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Specificity of the enzyme-linked immunosorbent assay (ELISA) for Toxoplasma IgG antibody
TRANSACTIONS OF THE ROYAI.SOCIETY OF TROPICAL MEDICINEANDHYGIENE(1984)78, 661-662
Specificity
of the enzyme-linked Toxoplasma
immunosorbent IgG anti body
661
assay
(EL.ISA) for
R. J. DAHL' AND ALAN M. JOHNSON' ‘School of Pharmacy, South Australian Institute of Technology, Adelaide; LDept. of Clinical Flinders Medical Centre, Bedford Park, South Australia 5042, Australia
Microbiology,
Summary An ELISA developed for Toxoplasma IgG antibody was demonstrated to be more sensitive than the current reference tests, the indirect haemagglutination antibody test (IHAT) and the indirect immunofluorescence test (IIFT). No additional cross reactions were found in the ELISA with sera from patients with other parasitic infections, and there was no interference due to the presence of either rheumatoid factor or antinuclear antibodies. Introduction The clinical diagnosis of toxoplasmosis is usually confirmed by serological testing. The ELBA has been found to be more sensitive than other assays for Toxoplasma antibody (NAOT & REMINGTON, 1981) but with the increase in sensitivity the possibility of loss of specificity arises. Therefore, we investigated the specificity of the ELISA for T. gondii antibody by testing serum from patients who were seronegative for Toxoplasma, but who were infected with one of a range of unrelated parasites. Sera containing rheumatoid factor or antinuclear antibodies were also included as such sera have been shown to produce false positive results in the IIFT and the ELISA for Toxoplasma IgM antibodies (NAOT et al., 1981). Materials and Methods Reference serological tests The IIFT and IHAT were performed described (JOHNSONet al., 1980, 1981).
as previously
Antigen for ELISA Tachyzoites of the RH strain of T. gondii were obtained from infected mice when parasite numbers were maximal; host cell contamination was removed by filtration through a
Table I-Results
No. of sera tested
nuclepore polycarbonate membrane of 3 pm porosity (DAHL & JOHNSON, 1983). The organisms were ruptured by ultrasonication, cell debris was removed by centrifugation at
3000 g for 5 min and the supernatant fluid (stock antigen) was stored at -20°C. ELZSA This test was performed as previously described (VOLLER et al., 1976; WALLS et al., 1977). Briefly, microtitre plates were sensitized with the stock antigen preparation diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.8) at a protein concentration of 2.5 pgiml. Plates were incubated (18 hours, 4°C) then washed with phosphate buffered saline (PBS) pH 7.2 containing 0.5% Tween 20 to remove excess antigen. The plate was then pre-coated with 1% BSA (bovifie serum albumin) in PBS-Tween 20 for one hour at 37°C. After washing, test sera diluted 11200in PBS-Tween 20 containing 1% BSA were incubated for one hour at 37°C and then washed again. Alkaline phosphatase conjugated anti-human IgG (Calbiochem-Behring, W. Germany) diluted 1140 was added and incubated for one hour at 37°C. After washing, 0.1% p-nitrophenol phosphate was added, the plate was incubated for 45 min at 22”C, and the reaction was stopped by the addition of 2M NaOH. The ODb10 was measured on a Dynatech Micro-ELISA reader. *Address for Reprints
of testing various sera for Toxoplasma IgG antibody by reference tests and ELBA No. of sera negative by reference tests
Type of sera
No. of sera positive &A
Sera obtained from:
Wuchereria bancrofti Loa loa Onchocerca volvulus Schistosoma japonicum
6 : 3
!I 0 0
Laboratory of Immunoparasitology, Walter & Eliza Hall Inst. Med. Res., Victoria
21
Echinococcus granulosus
7
0
16
Toxocara canis
10
0
16
Entamoeba histolytica
9
0
Veterinary Clinical Centre, University of Melbourne Commonwealth Institute of Health, University of Sydney Microbiology Department, Queen Elizabeth Hospital, South Australia.
26
Antinuclear
antibody
15
0
54
Rheumatoid
factor positive
28
0
9 : 6
positive
Department of Medical Centre.
Immunology,
Flinders
662
SPECIFICITY OF
ELBA FOR Toxoplasma IGG ANTIBODY
Standardization of the ELISA 25 sera negative for Toxoplasma antibody (IHAT<
1164, IIFT<1/16) and 73 sera with IHAT titres from l/64 to l/8192 and with IIFT titres from l/16 to l/2048 were tested to determine negative cut-off OD410and correlation with the ELISA. Titres 31164 in the IHAT and ~1116 in the IIFT were considered positive, and sera with such titres were found to have a mean ODdI of 20.20 in the ELISA
Sensitivity
The WHO International Standard for Toxoplasma antibody (1000 IU/ml) was tested in the IIFT, IHAT and ELISA, to determine the sensitivity of each type of test. Sera Sera from patients with one of seven parasitic infections, and sera containing rheumatoid factor or antinuclear factor were tested in the reference serological tests. Those sera negative in the reference tests were then assayed in the ELISA Results In the IIFT and IHAT, the WHO International Standard for Toxoplasma antibody gave positive reactions for the l/1024 and 11512 dilutions respectively, suggesting that the sensitivity of these tests is about lIU/ml and 2IU/ml. A l/3200 dilution of the International Standard gave an ODa10 of 0.21 in the ELISA suggesting that the sensitivity of this test is about 0.3 IUlml. The results in Table I show that of the sera that were negative in the reference tests, none were found to be positive in the ELISA. Discussion The ELISA is more sensitive than the IIFT and IHAT for Toxoalasma IeG antibodv. Because of this increased sensitivity, weynvestigated the possibility of loss of specificity. Using the ELISA, cross reactions have been reported between Onchocerca volvulus and sera containing antibodies to the intestinal helminths Ascaris, Trichuris and Necator (LUJAN et al., 1983) and also between Echinococcus granulosus and sera from patients with onchocerciasis, Loa Zoa, Bancroftian filariasis and trichinosis (SPIESER, 1980; RICKARD et aZ., 1984). Cross reactions have previously been reported between Toxoplasma gondii and sera containing antibodies to Sarcocystis and Typanosoma cruzi in the Sabin-Feldman dye test (AWAD & LAINSON, 1954; VAN THIEL, 1958). Cross reactions have also been observed between Toxoplasma gondii and antibodies to Besnoitia jellisoni in the complement fixation test and the IHAT (SUGGS et al., 1968). The results obtained here show that sera negative in both the IIFT and IHAT for Toxoplasma antibody, but positive for one of a number of other parasitic diseases, gave no additional cross reactions in the ELISA. Also, no interference was found due to the presence of kither rheumatoid factor or antinuclear antibodies. It can be concluded that the ELISA is highly sensitive and the specificity is comparable with that of the IIFT and IHAT, at least for the range of parasites tested. The results presented here suggest that the ELISA should become a reliable routine test for the measurement of IgG antibodies to T. gondii.
Acknowledgements We wish to thank the following for the supply of sera used in this research: Drs. G. F. Mitchell and M. D. Rickard. and Messrs. A. Caon and J. Walker. Miss Kathy Mainwood skilfully typed the manuscript. References Awad, F. I. & Lainson, R. (1954). A note on the serology of sarcosnoridiosis and toxonlasmosis. Bournal of Clinical Pathoiogy, 7, 152-156. ” ” Dahl, R. J. & Johnson, A. M. (1983). Purification of Toxoplasma gondii from host cells. Journal of Clinical Pathology, 36, 606-604.
Johnson, A. M., Roberts, H. & McDonald, P. J. (1980). Age-sex distribution of Toxoplasmaantibody in the South Australian population. Journal of Hygiene, 84, 315-320. Johnson, A. M., McNamara, P. J., Neoh, S. H., McDonald, P. J. & Zola, H. (1981). Hybridomas secreting monoclonal antibody to Toxoplasma gondii. Australian Journal of Experimental Biology and Medical Science, 59,
303-306. Lujan, R. L., Collins, W. E., Stanfill, I’. S., Campbell, C. C., Collins, R. C., Brogdon, W. & Huong, A. Y. (1983). Enzyme-linked Immunosorbent Assay (ELISA) for serodiagnosis of Guatemalan onchocerciasis: Comparison with the indirect fluorescent antibody (IFA) test. American Journal of Tropical Medicine and Hygiene, 32, 747-752. Naot, Y. & Remington, J. S. (1981). Use of enzyme-linked immunosorbent assay (ELISA) for detection of monoclonal antibodies: Experience with antigens of Toxoplasma gondii. Journal
of Immunological Methods, 43,
333-341. Naot. Y.. Barnett. E. V. & Remington, I. S. (1981). Method for avoiding filse-positive res& &&r&g in’immunoglobulin M enzyme-linked immunosorbent assaysdue to presence of both rheumatoid factor and antinuclear antibodies. Journal of Clinical Microbiology, 14, 73-78. Rickard, M. D., Honey, R. D., Brumley, J. L. & Mitchell, G. F. (1984). Serological diagnosis and post-operative surveillance of human hydatid diseaseII. The enzymelinked immunosorbent assay (ELISA) using various antigens. Pathology, 16, 211-215. Snieser. F. (1980). ADDlication of the enzvme-linked im* muioso&ent &say-&LISA) for the diagnosis of filariasis and echinococcosis.Tropenmedizinund Parasitologic, 29, 95-102. Suggs, M., Walls, K. W. & Kagan, I. G. (1968). Comparative antieenic studv of Besnoitia ielliscmi. B. aanamenis and five”Toxoplaska gondii isola&s. Jot&al if Zmmunology, 101, 166-175. Van Tluel, I’. H. (1958). The problem of the specificity of the Methylene blue dye test. Ant&e Van Leeuwenhoek, 24, 113-133. (In German). Voller, A., Bidwell, D. E., Bartlett, A., Fleck, D. G., Perkins, M. & Oladehin, B. (1976). A microplate enzyme-immunoassayfor Toxoplasma antibody. Journal of Clinical Pathology, 29, 150-153. Walls, K. W., Bullock, S. L. & English, D. K. (1977). Use of the enzyme-linked immunosorbent assay(ELISA) and its microadaption for the serodiagnosisof toxoplasmosis. Journal of Clinical Microbiology,
5, 273-277.
Accepted for publication 12th January,
1984.
Specificity
of the enzyme-linked Toxoplasma
immunosorbent IgG anti body
661
assay
(EL.ISA) for
R. J. DAHL' AND ALAN M. JOHNSON' ‘School of Pharmacy, South Australian Institute of Technology, Adelaide; LDept. of Clinical Flinders Medical Centre, Bedford Park, South Australia 5042, Australia
Microbiology,
Summary An ELISA developed for Toxoplasma IgG antibody was demonstrated to be more sensitive than the current reference tests, the indirect haemagglutination antibody test (IHAT) and the indirect immunofluorescence test (IIFT). No additional cross reactions were found in the ELISA with sera from patients with other parasitic infections, and there was no interference due to the presence of either rheumatoid factor or antinuclear antibodies. Introduction The clinical diagnosis of toxoplasmosis is usually confirmed by serological testing. The ELBA has been found to be more sensitive than other assays for Toxoplasma antibody (NAOT & REMINGTON, 1981) but with the increase in sensitivity the possibility of loss of specificity arises. Therefore, we investigated the specificity of the ELISA for T. gondii antibody by testing serum from patients who were seronegative for Toxoplasma, but who were infected with one of a range of unrelated parasites. Sera containing rheumatoid factor or antinuclear antibodies were also included as such sera have been shown to produce false positive results in the IIFT and the ELISA for Toxoplasma IgM antibodies (NAOT et al., 1981). Materials and Methods Reference serological tests The IIFT and IHAT were performed described (JOHNSONet al., 1980, 1981).
as previously
Antigen for ELISA Tachyzoites of the RH strain of T. gondii were obtained from infected mice when parasite numbers were maximal; host cell contamination was removed by filtration through a
Table I-Results
No. of sera tested
nuclepore polycarbonate membrane of 3 pm porosity (DAHL & JOHNSON, 1983). The organisms were ruptured by ultrasonication, cell debris was removed by centrifugation at
3000 g for 5 min and the supernatant fluid (stock antigen) was stored at -20°C. ELZSA This test was performed as previously described (VOLLER et al., 1976; WALLS et al., 1977). Briefly, microtitre plates were sensitized with the stock antigen preparation diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.8) at a protein concentration of 2.5 pgiml. Plates were incubated (18 hours, 4°C) then washed with phosphate buffered saline (PBS) pH 7.2 containing 0.5% Tween 20 to remove excess antigen. The plate was then pre-coated with 1% BSA (bovifie serum albumin) in PBS-Tween 20 for one hour at 37°C. After washing, test sera diluted 11200in PBS-Tween 20 containing 1% BSA were incubated for one hour at 37°C and then washed again. Alkaline phosphatase conjugated anti-human IgG (Calbiochem-Behring, W. Germany) diluted 1140 was added and incubated for one hour at 37°C. After washing, 0.1% p-nitrophenol phosphate was added, the plate was incubated for 45 min at 22”C, and the reaction was stopped by the addition of 2M NaOH. The ODb10 was measured on a Dynatech Micro-ELISA reader. *Address for Reprints
of testing various sera for Toxoplasma IgG antibody by reference tests and ELBA No. of sera negative by reference tests
Type of sera
No. of sera positive &A
Sera obtained from:
Wuchereria bancrofti Loa loa Onchocerca volvulus Schistosoma japonicum
6 : 3
!I 0 0
Laboratory of Immunoparasitology, Walter & Eliza Hall Inst. Med. Res., Victoria
21
Echinococcus granulosus
7
0
16
Toxocara canis
10
0
16
Entamoeba histolytica
9
0
Veterinary Clinical Centre, University of Melbourne Commonwealth Institute of Health, University of Sydney Microbiology Department, Queen Elizabeth Hospital, South Australia.
26
Antinuclear
antibody
15
0
54
Rheumatoid
factor positive
28
0
9 : 6
positive
Department of Medical Centre.
Immunology,
Flinders
662
SPECIFICITY OF
ELBA FOR Toxoplasma IGG ANTIBODY
Standardization of the ELISA 25 sera negative for Toxoplasma antibody (IHAT<
1164, IIFT<1/16) and 73 sera with IHAT titres from l/64 to l/8192 and with IIFT titres from l/16 to l/2048 were tested to determine negative cut-off OD410and correlation with the ELISA. Titres 31164 in the IHAT and ~1116 in the IIFT were considered positive, and sera with such titres were found to have a mean ODdI of 20.20 in the ELISA
Sensitivity
The WHO International Standard for Toxoplasma antibody (1000 IU/ml) was tested in the IIFT, IHAT and ELISA, to determine the sensitivity of each type of test. Sera Sera from patients with one of seven parasitic infections, and sera containing rheumatoid factor or antinuclear factor were tested in the reference serological tests. Those sera negative in the reference tests were then assayed in the ELISA Results In the IIFT and IHAT, the WHO International Standard for Toxoplasma antibody gave positive reactions for the l/1024 and 11512 dilutions respectively, suggesting that the sensitivity of these tests is about lIU/ml and 2IU/ml. A l/3200 dilution of the International Standard gave an ODa10 of 0.21 in the ELISA suggesting that the sensitivity of this test is about 0.3 IUlml. The results in Table I show that of the sera that were negative in the reference tests, none were found to be positive in the ELISA. Discussion The ELISA is more sensitive than the IIFT and IHAT for Toxoalasma IeG antibodv. Because of this increased sensitivity, weynvestigated the possibility of loss of specificity. Using the ELISA, cross reactions have been reported between Onchocerca volvulus and sera containing antibodies to the intestinal helminths Ascaris, Trichuris and Necator (LUJAN et al., 1983) and also between Echinococcus granulosus and sera from patients with onchocerciasis, Loa Zoa, Bancroftian filariasis and trichinosis (SPIESER, 1980; RICKARD et aZ., 1984). Cross reactions have previously been reported between Toxoplasma gondii and sera containing antibodies to Sarcocystis and Typanosoma cruzi in the Sabin-Feldman dye test (AWAD & LAINSON, 1954; VAN THIEL, 1958). Cross reactions have also been observed between Toxoplasma gondii and antibodies to Besnoitia jellisoni in the complement fixation test and the IHAT (SUGGS et al., 1968). The results obtained here show that sera negative in both the IIFT and IHAT for Toxoplasma antibody, but positive for one of a number of other parasitic diseases, gave no additional cross reactions in the ELISA. Also, no interference was found due to the presence of kither rheumatoid factor or antinuclear antibodies. It can be concluded that the ELISA is highly sensitive and the specificity is comparable with that of the IIFT and IHAT, at least for the range of parasites tested. The results presented here suggest that the ELISA should become a reliable routine test for the measurement of IgG antibodies to T. gondii.
Acknowledgements We wish to thank the following for the supply of sera used in this research: Drs. G. F. Mitchell and M. D. Rickard. and Messrs. A. Caon and J. Walker. Miss Kathy Mainwood skilfully typed the manuscript. References Awad, F. I. & Lainson, R. (1954). A note on the serology of sarcosnoridiosis and toxonlasmosis. Bournal of Clinical Pathoiogy, 7, 152-156. ” ” Dahl, R. J. & Johnson, A. M. (1983). Purification of Toxoplasma gondii from host cells. Journal of Clinical Pathology, 36, 606-604.
Johnson, A. M., Roberts, H. & McDonald, P. J. (1980). Age-sex distribution of Toxoplasmaantibody in the South Australian population. Journal of Hygiene, 84, 315-320. Johnson, A. M., McNamara, P. J., Neoh, S. H., McDonald, P. J. & Zola, H. (1981). Hybridomas secreting monoclonal antibody to Toxoplasma gondii. Australian Journal of Experimental Biology and Medical Science, 59,
303-306. Lujan, R. L., Collins, W. E., Stanfill, I’. S., Campbell, C. C., Collins, R. C., Brogdon, W. & Huong, A. Y. (1983). Enzyme-linked Immunosorbent Assay (ELISA) for serodiagnosis of Guatemalan onchocerciasis: Comparison with the indirect fluorescent antibody (IFA) test. American Journal of Tropical Medicine and Hygiene, 32, 747-752. Naot, Y. & Remington, J. S. (1981). Use of enzyme-linked immunosorbent assay (ELISA) for detection of monoclonal antibodies: Experience with antigens of Toxoplasma gondii. Journal
of Immunological Methods, 43,
333-341. Naot. Y.. Barnett. E. V. & Remington, I. S. (1981). Method for avoiding filse-positive res& &&r&g in’immunoglobulin M enzyme-linked immunosorbent assaysdue to presence of both rheumatoid factor and antinuclear antibodies. Journal of Clinical Microbiology, 14, 73-78. Rickard, M. D., Honey, R. D., Brumley, J. L. & Mitchell, G. F. (1984). Serological diagnosis and post-operative surveillance of human hydatid diseaseII. The enzymelinked immunosorbent assay (ELISA) using various antigens. Pathology, 16, 211-215. Snieser. F. (1980). ADDlication of the enzvme-linked im* muioso&ent &say-&LISA) for the diagnosis of filariasis and echinococcosis.Tropenmedizinund Parasitologic, 29, 95-102. Suggs, M., Walls, K. W. & Kagan, I. G. (1968). Comparative antieenic studv of Besnoitia ielliscmi. B. aanamenis and five”Toxoplaska gondii isola&s. Jot&al if Zmmunology, 101, 166-175. Van Tluel, I’. H. (1958). The problem of the specificity of the Methylene blue dye test. Ant&e Van Leeuwenhoek, 24, 113-133. (In German). Voller, A., Bidwell, D. E., Bartlett, A., Fleck, D. G., Perkins, M. & Oladehin, B. (1976). A microplate enzyme-immunoassayfor Toxoplasma antibody. Journal of Clinical Pathology, 29, 150-153. Walls, K. W., Bullock, S. L. & English, D. K. (1977). Use of the enzyme-linked immunosorbent assay(ELISA) and its microadaption for the serodiagnosisof toxoplasmosis. Journal of Clinical Microbiology,
5, 273-277.
Accepted for publication 12th January,
1984.