Dreesman, and J. L. Melnick. 1975. Detection of hepatitis A viral antigen by radioimmunoassay. J. Immunol. 115:1464-1466. 8. Lehmann, N. I., and I. D. Gust. 1977. The prevalence of antibody to hepatitis A virus in two populations in Victoria. Med. J. Aust. 2:731-732. 9. Locarnini, S. A., A. G. Coulepis, A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1978. Purification of hepatitis A virus from human feces. Intervirology 10:300308. 10. Locarnini, S. A., A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1977. The antibody response following hepatitis A infection. Intervirology 8:309-318. 11. Locarnini, S. A., A. A. Ferris, A. C. Stott, and I. D. Gust. 1974. The relationship between a 27 nm viruslike particle and hepatitis A as demonstrated by immune electron microscopy. Intervirology 4:110-118. 12. Locarnini, S. A., S. M. Garland, N. I. Lehmann, R. C. Pringle, and I. D. Gust. 1978. Solid-phase enzymelinked immunosorbent assay for detection of hepatitis A virus. J. Clin. Microbiol. 8:277-282. 13. Locarnini, S. A., L. D. Gust, A. A. Ferris, A. C. Stott, and M. L. Wong. 1976. A prospective study of acute viral hepatitis with particular reference to hepatitis type A. Bull. W.H.O. 54:199-206. 14. McKinnon, G. T., and R. C. Pringle. 1974. A serological study of antibody to Epstein-Barr virus in an Australian population. Med. J. Aust. 2:243-246. 15. Mathiesen, L. R., S. M. Feinstone, D. C. Wong, P. Skinhoej, and R. H. Purcell. 1978. Enzyme-linked
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