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Rapid detection of rotavirus by using colloidal gold particles labeled with monoclonal antibody
ELSEVIER
Journal of Virological Methods 47 (1994) 51-58
Journal of Virological Methods
Rapid detection of rotavirus by using colloidal gold particles labeled with monoclonal antibody V.S. Dar*,“, S. Ghosh”, S. Broorb “Hybridoma Laboratory, National Institute of Immunology, Shahid Jeet Singh Road, New Delhi 110067, India, “Virology Section, Department of Microbiology, All India Institute of Medical Sciences, New Delhi IlOO29, India
(Accepted 14 September 1993)
Abstract A rapid single-step, sensitive and specific immunogold assay is described for detection of rotavirus. Murine monoclonal antibody (MCA) to the group antigen of rotavirus was dotted on a nitrocellulose paper strip. This was used to capture the rotavirus antigen which was revealed using a conjugate consisting of the MCA conjugated to gold particles. This assay detects 5 x 10’ particles of rotavirus. It also accutely detected rotavirus from fecal samples, but failed to show any reaction with microscopically confirmed fecal samples containing G. lumblia and E. histolytica. The time taken for the test was approximately 10 min. Its versatality, simplicity, sensitivity and specificity makes it useful for third world countries. Key words: Immunogold
assay; Viral diagnosis;
Rotavirus
1. Introduction Of the five distinct groups (A-E) of rotaviruses, group A is at present by far the most frequently isolated in cases of diarrhea in a wide variety of mammalian species (Flewett and Woode, 1978; Kapikian et al., 1986). As a consequence group A rotavirus is a major pathogen responsible for an estimated 2-5 million infant deaths per annum predominantly in the third world (Agarwal, 1979). Rotavirus accounts for .57% of acute diarrhea in children attending hospitals in India (Broor et al., 1985, Paniker et al., 1982; Broor et al., 1993). The development of rapid and sensitive procedures for the detection of these *Corresponding author.
Fax: +91 11 6862125.
0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(93)E0127-N
52
V.S. Dar et al./Journal
of Virological Methods
47 (1994) 51-58
viruses is a necessary prerequisite to control the disease. Electron microscopy and immune electron microscopy were for some time the only methods available for the diagnosis of rotavirus. Numerous assays were developed later for detecting rotavirus in feces; latex agglutination (Bryden, 1981) and enzyme linked immunosorbant assays ( ELISA) (Westmoreland et al., 1987; Beards and Bryden, 1981) are generally recommended for routine diagnosis. In this communication we report a simple single-step diagnostic assay using colloidal gold particles labeled with monoclonal antibody targeted at the group A common antigen VP6 (Estes and Cohen, 1989). The immunogold assay employs specific monoclonal antibody dotted on to a nitrocellulose strip which acts as capture antibody. When rotavirus containing samples and MCA-colloidal gold conjugate are mixed, immune complexes are formed and move up by capillary action through the nitrocellulose membrane, and are then retained by the capture antibody as the immobilizing support. Deposition of this complex appears in the form of a burgundy-red dot due to concentration of colloidal gold, the intensity of which varies according to the antigen concentration in the test sample. The absence of a dot indicates a negative result (Gupta et al. 1990). This assay allows rapid testing of a large number of samples and can be carried out under field conditions within a few minutes and read visually.
2. Materials
and methods
2.1. Pure cultures group A rotaviral strains DSl, Wa, SA 11 and Hochi were Virus. Culture-adapted obtained from Dr. H. Greenberg (Stanford, CA). These were grown in MA104 cell line (National Facility for Animal Tissue Culture, Pune, India) in Eagle’s MEM (Himedia, India ) with 10% fetal calf serum. Virus particles were quantitated by absorbance at 260 nm (Smith et al., 1969) Cultures of ECHO (strain 08), Polio and Coxsackie (strains B6 and B5) viruses were obtained from the Virology section, Deparment of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi. These were grown in BGMK cell line in Eagle’s MEM with fetal calf serum. Parasite. E. histolytica strain NIH:200 was obtained from Dr. J.C. Samantaray, Department of Microbiology, AIIMS, New Delhi. These were grown in TYI-S-33 medium. Pure isolates of Escherichia coli, Shigella dysentriae type 1 and SalmonelBacteria. la typhimurium were obtained from the Bacteriology section of the department of Microbiology at the AIIMS, New Delhi and were grown in nutrient broth. 2.2. Clinical samples Fecal samples
were collected
from the pediatric
emergency
clinic at LNJP hospi-
V.S. Dar et al./Journal of Virological
Methods 47 (1994) 51-58
tal, New Delhi and from the Parasitology unit of the All India Institute Sciences, New Delhi. The samples were taken from patients suffering diarrhea or gastroenteritis. 2.3. Production
and characterization
of monoclonal
53 of Medical from acute
antibodies
6-8 weeks old inbred BALB/c mice (N.I.I. small animal facility ) were Fusion. immunized with 100 pg of Wa infected MA104 cell lysate (Lee et al., 1981) intraperitoneally with complete Freunds adjuvant. Three weeks later they were immunized with 100 pg of the same antigen in incomplete Freunds adjuvant followed by an intravenous booster in saline after two weeks. Three days after the last booster the mice were killed and the spleens removed for fusion. Fusion for producing monoclonal antibodies was carried out as described elsewhere (Kohler and Milstein, 1979). The wells positive for growth of hybrid cells were screened for anti-rotavirus antibodies. Hybrids from positive wells were cloned and subcloned by limiting dilution to obtain stable cell lines secreting MCA. These were subsequently grown as ascites in inbred BALB/c mice and were purified by salt fractionation ( Weir, 1979). Culture supernatants of hybrids were Screening of monoclonal antibodies (MCA) screened with the four serotypes of rotavirus (14)represented by Wa, DSl, SAll and Hochi in ELISA. Microtitre EIA plates were coated with 1:50 anti-rotavirus polyclonal rabbit serum (Dakopatts) in 50 mM carbonate buffer, pH 9.5 for 1 h at 37°C. The plates were washed with PBS (50 mM phosphate buffer containing 0.09% saline, pH 7.4) and blocked with 1% milk protein (Lactogen formula 2) for 30 min at 37OC. These were again washed with PBS containing 0.05% Tween 20 (PBS-T). lo8 particles per ml of cell-free virus from tissue culture of each serotype, viz. SA 11, Wa, DSl and Hochi and uninfected MA104 cell lysate were added in separate wells in duplicate. After incubation for 1 h at 37°C 100 ~1 of the culture supernatants from growing hybridomas were added per well and allowed to incubate for an h at 37°C followed by washing and addition of anti mouse immunoglobulin conjugated to horse radish peroxidase (Dakopatts) at a concentration of 1: 1000. Plates were washed and the enzyme activity was revealed by adding 100 ~1 per well of freshly prepared O-phenylene diamine solution (1 mg dissolved in citrate phosphate buffer pH 5.0 containing 0.03% H202). The reaction was stopped using 50 ~1 of 5 N H2S04 and absorbance was read at 492 nm in a Biotek ELISA reader. Zmmunoblot. Different serotypes of rotavirus infected and uninfected cell lysates were run on 10% SDS-PAGE and transferred on to nitrocellulose paper (Towbin et al. 1979). Culture supernatant of MCA secreting clone (CDlbll) was incubated with strips bearing transferred proteins for 1 h. This was followed by washing and incubation with with 1:500 dilution of antimouse immunoglobulin-HRP conjugate (Dakopatts) in PBS-T. Color was developed using 4-chloronaphthol. SpeciJicity histolytica,
of MCA. The specificity of MCA was tested by ELISA. Sonicated E. E. coli, S. typhimurium and S. dysentriae type 1 antigen and infected and
54
V.S. Dar et al./Journal of Virological Methods 47 (1994) 51-58
sonicated cell pellets of ECHO, Coxsackie (B6 and B5) and polio virus were used for coating the wells. After blocking the plate for non specific reaction as described above, 100 ~1 of culture supernatants of the MCA were added to the wells. Each antibody was tested against the entire set of coated pathogens. The rest of the test was performed as described above. 2.4. Immunogold
assay
MCA-colloidal gold conjugation. 50 ml of 0.02% gold chloride (Amresco, OH, USA) in distilled water was put to boil. As soon as it began to boil, 1.2 ml of 1% sodium citrate was added with constant stirring. Color changed from gray to blue to purple violet within 45-60 s. After the color change, the solution was taken off the heat, stirred for 2-3 min and allowed to cool at room temperature. This colloidal gold solution was scanned between 450 and 700 nm by using a spectrophotometer. The batch having lmax between 525 and 535 nm was subsequently used for preparing the conjugate with MCA. To 1 ml of colloidal gold solution in a glass tube, 612 ~1 of 1% K2C03 was added and the pH was adjusted to 6.5. This was followed by addition of 6 pg of MCA and 6 ~1 of 20% BSA (final concentration approx. 0.1%) and the contents were transferred in an Eppendorf tube to be centrifuged for 5 min in a microfuge at 5000 rpm. The entire supernatant except for 50 ~1 was discarded and the pellet was resuspended in it (Martin et al., 1990). Assay. 10% (v/v) fecal suspension was made in PBS, pH 7.4. The suspension was clarified by low-speed centrifugation and supernatant was collected. For the assay 1 pg MCA (CDlbl 1) was dotted on to nitrocellulose paper strip of 0.5 x 5 cm dimension and 8 microns pore size (Advanced Microdevices, India). It was left to dry at room temperature. To a flat bottomed microtitration plate, MCA colloidal gold cojugate was added followed by 5 ~1 each of 10% polyethylene glycol 6000 and 20% BSA. To this was added 30 ~1 of clarified fecal extract and the contents were mixed well. A nitrocellulose strip that was dotted with antibody was put vertically in the well. The solution was allowed to rise by capillary action. The samples positive for rotavirus showed a burgundy-red dot at the region where the antibody was dotted as compared to its absence in negative samples. Sensitivity. The assay was carried out as above except that the fecal sample suspension was substituted by different concentrations of tissue culture derived SAl 1 virus.
3. Results Four hybrid cell clones secreting MCA which reacted to all the four most prevalent of human rotavirus serotypes (Flores et al., 1986) were generated (Fig. 1). MCA CD1 bl 1 was selected for use in the immunogold assay because of its good reactivity as compared to the other three MCA. When tested for specificity in ELISA, CDlbl 1 did not show any cross reaction with other enteroviruses like ECHO, Cox-
V.S. Dar et al./Journal 0.D
490
of Virological Methods 47 (1994) 51-58
nm
CDlbll
BA3fff7
BAZcf4
Monoclonal
Fig. 1. Screening test the reactivity
55
m
DS 1
EXE4Wa
m
Hochi
0
of MCA to rotavirus. of MCA to serotypes
CDlbfQ
antibodies
0
SA11
MA104
ELBA was carried out as described in Materials and Methods 14 of cell-free rotavirus and uninfected MA104 cell lysate.
to
sackie and polio viruses, E. histolytica an enteric parasite and enteric bacteria like E. coli, S. typhimurium and S. dysentrieae type 1 (Fig. 3). As seen in Western blot (Fig. 2) this MCA reacts to the 45 kDa antigen of the four serotypes of group A rotaviruses represented by Wa, DSI, SAl 1 and Hochi strains. The immunogold assay standardised using CDlbll was able to detect 5 x 10’ particles of rotavirus (Fig. 4) when tested on different concentrations of tissue culture derived SAll virus. Fecal samples containing other identified pathogens were also tested by the immunogold assay to establish test specificity in clinical situations (Fig. 5). This assay was carried out on three sets of fecal extracts. Each set consisted of five confirmed samples each containing (i) rotavirus, (ii) G. Zamblia and (iii) E. histolytica. The presence of rotavirus had been previously confirmed by PAGE and
45K-
Fig. 2. Immunoblot showing the reactivity of MCA Cdlbl 1 to VP6 (45 K ) core protein of the rotavirus. Lanes 2,3,4 and 5 show reactivity of CDlbll to DSI, Wa, SAll and Hochi, respectively. Lane 1 shows the reactivity of anti-rotavirus polyclonal serum to SAI 1 antigen.
56
VS. Dur et al./Journal
o.*c
of Virological Methods
I.D. 490nm
47 (1994) 51--S
_
0.1
0.1:
0.1
O.Of
C E:h
E&O
clew
C(S5)
Enteric
P
ix.0
S.d
S.1
Microorganisms
Fig. 3. Specificity of MCA CDlbll. Specificity was assed in ELISA where MCA was reacted to different enteric microorganisms like E. histolytica [E.h], ECHO virus [ECHO], Coxsackie virus, strains B6 [C(B6)], B5 [C(BS]), polio virus [PI, Escherichia coli [E.c], Shigella dysentriae [S.d] and Salmonella typhimurium
[s.t].
that of G. lamblia and E. histolytica by microscopy. Using this assay rotavirus was detected in all the five rotavirus containing fecal samples. No reaction was seen in case of E. histozytica and G. ~arnb~~~ containing samples.
Fig. 4. Sensitivity of this assay.
ofimmunogold
test. Quantitated
SAI 1 viral particles
were used to assess detection
limit
51
V.S. Dar et al./Journal of Virological Methods 47 (1994) 51-58
C Fig. 5. Immunogold Test on clinical samplesThree sets of five fecal samples were tested in this assay. Sets A, B and C represent the immunogold test performed on fecal samples containing rotavirus, E. histolytica and G. Iamblia, respectively. A positive reaction for rotavirus can be identified by the appearance of a burgundy-red dot.
4. Discussion
In contrast to ELISA the immunogold assay is a single-step assay. It is highly specific for rotavirus as it employs MCA. However, enough numbers of clinical samples need to be assayed to define its sensitivity in practice. However, it has been reported that the levels of group A virus found in clinical specimens falls in the range of 108-1012 particles/ml (Xu et al., 1990) and so even at the low end of this range, which is rarely seen, the immunogold assay would require less than 5 ~1 of infected feces to give a positive result. This assay has a great potential for use in third world countries where rapidity of diagnosis and relatively low cost of tests are prerequisite for widespread use. The relative simplicity of performance and easy interpretation makes it suitable for use by unskilled workers away from laboratory settings. Although latex agglutination tests are as rapid, they require trained persons for interpretation of results. In practice both ELISA and latex agglutination tests have inherent unclear interpretation of borderline cases. The immunogold assay has no such difficulties in interpretation. The results are read as a clear ‘dot’ or ‘no dot’. To the best of our knowledge such a test has not been reported for detection of rotaviruses so far. 5. References Agarwal, A. (1979) A cure for a killer - but how to deliver it? Nature (Lond.) 278, 389. Beards, G.M. and Bryden,A.S. (1981)Evaluation of a new enzyme-linked immunosorbant assay test for rotavirus antigen in faeces. J. Clin. Pathol. 34, 1388. Broor, S., Singh, V., Venkateshwaralu, K., Gautam, S., Mehta, S. and Mehta, S.K. (1985) Rotavirus
58
V.S. Dar et al./Journal
of Virological Methods 47 (1994) 51-58
diarrhoea in children in Chandigarh, India. J. Diarrhoel. Dis. Res. 3, 158. Broor,S., Hussain, M., Chatterjee, B., Chakraborty, A. and Seth, P.(1993) Temporal variation in the distribution of rotavirus electropherotypes in Delhi, India. J. Diarrhoeal. Dis. Res. II, 14. Bryden, A.S. (1987) An evaluation of commercial latex agglutination kits for rotavirus detection. Serodiagnos. Immunother. I, 13 1. Estes, M.K. and Cohen, J.(l989) Rotavirus gene structure and function. Microbial. Rev. 53, 410. Flewett,T.H. and Woode, G.N. (1978) The rotaviruses. Brief review. Arch. Virol. 57, 1. Flares, J., Nakagomi, T., Glass, R., Gorzilia, M., Askaa, J., Hoshino, Y., Perez-Schael, I. and Kapikian, A.Z. (1986) Role of rotaviruses in paediatric diarrhea. Pediatr. Infect. Dis. 5, 553. Gupta, R., Talwar, G.P. and Gupta, S.K. (1990) Rapid antibody capture assay for detection of group A streptococci using monoclonal antibody and colloidal gold monospecilic polyvalent antibody conjugate. J. Immunoassay, 13, 441. Kapikian, AZ., Flares, J., Hoshino, Y., Glass, RI., Midthun, K., Gorzilia, M. and Chanock, R.M. (1986) Rotavirus: the major etiologic agent of severe infantile diarrhea may be controllable by a < “ > Jennerian < e” > approach to vaccination. J. Infect. Dis. 153, 815. Kohler, G. and Milstein, C. (1979) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256, 495. Lee, P.N.K., Hayes, H.C. and Joklik, W.K. (1981) Characterization of anti-roevirus immunoglobulins secreted by cloned hybridoma cell lines. Virology 108, 134. Martin, J.M.C., Paques, M., Groat, T.A.M.V. and Beuvery, EC. (1990) Characterization of antibody labelled colloidal gold particles and their applicability in a sol particle immunoassay (SPIA). J. Immunoassay 11, 3 1. Paniker, C.J.K., Mathew, S. and Mathan, M. (1982) Rotavirus and acute diarrhoeal disease in children in a south Indian coastal town. Bull. WHO 60, 7. Smith, R.E., Zweerink, H.J. and Joklik, W.K.(1981) Polypeptide components of virions, top component and cores of reovirus type 3. Virology 108, 134. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Weir, D.M.(1979) Handbook of Experimental Immunology, Blackwell Scientific Publications, Oxford. Westermoreland D, Ashley, CR., Cam, E.0.(1987) Rapid and reliable routine diagnosis of rotavirus using a commercial monoclonal bases assay. J. Clin. Pathol. 40, 1384. Xu, L., Harbour, D. and McCrae, M.A. (1990) The application of polymerase chain reaction to the detection of rotavirus in faeces. J. Viral. Methods 27. 29.
Journal of Virological Methods 47 (1994) 51-58
Journal of Virological Methods
Rapid detection of rotavirus by using colloidal gold particles labeled with monoclonal antibody V.S. Dar*,“, S. Ghosh”, S. Broorb “Hybridoma Laboratory, National Institute of Immunology, Shahid Jeet Singh Road, New Delhi 110067, India, “Virology Section, Department of Microbiology, All India Institute of Medical Sciences, New Delhi IlOO29, India
(Accepted 14 September 1993)
Abstract A rapid single-step, sensitive and specific immunogold assay is described for detection of rotavirus. Murine monoclonal antibody (MCA) to the group antigen of rotavirus was dotted on a nitrocellulose paper strip. This was used to capture the rotavirus antigen which was revealed using a conjugate consisting of the MCA conjugated to gold particles. This assay detects 5 x 10’ particles of rotavirus. It also accutely detected rotavirus from fecal samples, but failed to show any reaction with microscopically confirmed fecal samples containing G. lumblia and E. histolytica. The time taken for the test was approximately 10 min. Its versatality, simplicity, sensitivity and specificity makes it useful for third world countries. Key words: Immunogold
assay; Viral diagnosis;
Rotavirus
1. Introduction Of the five distinct groups (A-E) of rotaviruses, group A is at present by far the most frequently isolated in cases of diarrhea in a wide variety of mammalian species (Flewett and Woode, 1978; Kapikian et al., 1986). As a consequence group A rotavirus is a major pathogen responsible for an estimated 2-5 million infant deaths per annum predominantly in the third world (Agarwal, 1979). Rotavirus accounts for .57% of acute diarrhea in children attending hospitals in India (Broor et al., 1985, Paniker et al., 1982; Broor et al., 1993). The development of rapid and sensitive procedures for the detection of these *Corresponding author.
Fax: +91 11 6862125.
0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(93)E0127-N
52
V.S. Dar et al./Journal
of Virological Methods
47 (1994) 51-58
viruses is a necessary prerequisite to control the disease. Electron microscopy and immune electron microscopy were for some time the only methods available for the diagnosis of rotavirus. Numerous assays were developed later for detecting rotavirus in feces; latex agglutination (Bryden, 1981) and enzyme linked immunosorbant assays ( ELISA) (Westmoreland et al., 1987; Beards and Bryden, 1981) are generally recommended for routine diagnosis. In this communication we report a simple single-step diagnostic assay using colloidal gold particles labeled with monoclonal antibody targeted at the group A common antigen VP6 (Estes and Cohen, 1989). The immunogold assay employs specific monoclonal antibody dotted on to a nitrocellulose strip which acts as capture antibody. When rotavirus containing samples and MCA-colloidal gold conjugate are mixed, immune complexes are formed and move up by capillary action through the nitrocellulose membrane, and are then retained by the capture antibody as the immobilizing support. Deposition of this complex appears in the form of a burgundy-red dot due to concentration of colloidal gold, the intensity of which varies according to the antigen concentration in the test sample. The absence of a dot indicates a negative result (Gupta et al. 1990). This assay allows rapid testing of a large number of samples and can be carried out under field conditions within a few minutes and read visually.
2. Materials
and methods
2.1. Pure cultures group A rotaviral strains DSl, Wa, SA 11 and Hochi were Virus. Culture-adapted obtained from Dr. H. Greenberg (Stanford, CA). These were grown in MA104 cell line (National Facility for Animal Tissue Culture, Pune, India) in Eagle’s MEM (Himedia, India ) with 10% fetal calf serum. Virus particles were quantitated by absorbance at 260 nm (Smith et al., 1969) Cultures of ECHO (strain 08), Polio and Coxsackie (strains B6 and B5) viruses were obtained from the Virology section, Deparment of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi. These were grown in BGMK cell line in Eagle’s MEM with fetal calf serum. Parasite. E. histolytica strain NIH:200 was obtained from Dr. J.C. Samantaray, Department of Microbiology, AIIMS, New Delhi. These were grown in TYI-S-33 medium. Pure isolates of Escherichia coli, Shigella dysentriae type 1 and SalmonelBacteria. la typhimurium were obtained from the Bacteriology section of the department of Microbiology at the AIIMS, New Delhi and were grown in nutrient broth. 2.2. Clinical samples Fecal samples
were collected
from the pediatric
emergency
clinic at LNJP hospi-
V.S. Dar et al./Journal of Virological
Methods 47 (1994) 51-58
tal, New Delhi and from the Parasitology unit of the All India Institute Sciences, New Delhi. The samples were taken from patients suffering diarrhea or gastroenteritis. 2.3. Production
and characterization
of monoclonal
53 of Medical from acute
antibodies
6-8 weeks old inbred BALB/c mice (N.I.I. small animal facility ) were Fusion. immunized with 100 pg of Wa infected MA104 cell lysate (Lee et al., 1981) intraperitoneally with complete Freunds adjuvant. Three weeks later they were immunized with 100 pg of the same antigen in incomplete Freunds adjuvant followed by an intravenous booster in saline after two weeks. Three days after the last booster the mice were killed and the spleens removed for fusion. Fusion for producing monoclonal antibodies was carried out as described elsewhere (Kohler and Milstein, 1979). The wells positive for growth of hybrid cells were screened for anti-rotavirus antibodies. Hybrids from positive wells were cloned and subcloned by limiting dilution to obtain stable cell lines secreting MCA. These were subsequently grown as ascites in inbred BALB/c mice and were purified by salt fractionation ( Weir, 1979). Culture supernatants of hybrids were Screening of monoclonal antibodies (MCA) screened with the four serotypes of rotavirus (14)represented by Wa, DSl, SAll and Hochi in ELISA. Microtitre EIA plates were coated with 1:50 anti-rotavirus polyclonal rabbit serum (Dakopatts) in 50 mM carbonate buffer, pH 9.5 for 1 h at 37°C. The plates were washed with PBS (50 mM phosphate buffer containing 0.09% saline, pH 7.4) and blocked with 1% milk protein (Lactogen formula 2) for 30 min at 37OC. These were again washed with PBS containing 0.05% Tween 20 (PBS-T). lo8 particles per ml of cell-free virus from tissue culture of each serotype, viz. SA 11, Wa, DSl and Hochi and uninfected MA104 cell lysate were added in separate wells in duplicate. After incubation for 1 h at 37°C 100 ~1 of the culture supernatants from growing hybridomas were added per well and allowed to incubate for an h at 37°C followed by washing and addition of anti mouse immunoglobulin conjugated to horse radish peroxidase (Dakopatts) at a concentration of 1: 1000. Plates were washed and the enzyme activity was revealed by adding 100 ~1 per well of freshly prepared O-phenylene diamine solution (1 mg dissolved in citrate phosphate buffer pH 5.0 containing 0.03% H202). The reaction was stopped using 50 ~1 of 5 N H2S04 and absorbance was read at 492 nm in a Biotek ELISA reader. Zmmunoblot. Different serotypes of rotavirus infected and uninfected cell lysates were run on 10% SDS-PAGE and transferred on to nitrocellulose paper (Towbin et al. 1979). Culture supernatant of MCA secreting clone (CDlbll) was incubated with strips bearing transferred proteins for 1 h. This was followed by washing and incubation with with 1:500 dilution of antimouse immunoglobulin-HRP conjugate (Dakopatts) in PBS-T. Color was developed using 4-chloronaphthol. SpeciJicity histolytica,
of MCA. The specificity of MCA was tested by ELISA. Sonicated E. E. coli, S. typhimurium and S. dysentriae type 1 antigen and infected and
54
V.S. Dar et al./Journal of Virological Methods 47 (1994) 51-58
sonicated cell pellets of ECHO, Coxsackie (B6 and B5) and polio virus were used for coating the wells. After blocking the plate for non specific reaction as described above, 100 ~1 of culture supernatants of the MCA were added to the wells. Each antibody was tested against the entire set of coated pathogens. The rest of the test was performed as described above. 2.4. Immunogold
assay
MCA-colloidal gold conjugation. 50 ml of 0.02% gold chloride (Amresco, OH, USA) in distilled water was put to boil. As soon as it began to boil, 1.2 ml of 1% sodium citrate was added with constant stirring. Color changed from gray to blue to purple violet within 45-60 s. After the color change, the solution was taken off the heat, stirred for 2-3 min and allowed to cool at room temperature. This colloidal gold solution was scanned between 450 and 700 nm by using a spectrophotometer. The batch having lmax between 525 and 535 nm was subsequently used for preparing the conjugate with MCA. To 1 ml of colloidal gold solution in a glass tube, 612 ~1 of 1% K2C03 was added and the pH was adjusted to 6.5. This was followed by addition of 6 pg of MCA and 6 ~1 of 20% BSA (final concentration approx. 0.1%) and the contents were transferred in an Eppendorf tube to be centrifuged for 5 min in a microfuge at 5000 rpm. The entire supernatant except for 50 ~1 was discarded and the pellet was resuspended in it (Martin et al., 1990). Assay. 10% (v/v) fecal suspension was made in PBS, pH 7.4. The suspension was clarified by low-speed centrifugation and supernatant was collected. For the assay 1 pg MCA (CDlbl 1) was dotted on to nitrocellulose paper strip of 0.5 x 5 cm dimension and 8 microns pore size (Advanced Microdevices, India). It was left to dry at room temperature. To a flat bottomed microtitration plate, MCA colloidal gold cojugate was added followed by 5 ~1 each of 10% polyethylene glycol 6000 and 20% BSA. To this was added 30 ~1 of clarified fecal extract and the contents were mixed well. A nitrocellulose strip that was dotted with antibody was put vertically in the well. The solution was allowed to rise by capillary action. The samples positive for rotavirus showed a burgundy-red dot at the region where the antibody was dotted as compared to its absence in negative samples. Sensitivity. The assay was carried out as above except that the fecal sample suspension was substituted by different concentrations of tissue culture derived SAl 1 virus.
3. Results Four hybrid cell clones secreting MCA which reacted to all the four most prevalent of human rotavirus serotypes (Flores et al., 1986) were generated (Fig. 1). MCA CD1 bl 1 was selected for use in the immunogold assay because of its good reactivity as compared to the other three MCA. When tested for specificity in ELISA, CDlbl 1 did not show any cross reaction with other enteroviruses like ECHO, Cox-
V.S. Dar et al./Journal 0.D
490
of Virological Methods 47 (1994) 51-58
nm
CDlbll
BA3fff7
BAZcf4
Monoclonal
Fig. 1. Screening test the reactivity
55
m
DS 1
EXE4Wa
m
Hochi
0
of MCA to rotavirus. of MCA to serotypes
CDlbfQ
antibodies
0
SA11
MA104
ELBA was carried out as described in Materials and Methods 14 of cell-free rotavirus and uninfected MA104 cell lysate.
to
sackie and polio viruses, E. histolytica an enteric parasite and enteric bacteria like E. coli, S. typhimurium and S. dysentrieae type 1 (Fig. 3). As seen in Western blot (Fig. 2) this MCA reacts to the 45 kDa antigen of the four serotypes of group A rotaviruses represented by Wa, DSI, SAl 1 and Hochi strains. The immunogold assay standardised using CDlbll was able to detect 5 x 10’ particles of rotavirus (Fig. 4) when tested on different concentrations of tissue culture derived SAll virus. Fecal samples containing other identified pathogens were also tested by the immunogold assay to establish test specificity in clinical situations (Fig. 5). This assay was carried out on three sets of fecal extracts. Each set consisted of five confirmed samples each containing (i) rotavirus, (ii) G. Zamblia and (iii) E. histolytica. The presence of rotavirus had been previously confirmed by PAGE and
45K-
Fig. 2. Immunoblot showing the reactivity of MCA Cdlbl 1 to VP6 (45 K ) core protein of the rotavirus. Lanes 2,3,4 and 5 show reactivity of CDlbll to DSI, Wa, SAll and Hochi, respectively. Lane 1 shows the reactivity of anti-rotavirus polyclonal serum to SAI 1 antigen.
56
VS. Dur et al./Journal
o.*c
of Virological Methods
I.D. 490nm
47 (1994) 51--S
_
0.1
0.1:
0.1
O.Of
C E:h
E&O
clew
C(S5)
Enteric
P
ix.0
S.d
S.1
Microorganisms
Fig. 3. Specificity of MCA CDlbll. Specificity was assed in ELISA where MCA was reacted to different enteric microorganisms like E. histolytica [E.h], ECHO virus [ECHO], Coxsackie virus, strains B6 [C(B6)], B5 [C(BS]), polio virus [PI, Escherichia coli [E.c], Shigella dysentriae [S.d] and Salmonella typhimurium
[s.t].
that of G. lamblia and E. histolytica by microscopy. Using this assay rotavirus was detected in all the five rotavirus containing fecal samples. No reaction was seen in case of E. histozytica and G. ~arnb~~~ containing samples.
Fig. 4. Sensitivity of this assay.
ofimmunogold
test. Quantitated
SAI 1 viral particles
were used to assess detection
limit
51
V.S. Dar et al./Journal of Virological Methods 47 (1994) 51-58
C Fig. 5. Immunogold Test on clinical samplesThree sets of five fecal samples were tested in this assay. Sets A, B and C represent the immunogold test performed on fecal samples containing rotavirus, E. histolytica and G. Iamblia, respectively. A positive reaction for rotavirus can be identified by the appearance of a burgundy-red dot.
4. Discussion
In contrast to ELISA the immunogold assay is a single-step assay. It is highly specific for rotavirus as it employs MCA. However, enough numbers of clinical samples need to be assayed to define its sensitivity in practice. However, it has been reported that the levels of group A virus found in clinical specimens falls in the range of 108-1012 particles/ml (Xu et al., 1990) and so even at the low end of this range, which is rarely seen, the immunogold assay would require less than 5 ~1 of infected feces to give a positive result. This assay has a great potential for use in third world countries where rapidity of diagnosis and relatively low cost of tests are prerequisite for widespread use. The relative simplicity of performance and easy interpretation makes it suitable for use by unskilled workers away from laboratory settings. Although latex agglutination tests are as rapid, they require trained persons for interpretation of results. In practice both ELISA and latex agglutination tests have inherent unclear interpretation of borderline cases. The immunogold assay has no such difficulties in interpretation. The results are read as a clear ‘dot’ or ‘no dot’. To the best of our knowledge such a test has not been reported for detection of rotaviruses so far. 5. References Agarwal, A. (1979) A cure for a killer - but how to deliver it? Nature (Lond.) 278, 389. Beards, G.M. and Bryden,A.S. (1981)Evaluation of a new enzyme-linked immunosorbant assay test for rotavirus antigen in faeces. J. Clin. Pathol. 34, 1388. Broor, S., Singh, V., Venkateshwaralu, K., Gautam, S., Mehta, S. and Mehta, S.K. (1985) Rotavirus
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