Clin. exp. Immunol. (1976) 26, 78-85.
Detection of antibody to respiratory syncytial virus by membrane fluorescence R. SCOTT,* M. 0. DE LANDAZURI,t P. S. GARDNER,*&J.J. T. OWENS * DepartmentofVirology, Royal Victoria Infirmary, Newcastle-upon-Tyne, t Centro Nacional de Investigaciones Medico Quirurgicas de la Seguridad Social Department of Immunology, Madrid, Spain and I Department of Anatomy, University of Newcastle upon Tyne, Newcastle-upon-Tyne
(Received 4 June 1976) SUMMARY
An indirect membrane fluorescent antibody technique was established to study HEp 2 cells infected with respiratory syncytial (RS) virus. It was possible to detect IgG and IgM antibody to RS virus in the sera of patients with respiratory infections using this technique. The technique was further applied to the detection of IgA antibody to the same virus in colostrum.
INTRODUCTION The indirect fluorescent antibody technique for the detection of RS virus, using cells aspirated from the nasopharynx of patients with respiratory infections, has been established as a routine diagnostic test in this laboratory (Gardner & McQuillin, 1968). The same technique has also been applied to the detection of RS virus in infected tissue culture cells (McQuillin & Gardner, 1968). In both cases, the cells are fixed in acetone and the resulting fluorescent picture is one of bright apple-green particulate cytoplasmic fluorescence. The present investigation was concerned with the study of RS virus infected HEp 2 cells by a membrane fluorescent antibody technique. Membrane fluorescence of viable cells provided an opportunity to study RS virus antigen in an unaltered condition, compared to methods employing fixed cells. The technique was also used in conjunction with an antibody-dependent cell cytotoxicity assay (De Landazuri et al., 1976 in preparation), and it was therefore necessary to study RS virus antigen present on the membrane of the infected cell. In RS virus infection membrane antigen is likely to be of paramount importance with respect to stimulation of the host immune response. Membrane fluorescence, therefore, provides an in vitro technique which should accurately reflect the surface antigens in vivo. A further aim of the present investigation was to evaluate membrane fluorescence as a method for detection of IgA, IgG and IgM antibody to RS virus in serum and colostrum. MATERIALS AND METHODS Patients. Specimens of sera were taken from patients, with respiratory infections, on admission to hospital and 1-2 weeks later. The sera were stored at -20'C until required for routine serological investigation by the complement fixation test (Bradstreet & Taylor, 1962), or examination by the membrane fluorescent antibody technique. The colostra specimens, examined by membrane fluorescence, were taken from a series of twenty-one women admitted to the Princess Mary Maternity Hospital in Newcastle-upon-Tyne. A comprehensive immunological investigation of the same colostra specimens has been reported elsewhere (Downham et al., 1976). Virus. Long strain RS virus was inoculated onto HEp 2 cells and, after several passages, the resulting virus suspension was divided into l-ml aliquots and stored at - 70'C until required. The virus pool was titrated on HEp 2 cells and a titre of TCD50 10`5 was recorded.
Correspondence: Dr R. Scott, Department of Virology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle-uponTyne.
78
Respiratory syncytial virus membrane fluorescence
79
Membrane fluorescent antibody test. A confluent monolayer of HEp 2 cells (approximately 107 cells), grown in 4-oz flat bottles, was inoculated with 2-5 ml of a 1/5 dilution of the virus stock. After an incubation period of 3 days, the cells were washed twice with a solution of 0 25% trypsin diluted 1/3 with versene. They were then incubated in this medium at 37'C for 2 min, by which time the cells had ceased to adhere to the glass surface. The resulting cell suspension was washed in growth medium comprising Eagle's minimal essential medium supplemented with 10% calf serum and antibiotics (penicillin 500 mg/ml, streptomycin 250 mg/ml and nystatin 125 mg/ml). The cells were then resuspended in the same medium at a concentration of 3 105 cells/ml. Coverslips were seeded with 1 ml of this cell suspension and the cells allowed to adhere by incubation at 370C for 4 hr in an atmosphere of 5% CO2 and air. They were rinsed in maintenance medium comprising medium 199 supplemented with 2% embryo calf serum and antibiotics (penicillin 500 mg/ml, streptomycin 250 mg/ml and nystatin 125 mg/ml), and mounted on rubber discs in plastic trays. Coverslips seeded with negative HEp 2 cells were treated in the same way. The specimens to be tested were diluted 1/10 in growth medium and 20 p1 was inoculated onto both RS-virus-infected and negative HEp 2 coverslips. After incubation at room temperature for 30 min, the coverslips were given three 2-min rinses in maintenance medium, 20 pl of the conjugate, containing antibody to human IgA/IgM/IgG labelled with fluorescein isothiocyanate (Wellcome Reagents Ltd) was then inoculated onto the coverslips, which were incubated for a further 30 min at room temperature. They were again given three 2-min rinses in maintenance medium, finally rinsed in distilled water, dehydrated in 70%, 95% and absolute alcohol for 30-sec intervals, and cleared in xylol for 30 sec, before mounting in Gurr's Uvinert mountant. They were examined with a Vickers M41 fluorescent microscope, using incident light from an HBO-200 mercury vapour light source. In preliminary experiments the optimal dilution of each conjugate was determined by using specimens containing high levels of RS virus specific IgA, IgM or IgG. The anti-IgA and IgG conjugates were used at 1/20 dilution and the anti-IgM conjugate at a dilution of 1/12 throughout the period of the present investigation. Initially, both RS-virus-infected and negative cells were stained with each of the conjugates alone as a control. The results were found to be negative and these controls were omitted from the remainder of the investigation. However, the specificity of fluorescence obtained with RSvirus-infected cells was controlled for each specimen of serum or colostrum examined by staining negative cells in parallel. All specimens were diluted 1/10 in growth medium and the resulting fluorescence was graded from +-+ + + + depending on the intensity. A typical + + + + reading can be seen in Fig. 1 for IgA. Membrane fluorescence for IgG was of similar appearance to that of IgA. A + + + + reading for IgM was not obtained for any of the specimens examined. Fig. 2 shows a typical + +-+ + + reading for IgM.
FIG. 1 FIG. 1. IgA membrane fluorescence. (Magnification x 500.)
FIG. 2 fluorescence. (Magnification x 500.) membrane FIG. 2. IgM
80
R. Scott et al.
Three-day R-S-virus infected cells were used for the preparation of coverslips as preliminary experiments showed that
70-90Y4 of cells exhibited + + + + fluorescence at that time. RESULTS IgG fluorescence Acute and convalescent specimens of sera, taken from eight patients with RS virus respiratory infections, as diagnosed by the indirect fluorescent antibody technique on cells from nasopharyngeal secretions and ten patients of comparable ages with respiratory infections from which no virus was isolated, were assayed for specific IgG antibody to RS virus (Tables 1 and 2). All patients in the RS virus group TABLE 1. Membrane fluorescent IgG antibody to RS virus in sera of patients with RS virus infections
Serum no. 7135 7241 7267 2978 3011 4658 4760 5113 5159 7071 7250 7070 7159 4663 4723 4804 2257 2299
No. of days between sera
Age
7 1
4*
11
7*
12
8*
7
9*
13
20*
7
3t
Membrane fluorescent IgG antibody to RS virus in 1/10 dilution of serum
Titre of complementfixing antibody
++ ++++ ++++ +
< 1/4 1/128 1/128 < 1/4 1/32 < 1/4 1/256 < 1/4 1/16 < 1/4 1/256
+++-++++ + ++++ +++-++++
++++ + + ++ + + ++
++++ +
6
15
3t+ +++ +++-++++ 4t +++-++++ + +++
1/8 1/32 < 1/4 1/16 1/16 1/8 1/256
* Age in months. t Age in years.
possessed IgG antibody to the virus in both acute and convalescent sera. An increase in intensity of IgG fluorescence was detected in the paired sera of all patients in this group. Specific IgG antibody to RS virus was also detected in eighteen out of twenty specimens of sera taken from those patients with respiratory infections from which no virus was isolated. In this group, however, there were no paired sera showing an increase in intensity of fluorescence. A comparison of the results obtained by membrane fluorescence and by complement fixation (Tables 1 and 2) gave an indication of the high sensitivity of the former technique. All individual sera from patients with RS virus infections had detectable levels of antibody by membrane fluorescence, whereas only twelve of the eighteen sera were positive by complement fixation. Similarly, in the group of patients with respiratory infections from which no virus was isolated, only two of the twenty sera were negative by membrane fluorescence compared with twenty negative sera by complement fixation. It is interesting to note, however, that the number of rises in antibody was the same by both membrane fluorescence and complement fixation.
Respiratory syncytial virus membrane fluorescence
81
TABLE 2. Membrane fluorescent IgG antibody to RS virus in sera of patients with respiratory infections from which no virus was isolated
Serum no.
No. of days between sera
Age
7
3*
18
3*
7
4*
9
5*
7
6*
4
6*
11
11*
7
18*
11
2t
10
2t
1813 1898 1226 1366 0609 0676 1704 1779 7598 9698 1921 1946 9774 9886 1804 1888 1560 1632 9780 9854
Membrane fluorescent IgG antibody to RS virus in 1/10 dilution of serum
Titre of complementfixing antibody < 1/8
Detection of antibody to respiratory syncytial virus by membrane fluorescence R. SCOTT,* M. 0. DE LANDAZURI,t P. S. GARDNER,*&J.J. T. OWENS * DepartmentofVirology, Royal Victoria Infirmary, Newcastle-upon-Tyne, t Centro Nacional de Investigaciones Medico Quirurgicas de la Seguridad Social Department of Immunology, Madrid, Spain and I Department of Anatomy, University of Newcastle upon Tyne, Newcastle-upon-Tyne
(Received 4 June 1976) SUMMARY
An indirect membrane fluorescent antibody technique was established to study HEp 2 cells infected with respiratory syncytial (RS) virus. It was possible to detect IgG and IgM antibody to RS virus in the sera of patients with respiratory infections using this technique. The technique was further applied to the detection of IgA antibody to the same virus in colostrum.
INTRODUCTION The indirect fluorescent antibody technique for the detection of RS virus, using cells aspirated from the nasopharynx of patients with respiratory infections, has been established as a routine diagnostic test in this laboratory (Gardner & McQuillin, 1968). The same technique has also been applied to the detection of RS virus in infected tissue culture cells (McQuillin & Gardner, 1968). In both cases, the cells are fixed in acetone and the resulting fluorescent picture is one of bright apple-green particulate cytoplasmic fluorescence. The present investigation was concerned with the study of RS virus infected HEp 2 cells by a membrane fluorescent antibody technique. Membrane fluorescence of viable cells provided an opportunity to study RS virus antigen in an unaltered condition, compared to methods employing fixed cells. The technique was also used in conjunction with an antibody-dependent cell cytotoxicity assay (De Landazuri et al., 1976 in preparation), and it was therefore necessary to study RS virus antigen present on the membrane of the infected cell. In RS virus infection membrane antigen is likely to be of paramount importance with respect to stimulation of the host immune response. Membrane fluorescence, therefore, provides an in vitro technique which should accurately reflect the surface antigens in vivo. A further aim of the present investigation was to evaluate membrane fluorescence as a method for detection of IgA, IgG and IgM antibody to RS virus in serum and colostrum. MATERIALS AND METHODS Patients. Specimens of sera were taken from patients, with respiratory infections, on admission to hospital and 1-2 weeks later. The sera were stored at -20'C until required for routine serological investigation by the complement fixation test (Bradstreet & Taylor, 1962), or examination by the membrane fluorescent antibody technique. The colostra specimens, examined by membrane fluorescence, were taken from a series of twenty-one women admitted to the Princess Mary Maternity Hospital in Newcastle-upon-Tyne. A comprehensive immunological investigation of the same colostra specimens has been reported elsewhere (Downham et al., 1976). Virus. Long strain RS virus was inoculated onto HEp 2 cells and, after several passages, the resulting virus suspension was divided into l-ml aliquots and stored at - 70'C until required. The virus pool was titrated on HEp 2 cells and a titre of TCD50 10`5 was recorded.
Correspondence: Dr R. Scott, Department of Virology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle-uponTyne.
78
Respiratory syncytial virus membrane fluorescence
79
Membrane fluorescent antibody test. A confluent monolayer of HEp 2 cells (approximately 107 cells), grown in 4-oz flat bottles, was inoculated with 2-5 ml of a 1/5 dilution of the virus stock. After an incubation period of 3 days, the cells were washed twice with a solution of 0 25% trypsin diluted 1/3 with versene. They were then incubated in this medium at 37'C for 2 min, by which time the cells had ceased to adhere to the glass surface. The resulting cell suspension was washed in growth medium comprising Eagle's minimal essential medium supplemented with 10% calf serum and antibiotics (penicillin 500 mg/ml, streptomycin 250 mg/ml and nystatin 125 mg/ml). The cells were then resuspended in the same medium at a concentration of 3 105 cells/ml. Coverslips were seeded with 1 ml of this cell suspension and the cells allowed to adhere by incubation at 370C for 4 hr in an atmosphere of 5% CO2 and air. They were rinsed in maintenance medium comprising medium 199 supplemented with 2% embryo calf serum and antibiotics (penicillin 500 mg/ml, streptomycin 250 mg/ml and nystatin 125 mg/ml), and mounted on rubber discs in plastic trays. Coverslips seeded with negative HEp 2 cells were treated in the same way. The specimens to be tested were diluted 1/10 in growth medium and 20 p1 was inoculated onto both RS-virus-infected and negative HEp 2 coverslips. After incubation at room temperature for 30 min, the coverslips were given three 2-min rinses in maintenance medium, 20 pl of the conjugate, containing antibody to human IgA/IgM/IgG labelled with fluorescein isothiocyanate (Wellcome Reagents Ltd) was then inoculated onto the coverslips, which were incubated for a further 30 min at room temperature. They were again given three 2-min rinses in maintenance medium, finally rinsed in distilled water, dehydrated in 70%, 95% and absolute alcohol for 30-sec intervals, and cleared in xylol for 30 sec, before mounting in Gurr's Uvinert mountant. They were examined with a Vickers M41 fluorescent microscope, using incident light from an HBO-200 mercury vapour light source. In preliminary experiments the optimal dilution of each conjugate was determined by using specimens containing high levels of RS virus specific IgA, IgM or IgG. The anti-IgA and IgG conjugates were used at 1/20 dilution and the anti-IgM conjugate at a dilution of 1/12 throughout the period of the present investigation. Initially, both RS-virus-infected and negative cells were stained with each of the conjugates alone as a control. The results were found to be negative and these controls were omitted from the remainder of the investigation. However, the specificity of fluorescence obtained with RSvirus-infected cells was controlled for each specimen of serum or colostrum examined by staining negative cells in parallel. All specimens were diluted 1/10 in growth medium and the resulting fluorescence was graded from +-+ + + + depending on the intensity. A typical + + + + reading can be seen in Fig. 1 for IgA. Membrane fluorescence for IgG was of similar appearance to that of IgA. A + + + + reading for IgM was not obtained for any of the specimens examined. Fig. 2 shows a typical + +-+ + + reading for IgM.
FIG. 1 FIG. 1. IgA membrane fluorescence. (Magnification x 500.)
FIG. 2 fluorescence. (Magnification x 500.) membrane FIG. 2. IgM
80
R. Scott et al.
Three-day R-S-virus infected cells were used for the preparation of coverslips as preliminary experiments showed that
70-90Y4 of cells exhibited + + + + fluorescence at that time. RESULTS IgG fluorescence Acute and convalescent specimens of sera, taken from eight patients with RS virus respiratory infections, as diagnosed by the indirect fluorescent antibody technique on cells from nasopharyngeal secretions and ten patients of comparable ages with respiratory infections from which no virus was isolated, were assayed for specific IgG antibody to RS virus (Tables 1 and 2). All patients in the RS virus group TABLE 1. Membrane fluorescent IgG antibody to RS virus in sera of patients with RS virus infections
Serum no. 7135 7241 7267 2978 3011 4658 4760 5113 5159 7071 7250 7070 7159 4663 4723 4804 2257 2299
No. of days between sera
Age
7 1
4*
11
7*
12
8*
7
9*
13
20*
7
3t
Membrane fluorescent IgG antibody to RS virus in 1/10 dilution of serum
Titre of complementfixing antibody
++ ++++ ++++ +
< 1/4 1/128 1/128 < 1/4 1/32 < 1/4 1/256 < 1/4 1/16 < 1/4 1/256
+++-++++ + ++++ +++-++++
++++ + + ++ + + ++
++++ +
6
15
3t+ +++ +++-++++ 4t +++-++++ + +++
1/8 1/32 < 1/4 1/16 1/16 1/8 1/256
* Age in months. t Age in years.
possessed IgG antibody to the virus in both acute and convalescent sera. An increase in intensity of IgG fluorescence was detected in the paired sera of all patients in this group. Specific IgG antibody to RS virus was also detected in eighteen out of twenty specimens of sera taken from those patients with respiratory infections from which no virus was isolated. In this group, however, there were no paired sera showing an increase in intensity of fluorescence. A comparison of the results obtained by membrane fluorescence and by complement fixation (Tables 1 and 2) gave an indication of the high sensitivity of the former technique. All individual sera from patients with RS virus infections had detectable levels of antibody by membrane fluorescence, whereas only twelve of the eighteen sera were positive by complement fixation. Similarly, in the group of patients with respiratory infections from which no virus was isolated, only two of the twenty sera were negative by membrane fluorescence compared with twenty negative sera by complement fixation. It is interesting to note, however, that the number of rises in antibody was the same by both membrane fluorescence and complement fixation.
Respiratory syncytial virus membrane fluorescence
81
TABLE 2. Membrane fluorescent IgG antibody to RS virus in sera of patients with respiratory infections from which no virus was isolated
Serum no.
No. of days between sera
Age
7
3*
18
3*
7
4*
9
5*
7
6*
4
6*
11
11*
7
18*
11
2t
10
2t
1813 1898 1226 1366 0609 0676 1704 1779 7598 9698 1921 1946 9774 9886 1804 1888 1560 1632 9780 9854
Membrane fluorescent IgG antibody to RS virus in 1/10 dilution of serum
Titre of complementfixing antibody < 1/8
