Journal of Helminthology (1977) 51, 143—148
The comparison of counterimmunoelectrophoresis with indirect haemagglutination test for detection of antibodies in experimentally infected guinea pigs with Toxocara canis* MOHAMMAD S. ENAYAT and MOHAMMAD PEZESHKI Pathobiology Department, School of Public Health and Institute of Public Health Research, PO Box 1310, University of Tehran, Iran
ABSTRACT The humoral response of guinea pigs infected with doses of 500,1000, and 1500 infective Toxocara canis ova were measured by counterimmunoelectrophoresis and indirect haemmagglutination tests one to five weeks after infection. The antigens used in these measurements were prepared from adult T. canis. Counterimmunoelectrophoresis showed lower titres than haemagglutination method, but it was performed rapidly and with ease. The possible use of these techniques for immunodiagnosis of human visceral larval migrans is discussed.
Since the description of human visceral larva migrans (VLM) syndrome by Beaver et al. (1952), need for a reliable and specific serodiagnostic test for detection of antibodies against migrating larvae of Toxocara canis has been emphasised by various groups of investigators (Bisseru and Woodruff, 1968; Kagan, 1968; Fernando et al, 1970; Krupp, 1974a). As detection and recognition of migrating Ascarid larvae in the tissue of infected man is immensely difficult, immunodiagnostic techniques could be of a great aid to physicians in the diagnosis of this infection. Most of the available immunological techniques have been used with variable success for detection of anti-toxocara antibody in experimentally infected animals and in suspected human cases (Kagan, 1974). Indirect fluorescent antibody test has been greatly used for this purpose by many investigators with encouraging results (Bisseru and Woodruff, 1968; Baufine-Ducracq et al., 1973; Viens et al., 1975). Counterimmunoelectrophoresis (CIEP) which has been extensively used for diagnosis of many other infectious diseases (Krupp, 1974b; Desowitz and Una, 1976), to our knowledge has not as yet been carried out for diagnosis of VLM. This report examines the relative usefulness of this technique for detection of antitoxocara antibody in experimentally infected guinea pigs and compares its application with indirect haemagglutination tests (IHA) which is being extensively used for this purpose. MATERIAL AND METHODS Antigen preparation Adult male and female worms were obtained from the intestine of young dogs killed by intracardiac injection of saturated magnesium sulphate solution. Worms were washed several times in phosphate buffered saline (PBS, pH 7-2) and finally with deionized water. They were then stored at — 20°C until use. For each preparation 25 worms weighing about 2 g were processed similarly as described by Algeboori and Ivey (1970) with small modifica* A section of this work was submitted in partial fulfilment of the requirements for an MSPH degree by Mohammad Pezeshki 143
M. S. ENAYAT and M. PEZESHKI
tions. Two preparations were obtained. The worms were first cut into small pieces and then manually homogenized in 10 ml of PBS. This preparation was further processed in an electrically operated glass homogenizer (Karl Kolb, Frankfurt, West Germany) in small aliquots, firstly at slow and then faster settings. The whole process was carried out at 4°C. This crude homogenate was centrifuged at 10 000 g for 30 min at 4°C. The supernatent fluid provided the first antigenic extract (Ei). The sediment, resuspended in PBS, was ultrasonicated and centrifuged as before. The supernate from this centrifugation gave the second antigenic extract (E2). The protein concentration of each fraction was measured by Biuret method and was adjusted to 5 mg/ml for Ei and 3 mg/ml for E2. Both antigenic preparations were stored in 1 ml aliquots at — 70°C until use.
Sera Three groups of 5 female guinea pigs weighing 500 to 1000 g, were infected per oz with 500, 1000 and 1500 embryonated ova. The eggs used were collected from the terminal protions of the uteri of mature female worms and were incubated at 30°C in 1 % formation in PBS for over 30 days. By this time active and infective second stage larva were obtained and these were used for infection. Similar groups of non-infected animals were used as controls. One guinea pig from each infected and control group was killed weekly for five weeks and after the confirmation of infection, their sera were collected and stored at — 70°C until use. Antibody titre against T. canis in these sera were then measured by indirect haemagglutination and counterirnmunoelectrophoresis, using the two antigenic preparations. In a trial study, ultrasonicated antigenic preparation (E2) provided the most reproducable results with both techniques, and this preparation was chosen to be mainly used in this study.
Indirect Haemagglutination test (IHA) Freshly prepared tanned sheep red blood cells were used for indirect haemagglutination test which was carried out according to Herbert (1967) in V-shaped microtitre plates. A dilution of 1:10000 tannic acid was used for coating the red blood cells. Optimal concentration for each antigen was determined using box titration with a strongly positive guinea pig serum. The optimal concentration for antigen E2 was found to be 1:128. Fraction Ei failed to be of particular use in this test.
Counterimmunoelectrophoresis (CIEP) Counterirnmunoelectrophoresis was performed on 10 x 10 cm glass plates covered with 14 ml of 1-5% Agar Noble (Difco laboratories, Michigan, U.S.A.) in barbitol buffer pH 8-6, 0-075 M. The buffer used in the tank was the same as the latter. Parallel rows of wells 3 mm in diameter were cut 4 mm apart in agar. On each plate 10 pairs of wells were cut out. Serum samples to be tested were placed in the row of wells near the anode, and antigens were placed in the wells near the cathode. Electrophoresis was carried out in a water cooled LKB electrophoresis tank at room temperature, with constant voltage of 2V/cm for 60 min. The plates were then washed in several changes of saline and then with deionized water, dried and stained with naphthalene black 12B in 5% acetic acid. Faintly positive reactions were intensified by washing the plates prior to staining with 2 % solution of tannic acid. The optimal antigen concentration for both Ei and E2 was found to be 1:4. 144
Tests for Toxocara canis
RESULTS The humoral response of three groups of guinea pigs infected with 500, 1000 and 1500 infective ova of T. canis was measured using two antigenic extracts from adult worms by indirect haemagglutination and counterimmunoelectrophoresis. These experiments were performed twice and similar results were obtained. Results from one set of these experiments are given here.
1:2560 1:1280 1:640
lution
1 320 .
1:160
•
Serum
•a
1:80
1:40
1:20
1:10
2
3 Time (weeks)
4
FIG. 1. Indirect haemagglutination (IHA) titres of sera from guinea pigs infected orally with 500 (-•-•-•-•-•-•), 1000 ( ), and 1500 ( ) infective ova of Toxocara canis.
145
M. S. ENAYAT and M. PEZESHKI 1:256
T
1:128 -
1.64
1:32 o •H
3
1:16
£
1:8
1:4
1:2
1:1
1
2 Time
3 (weeks)
FIG. 2. Counterimmunoelectrophoresis (CIEP) titres of sera from guinea pigs infected orally with 500 (-•-•-•-•-•-), 1000 ( ), and 1500 (-) infective ova of Toxocara canis.
Initially, an observable precipitation reaction was performed between the two antigenic preparations and a strongly positive reference serum from a heavily infected guinea pig using micro-immunodiffusion method. After confirmation of antigen antibody reaction by precipitation, each of the two antigenic preparations was tested with the same reference serum by both methods. The Ei preparation failed to give reproducable results with IHA, however both preparations 146
Tests for Toxocara canis
produced satisfactory results with CIEP. Therefore, E2 preparation was used in this study for the comparison of the sensitivity of the two methods. The results given in Fig. 1 show the titration of anti-Toxocara antibody in the infected guinea pigs with IHA test. Titres of 1:20 to 1:40 were observed for all the three groups of infected guinea pigs after the first week of infection. The titres were increased to a high of 1:256O after four weeks of infection. A week later the titres fell to 1:1280 to 1:640. With CIEP method lower antibody titres were obtained (Fig. 2). No antibody could be detected on the first week of infection. 1:16 titre was obtained in 1500 ova infected guinea pigs two weeks after infection and 1:8 in the other two groups. The titre increased to 1:128 on the third week for 1500 ova infected guinea pigs which was maintained for another two weeks. The titres for 1000 ova infected group increased to 1:64 and 1:128 and then fell to 1:64 after five weeks of infection. 500 ova infected group gave the lowest titres and showed only a titre of 1:32 after 5 weeks of infection. Sera from control group of guinea pigs gave no reaction with both antigens using either of the two methods. DISCUSSION The usefulness and advantages of adult worm antigen over larval extract of T. canis for use in immunodiagnoses of human V.L.M. has been questioned. However, by using a purified extract from either of these two sources (Collins and Ivey, 1975), it is possible to detect antibody to T. canis in infected human and experimental animals. The sonicated adult antigenic extract preparation (E2) used in this investigation seemed to be more superior than the initial crude homogenate (Ei). Although the infective doses of T. canis ova used in this study is higher than some of those used by Collins and Ivey (1975) (0-01, 0 1 and 10 ova per gram body weight), but similar to their findings the antibody titres at the peaks amongst the three groups of guinea pigs reflected the dose of infective agents. After three weeks of infection both techniques detected varying amounts of antibody in each group but higher antibody titres always corresponded to higher infection. Major differences in the immune response with regard to dose of infective ova in animal model needs further evaluation. A maximum number of 4 precipitin arcs were produced by CIEP in guinea pigs infected with the highest number of ova. In this way it seems likely that CIEP can give some information regarding the intensity of infection, since in addition to the antibody titre, this method also provides information about the minimum number of specific antigen antibody reactions. This is an advantage over IHA which only measures the antibody titres. In addition CIEP seems to be a valuable serological aid for diagnosis of V.L.M. since it could be performed rapidly and with ease. It is however less sensitive than IHA. No false positive reaction was observed with either of the techniques when testing fifteen guinea pigs in the control groups. The result of this study shows that CIEP and IHA both can be used for detection of anti-toxocara antibodies. However for diagnosis of human V.L.M., a specific and purified T. canis antigen is required since many investigators have reported the existence of common antigens between Ascaris and Toxocara (Kagan et al, 1959; Jung and Pacheco 1960; Aljeboori and Ivey, 1970). When specific antigen for use in these assays become available, the results of such tests can then be used together with other pathological and clinical information for diagnosis of human V.L.M. 147
M. S. ENAYAT and M. PEZESHKI ACKNOWLEDGEMENTS This work was supported in parts by a grant from Ministry of Higher Education and Science and by School of Public Health and Institute of Public Health Research, Tehran, Iran.
REFERENCES ALJEBOORI, T. I. and IVEY, M. H. (1970) An improved hemagglutination technique for detecting antibody against Toxocara canis. The American Journal of Tropical Medicine and Hygiene, 19, 244-248. BAUFINE-DUCRACQ, H., CONGINEAV, P. and BEAUVAIS, B. (1973) Diagnostic par la reaction rimmunofluorescence du syndrome de larva migrans visceral. Bulletin de la Societi de Pathologie Exotique, 66, 746-751. BEAVER, P. C , SYNDER, C. H., CARRERA, G. M., DENT, J. H. and LAFFERTY, J. W. (1952) Chronic eosinophilia due to visceral larva migrans. Report of three cases. Pediatrics, 55, 7-19. BISSERU, B. and WOODRUFF, A. W. (1968) The detection of circulating antibody in human Toxocara infections using the indirect fluorescent antibody test. Journal of Clinical Pathology, 21, 449^155. COLLINS, R. F. and IVEY, M. H. (1975) Specificity and sensitivity of skin test reactions to extracts of Toxocara canis and Ascaris suum. I. Skin tests done on infected guinea pigs. The American Journal of Tropical Medicine and Hygiene, 24, 455-459. DESOWITZ, R. S. and UNA, S. R. (1976) The detection of antibodies in human and animal filariasis by counterimmunoelectrophoresis with Dirofilaria immitis antigen. Journal of Helminthology, 50, 53-57. HERBERT, W. J. (1967) Passive haemagglutination. In Handbook of Experimental Immunology (ed D. M. Weir), pp. 720-744 Blackwell: Oxford. JUNG, R. C. and PACHECO, C. (1960) Use of a hemagglutination test in visceral larva migrans. The American Journal of Tropical Medicine and Hygiene, 9,185-191. KAGAN, I. G., NORMAN, L. and ALLAIN, D. S. (1959) Studies on the serology of visceral larva migrans. I. Hemagglutination and flocculation tests with purified Ascaris antigens. Journal of Immunology, 83, 297-301. KAGAN, I. G. (1968) Serological diagnosis of Visceral Larva Migrans. Clinical Pediatrics, 7, 508-509. KAGAN, I. G. (1974) Advances in the immunodiagnosis of parasite infections. Zeitschrift fur Parasitenkunde, 45, 163-195. KRUPP, IRIS M. (1974a) Hemagglutination test for the detection of antibodies specific for Ascaris and Toxocara antigens in patients with suspected visceral larva migrans. The American Journal of Tropical Medicine and Hygiene, 23, 378-384. KRUPP, IRIS M. (1974b) Comparison of counterimmunoelectrophoresis with other serological tests in the diagnosis of amebiasis. The American Journal of Tropical Medicine and Hygiene, 23, 27-30. VIENS, P., STRYKAWSKI, H., RICHARDS, R. and SONEA, S. (1975) A modified immunofluorescent antibody technique for the serodiagnosis of human toxocaral migrans. Canadian Journal of Public Health, 66, 237-240. Accepted 8 September, 1976.
148
The comparison of counterimmunoelectrophoresis with indirect haemagglutination test for detection of antibodies in experimentally infected guinea pigs with Toxocara canis* MOHAMMAD S. ENAYAT and MOHAMMAD PEZESHKI Pathobiology Department, School of Public Health and Institute of Public Health Research, PO Box 1310, University of Tehran, Iran
ABSTRACT The humoral response of guinea pigs infected with doses of 500,1000, and 1500 infective Toxocara canis ova were measured by counterimmunoelectrophoresis and indirect haemmagglutination tests one to five weeks after infection. The antigens used in these measurements were prepared from adult T. canis. Counterimmunoelectrophoresis showed lower titres than haemagglutination method, but it was performed rapidly and with ease. The possible use of these techniques for immunodiagnosis of human visceral larval migrans is discussed.
Since the description of human visceral larva migrans (VLM) syndrome by Beaver et al. (1952), need for a reliable and specific serodiagnostic test for detection of antibodies against migrating larvae of Toxocara canis has been emphasised by various groups of investigators (Bisseru and Woodruff, 1968; Kagan, 1968; Fernando et al, 1970; Krupp, 1974a). As detection and recognition of migrating Ascarid larvae in the tissue of infected man is immensely difficult, immunodiagnostic techniques could be of a great aid to physicians in the diagnosis of this infection. Most of the available immunological techniques have been used with variable success for detection of anti-toxocara antibody in experimentally infected animals and in suspected human cases (Kagan, 1974). Indirect fluorescent antibody test has been greatly used for this purpose by many investigators with encouraging results (Bisseru and Woodruff, 1968; Baufine-Ducracq et al., 1973; Viens et al., 1975). Counterimmunoelectrophoresis (CIEP) which has been extensively used for diagnosis of many other infectious diseases (Krupp, 1974b; Desowitz and Una, 1976), to our knowledge has not as yet been carried out for diagnosis of VLM. This report examines the relative usefulness of this technique for detection of antitoxocara antibody in experimentally infected guinea pigs and compares its application with indirect haemagglutination tests (IHA) which is being extensively used for this purpose. MATERIAL AND METHODS Antigen preparation Adult male and female worms were obtained from the intestine of young dogs killed by intracardiac injection of saturated magnesium sulphate solution. Worms were washed several times in phosphate buffered saline (PBS, pH 7-2) and finally with deionized water. They were then stored at — 20°C until use. For each preparation 25 worms weighing about 2 g were processed similarly as described by Algeboori and Ivey (1970) with small modifica* A section of this work was submitted in partial fulfilment of the requirements for an MSPH degree by Mohammad Pezeshki 143
M. S. ENAYAT and M. PEZESHKI
tions. Two preparations were obtained. The worms were first cut into small pieces and then manually homogenized in 10 ml of PBS. This preparation was further processed in an electrically operated glass homogenizer (Karl Kolb, Frankfurt, West Germany) in small aliquots, firstly at slow and then faster settings. The whole process was carried out at 4°C. This crude homogenate was centrifuged at 10 000 g for 30 min at 4°C. The supernatent fluid provided the first antigenic extract (Ei). The sediment, resuspended in PBS, was ultrasonicated and centrifuged as before. The supernate from this centrifugation gave the second antigenic extract (E2). The protein concentration of each fraction was measured by Biuret method and was adjusted to 5 mg/ml for Ei and 3 mg/ml for E2. Both antigenic preparations were stored in 1 ml aliquots at — 70°C until use.
Sera Three groups of 5 female guinea pigs weighing 500 to 1000 g, were infected per oz with 500, 1000 and 1500 embryonated ova. The eggs used were collected from the terminal protions of the uteri of mature female worms and were incubated at 30°C in 1 % formation in PBS for over 30 days. By this time active and infective second stage larva were obtained and these were used for infection. Similar groups of non-infected animals were used as controls. One guinea pig from each infected and control group was killed weekly for five weeks and after the confirmation of infection, their sera were collected and stored at — 70°C until use. Antibody titre against T. canis in these sera were then measured by indirect haemagglutination and counterirnmunoelectrophoresis, using the two antigenic preparations. In a trial study, ultrasonicated antigenic preparation (E2) provided the most reproducable results with both techniques, and this preparation was chosen to be mainly used in this study.
Indirect Haemagglutination test (IHA) Freshly prepared tanned sheep red blood cells were used for indirect haemagglutination test which was carried out according to Herbert (1967) in V-shaped microtitre plates. A dilution of 1:10000 tannic acid was used for coating the red blood cells. Optimal concentration for each antigen was determined using box titration with a strongly positive guinea pig serum. The optimal concentration for antigen E2 was found to be 1:128. Fraction Ei failed to be of particular use in this test.
Counterimmunoelectrophoresis (CIEP) Counterirnmunoelectrophoresis was performed on 10 x 10 cm glass plates covered with 14 ml of 1-5% Agar Noble (Difco laboratories, Michigan, U.S.A.) in barbitol buffer pH 8-6, 0-075 M. The buffer used in the tank was the same as the latter. Parallel rows of wells 3 mm in diameter were cut 4 mm apart in agar. On each plate 10 pairs of wells were cut out. Serum samples to be tested were placed in the row of wells near the anode, and antigens were placed in the wells near the cathode. Electrophoresis was carried out in a water cooled LKB electrophoresis tank at room temperature, with constant voltage of 2V/cm for 60 min. The plates were then washed in several changes of saline and then with deionized water, dried and stained with naphthalene black 12B in 5% acetic acid. Faintly positive reactions were intensified by washing the plates prior to staining with 2 % solution of tannic acid. The optimal antigen concentration for both Ei and E2 was found to be 1:4. 144
Tests for Toxocara canis
RESULTS The humoral response of three groups of guinea pigs infected with 500, 1000 and 1500 infective ova of T. canis was measured using two antigenic extracts from adult worms by indirect haemagglutination and counterimmunoelectrophoresis. These experiments were performed twice and similar results were obtained. Results from one set of these experiments are given here.
1:2560 1:1280 1:640
lution
1 320 .
1:160
•
Serum
•a
1:80
1:40
1:20
1:10
2
3 Time (weeks)
4
FIG. 1. Indirect haemagglutination (IHA) titres of sera from guinea pigs infected orally with 500 (-•-•-•-•-•-•), 1000 ( ), and 1500 ( ) infective ova of Toxocara canis.
145
M. S. ENAYAT and M. PEZESHKI 1:256
T
1:128 -
1.64
1:32 o •H
3
1:16
£
1:8
1:4
1:2
1:1
1
2 Time
3 (weeks)
FIG. 2. Counterimmunoelectrophoresis (CIEP) titres of sera from guinea pigs infected orally with 500 (-•-•-•-•-•-), 1000 ( ), and 1500 (-) infective ova of Toxocara canis.
Initially, an observable precipitation reaction was performed between the two antigenic preparations and a strongly positive reference serum from a heavily infected guinea pig using micro-immunodiffusion method. After confirmation of antigen antibody reaction by precipitation, each of the two antigenic preparations was tested with the same reference serum by both methods. The Ei preparation failed to give reproducable results with IHA, however both preparations 146
Tests for Toxocara canis
produced satisfactory results with CIEP. Therefore, E2 preparation was used in this study for the comparison of the sensitivity of the two methods. The results given in Fig. 1 show the titration of anti-Toxocara antibody in the infected guinea pigs with IHA test. Titres of 1:20 to 1:40 were observed for all the three groups of infected guinea pigs after the first week of infection. The titres were increased to a high of 1:256O after four weeks of infection. A week later the titres fell to 1:1280 to 1:640. With CIEP method lower antibody titres were obtained (Fig. 2). No antibody could be detected on the first week of infection. 1:16 titre was obtained in 1500 ova infected guinea pigs two weeks after infection and 1:8 in the other two groups. The titre increased to 1:128 on the third week for 1500 ova infected guinea pigs which was maintained for another two weeks. The titres for 1000 ova infected group increased to 1:64 and 1:128 and then fell to 1:64 after five weeks of infection. 500 ova infected group gave the lowest titres and showed only a titre of 1:32 after 5 weeks of infection. Sera from control group of guinea pigs gave no reaction with both antigens using either of the two methods. DISCUSSION The usefulness and advantages of adult worm antigen over larval extract of T. canis for use in immunodiagnoses of human V.L.M. has been questioned. However, by using a purified extract from either of these two sources (Collins and Ivey, 1975), it is possible to detect antibody to T. canis in infected human and experimental animals. The sonicated adult antigenic extract preparation (E2) used in this investigation seemed to be more superior than the initial crude homogenate (Ei). Although the infective doses of T. canis ova used in this study is higher than some of those used by Collins and Ivey (1975) (0-01, 0 1 and 10 ova per gram body weight), but similar to their findings the antibody titres at the peaks amongst the three groups of guinea pigs reflected the dose of infective agents. After three weeks of infection both techniques detected varying amounts of antibody in each group but higher antibody titres always corresponded to higher infection. Major differences in the immune response with regard to dose of infective ova in animal model needs further evaluation. A maximum number of 4 precipitin arcs were produced by CIEP in guinea pigs infected with the highest number of ova. In this way it seems likely that CIEP can give some information regarding the intensity of infection, since in addition to the antibody titre, this method also provides information about the minimum number of specific antigen antibody reactions. This is an advantage over IHA which only measures the antibody titres. In addition CIEP seems to be a valuable serological aid for diagnosis of V.L.M. since it could be performed rapidly and with ease. It is however less sensitive than IHA. No false positive reaction was observed with either of the techniques when testing fifteen guinea pigs in the control groups. The result of this study shows that CIEP and IHA both can be used for detection of anti-toxocara antibodies. However for diagnosis of human V.L.M., a specific and purified T. canis antigen is required since many investigators have reported the existence of common antigens between Ascaris and Toxocara (Kagan et al, 1959; Jung and Pacheco 1960; Aljeboori and Ivey, 1970). When specific antigen for use in these assays become available, the results of such tests can then be used together with other pathological and clinical information for diagnosis of human V.L.M. 147
M. S. ENAYAT and M. PEZESHKI ACKNOWLEDGEMENTS This work was supported in parts by a grant from Ministry of Higher Education and Science and by School of Public Health and Institute of Public Health Research, Tehran, Iran.
REFERENCES ALJEBOORI, T. I. and IVEY, M. H. (1970) An improved hemagglutination technique for detecting antibody against Toxocara canis. The American Journal of Tropical Medicine and Hygiene, 19, 244-248. BAUFINE-DUCRACQ, H., CONGINEAV, P. and BEAUVAIS, B. (1973) Diagnostic par la reaction rimmunofluorescence du syndrome de larva migrans visceral. Bulletin de la Societi de Pathologie Exotique, 66, 746-751. BEAVER, P. C , SYNDER, C. H., CARRERA, G. M., DENT, J. H. and LAFFERTY, J. W. (1952) Chronic eosinophilia due to visceral larva migrans. Report of three cases. Pediatrics, 55, 7-19. BISSERU, B. and WOODRUFF, A. W. (1968) The detection of circulating antibody in human Toxocara infections using the indirect fluorescent antibody test. Journal of Clinical Pathology, 21, 449^155. COLLINS, R. F. and IVEY, M. H. (1975) Specificity and sensitivity of skin test reactions to extracts of Toxocara canis and Ascaris suum. I. Skin tests done on infected guinea pigs. The American Journal of Tropical Medicine and Hygiene, 24, 455-459. DESOWITZ, R. S. and UNA, S. R. (1976) The detection of antibodies in human and animal filariasis by counterimmunoelectrophoresis with Dirofilaria immitis antigen. Journal of Helminthology, 50, 53-57. HERBERT, W. J. (1967) Passive haemagglutination. In Handbook of Experimental Immunology (ed D. M. Weir), pp. 720-744 Blackwell: Oxford. JUNG, R. C. and PACHECO, C. (1960) Use of a hemagglutination test in visceral larva migrans. The American Journal of Tropical Medicine and Hygiene, 9,185-191. KAGAN, I. G., NORMAN, L. and ALLAIN, D. S. (1959) Studies on the serology of visceral larva migrans. I. Hemagglutination and flocculation tests with purified Ascaris antigens. Journal of Immunology, 83, 297-301. KAGAN, I. G. (1968) Serological diagnosis of Visceral Larva Migrans. Clinical Pediatrics, 7, 508-509. KAGAN, I. G. (1974) Advances in the immunodiagnosis of parasite infections. Zeitschrift fur Parasitenkunde, 45, 163-195. KRUPP, IRIS M. (1974a) Hemagglutination test for the detection of antibodies specific for Ascaris and Toxocara antigens in patients with suspected visceral larva migrans. The American Journal of Tropical Medicine and Hygiene, 23, 378-384. KRUPP, IRIS M. (1974b) Comparison of counterimmunoelectrophoresis with other serological tests in the diagnosis of amebiasis. The American Journal of Tropical Medicine and Hygiene, 23, 27-30. VIENS, P., STRYKAWSKI, H., RICHARDS, R. and SONEA, S. (1975) A modified immunofluorescent antibody technique for the serodiagnosis of human toxocaral migrans. Canadian Journal of Public Health, 66, 237-240. Accepted 8 September, 1976.
148
