JOURNAL OF CLINICAL MICROBIOLOGY,
July 1992, P.
1899-1901
Vol. 30, No. 7
0095-1137/92/071899-03$02.00/0 Copyright © 1992, American Society for Microbiology
Detection of Rubella Virus-Specific Polymeric Immunoglobulin A by Enzyme-Linked Immunosorbent Assay in Combination with Streptococcal Pretreatment of Serum KIMIKO KAWANO1 AND YOICHI MINAMISHIMA2* Miyazaki Prefectural Institute for Public Health and Environment' and Department of
Microbiology, Miyazaki Medical College, Kiyotake,2 Miyazaki, Japan Received 15 November 1991/Accepted 6 April 1992
An enzyme-linked immunosorbent assay combined with streptococcal treatment of serum was assessed for its serum polymeric immunoglobulin A. This technique detects rubella virus-specific polymeric immunoglobulin A antibody, which appears for only a short time after infection, and it is useful for serodiagnosis of recent rubella virus infection.
ability to detect
Virus-specific polymeric immunoglobulin A (IgA) antibodies have been detected only in early sera after several viral infections (2-5, 9). Detection of polymeric IgA antibody has been applied to differentiation of primary from nonprimary infections with herpes simplex virus (3). However, the techniques employed for detecting polymeric IgA antibody include sucrose gradient centrifugation (3, 5) and chromatography (2, 4, 9), and they are not practical for routine tests. On the other hand, treatment of serum with Streptococcus pyogenes AW43 cells removes all the subclasses of IgA but leaves some polymeric IgA (6-8). In the present study, we quantified polymeric IgA and monomeric IgA left in serum after treatment with AW43 cells and examined whether the AW43 treatment is useful to detect polymeric IgA specific to rubella virus. The AW43 strain was kindly supplied by G. Kronvall (Department of Medical Microbiology, University of Lund, Lund, Sweden). Preparation of the bacterial cells and treatment of sera with the cells were carried out by the method of Kronvall et al. (7). Six serum specimens from four people with a history of rubella were analyzed for IgA and rubella virus-specific antibodies. Two-tenths of a milliliter of serum diluted 1:8 was treated with 100 mg (wet weight) of AW43 cells for 1 h at room temperature with constant shaking and then centrifuged at 1,670 x g for 20 min. Three-tenths of a milliliter of the treated and untreated sera was fractionated by sucrose gradient centrifugation at 35,000 rpm for 16 h at 4°C in a swing bucket rotor (RPS 55T-2-244; Hitachi Koki Co., Ltd., Ibaragi, Japan) (5). Next, the concentration of IgA in each sample was quantified by a capture enzyme-linked immunosorbent assay (ELISA). Briefly, 50 ,ul of goat antibody against human IgA(a) (catalog no. 4101; Tago Inc., Burlingame, Calif.) diluted 1:2,000 with carbonate-bicarbonate buffer (pH 9.6) was added to the wells of a polystyrene plate (Immunoplate II; Nunc, Roskilde, Denmark). The wells were kept at 4°C overnight and washed with phosphatebuffered saline containing 0.05% Tween 20. After the wells were saturated with 1% bovine serum albumin (200 ,ul), they were inoculated with serially diluted samples (50 ,lI) and incubated at room temperature for 1 h. After the wells were washed, 50 j,l of peroxidase-conjugated goat antibody
*
against human IgA(a) (catalog no. 2391; Tago) (1:2,000 dilution) was reacted with the captured IgA for 1 h. Finally, 100 ,ul of a substrate solution consisting of hydrogen peroxide (0.006%) and ABTS [2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid); 0.8 mg/ml in a mixture of citric acid and Na2HPO4, pH 4.8] was added to each well, and the color reaction was stopped by addition of 1.25% NaF (50 ,ul). The optical density (OD) at 414 nm was determined by throughthe-plate reading with a Titertek Multiskan MC (Flow Laboratories Inc., Helsinki, Finland). On the basis of correlation between IgA concentration and OD for the reference serum, the concentration of IgA in the samples was calculated from the scored OD. Additionally, IgM and IgA antibodies specific to rubella virus were titrated with Enzygnost Rubella Test reagents (Behringwerke AG, Marburg, Germany). A slight modification was made for IgA. Namely, IgA antibody was titrated with peroxidase-conjugated goat antibody against human IgA(a) and hydrogen peroxide-ABTS, and the OD was measured at 414 nm. The sera diluted to 1:8 contained 23.5 + 4.09 mg of IgA per dl (mean standard deviation; n = 6) before AW43 treat0.23 ment, but after treatment they contained only 1.1 mg/dl (n = 6). As illustrated in Fig. 1, those sera showed a peak level of IgA at the 7S position before treatment. After treatment, the peak shifted to approximately 10S, and a trace of IgA was left at 7S in certain sera (Fig. 1D and E). Rubella virus-specific IgA and IgM antibody activities are shown in Fig. 1 along with the OD for each fraction diluted to 1:5. Rubella virus-specific IgA antibody activity of the nonAW43-treated sera from patients at 13, 22, and 23 days after onset had peaks at approximately 10S but not at the 7S position, where the highest concentration of IgA was detected (Fig. 1B to D). This indicated the presence of polymeric IgA with a high level of antibody activity in those sera. After AW43 treatment, the rubella virus-specific IgA antibody activity was detected in the fractions around 10S but not in the 7S fraction (Fig. 1B to D). In contrast, the non-AW43-treated ±
±
sera
at 1 or 4 years after the onset of rubella contained rubella
virus-specific IgA antibodies in some fractions peaking at 7S, but none in the fraction at approximately 10S (Fig. 1E and F). After the AW43 treatment, those antibodies were completely lost from all the fractions (Fig. 1E and F). On the other hand, no antibody activity was found in any fraction of a patient's serum at 3 days (Fig. 1A). Thus, AW43 treatment of serum leaves mainly polymeric IgA and a trace of monomeric IgA in
Corresponding author. 1899
1900
J. CLIN. MICROBIOL.
NOTES C. 22 days
I
la
EI
I.
41
10-
F . 4 year!
E. 1 year
D. 23 days
-2.0
I
PreI
Pre
Pre
I I It
-1.0
.a.
'I
40
0
I
0
0
us
a-
to
_
E
1.005-1
Poslt
Posit
PostI -
.
2.0 E-
I
I.
a
1.0
I
.0
I
lh
1'Z\I
cr
wfo
I * an iO l -.|
s5s
11S
75
Fractions
115 7S FractIons
195
195
11S
7S
Fractions
FIG. 1. Sedimentation patterns of IgA, rubella virus-specific IgA antibody, and rubella virus-specific IgM antibody in sera at various intervals after rubella virus infection, before and after treatment with AW43 cells. The six serum specimens were collected from four people with a history of rubella. Serum samples A and C were from one person, B was from another person, D was from a third person, and E and F were from a fourth person.
some cases, but the antibody activity was associated with only polymeric IgA. In another set of experiments, AW43 treatment was combined with an ELISA using Enzygnost Rubella Test reagents, and the combination was assessed as a diagnostic method for rubella. Twenty-six serum specimens collected from 14 patients at various intervals after the onset of rubella were tested for polymeric IgA and IgM antibody specific to rubella virus. Each antibody activity is shown by the OD at 414 nm of serum diluted to 1:40 (Fig. 2). Rubella virusspecific IgA antibody in the untreated sera remained detectable for years (Fig. 2A). After treatment of the sera with AW43, five patients had nearly undetectable levels of polymeric anti-rubella virus IgA in the first few days after infection (Fig. 2B). Those five patients subsequently produced polymeric IgA antibody when reevaluated, as evidenced by ODs of 0.626 (10 days after onset of the disease), 0.602 (22 days), 0.242 (22 days), 0.179 (23 days), and 0.124 (43 days) (Fig. 2B). Thus, seroconversion for polymeric IgA antibody occurred in those five patients, and the polymeric IgA antibodies were produced in several days after onset of rubella and were detectable only for the first month. Sera from 10 people without a history of rubella were negative for
rubella virus-specific antibody by this method. Rubella virus-specific IgM antibody was detectable for 3 to 4 months (Fig. 2C). As described above, detection by ELISA of rubella virusspecific polymeric IgA antibody after treatment of serum with AW43 cells is useful for serodiagnosis of the early stage of rubella virus infection. This technique is also applicable to detection of polymeric IgA in other viral infections, although it may not detect the total amount of polymeric IgA antibody. The possibility that AW43 cells bind some polymeric IgA cannot be excluded, and this may limit the sensitivity of this technique. However, it has the advantage of simplicity over sucrose gradient centrifugation and chromatography. Recently, a novel technique to detect polymeric IgA specific to Escherichia coli lipopolysaccharide by using a secretory component with selectively binds to polymeric IgA was developed (1). However, the technique requires a radiolabelled reagent and is not practical. Our method is practical for routine tests to detect polymeric IgA antibody which appears in the early stage of virus infections, especially when the antibody has a high level of activity, as is the case with anti-rubella virus antibody.
O.A_*
VOL. 30, 1992
NOTES
We are much obliged to G. Kronvall for providing the bacterial strain. We also thank Hoechst Japan Ltd. for supplying Enzygnost Rubella Test reagents.
OD1 4
so
1.0-
0
A.
19A 0
-
0
0.2n
1. 0
.0
0
2.
I-
10 20 30 40 so 60 70 *0 90 100
0
150 200 25001
22 4
00414 1.0
0.2*
3.
B. 19A post treatment with AW43 (Polymeric 19A)
4.
0 tO 20 30 40 50
s0
70
60
Nooio
90
20 2i06 1
5.
OD40
C. IgM
*
1.0
02-
*
6.
2.
7.
|
. ! 0.b **
0 10 20 30 40 50 60 70
FIG.
Rubella
1901
D
a
50 90 y
100
150 2002W0 300 1
s
virus-specific antibody
determined
2 3 4 Years
by
8.
ELISA
for 26 serum specimens taken from 14 patients at various intervals after rubella virus infection. (A) IgA antibody before AW43 treatment (total IgA); (B) IgA antibody after AW43 treatment (polymeric IgA); (C) IgM antibody before AW43 treatment.
9.
REFERENCES Bartholomeus, R. C. A., J. T. LaBrooy, P. L. Ey, A. C. Di Matteo, D. A. Daniels, C. S. Anderson, and D. Rowley. 1989. Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component. J. Immunol. Methods 117:247-255. Brown, T. A., B. R. Murphy, J. RadI, J. J. Haaiman, and J. Mestecky. 1985. Subclass distribution and molecular form of immunoglobulin A hemagglutinin antibodies in sera and nasal secretions after experimental secondary infection with influenza A virus in humans. J. Clin. Microbiol. 22:259-264. Hashido, M., T. Kawana, and S. Inouye. 1989. Differentiation of primary from nonprimary genital herpesvirus infections by detection of polymeric immunoglobulin A activity. J. Clin. Microbiol. 27:2609-2611. Hikata, M., K. Tachibana, M. Imai, S. Naito, A. Oinuma, F. Tsuda, Y. Miyakawa, and M. Mayumi. 1986. Immunoglobulin A antibody against hepatitis B core antigen of polymeric and monomeric forms, as well as of IgAl and IgA2 subclasses, in acute and chronic infection with hepatitis B virus. Hepatology 6:652-657. Inouye, S., R. Kono, and Y. Takeuchi. 1978. Oligomeric immunoglobulin A antibody response to rubella virus infection. J. Clin. Microbiol. 8:1-6. Kawano, K., S. Yamamoto, and Y. Minamishima. 1986. Detection of early hemagglutination inhibitory antibodies to rubella virus by pretreatment of sera with streptococcal cells. J. Med. Virol. 19:101-110. Kronvall, G., A. Simmons, E. B. Myhre, and S. Jonsson. 1979. Specific absorption of human serum albumin, immunoglobulin A, and immunoglobulin G with selected strains of group A and G streptococci. Infect. Immun. 25:1-10. Lofgren, B., E. Nordenfelt, S. Jonsson, and G. Kronvall. 1980. New bacterial absorption method for determination of hepatitis A IgM and IgA antibodies. J. Med. Virol. 6:37-44. Ponzi, A. N., C. Merlino, A. Angeretti, and R. Penna. 1985. Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections. J. Clin. Microbiol. 22:505-509.
July 1992, P.
1899-1901
Vol. 30, No. 7
0095-1137/92/071899-03$02.00/0 Copyright © 1992, American Society for Microbiology
Detection of Rubella Virus-Specific Polymeric Immunoglobulin A by Enzyme-Linked Immunosorbent Assay in Combination with Streptococcal Pretreatment of Serum KIMIKO KAWANO1 AND YOICHI MINAMISHIMA2* Miyazaki Prefectural Institute for Public Health and Environment' and Department of
Microbiology, Miyazaki Medical College, Kiyotake,2 Miyazaki, Japan Received 15 November 1991/Accepted 6 April 1992
An enzyme-linked immunosorbent assay combined with streptococcal treatment of serum was assessed for its serum polymeric immunoglobulin A. This technique detects rubella virus-specific polymeric immunoglobulin A antibody, which appears for only a short time after infection, and it is useful for serodiagnosis of recent rubella virus infection.
ability to detect
Virus-specific polymeric immunoglobulin A (IgA) antibodies have been detected only in early sera after several viral infections (2-5, 9). Detection of polymeric IgA antibody has been applied to differentiation of primary from nonprimary infections with herpes simplex virus (3). However, the techniques employed for detecting polymeric IgA antibody include sucrose gradient centrifugation (3, 5) and chromatography (2, 4, 9), and they are not practical for routine tests. On the other hand, treatment of serum with Streptococcus pyogenes AW43 cells removes all the subclasses of IgA but leaves some polymeric IgA (6-8). In the present study, we quantified polymeric IgA and monomeric IgA left in serum after treatment with AW43 cells and examined whether the AW43 treatment is useful to detect polymeric IgA specific to rubella virus. The AW43 strain was kindly supplied by G. Kronvall (Department of Medical Microbiology, University of Lund, Lund, Sweden). Preparation of the bacterial cells and treatment of sera with the cells were carried out by the method of Kronvall et al. (7). Six serum specimens from four people with a history of rubella were analyzed for IgA and rubella virus-specific antibodies. Two-tenths of a milliliter of serum diluted 1:8 was treated with 100 mg (wet weight) of AW43 cells for 1 h at room temperature with constant shaking and then centrifuged at 1,670 x g for 20 min. Three-tenths of a milliliter of the treated and untreated sera was fractionated by sucrose gradient centrifugation at 35,000 rpm for 16 h at 4°C in a swing bucket rotor (RPS 55T-2-244; Hitachi Koki Co., Ltd., Ibaragi, Japan) (5). Next, the concentration of IgA in each sample was quantified by a capture enzyme-linked immunosorbent assay (ELISA). Briefly, 50 ,ul of goat antibody against human IgA(a) (catalog no. 4101; Tago Inc., Burlingame, Calif.) diluted 1:2,000 with carbonate-bicarbonate buffer (pH 9.6) was added to the wells of a polystyrene plate (Immunoplate II; Nunc, Roskilde, Denmark). The wells were kept at 4°C overnight and washed with phosphatebuffered saline containing 0.05% Tween 20. After the wells were saturated with 1% bovine serum albumin (200 ,ul), they were inoculated with serially diluted samples (50 ,lI) and incubated at room temperature for 1 h. After the wells were washed, 50 j,l of peroxidase-conjugated goat antibody
*
against human IgA(a) (catalog no. 2391; Tago) (1:2,000 dilution) was reacted with the captured IgA for 1 h. Finally, 100 ,ul of a substrate solution consisting of hydrogen peroxide (0.006%) and ABTS [2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid); 0.8 mg/ml in a mixture of citric acid and Na2HPO4, pH 4.8] was added to each well, and the color reaction was stopped by addition of 1.25% NaF (50 ,ul). The optical density (OD) at 414 nm was determined by throughthe-plate reading with a Titertek Multiskan MC (Flow Laboratories Inc., Helsinki, Finland). On the basis of correlation between IgA concentration and OD for the reference serum, the concentration of IgA in the samples was calculated from the scored OD. Additionally, IgM and IgA antibodies specific to rubella virus were titrated with Enzygnost Rubella Test reagents (Behringwerke AG, Marburg, Germany). A slight modification was made for IgA. Namely, IgA antibody was titrated with peroxidase-conjugated goat antibody against human IgA(a) and hydrogen peroxide-ABTS, and the OD was measured at 414 nm. The sera diluted to 1:8 contained 23.5 + 4.09 mg of IgA per dl (mean standard deviation; n = 6) before AW43 treat0.23 ment, but after treatment they contained only 1.1 mg/dl (n = 6). As illustrated in Fig. 1, those sera showed a peak level of IgA at the 7S position before treatment. After treatment, the peak shifted to approximately 10S, and a trace of IgA was left at 7S in certain sera (Fig. 1D and E). Rubella virus-specific IgA and IgM antibody activities are shown in Fig. 1 along with the OD for each fraction diluted to 1:5. Rubella virus-specific IgA antibody activity of the nonAW43-treated sera from patients at 13, 22, and 23 days after onset had peaks at approximately 10S but not at the 7S position, where the highest concentration of IgA was detected (Fig. 1B to D). This indicated the presence of polymeric IgA with a high level of antibody activity in those sera. After AW43 treatment, the rubella virus-specific IgA antibody activity was detected in the fractions around 10S but not in the 7S fraction (Fig. 1B to D). In contrast, the non-AW43-treated ±
±
sera
at 1 or 4 years after the onset of rubella contained rubella
virus-specific IgA antibodies in some fractions peaking at 7S, but none in the fraction at approximately 10S (Fig. 1E and F). After the AW43 treatment, those antibodies were completely lost from all the fractions (Fig. 1E and F). On the other hand, no antibody activity was found in any fraction of a patient's serum at 3 days (Fig. 1A). Thus, AW43 treatment of serum leaves mainly polymeric IgA and a trace of monomeric IgA in
Corresponding author. 1899
1900
J. CLIN. MICROBIOL.
NOTES C. 22 days
I
la
EI
I.
41
10-
F . 4 year!
E. 1 year
D. 23 days
-2.0
I
PreI
Pre
Pre
I I It
-1.0
.a.
'I
40
0
I
0
0
us
a-
to
_
E
1.005-1
Poslt
Posit
PostI -
.
2.0 E-
I
I.
a
1.0
I
.0
I
lh
1'Z\I
cr
wfo
I * an iO l -.|
s5s
11S
75
Fractions
115 7S FractIons
195
195
11S
7S
Fractions
FIG. 1. Sedimentation patterns of IgA, rubella virus-specific IgA antibody, and rubella virus-specific IgM antibody in sera at various intervals after rubella virus infection, before and after treatment with AW43 cells. The six serum specimens were collected from four people with a history of rubella. Serum samples A and C were from one person, B was from another person, D was from a third person, and E and F were from a fourth person.
some cases, but the antibody activity was associated with only polymeric IgA. In another set of experiments, AW43 treatment was combined with an ELISA using Enzygnost Rubella Test reagents, and the combination was assessed as a diagnostic method for rubella. Twenty-six serum specimens collected from 14 patients at various intervals after the onset of rubella were tested for polymeric IgA and IgM antibody specific to rubella virus. Each antibody activity is shown by the OD at 414 nm of serum diluted to 1:40 (Fig. 2). Rubella virusspecific IgA antibody in the untreated sera remained detectable for years (Fig. 2A). After treatment of the sera with AW43, five patients had nearly undetectable levels of polymeric anti-rubella virus IgA in the first few days after infection (Fig. 2B). Those five patients subsequently produced polymeric IgA antibody when reevaluated, as evidenced by ODs of 0.626 (10 days after onset of the disease), 0.602 (22 days), 0.242 (22 days), 0.179 (23 days), and 0.124 (43 days) (Fig. 2B). Thus, seroconversion for polymeric IgA antibody occurred in those five patients, and the polymeric IgA antibodies were produced in several days after onset of rubella and were detectable only for the first month. Sera from 10 people without a history of rubella were negative for
rubella virus-specific antibody by this method. Rubella virus-specific IgM antibody was detectable for 3 to 4 months (Fig. 2C). As described above, detection by ELISA of rubella virusspecific polymeric IgA antibody after treatment of serum with AW43 cells is useful for serodiagnosis of the early stage of rubella virus infection. This technique is also applicable to detection of polymeric IgA in other viral infections, although it may not detect the total amount of polymeric IgA antibody. The possibility that AW43 cells bind some polymeric IgA cannot be excluded, and this may limit the sensitivity of this technique. However, it has the advantage of simplicity over sucrose gradient centrifugation and chromatography. Recently, a novel technique to detect polymeric IgA specific to Escherichia coli lipopolysaccharide by using a secretory component with selectively binds to polymeric IgA was developed (1). However, the technique requires a radiolabelled reagent and is not practical. Our method is practical for routine tests to detect polymeric IgA antibody which appears in the early stage of virus infections, especially when the antibody has a high level of activity, as is the case with anti-rubella virus antibody.
O.A_*
VOL. 30, 1992
NOTES
We are much obliged to G. Kronvall for providing the bacterial strain. We also thank Hoechst Japan Ltd. for supplying Enzygnost Rubella Test reagents.
OD1 4
so
1.0-
0
A.
19A 0
-
0
0.2n
1. 0
.0
0
2.
I-
10 20 30 40 so 60 70 *0 90 100
0
150 200 25001
22 4
00414 1.0
0.2*
3.
B. 19A post treatment with AW43 (Polymeric 19A)
4.
0 tO 20 30 40 50
s0
70
60
Nooio
90
20 2i06 1
5.
OD40
C. IgM
*
1.0
02-
*
6.
2.
7.
|
. ! 0.b **
0 10 20 30 40 50 60 70
FIG.
Rubella
1901
D
a
50 90 y
100
150 2002W0 300 1
s
virus-specific antibody
determined
2 3 4 Years
by
8.
ELISA
for 26 serum specimens taken from 14 patients at various intervals after rubella virus infection. (A) IgA antibody before AW43 treatment (total IgA); (B) IgA antibody after AW43 treatment (polymeric IgA); (C) IgM antibody before AW43 treatment.
9.
REFERENCES Bartholomeus, R. C. A., J. T. LaBrooy, P. L. Ey, A. C. Di Matteo, D. A. Daniels, C. S. Anderson, and D. Rowley. 1989. Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component. J. Immunol. Methods 117:247-255. Brown, T. A., B. R. Murphy, J. RadI, J. J. Haaiman, and J. Mestecky. 1985. Subclass distribution and molecular form of immunoglobulin A hemagglutinin antibodies in sera and nasal secretions after experimental secondary infection with influenza A virus in humans. J. Clin. Microbiol. 22:259-264. Hashido, M., T. Kawana, and S. Inouye. 1989. Differentiation of primary from nonprimary genital herpesvirus infections by detection of polymeric immunoglobulin A activity. J. Clin. Microbiol. 27:2609-2611. Hikata, M., K. Tachibana, M. Imai, S. Naito, A. Oinuma, F. Tsuda, Y. Miyakawa, and M. Mayumi. 1986. Immunoglobulin A antibody against hepatitis B core antigen of polymeric and monomeric forms, as well as of IgAl and IgA2 subclasses, in acute and chronic infection with hepatitis B virus. Hepatology 6:652-657. Inouye, S., R. Kono, and Y. Takeuchi. 1978. Oligomeric immunoglobulin A antibody response to rubella virus infection. J. Clin. Microbiol. 8:1-6. Kawano, K., S. Yamamoto, and Y. Minamishima. 1986. Detection of early hemagglutination inhibitory antibodies to rubella virus by pretreatment of sera with streptococcal cells. J. Med. Virol. 19:101-110. Kronvall, G., A. Simmons, E. B. Myhre, and S. Jonsson. 1979. Specific absorption of human serum albumin, immunoglobulin A, and immunoglobulin G with selected strains of group A and G streptococci. Infect. Immun. 25:1-10. Lofgren, B., E. Nordenfelt, S. Jonsson, and G. Kronvall. 1980. New bacterial absorption method for determination of hepatitis A IgM and IgA antibodies. J. Med. Virol. 6:37-44. Ponzi, A. N., C. Merlino, A. Angeretti, and R. Penna. 1985. Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections. J. Clin. Microbiol. 22:505-509.
