Microbiol. Immunol . Vol. 23 (8), 801-804, 1979
Application Type
Specific
of Indirect Antibody
Hemagglutination to Herpes
Simplex
Test
to Measurement
Virus
in Human
of Sera
Masami OHASHI and Yoshikatsu OZAKI* *Departmentof Microbiology, Shiga Universityof Medical Science,Schoolof Medicine, Ohtsu, Shiga (Received for publication, January 9, 1979)
Herpes simplex virus (HSV) has been differentiated into type 1 (HSV-1) and type 2 (HSV-2). Type 1 virus is usually isolated from non-genital lesions while type 2 virus is found in genital lesions. In addition to such correlation of the site of lesions with the type of virus, it has been shown in seroepidemiological studies that HSV-2 plays a possible etiological role in carcinoma of the cervix (3, 6, 7). However, one of the main problems involved in the survey of human sera is the lack of a precise method of identifying the type specific antibody as there are common antigens between HSV1 and HSV-2. The neutralization tests, including kinetic, plaque reduction and microneutralization methods, are usually employed to detect antibody to HSV in human sera, but these tests require tissue culture systems and are time-consuming. The indirect hemagglutination (IHA) test described by Fuccillo et al (1) is convenient and quite sensitive in detecting antibody to HSV and measuring the level. We report herein that the micro-IHA test is suitable for detecting antibody levels to HSV in human sera and for detecting the type specific antibody in sera absorbed by heterotypic viral antigens. Twenty-seven serum samples were selected from patients with cervical cancer and control women who were included in seroepidemiological study for cancer of the cervix (4). The criterion for the selection was based on the titer and type specificity of antibody which was calculated from the results obtained by microneutralization tests. Seibert strain of HSV-1 and UW-268 strain of HSV-2 were used throughout the experiments. The microneutralization test and micro-IHA test were performed according to the procedures described by Rawls et al (5) and Lerner et al (2), respectively. Vero cell cultures were used for microneutralization test and preparation of antigen for sensitization of sheep red blood cells. In preparation of antigen, Vero cells were infected with either HSV-1 or HSV-2 at an input multiplicity of approximately 2.0 plaque forming units (PFU) per cell and the infected cells were collected after incubation at 37 C for 24 hr by scraping with a rubber policeman. The cell suspension was spun down by centrifugation at 2,000 rpm for 5 min, and the packed cells were resuspended in phosphate buffered saline (PBS), pH 7.2, to yield a 10% cell suspension. The cell suspension was frozen and thawed once and then homogenized in a teflon homogenizer. The homogenate was centrifuged at 3,000 rpm for 30 min to remove cell debris, and the supernatant fluid was used as the antigen . 801
802
M.
OHASHI
AND Y. OZAKI
Fig. 1. Correlation between II/I indices obtained from microneutraliza tion and micro-IHA tests. Table
1.
Comparison micro-IHA
and
of mean antibody microneutralization
titers
between tests
the
Tannic acid treated sheep red blood cells were sensitized by mixing with properly diluted antigen and incubating at 37 C for 15 min. The sensitized cells were washed with PBS twice and diluted to give a 1.0% suspension with PBS containing 1.0% normal rabbit serum. The micro-IHA test was carried out using the V-bottom microplate. 0.025 ml of sensitized sheep red blood cell suspension was mixed with an equal volume of serial two-fold dilutions of serum in each well of the microplate, and incubated at a room temperature for 90 min. Diluent and antigen free tannic acid treated sheep red blood cells alone served as the control in all tests. Antibody titer was expressed as a reciprocal of the highest dilution giving a definite positive agglutination. The type specificity in each serum was expressed as II/I index as proposed by Rawls et al (5). This was given by the following formula : To determine whether IHA test is a suitable method for detection of type specific antibody in human sera, II/I index in each serum was compared between microIHA and microneutralization tests. As shown in Fig. 1, the IHA test gave comparable results to those obtained by the microneutralization test, except that there was a trend toward somewhat higher values of antibody titer against HSV-1 in the
803
NOTES Table
2.
Effect
of absorption
on
antibody
titers
as measured
by
the
IHA
test
IHA test. This correlation was also seen in a comparison of mean antibody titers (Table 1). In some cases, (not presented) even an amount of antibody too small to be detected by the microneutralization test could be detected by the IHA test. This would indicate that the IHA test may be a more sensitive method than the microneutralization test. The next experiment was designed to determine if the type specific antibody is detectable by the IHA test using human sera absorbed with heterotypic viral antigen . Ten serum samples were chosen on the basis of values of the II/I index, which ranged from 49 to 150. The procedures for absorption were as follows: Vero cells grown in a 500-ml culture bottle were infected with HSV-1 or HSV-2 at an input multiplicity of about 1.0 PFU per cell. The infected cells were harvested when a definite cytopathic effect was noted, usually 24 hr after infection. The cells were suspended in PBS to give a density of 5x 107cells per ml. A 0.5-ml aliquot of serum was added to 4.5 ml of a suspension of infected cells. The mixture was homogenized by a teflon homogenizer and kept at 37 C for 60 min and then at 4 C overnight. The mixture was then centrifuged at 2,000 rpm for 20 minto remove large cell debris. The supernatant was centrifuged at 40,000 rpm for 60 min, and the clarified supernatant was used as a 1: 10 dilution of the absorbed serum after heating at 56 C for 30 min. The results are shown in Table 2. In the cases of sera of which the II/I index was less than 100, absorption with HSV-1 resulted in disappearance not only of antibody against HSV-1 but also of that against HSV-2. In contrast, treatment with HSV-2 absorbed homotypic antibody completely but left heterotypic antibody (antibody to HSV-1). These results suggest that antibody measured by HSV-2 antigen sensitized sheep red blood cells is not type specific but common antibody against both types of virus. On the other hand, in cases when the II/I index was over 100, the sera probably contained type specific antibody as well as type common antibody, because even after absorption with HSV-2, some heterotypic antibody (HSV-1 antibody) remained. Thus the micro-IHA test can be applied to quantitative survey for HSV antibody in human sera, not only for accurate determinations but also as a
804
M.
comparison type
with
specific Thanks
results
antibody are
due
to M.
of the in sera Ohara
OHASHI
AND Y. OZAKI
microneutralization absorbed for
assistance
by
test . heterotypic
in preparation
The viral
of the
IHA
test
antigen manuscript
can
also detect
. .
REFERENCES
1) Fuccillo, D.A., Moder, F.L., Catalano, L.W., Vincent, M.M., and Sever, J.L. 1970. Herpesvirus hominis types 1 and 2: A specific microindirect hemagglutination test. Proc. Soc. Exp. Biol. Med. 133: 735-739 . 2) Lerner, A.M., Lauter, C.B., Nolan, D.C., and Shippey, M. J. 1972. Passive hemagglutinating antibodies in cerebrospinal fluids in herpesvirus hominis encephalitis. Proc. Soc. Exp. Biol. Med . 140: 1460-1466 . 3) Nahmias, A.J., Josey, W.E., Naib, Z.M., Luce, C.F., and Gust, B.A. 1970. Antibodies to herpesvirus hominis type 1 and 2 in human. II. Women with cervical cancer. Am. J. Epidemiol . 91: 547-552 . 4) Ozaki, Y., Ishiguro, T., Ohashi, M., Sawaragi, I., and Ito, Y. 1978. Antibodies to herpesvirus type 1 and type 2 among Japanese cervical cancer patients. Gann 69: 119-122 . 5) Rawls, W.E., Iwamoto, K., Adam, E., and Melnick, J.L. 1970. Measurement of antibodies to herpesvirus type 1 and 2 in human sera. J. Immunol. 104: 599-606 . 6) Rawls, W.E., Tompkins, W.A.F., and Melnick, J.L. 1969. The association of herpesvirus type 2 and carcinoma of the uterine cervix. Am. J. Epidemiol. 89: 547-554 . 7) Royston, I., and Aurelian L. 1970. The association of genital herpesvirus with cervical atypia and carcinoma in situ. Am. J. Epidemiol. 91: 531-538 .
Application Type
Specific
of Indirect Antibody
Hemagglutination to Herpes
Simplex
Test
to Measurement
Virus
in Human
of Sera
Masami OHASHI and Yoshikatsu OZAKI* *Departmentof Microbiology, Shiga Universityof Medical Science,Schoolof Medicine, Ohtsu, Shiga (Received for publication, January 9, 1979)
Herpes simplex virus (HSV) has been differentiated into type 1 (HSV-1) and type 2 (HSV-2). Type 1 virus is usually isolated from non-genital lesions while type 2 virus is found in genital lesions. In addition to such correlation of the site of lesions with the type of virus, it has been shown in seroepidemiological studies that HSV-2 plays a possible etiological role in carcinoma of the cervix (3, 6, 7). However, one of the main problems involved in the survey of human sera is the lack of a precise method of identifying the type specific antibody as there are common antigens between HSV1 and HSV-2. The neutralization tests, including kinetic, plaque reduction and microneutralization methods, are usually employed to detect antibody to HSV in human sera, but these tests require tissue culture systems and are time-consuming. The indirect hemagglutination (IHA) test described by Fuccillo et al (1) is convenient and quite sensitive in detecting antibody to HSV and measuring the level. We report herein that the micro-IHA test is suitable for detecting antibody levels to HSV in human sera and for detecting the type specific antibody in sera absorbed by heterotypic viral antigens. Twenty-seven serum samples were selected from patients with cervical cancer and control women who were included in seroepidemiological study for cancer of the cervix (4). The criterion for the selection was based on the titer and type specificity of antibody which was calculated from the results obtained by microneutralization tests. Seibert strain of HSV-1 and UW-268 strain of HSV-2 were used throughout the experiments. The microneutralization test and micro-IHA test were performed according to the procedures described by Rawls et al (5) and Lerner et al (2), respectively. Vero cell cultures were used for microneutralization test and preparation of antigen for sensitization of sheep red blood cells. In preparation of antigen, Vero cells were infected with either HSV-1 or HSV-2 at an input multiplicity of approximately 2.0 plaque forming units (PFU) per cell and the infected cells were collected after incubation at 37 C for 24 hr by scraping with a rubber policeman. The cell suspension was spun down by centrifugation at 2,000 rpm for 5 min, and the packed cells were resuspended in phosphate buffered saline (PBS), pH 7.2, to yield a 10% cell suspension. The cell suspension was frozen and thawed once and then homogenized in a teflon homogenizer. The homogenate was centrifuged at 3,000 rpm for 30 min to remove cell debris, and the supernatant fluid was used as the antigen . 801
802
M.
OHASHI
AND Y. OZAKI
Fig. 1. Correlation between II/I indices obtained from microneutraliza tion and micro-IHA tests. Table
1.
Comparison micro-IHA
and
of mean antibody microneutralization
titers
between tests
the
Tannic acid treated sheep red blood cells were sensitized by mixing with properly diluted antigen and incubating at 37 C for 15 min. The sensitized cells were washed with PBS twice and diluted to give a 1.0% suspension with PBS containing 1.0% normal rabbit serum. The micro-IHA test was carried out using the V-bottom microplate. 0.025 ml of sensitized sheep red blood cell suspension was mixed with an equal volume of serial two-fold dilutions of serum in each well of the microplate, and incubated at a room temperature for 90 min. Diluent and antigen free tannic acid treated sheep red blood cells alone served as the control in all tests. Antibody titer was expressed as a reciprocal of the highest dilution giving a definite positive agglutination. The type specificity in each serum was expressed as II/I index as proposed by Rawls et al (5). This was given by the following formula : To determine whether IHA test is a suitable method for detection of type specific antibody in human sera, II/I index in each serum was compared between microIHA and microneutralization tests. As shown in Fig. 1, the IHA test gave comparable results to those obtained by the microneutralization test, except that there was a trend toward somewhat higher values of antibody titer against HSV-1 in the
803
NOTES Table
2.
Effect
of absorption
on
antibody
titers
as measured
by
the
IHA
test
IHA test. This correlation was also seen in a comparison of mean antibody titers (Table 1). In some cases, (not presented) even an amount of antibody too small to be detected by the microneutralization test could be detected by the IHA test. This would indicate that the IHA test may be a more sensitive method than the microneutralization test. The next experiment was designed to determine if the type specific antibody is detectable by the IHA test using human sera absorbed with heterotypic viral antigen . Ten serum samples were chosen on the basis of values of the II/I index, which ranged from 49 to 150. The procedures for absorption were as follows: Vero cells grown in a 500-ml culture bottle were infected with HSV-1 or HSV-2 at an input multiplicity of about 1.0 PFU per cell. The infected cells were harvested when a definite cytopathic effect was noted, usually 24 hr after infection. The cells were suspended in PBS to give a density of 5x 107cells per ml. A 0.5-ml aliquot of serum was added to 4.5 ml of a suspension of infected cells. The mixture was homogenized by a teflon homogenizer and kept at 37 C for 60 min and then at 4 C overnight. The mixture was then centrifuged at 2,000 rpm for 20 minto remove large cell debris. The supernatant was centrifuged at 40,000 rpm for 60 min, and the clarified supernatant was used as a 1: 10 dilution of the absorbed serum after heating at 56 C for 30 min. The results are shown in Table 2. In the cases of sera of which the II/I index was less than 100, absorption with HSV-1 resulted in disappearance not only of antibody against HSV-1 but also of that against HSV-2. In contrast, treatment with HSV-2 absorbed homotypic antibody completely but left heterotypic antibody (antibody to HSV-1). These results suggest that antibody measured by HSV-2 antigen sensitized sheep red blood cells is not type specific but common antibody against both types of virus. On the other hand, in cases when the II/I index was over 100, the sera probably contained type specific antibody as well as type common antibody, because even after absorption with HSV-2, some heterotypic antibody (HSV-1 antibody) remained. Thus the micro-IHA test can be applied to quantitative survey for HSV antibody in human sera, not only for accurate determinations but also as a
804
M.
comparison type
with
specific Thanks
results
antibody are
due
to M.
of the in sera Ohara
OHASHI
AND Y. OZAKI
microneutralization absorbed for
assistance
by
test . heterotypic
in preparation
The viral
of the
IHA
test
antigen manuscript
can
also detect
. .
REFERENCES
1) Fuccillo, D.A., Moder, F.L., Catalano, L.W., Vincent, M.M., and Sever, J.L. 1970. Herpesvirus hominis types 1 and 2: A specific microindirect hemagglutination test. Proc. Soc. Exp. Biol. Med. 133: 735-739 . 2) Lerner, A.M., Lauter, C.B., Nolan, D.C., and Shippey, M. J. 1972. Passive hemagglutinating antibodies in cerebrospinal fluids in herpesvirus hominis encephalitis. Proc. Soc. Exp. Biol. Med . 140: 1460-1466 . 3) Nahmias, A.J., Josey, W.E., Naib, Z.M., Luce, C.F., and Gust, B.A. 1970. Antibodies to herpesvirus hominis type 1 and 2 in human. II. Women with cervical cancer. Am. J. Epidemiol . 91: 547-552 . 4) Ozaki, Y., Ishiguro, T., Ohashi, M., Sawaragi, I., and Ito, Y. 1978. Antibodies to herpesvirus type 1 and type 2 among Japanese cervical cancer patients. Gann 69: 119-122 . 5) Rawls, W.E., Iwamoto, K., Adam, E., and Melnick, J.L. 1970. Measurement of antibodies to herpesvirus type 1 and 2 in human sera. J. Immunol. 104: 599-606 . 6) Rawls, W.E., Tompkins, W.A.F., and Melnick, J.L. 1969. The association of herpesvirus type 2 and carcinoma of the uterine cervix. Am. J. Epidemiol. 89: 547-554 . 7) Royston, I., and Aurelian L. 1970. The association of genital herpesvirus with cervical atypia and carcinoma in situ. Am. J. Epidemiol. 91: 531-538 .
