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Assessment of enzyme linked immunosorbent assay based diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii in human serum
~)
Pergamon
Comp. lmmun. Microbiol. Infect. Dis'. Vol. 20, No. 4, pp. 309-314, 1997 Published by ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0147-9571(97)00011-8 o147-9571/97 $17.0o + 0.00
ASSESSMENT OF ENZYME LINKED IMMUNOSORBENT ASSAY BASED DIAGNOSTIC KITS (TOXOKIT-G A N D TOXOKIT-M) FOR THE DETECTION OF IgG A N D IgM ANTIBODIES TO T O X O P L A S M A G O N D H IN H U M A N SERUM G . M . B H O P A L E , ~ S. R . N A I K , ' * G . G . B H A V E , 2 S. S. N A I K 3 a n d A. G O G A T E 4 'Laboratory of Parasitology and Immunodiagnostics, Research and Development Division, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India, 2Department of Microbiology, G.S. Medical College, Parel, Bombay 400 012, India, 3Department of Endocrinology, KEM Hospital and Research Centre, Pune 411 011, India and "Department of Microbiology, L.T. Medical College, Sion, Bombay 400 022, India
(ReceivedJbr publication 10 April 1997)
Abstract--An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), (Flow Lab., U.K.) and Toxoplasma lgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies. Published by Elsevier Science Ltd
TOXO-
TEK-G
Key words: Toxoplasma gondiL IgG and IgM antibodies, ELISA, penicillinase, diagnostic kits
R~sum6--Un test (ELISA) utilisant le pbnicillinase a ~t6 d6volopp6 sous forme de "coffret diagnostic (Toxokit G e t Toxokit M) pour la dbtection d'anticorps IgM et IgM de Toxoplasma gondii. La performance des deux coffrets a 6t6 compar6e avec d'autres coffrets disponsibles dans le commerce, par exemple Enzygnost-Toxoplasmosis/IgG (Behring-AIlemagne) et Toxotek-G (Laboratoire FIow-U.K.) et Toxoplasma IgM, microessai (Diamedix Corp. USA) en testant des ~chantillons de s~rums humains. Les r6sultats ont montr~ une bonne correlation entre les coffrets de diagnostics. Toxokit G montre une sensibilit6 et une sp6cificit6 respectivement de 86,66 et de 96,05% Le principal avantage de Toxokit G r6side dans le fait que le r~sultat final peut ~tre visualisb sans intervention sophistiqu6e. Toxokit-M a montr6 une sensibilit+ et une sp+cificit6 de 100% et les r6sultats n'ont pas 6tO affect6s par la prbsence d'anticorps antitoxoplasma de type IgG, de facteurs rhumatoides ou d'anticorps antinucl6aires. Published by Elsevier Science Ltd
Mots clefs: Toxoplasma gondii, IgG et lgM anticorps, ELISA, penicillinase, diagnostique kits *Author for correspondence. 309
310
G.M. Bhopaleet al.
INTRODUCTION Toxoplasmosis, caused by the intracellular protozoan parasite, Toxoplasma gondii, is widely distributed throughout the world [1, 2]. The disease exhibits various clinical manifestations [1, 3] and therefore, poses difficulty in diagnosis. Generally, for the diagnosis of toxoplasmosis, of the several serological tests, viz. dye test, haemagglutination test and immunofluorescence, enzyme linked immunosorbent assay (ELISA) is most reliable because of its sensitivity and specificity [4]. Furthermore, ELISA can be used for the detection of different type of antibodies [5]. Detection of antitoxoplasma IgG and IgM antibodies have been routinely used in many clinical laboratories to determine the probable immune status of individuals [4, 6]. In view of the above fact, we have developed ELISA based diagnostic kits (Toxokit-G and Toxokit-M) using penicillinase as an enzyme marker for the diagnosis of toxoplasmosis. The present communication reports the comparative results of toxoplasma-suspected human samples tested in double blind fashion for the detection of IgG and IgM antibodies to T. gondii by Toxokit-G and Toxokit-M, respectively, alongwith commercially available diagnostic kits at different clinical laboratories. MATERIALS AND METHODS Human serum samples suspected to have toxoplasma infection were tested by using Toxokit-G, Toxokit-M, Enzygnost/Toxoplasmosis IgG (Behring Co., Germany) TOXOTEK-G (Flow lab., U.K.) and Toxoplasma IgM (Microassay, Diamedix Corp., U.S.A.) at different laboratories, viz. Department of Endocrinology, KEM Hospital and Research Centre, Pune 411 011, India; Department of Microbiology, G.S. Medical College and KEM hospital, Bombay 400 012, India; Department of Microbiology, L.T. Medical College and Sion Hospital, Bombay 400 022, India; and Laboratory of Parasitology and Immunodiagnostics, Research and Development Division, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India. The serum samples were collected from the out patient department/central pathological laboratory of afore-mentioned hospitals and tested by kits in double blind fashion for the detection of IgG and IgM antibodies to T. gondii. Toxokit-G is based on indirect ELISA using penicillinase as an enzyme marker. The wells of polyvinyl chloride microtitre plates were coated with T. gondii antigen. After incubation of patient sera samples, enzyme-conjugate (penicillinase-antihuman IgG) was added. Penicillinase activity was determined by using starch iodine-penicillin V solution. Penicillin V reacts with penicillinase and is hydrolysed to penicilloic acid which absorbs iodine from the reaction mixture leading to discolourization in the presence of specific IgG antibody in the well. The end results were read visually by comparing the colour of the test samples with negative, positive and buffer control wells. Enzygnost/ Toxoplasmosis IgG kit of Behring Co. is also based on indirect ELISA. Toxoplasma antigen was coated on the wells of polystyrene microtitre plate. After incubation of patient sera samples, conjugate (antihuman IgG-peroxidase) was added. Peroxidase activity was determined by adding a mixture of tetramethyl benzidine and hydrogen peroxide. After stopping the reaction with sulphuric acid, absorbence values were determined at 450 nm by ELISA reader. Equivocal range (cut off value) was calculated following the instructions of the kit manual.
A s s e s s m e n t o f T o x o k i t - G and T o x o k i t - M
311
Table 1. Stepwise procedure adopted for the detection of lgG antibodies to T. gondii Stepwise procedure Addition of serum First incubation 60 + 2 min at 37°C Wash with washing solution Addition of conjugate Second incubation 60 + 2 min at 3T'C Wash with washing solution Addition of substrate and chromogen Stop the reaction Reader (30 + 2 min after substrate addition)
Toxokit-G*
Kits used for study procedure Behring Co. kit*
Flow Lab. kit*
Serum dilution (l:100), 200 #1/well
Serum dilution (1:231), 200 #1/well
Serum dilution (1:500) 100 #l/well
Manually 5 times with 3 rain interval Conjugate dilution 1:100, 200 #1/well
Manually 5 times with 3 min interval Conjugate dilution 1:50, 100 #l/ml
Manually 5 times times with 3 minutes interval Conjugate undiluted 100 #l/well
Manually 5 times with 3 rain interval Starch iodine + penicillin V 200 #l/well -Visuallyt
Manually 5 times with 3 rain intervals H202 + TMB 100 #l/well
Manually 5 times with 3 rain intervals H202 (I00 #l/well) TMB ( 100 #l/well) Add 200 #l/well H2SO4 ELISA reader at 450 nm§
Add 100 #l/well H2SO4 ELISA reader at 450 nm~
*Test carried out as per the kit manual. tThe results were read visually by comparing the colour of the test sample with negative, positive and buffer control wells. A test sample was interpreted as positive when it exhibits distinct light blue colour as compared with the corresponding dark blue colour of negative and buffer control wells. :~Calculated cut off value (equivocal range) of sample. An absorbence below the equivocal range was negative whereas above was considered as positive for toxoplasmosis. §The absorbence of a sample below the cut off value (20 IU/ml) was considered as negative whereas above was considered as positive for toxoplasmosis.
T O X O T E K - G kit (Flow lab., U.K.) is also based on indirect ELISA. The wells of polystyrene microtitre plates coated with T. gondii antigen were incubated with the patient's serum. Conjugate (antihuman IgG-peroxidase) was then added to the wells. The presence of peroxidase was detected by adding the mixture of tetramethyl benzidine and hydrogen peroxide. After stopping the reaction with sulphuric acid, absorbence values were determined at 450 nm by ELISA reader. The end result of the sample was interpreted as per the kit manual (Table 1). Toxokit-M is based on the principle of antibody class capture ELISA. Patients' serum samples were added into antihuman IgM precoated polyvinyl microtitre plate wells, After incubation, antigen (T. gondii) was added. Thereafter, penicillinase conjugated with F'(ab)2 of antitoxoplasma IgG was added. Penicillinase activity was determined using starch-iodine-penicillin V solution. Absorbence values were determined on ELISA reader at 620 nm. The absorbence value of test samples below and above the cut off value was interpreted as positive and negative for IgM antibodies, respectively. Diamedic kit is based on indirect ELISA, Toxoplasma antigen coated on the wells of polystyrene microtitre plate to which a patient's serum sample was added and then incubated. After incubation, enzyme conjugate (alkaline phosphatase antihuman IgM) was added. Phosphatase activity was determined on ELISA reader at 405 nm using p-nitrophenyl phosphate. Enzyme Unit/ml was calculated as per the instruction of the kit manual (Table 2). Initially, the reference sera samples supplied in the commercially diagnostic kits were counter checked using the WHO reference standard sample. Thereafter, we have used the W H O reference standard for standardization of Toxokit-G and Toxokit-M for determining IgG and IgM antibodies to T. gondii. The end results of Toxokit-G and Toxokit-M were compared with other commercially diagnostic kits (Enzygnost/
G . M. B h o p a l e et al.
312
Table 2. Stepwise procedure followed for the detection of lgM antibodies to T. gondii Stepwise procedure
Kits used for the study Toxokit-M*
Diamedix Co. kit*
Serum dilution l:41, 100 t~l/well Serum dilution 1:50, 200 ~d/well Room temperature (20-30 C) for 30 min 3 7 C for 45 rain Manually 5 times with 2 rain interval Manually 5 times with 2 rain interval Antigen dilution 1:10, 200 ~tl/well 3 7 C for 45 rain Manually 5 times with 2 rain interval Conjugate dilution 1:100,200 ld/well Diluted conjugate 100 ~ul/well 37"C for 45 rain Room temperature (20-30"~C) for 30 min Manually 5 times with 2 rain interval Manually 5 times with 2 rain interval Starch iodine + penicillin V, 200 td/well p-Nitrophenyl phosphate 100 ~d/well Add 100 ,ul/well stop solution ELISA reader at 620 n m t EL1SA reader at 405 nm$
Addition of serum Incubation Wash with washing solution Addition of antigen Incubation Wash with washing solution Addition of conjugate Incubation Wash with washing solution Addition of substrate Stop the reaction Reading (30 min after substrate addition
*Test carried out as per the kit manual. tOptical density of test sample below the cut off value (1.412) was interpreted as positive for toxoplasmosis. ~:Determine EU/ml value of each test samples, Greater than 80 EU/ml indicates positive and less than 40 EU/ml indicates negative for toxoplasmosis. The sensitivity and specificity of Toxokit-G and Toxokit-M were determined using the following formulae. Percentage of sensitivity = (True positive/True positive + False negative) × 100 Percentage of specificity = (True negative/True negative + False positive)x 100
Toxoplasmosis IgG, Behring Co., Germany; TOXOTEK-G, Flow Lab., U.K.; and Toxoplasma IgG microasssay, Diamedix Corp., U.S.A.) to determine the true/false positive or true/false negative for toxoplasmosis. RESULTS In the present study, out of 138 toxoplasma-suspected human samples tested, 58 samples showed positive for antitoxoplasma IgG antibodies by Toxokit-G. When the results were compared with those obtained by the Behring Co. kit and the Flow Lab. kit, 90.5% of samples gave identical results. However, three false positive and 10 false negative results were also observed with our kit as compared with Behring or Flow Lab. kits (Table 3). Sixty-seven samples were tested by Toxokit-M and Toxoplasm lgM microassay (Diamedix Corp., U.S.A.) for the presence of IgM antibodies to T. gondii (Table 4). Six samples showed positive by both Toxokit-M and Diamedix Corp. kits. It was also observed that five samples (positive for rheumatoid factor and antinuclear antibodies) showed negative for IgM antibodies by both Toxokit-M and Diamedix Corp. kit. Table 3. Results of toxoplasmosis-suspected human serum samples tested by Toxokit-G, the Behring Co. kit (Germany) and the Flow Lab. kit ( U K . ) Name of lab.
No. of sera tested
Toxokit-G (+)
70
24
14 29
8 13 (2)* 13 ( 1)*
KEM, Pune LTMC, Bombay GSMC. Bombay
25
Behring Co. kit (-) 46 (7)t 6 16 (3)t 12
(+)
Flow Lab. kit
(-)
(+)
(-)
--
31
39
-
-
8 14
6 15
12
13
Figures in parentheses indicates result for toxoplasmosis shown as false positive (*) and false negative (t).
Assessment of Toxokit-G and Toxokit-M
313
Table 4. Results of toxoplasmosis-suspected human serum samples tested by Toxokit-M and the Diamedix Corp. kit (U.S.A.) Group Suspected sera samples Antitoxoplasma IgG Positive sera samples Rheumatoid factor and/or antinuclear Antibodies positive sera samples
Number of sera tested
Toxokit-M
Diomedix Corp. kit
Positive
Negative
Positive
Negative
24 37
0 6
24 31
0 6
24 31
6
0
0
0
0
DISCUSSION The present evaluation results of Toxokit-G and Toxokit-M showed a good correlation with that of commercially available kits. The Toxokit-G has shown 86.7 and 96.0% sensitivity and specificity respectively due to the false negative and false positive results. However, test sera samples (false negative and false positive) had an absorbence value very close to the cut off value of the Behring, kit as well as the Flow Lab. kit. The Behring and Flow Lab. kits require ELISA reader for measuring the absorbence of samples for determining the end results. Whereas the end results of Toxokit-G can be assessed visually without using sophisticated instruments. Thus, our studies suggest that there is fairly good agreement between the results of Toxokit-G and other commercially available diagnostic kits for the detection of IgG antibodies to T. gondii. Hence we conclude and recommend that Toxokit-G is a very useful diagnostic aid for epidemiological studies of prevalence of toxoplasma infection. The early diagnosis of acute toxoplasmosis can be possible by the detection of IgM antibodies to T. gondii. The simultaneous determination of the IgG and IgM antibodies shall facilitate and help assessment of the actual immune status of the patients. It has been reported that false positive results may occur for toxoplasmosis in the presence of either rheumatoid factor or antinuclear antibodies in the patient's serum [7, 8]. False negative results are also reported in the presence of high concentration of antitoxoplasma IgG [9]. In order to rule out such interfering factors, we have developed Toxokit-M based on antibody class capture ELISA using a conjugate of F~(ab)2 of antitoxoplasma IgG antibody with penicillinase. We have also tested a few positive samples of rheumatoid factor as well as antinuclear antibodies for toxoplasmosis using developed Toxokit-M, and subsequently confirmed that such serum samples did not give false positive results. Therefore, we claim that Toxokit-M is specific for the detection of IgM antibodies to T. gondii. The choice of penicillinase as an enzyme marker was due to its low cost and greater sensitivity than other conventionally used enzymes such as peroxidase, alkaline phosphatase or B-galactosidase in ELISA [10]. In addition, absence of penicillinase in biological fluids and use of a non-carcinogenic substrate, penicillin V, are also major advantages in the penicillinase ELISA system. Furthermore, penicillinase has also been successfully used in ELISA for the detection of hormones [11-13] and infectious diseases [I 1, 14-16]. In conclusion, our evaluation studies clearly indicate that Toxokit-G and Toxokit-M are simple, sensitive and specific for the detection of IgG and IgM antibodies to T. gon-
314
G. M, Bhopale et al.
dii. T h e k i t s c a n b e u s e d i n t h e p a t h o l o g i c a l l a b o r a t o r i e s f o r t h e d i a g n o s i s o f t o x o p l a s m o s i s as well as f o r t h e i m m u n e s t a t u s o f t h e p a t i e n t s . Acknowledgements--The research work was partly supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, India.
REFERENCES 1. Jackson M. H. and Hutchison W. M. The prevalence and source of toxoplasma infection in the environment. In: Advanees in Parasitology. Baker, J. R. and Muller, R. (Eds). pp. 55 86. Academic Press, London (1989). 2. Bhopale, G. M. and Naik, S. R. Toxoplasmosis in India (1992) Indian Practitioner 45, 237 -245. 3. Frenkel J. K. Toxoplasmosis. In: Pathology of Protozoal and Hehninthic Diseases with Clinieal Correlation. Marcial-Rojas, R. A. (Ed.). pp. 254-280. Williams and Wilkines, Baltimore (1971). 4. Carlier, Y., Bout, D., Dessaiut, J. B., Capron, A., Vanknapen, F., Ruitenberg, E. J., Requist, R. J. and Huldt, G. Evaluation of the enzyme linked immunosorbent assay and other serological test for the diagnosis of toxoplasmosis (1980) Bulletin--WHO 58, 99-105. 5. Turunen, H., Vuorio, K. A. and Leinikki, P. O. Detection of lgG, IgM and lgA antibody response in human toxoplasmosis by Enzyme Linked Immunosorbent Assay (ELISA) (1983) Scandinavian Journal o! InJectious Diseases 15, 307-311. 6. Payne, R. A., Joynson, D. H. M., Balfour, A. H., Harford, J. P., Fleck, D. G., Mythen, M. and Saunders, R. J. Public Health Laboratory Service Enzyme Linked Immunosorbent Assay for detecting toxoplasma IgM antibody (1987) Journal o[" Clinieal Pathology 40, 276-281. 7. Comargo, M. E., Jesen, P. G. and Rocca, A. Rheumatoid factors as a cause for false positive lgM antitoxoplasma fluorescent tests. A technique for specific results (1992) Reviews of the lnstitiute ~[' Medicine and Toxoplasrnosis- Sao Paulo 14, 310-313. 8. Araujo, F. G., Barnett, E.V., Gentry, L. O. and Remington, J. S. False positive antitoxoplasma fluorescent antibody test in patients with antinuclear antibodies (1991) Applied Microbiology 22, 270-275. 9. Cohen, I. R., Nouins, L. C. and Julian, A. J. Competition between and effectiveness of IgG and IgM antibodies in indirect fluorescent antibody and other tests (1967) Journal of Immunology 98, 143-149. 10. Sauer, M. J., Foulkes, J. A. and O Neill, P. M. A comparison of alkaline phosphatase, B-glactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtiter plate enzyme immunoassay (1989) Journal of Steroid Biochemistry 33, 423-431. 11. Bhopale, G . M . and Naik, S. R. Penicillinase as a potential enzyme marker for Enzyme Linked Immunosorbent Assay and its application in immunodiagnostics (1993) Hindustan Antibiotic Bulletin 35, 157 172. 12. Shah, H. P. and Joshi, U. M. A simple and reliable Enzyme Linked Immunosorbent Assay (ELISA) for measuring estrone 3-glucuronide in urine (1982) Steroid Biochemist O, 16, 283-286. 13. Shrinivasan, T. G., Kumari, G. L. and Rao, P. N. Enzyme immunoassay of cortisol in human plasma using penicillinase as label (1988) Cliniea Chimiea Acta 117, 483 492. 14. Wagle, N. M., Desphande, C. K. and Bhave, G. G. Enzyme Linked lmmunosorbent Assay using penicillinase in serodiagnosis of toxoplasmosis (1985) Indian Journal o/" Medicinal Research 81,275-280. 15. Deodhar, L., Gogate, A., Joshi, U. and Sonawala, M. Comparison of EL1SA with metabolism urea!vticum antibodies in nongonococcal urethritis (1988) Indian Journal gf Medieinal Researeh 88, 20 22. 16. Ramaprasad, P., Prasad, G. B. K. S. and Harinath, B. C. Microfilaramia, filarial antibody and immune complex levels in human filariasis before during and after DEC therapy (1988) Acta Tropica 45, 245-255.
Pergamon
Comp. lmmun. Microbiol. Infect. Dis'. Vol. 20, No. 4, pp. 309-314, 1997 Published by ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0147-9571(97)00011-8 o147-9571/97 $17.0o + 0.00
ASSESSMENT OF ENZYME LINKED IMMUNOSORBENT ASSAY BASED DIAGNOSTIC KITS (TOXOKIT-G A N D TOXOKIT-M) FOR THE DETECTION OF IgG A N D IgM ANTIBODIES TO T O X O P L A S M A G O N D H IN H U M A N SERUM G . M . B H O P A L E , ~ S. R . N A I K , ' * G . G . B H A V E , 2 S. S. N A I K 3 a n d A. G O G A T E 4 'Laboratory of Parasitology and Immunodiagnostics, Research and Development Division, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India, 2Department of Microbiology, G.S. Medical College, Parel, Bombay 400 012, India, 3Department of Endocrinology, KEM Hospital and Research Centre, Pune 411 011, India and "Department of Microbiology, L.T. Medical College, Sion, Bombay 400 022, India
(ReceivedJbr publication 10 April 1997)
Abstract--An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), (Flow Lab., U.K.) and Toxoplasma lgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies. Published by Elsevier Science Ltd
TOXO-
TEK-G
Key words: Toxoplasma gondiL IgG and IgM antibodies, ELISA, penicillinase, diagnostic kits
R~sum6--Un test (ELISA) utilisant le pbnicillinase a ~t6 d6volopp6 sous forme de "coffret diagnostic (Toxokit G e t Toxokit M) pour la dbtection d'anticorps IgM et IgM de Toxoplasma gondii. La performance des deux coffrets a 6t6 compar6e avec d'autres coffrets disponsibles dans le commerce, par exemple Enzygnost-Toxoplasmosis/IgG (Behring-AIlemagne) et Toxotek-G (Laboratoire FIow-U.K.) et Toxoplasma IgM, microessai (Diamedix Corp. USA) en testant des ~chantillons de s~rums humains. Les r6sultats ont montr~ une bonne correlation entre les coffrets de diagnostics. Toxokit G montre une sensibilit6 et une sp6cificit6 respectivement de 86,66 et de 96,05% Le principal avantage de Toxokit G r6side dans le fait que le r~sultat final peut ~tre visualisb sans intervention sophistiqu6e. Toxokit-M a montr6 une sensibilit+ et une sp+cificit6 de 100% et les r6sultats n'ont pas 6tO affect6s par la prbsence d'anticorps antitoxoplasma de type IgG, de facteurs rhumatoides ou d'anticorps antinucl6aires. Published by Elsevier Science Ltd
Mots clefs: Toxoplasma gondii, IgG et lgM anticorps, ELISA, penicillinase, diagnostique kits *Author for correspondence. 309
310
G.M. Bhopaleet al.
INTRODUCTION Toxoplasmosis, caused by the intracellular protozoan parasite, Toxoplasma gondii, is widely distributed throughout the world [1, 2]. The disease exhibits various clinical manifestations [1, 3] and therefore, poses difficulty in diagnosis. Generally, for the diagnosis of toxoplasmosis, of the several serological tests, viz. dye test, haemagglutination test and immunofluorescence, enzyme linked immunosorbent assay (ELISA) is most reliable because of its sensitivity and specificity [4]. Furthermore, ELISA can be used for the detection of different type of antibodies [5]. Detection of antitoxoplasma IgG and IgM antibodies have been routinely used in many clinical laboratories to determine the probable immune status of individuals [4, 6]. In view of the above fact, we have developed ELISA based diagnostic kits (Toxokit-G and Toxokit-M) using penicillinase as an enzyme marker for the diagnosis of toxoplasmosis. The present communication reports the comparative results of toxoplasma-suspected human samples tested in double blind fashion for the detection of IgG and IgM antibodies to T. gondii by Toxokit-G and Toxokit-M, respectively, alongwith commercially available diagnostic kits at different clinical laboratories. MATERIALS AND METHODS Human serum samples suspected to have toxoplasma infection were tested by using Toxokit-G, Toxokit-M, Enzygnost/Toxoplasmosis IgG (Behring Co., Germany) TOXOTEK-G (Flow lab., U.K.) and Toxoplasma IgM (Microassay, Diamedix Corp., U.S.A.) at different laboratories, viz. Department of Endocrinology, KEM Hospital and Research Centre, Pune 411 011, India; Department of Microbiology, G.S. Medical College and KEM hospital, Bombay 400 012, India; Department of Microbiology, L.T. Medical College and Sion Hospital, Bombay 400 022, India; and Laboratory of Parasitology and Immunodiagnostics, Research and Development Division, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India. The serum samples were collected from the out patient department/central pathological laboratory of afore-mentioned hospitals and tested by kits in double blind fashion for the detection of IgG and IgM antibodies to T. gondii. Toxokit-G is based on indirect ELISA using penicillinase as an enzyme marker. The wells of polyvinyl chloride microtitre plates were coated with T. gondii antigen. After incubation of patient sera samples, enzyme-conjugate (penicillinase-antihuman IgG) was added. Penicillinase activity was determined by using starch iodine-penicillin V solution. Penicillin V reacts with penicillinase and is hydrolysed to penicilloic acid which absorbs iodine from the reaction mixture leading to discolourization in the presence of specific IgG antibody in the well. The end results were read visually by comparing the colour of the test samples with negative, positive and buffer control wells. Enzygnost/ Toxoplasmosis IgG kit of Behring Co. is also based on indirect ELISA. Toxoplasma antigen was coated on the wells of polystyrene microtitre plate. After incubation of patient sera samples, conjugate (antihuman IgG-peroxidase) was added. Peroxidase activity was determined by adding a mixture of tetramethyl benzidine and hydrogen peroxide. After stopping the reaction with sulphuric acid, absorbence values were determined at 450 nm by ELISA reader. Equivocal range (cut off value) was calculated following the instructions of the kit manual.
A s s e s s m e n t o f T o x o k i t - G and T o x o k i t - M
311
Table 1. Stepwise procedure adopted for the detection of lgG antibodies to T. gondii Stepwise procedure Addition of serum First incubation 60 + 2 min at 37°C Wash with washing solution Addition of conjugate Second incubation 60 + 2 min at 3T'C Wash with washing solution Addition of substrate and chromogen Stop the reaction Reader (30 + 2 min after substrate addition)
Toxokit-G*
Kits used for study procedure Behring Co. kit*
Flow Lab. kit*
Serum dilution (l:100), 200 #1/well
Serum dilution (1:231), 200 #1/well
Serum dilution (1:500) 100 #l/well
Manually 5 times with 3 rain interval Conjugate dilution 1:100, 200 #1/well
Manually 5 times with 3 min interval Conjugate dilution 1:50, 100 #l/ml
Manually 5 times times with 3 minutes interval Conjugate undiluted 100 #l/well
Manually 5 times with 3 rain interval Starch iodine + penicillin V 200 #l/well -Visuallyt
Manually 5 times with 3 rain intervals H202 + TMB 100 #l/well
Manually 5 times with 3 rain intervals H202 (I00 #l/well) TMB ( 100 #l/well) Add 200 #l/well H2SO4 ELISA reader at 450 nm§
Add 100 #l/well H2SO4 ELISA reader at 450 nm~
*Test carried out as per the kit manual. tThe results were read visually by comparing the colour of the test sample with negative, positive and buffer control wells. A test sample was interpreted as positive when it exhibits distinct light blue colour as compared with the corresponding dark blue colour of negative and buffer control wells. :~Calculated cut off value (equivocal range) of sample. An absorbence below the equivocal range was negative whereas above was considered as positive for toxoplasmosis. §The absorbence of a sample below the cut off value (20 IU/ml) was considered as negative whereas above was considered as positive for toxoplasmosis.
T O X O T E K - G kit (Flow lab., U.K.) is also based on indirect ELISA. The wells of polystyrene microtitre plates coated with T. gondii antigen were incubated with the patient's serum. Conjugate (antihuman IgG-peroxidase) was then added to the wells. The presence of peroxidase was detected by adding the mixture of tetramethyl benzidine and hydrogen peroxide. After stopping the reaction with sulphuric acid, absorbence values were determined at 450 nm by ELISA reader. The end result of the sample was interpreted as per the kit manual (Table 1). Toxokit-M is based on the principle of antibody class capture ELISA. Patients' serum samples were added into antihuman IgM precoated polyvinyl microtitre plate wells, After incubation, antigen (T. gondii) was added. Thereafter, penicillinase conjugated with F'(ab)2 of antitoxoplasma IgG was added. Penicillinase activity was determined using starch-iodine-penicillin V solution. Absorbence values were determined on ELISA reader at 620 nm. The absorbence value of test samples below and above the cut off value was interpreted as positive and negative for IgM antibodies, respectively. Diamedic kit is based on indirect ELISA, Toxoplasma antigen coated on the wells of polystyrene microtitre plate to which a patient's serum sample was added and then incubated. After incubation, enzyme conjugate (alkaline phosphatase antihuman IgM) was added. Phosphatase activity was determined on ELISA reader at 405 nm using p-nitrophenyl phosphate. Enzyme Unit/ml was calculated as per the instruction of the kit manual (Table 2). Initially, the reference sera samples supplied in the commercially diagnostic kits were counter checked using the WHO reference standard sample. Thereafter, we have used the W H O reference standard for standardization of Toxokit-G and Toxokit-M for determining IgG and IgM antibodies to T. gondii. The end results of Toxokit-G and Toxokit-M were compared with other commercially diagnostic kits (Enzygnost/
G . M. B h o p a l e et al.
312
Table 2. Stepwise procedure followed for the detection of lgM antibodies to T. gondii Stepwise procedure
Kits used for the study Toxokit-M*
Diamedix Co. kit*
Serum dilution l:41, 100 t~l/well Serum dilution 1:50, 200 ~d/well Room temperature (20-30 C) for 30 min 3 7 C for 45 rain Manually 5 times with 2 rain interval Manually 5 times with 2 rain interval Antigen dilution 1:10, 200 ~tl/well 3 7 C for 45 rain Manually 5 times with 2 rain interval Conjugate dilution 1:100,200 ld/well Diluted conjugate 100 ~ul/well 37"C for 45 rain Room temperature (20-30"~C) for 30 min Manually 5 times with 2 rain interval Manually 5 times with 2 rain interval Starch iodine + penicillin V, 200 td/well p-Nitrophenyl phosphate 100 ~d/well Add 100 ,ul/well stop solution ELISA reader at 620 n m t EL1SA reader at 405 nm$
Addition of serum Incubation Wash with washing solution Addition of antigen Incubation Wash with washing solution Addition of conjugate Incubation Wash with washing solution Addition of substrate Stop the reaction Reading (30 min after substrate addition
*Test carried out as per the kit manual. tOptical density of test sample below the cut off value (1.412) was interpreted as positive for toxoplasmosis. ~:Determine EU/ml value of each test samples, Greater than 80 EU/ml indicates positive and less than 40 EU/ml indicates negative for toxoplasmosis. The sensitivity and specificity of Toxokit-G and Toxokit-M were determined using the following formulae. Percentage of sensitivity = (True positive/True positive + False negative) × 100 Percentage of specificity = (True negative/True negative + False positive)x 100
Toxoplasmosis IgG, Behring Co., Germany; TOXOTEK-G, Flow Lab., U.K.; and Toxoplasma IgG microasssay, Diamedix Corp., U.S.A.) to determine the true/false positive or true/false negative for toxoplasmosis. RESULTS In the present study, out of 138 toxoplasma-suspected human samples tested, 58 samples showed positive for antitoxoplasma IgG antibodies by Toxokit-G. When the results were compared with those obtained by the Behring Co. kit and the Flow Lab. kit, 90.5% of samples gave identical results. However, three false positive and 10 false negative results were also observed with our kit as compared with Behring or Flow Lab. kits (Table 3). Sixty-seven samples were tested by Toxokit-M and Toxoplasm lgM microassay (Diamedix Corp., U.S.A.) for the presence of IgM antibodies to T. gondii (Table 4). Six samples showed positive by both Toxokit-M and Diamedix Corp. kits. It was also observed that five samples (positive for rheumatoid factor and antinuclear antibodies) showed negative for IgM antibodies by both Toxokit-M and Diamedix Corp. kit. Table 3. Results of toxoplasmosis-suspected human serum samples tested by Toxokit-G, the Behring Co. kit (Germany) and the Flow Lab. kit ( U K . ) Name of lab.
No. of sera tested
Toxokit-G (+)
70
24
14 29
8 13 (2)* 13 ( 1)*
KEM, Pune LTMC, Bombay GSMC. Bombay
25
Behring Co. kit (-) 46 (7)t 6 16 (3)t 12
(+)
Flow Lab. kit
(-)
(+)
(-)
--
31
39
-
-
8 14
6 15
12
13
Figures in parentheses indicates result for toxoplasmosis shown as false positive (*) and false negative (t).
Assessment of Toxokit-G and Toxokit-M
313
Table 4. Results of toxoplasmosis-suspected human serum samples tested by Toxokit-M and the Diamedix Corp. kit (U.S.A.) Group Suspected sera samples Antitoxoplasma IgG Positive sera samples Rheumatoid factor and/or antinuclear Antibodies positive sera samples
Number of sera tested
Toxokit-M
Diomedix Corp. kit
Positive
Negative
Positive
Negative
24 37
0 6
24 31
0 6
24 31
6
0
0
0
0
DISCUSSION The present evaluation results of Toxokit-G and Toxokit-M showed a good correlation with that of commercially available kits. The Toxokit-G has shown 86.7 and 96.0% sensitivity and specificity respectively due to the false negative and false positive results. However, test sera samples (false negative and false positive) had an absorbence value very close to the cut off value of the Behring, kit as well as the Flow Lab. kit. The Behring and Flow Lab. kits require ELISA reader for measuring the absorbence of samples for determining the end results. Whereas the end results of Toxokit-G can be assessed visually without using sophisticated instruments. Thus, our studies suggest that there is fairly good agreement between the results of Toxokit-G and other commercially available diagnostic kits for the detection of IgG antibodies to T. gondii. Hence we conclude and recommend that Toxokit-G is a very useful diagnostic aid for epidemiological studies of prevalence of toxoplasma infection. The early diagnosis of acute toxoplasmosis can be possible by the detection of IgM antibodies to T. gondii. The simultaneous determination of the IgG and IgM antibodies shall facilitate and help assessment of the actual immune status of the patients. It has been reported that false positive results may occur for toxoplasmosis in the presence of either rheumatoid factor or antinuclear antibodies in the patient's serum [7, 8]. False negative results are also reported in the presence of high concentration of antitoxoplasma IgG [9]. In order to rule out such interfering factors, we have developed Toxokit-M based on antibody class capture ELISA using a conjugate of F~(ab)2 of antitoxoplasma IgG antibody with penicillinase. We have also tested a few positive samples of rheumatoid factor as well as antinuclear antibodies for toxoplasmosis using developed Toxokit-M, and subsequently confirmed that such serum samples did not give false positive results. Therefore, we claim that Toxokit-M is specific for the detection of IgM antibodies to T. gondii. The choice of penicillinase as an enzyme marker was due to its low cost and greater sensitivity than other conventionally used enzymes such as peroxidase, alkaline phosphatase or B-galactosidase in ELISA [10]. In addition, absence of penicillinase in biological fluids and use of a non-carcinogenic substrate, penicillin V, are also major advantages in the penicillinase ELISA system. Furthermore, penicillinase has also been successfully used in ELISA for the detection of hormones [11-13] and infectious diseases [I 1, 14-16]. In conclusion, our evaluation studies clearly indicate that Toxokit-G and Toxokit-M are simple, sensitive and specific for the detection of IgG and IgM antibodies to T. gon-
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dii. T h e k i t s c a n b e u s e d i n t h e p a t h o l o g i c a l l a b o r a t o r i e s f o r t h e d i a g n o s i s o f t o x o p l a s m o s i s as well as f o r t h e i m m u n e s t a t u s o f t h e p a t i e n t s . Acknowledgements--The research work was partly supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, India.
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