440 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE
AND HYGIENE.
Vol. 69.No.4.
1975.
CORRESPONDENCE To the Editor APPLICATION OF ELBA-ENZYMELINKED IMMUNOSORBENT ASSAY-IN THE SERO-DIAGNOSIS OF AMOEBIASIS SIR,-III our laboratory the agargel diffusion test and indirect fluorescent antibody (IFA) test are routinely
used in the serodiagnosis of clinical amoebiasis. Both tests have disadvantages, so that we have been looking for a test that is easily performed, highly sensitive, quantitative and objective. We believe we have found such a test in the ELISA that was developed by ENGVALL and PERLMANN (1972) and has been applied for the serodiagnosis of trichinosis in pigs by RUITENBERG et al. (1974). Both groups of authors performed the assay in polystyrene tubes in which determination of enzyme activity could easily be measured in a spectrophotometer. Recently RUITENBERG et al. (1975) described the ELISA using a micro titration plate. We started with the tube test, but rapidly switched to the micro method using polystyrene Microtiter@ plates (Dynatech International Group of Companies, Alexandria, Va, U.S.A.). Lyophilized amoebic antigen is prepared in our laboratory from the axenically cultivated HK-9 strain (kindly provided by Dr. L. S. Diamond, NIH, Bethesda, U.S.A.) according to the method of LANGE and DIAMOND (1969). The cups of the microtitre plate are coated with antigen, dissolved in 0.1 M sodium carbonate buffer (pH 9.6) with 0.02% NaN, (200 ~1. per cup, containing 5 pg amoebic protein), during incubation in a water bath at 37°C for 3 hours. After thorough rinsing with tap water the serum dilutions are applied (200 ~1.per cup). The dilutions are made in phosphate buffered saline (PBS) with 05% bovine serum albumin (BSA) and 0*020,/,Tween 20. A positive and a negative control serum are always included per plate. After 3 hours incubation at 37°C and rinsing with tap water the cups are filled with 200 ~1.conjugate. Horseradish peroxidase conjugated to sheep anti-human immunoglobulin (Institut Pasteur, Paris) is diluted l:l,OOO in PBS with 0.5% BSA and 0.02% Tween 20. After incubation at 37°C for 30 minutes or longer (overnight) the cups are rinsed again and filled with 200 ~1.substrate. The substrate used is 5-amino-2-hydroxy benzoic acid (Merck, Germany) 0.08% w/v in distilled water (pH is adjusted to 6.0 with 1 N NaOH) with 0.005% H,O,. Results can be read visually after 10 minutes at room temperature. A positive reaction is demonstrated by a brown coloured reaction product in the solution. Usually, the reaction is stopped with 1 N NaOH (20 ~1. per cup) after 60 minutes. The modification to micro scale seemsto make a promising test for routine work, also under field conditions. Sera from patients with liver amoebiasis show ELISA titres of 10,000 and higher, whereas patients with intestinal symptoms probably caused by amoebae have lower titres. In the first group a positive agargel test is found and IFA titres are usually between 80 and 640; in the second group the agargel test is mostly negative and IFA titres are below 80. The evaluation of the reliability of this test and the determination of diagnostically significant titres in the ELISA is the aim of future research. Also, results obtained with the 3 different serological techniques need to be compared more extensively. We are, etc., H. J. BOS, Miss A. A. VANDEN EIJK. Laboratory
of Parasitology,
University of Leiden, Rapenburg 33, Leiden, The Netherlands. P.
National Institute of Public Health, Bilthoven, 3 April, 1975
A. STEERENBERG.
The Netherlands. REFERENCES
E., & PERLMANN, P. (1972). J. Immun., 109, 129. LUNDE,M. N., & DIAMOND, L. S. (1969). Am. 3. trap. Med. Hyg., 18, 1. RUITENBERG, E. J., STEERENBERG, P. A., & BROSI, B. J. M.(1975). Medikon,4, 30. Bws, J., LJUNGSTR~M, I., & ENGVALL, I. (1974). Proc. 3rd Int. Congr. Parasit., -, -, -, 3, 1203.
ENGVALL,
PROPOSALFOR ESTABLISHMENT OF A CENTRALFACILITY TO PRODUCEANTIGENFORSEROLOGIC MALARIAL ANTIBODY
TESTSFOR
&.,-The problem of supplies of nonhuman primates for biomedical purposes is rapidly becoming more and more acute (MUCHENHIRN. 1975). It has become a tonic of national and international concern. In suite of attempts to resolve the related dimculties, no easy solutions are apparent. This problem is especially acute in the field of human malaria serology (WHO, 1974). The simian primate most useful for the production of human malaria antigen is the South American night monkey, Aotus trivergatus. Many laboratories, in trying to maintain colonies of these animals for malaria research, have found that they are very fragile. They seem to succumb to a number of infections, very often before they can be placed on test or before tests are completed. Initially, this was our experience. In 1967, after having lost a number of animals to intercurrent infection, certain types of debility led us to conclude that much of the difficulty might be caused by diet. Our colony was then placed on a diet including fresh milk, fruit, pablum, and an occasional feeding with a good grade of raw ground beef. Other care includes a constant temperature of 26-27” C in the monkey quarters. All new animals are kept in a separate room and
AND HYGIENE.
Vol. 69.No.4.
1975.
CORRESPONDENCE To the Editor APPLICATION OF ELBA-ENZYMELINKED IMMUNOSORBENT ASSAY-IN THE SERO-DIAGNOSIS OF AMOEBIASIS SIR,-III our laboratory the agargel diffusion test and indirect fluorescent antibody (IFA) test are routinely
used in the serodiagnosis of clinical amoebiasis. Both tests have disadvantages, so that we have been looking for a test that is easily performed, highly sensitive, quantitative and objective. We believe we have found such a test in the ELISA that was developed by ENGVALL and PERLMANN (1972) and has been applied for the serodiagnosis of trichinosis in pigs by RUITENBERG et al. (1974). Both groups of authors performed the assay in polystyrene tubes in which determination of enzyme activity could easily be measured in a spectrophotometer. Recently RUITENBERG et al. (1975) described the ELISA using a micro titration plate. We started with the tube test, but rapidly switched to the micro method using polystyrene Microtiter@ plates (Dynatech International Group of Companies, Alexandria, Va, U.S.A.). Lyophilized amoebic antigen is prepared in our laboratory from the axenically cultivated HK-9 strain (kindly provided by Dr. L. S. Diamond, NIH, Bethesda, U.S.A.) according to the method of LANGE and DIAMOND (1969). The cups of the microtitre plate are coated with antigen, dissolved in 0.1 M sodium carbonate buffer (pH 9.6) with 0.02% NaN, (200 ~1. per cup, containing 5 pg amoebic protein), during incubation in a water bath at 37°C for 3 hours. After thorough rinsing with tap water the serum dilutions are applied (200 ~1.per cup). The dilutions are made in phosphate buffered saline (PBS) with 05% bovine serum albumin (BSA) and 0*020,/,Tween 20. A positive and a negative control serum are always included per plate. After 3 hours incubation at 37°C and rinsing with tap water the cups are filled with 200 ~1.conjugate. Horseradish peroxidase conjugated to sheep anti-human immunoglobulin (Institut Pasteur, Paris) is diluted l:l,OOO in PBS with 0.5% BSA and 0.02% Tween 20. After incubation at 37°C for 30 minutes or longer (overnight) the cups are rinsed again and filled with 200 ~1.substrate. The substrate used is 5-amino-2-hydroxy benzoic acid (Merck, Germany) 0.08% w/v in distilled water (pH is adjusted to 6.0 with 1 N NaOH) with 0.005% H,O,. Results can be read visually after 10 minutes at room temperature. A positive reaction is demonstrated by a brown coloured reaction product in the solution. Usually, the reaction is stopped with 1 N NaOH (20 ~1. per cup) after 60 minutes. The modification to micro scale seemsto make a promising test for routine work, also under field conditions. Sera from patients with liver amoebiasis show ELISA titres of 10,000 and higher, whereas patients with intestinal symptoms probably caused by amoebae have lower titres. In the first group a positive agargel test is found and IFA titres are usually between 80 and 640; in the second group the agargel test is mostly negative and IFA titres are below 80. The evaluation of the reliability of this test and the determination of diagnostically significant titres in the ELISA is the aim of future research. Also, results obtained with the 3 different serological techniques need to be compared more extensively. We are, etc., H. J. BOS, Miss A. A. VANDEN EIJK. Laboratory
of Parasitology,
University of Leiden, Rapenburg 33, Leiden, The Netherlands. P.
National Institute of Public Health, Bilthoven, 3 April, 1975
A. STEERENBERG.
The Netherlands. REFERENCES
E., & PERLMANN, P. (1972). J. Immun., 109, 129. LUNDE,M. N., & DIAMOND, L. S. (1969). Am. 3. trap. Med. Hyg., 18, 1. RUITENBERG, E. J., STEERENBERG, P. A., & BROSI, B. J. M.(1975). Medikon,4, 30. Bws, J., LJUNGSTR~M, I., & ENGVALL, I. (1974). Proc. 3rd Int. Congr. Parasit., -, -, -, 3, 1203.
ENGVALL,
PROPOSALFOR ESTABLISHMENT OF A CENTRALFACILITY TO PRODUCEANTIGENFORSEROLOGIC MALARIAL ANTIBODY
TESTSFOR
&.,-The problem of supplies of nonhuman primates for biomedical purposes is rapidly becoming more and more acute (MUCHENHIRN. 1975). It has become a tonic of national and international concern. In suite of attempts to resolve the related dimculties, no easy solutions are apparent. This problem is especially acute in the field of human malaria serology (WHO, 1974). The simian primate most useful for the production of human malaria antigen is the South American night monkey, Aotus trivergatus. Many laboratories, in trying to maintain colonies of these animals for malaria research, have found that they are very fragile. They seem to succumb to a number of infections, very often before they can be placed on test or before tests are completed. Initially, this was our experience. In 1967, after having lost a number of animals to intercurrent infection, certain types of debility led us to conclude that much of the difficulty might be caused by diet. Our colony was then placed on a diet including fresh milk, fruit, pablum, and an occasional feeding with a good grade of raw ground beef. Other care includes a constant temperature of 26-27” C in the monkey quarters. All new animals are kept in a separate room and
