EXPERIMENTAL
37, 131-137
PARASITOLOGY
Trypanosoma
(19%)
cruzi: lmmunoperoxidase for Serologic Diagnosis
Antibody
Test
A. WALTER FERREIRA, M. E. CAMARGO, AND 0. S. NAKAHARA Institute
de Medicina
Tropical
de Sa’o Puulo, S&o Paulo, Brasil
(Submitted for publication
May 24, 1973)
FERHEIRA, A. WALTER, CAMARGO, M. E., AMI NAKAHARA, 0. S. 1975. Trypanosoma cruzi: Immunoperoxiclase antibody test for serologic diagnosis. Experimental Parasitology 37, 131-137. An indirect antiglobulin immunoperoxidase test was developed for the serological diagnosis of American Trypanosomiasis. Purified rabbit antihuman IgG was labeled with the enzyme and the conjugate so obtained was characterized according to its immune and enzymatic activities, with the help of such parameters as the authors have recently described. For a maximal sensitivity in the tests, high antibody levels and a high-labeling ratio were chosen, as well as dilutions of conjugate ensuring maximal reactivity. Positive tests were found in all 90 serum samples from patients with Chagas’ disease and titers did not differ significantly from those observed in immunofluorescence tests done in parallel. The specificity of both tests was also similar, as indicated by the results found for serum samples from 60 patients with other diseases, parasitic or not, showing high levels of antibodies against other infective agents or autoantibodies, and in 15 normals. INDEX DESCRIPTORS: F’eroxidase antiglobulin test; Trypanosoma crzlzi; Antibodies; Techniques; American trypanosomiasis; Serology; Immunity.
There is a great need of practical tests for the serological diagnosis of infections by Trypanosoma cruzi, in view of its widespread incidence in many areas of Latin America and the risks of contamination through blood transfusion from infected donors. Also, easily performed serologic tests are necessary for surveys, in order to determine the prevalence of the infection or to evaluate measures intended to eradicate transmission. In such a context, the immunofluorescence antibody test represented a significant contribution since it is at once sensitive, specific and practical for routine and Muschell 1959; purposes, (Fife Camargo 1966). As many as 20 tests can be performed on a single microscope slide and not only are serum samples satisfactory for the test but also eluates from fingertip
blood collected on filter paper, (Souza and Camargo 1966), making it favourable for seroepidemiology studies. However, the requirement of a fluorescence microscope certainly reduces the possibility of employing this test for routine purposes in small laboratories and blood banks. The recent development of immunoenzymatic tests (Ram 1966; Nakane and Pearce 1966), may represent a more practical solution since preparations can be observed through a standard microscope and are also more stable than the fluorescence test slides. In this paper we describe the antiglobulin immunoperoxidase test for T. cruzi antibodies and present results in parallel with immunofluorescence tests of the same samples. Serum samples were examined from patients with Chagas’ dis131
Copyright All rights
0 1975 by Academic of reproduction in any
Press, Inc. form reserved.
132
FERREJRA,
CAMARGO
AND NAKAHARA
FIG. 1. Sephadex G-100 chromatogram of the peroxidase in 280 nm and (----) 403 nm (. . * * .).
ease, other parasitic and nonparasitic eases, and from healthy individuals. MATERIAL
Enzymatic
dis-
AND METHODS
Conjugates
Immunoperoxidase conjugates were prepared from rabbit serum against human IgG. Animals were inoculated with a purified IgG fraction, isolated from a pool of human serum according to a modification of the technique of Sober et al. (1956). This technique includes precipitation with 1.56 M ammonium sulphate and chromatographic isolation through a DEAE Sephadex A-50 column. The IgG fraction was eluted with Tris-HCI buffer, pH 8, between 0.01 and 0.03 M. Purity of the concentrated eluate was ascertained by immunoelectrophoretic analysis (Ferri and Cossermelli 1964). Animals were injected intramuscularly with 5 mg protein in complete Freund’s adjuvant followed a week later by a similar dose in incomplete adjuvant. Intravenous inoculations were then given twice a week for two weeks with 2 mg globulins in saline solution. The rabbits’ immune serum from bleedings 10 days after the last injection was specific for IgG as shown by a immunoelectrophoretic precipitation line against total human
conjugation
mixture.
Densitometries
serum and against a purified IgG fraction. An immunodiffusion titer (Beutner et al. 1968), of 1:64 was found against this fraction. Horseradish peroxidase, (tipe II, Sigma Chemical Co.), was conjugated to the globulin fraction precipitated from the immune serum by 1.56 M ammonium sulphate. Glutaraldehyde was used as the conjugating agent, as described by Avrameas (1969). For this purpose, 12 mg peroxidase dissolved in 1.0 ml of phosphate buffer 0.1 M, pH 6.8, were added to a volume of globulin saline containing 6 mg protein. The mixture was stirred at room temperature and 0.05 ml of a 1% glutaraldehyde solution were added dropwise. Stirring was maintained for 2 hr and to remove any free enzyme from the conjugate, the mixture was precipitated with 1.56 M ammonium sulphate and dialysed for 24 hr at 4 C against PBS ( NaCl 0.15 M, phosphates 0.01 hl, pH 7.2). Better results could be obtained by chromatography in Sephadex G-100, which allowed not only to remove free peroxidase but also to separate labeled and unlabeled globulins. For this purpose, the mixture was pipetted on a 40 cm x 1.5 cm Sephadex G-100 column and eluted with PBS at a rate of eight drops a minute, 20 drops being collected per tube. Densi-
SERO
DIAGNOSIS
OF
tometries were evaluated for each tube at 280 nm and 403 nm in a Beckman-Due espectrophotometer (Fig. 1). With the help of immunodiffusion of eluates against human globulins and against a rabbit antiperoxidase serum, it was determined that conjugate was present in Tubes l-8, followed by unlabeled globulins in successive tubes. Most free enzyme eluted between Tubes 32 and 45. The conjugates were characterized by the intensity of labeling measured by the molar enzyme-protein ratio (E/P) and by the relative enzymatic activity as defined by Ferreira et al. (in press). For this purpose, protein concentration was determined by the biuret technique, standardizecl with a Kjehldahl analysed human globulin solution. From the protein values obtained for the conjugates, the respective amounts corresponding to the enzyme were discounted. For the peroxidase determination, a batophenanthroline reaction for Fe was employed as already described by us, (Ferreira et al., in press). This technique was preferred to the more simple one by absorbance determination, ( Avrameas, 1969) because proteins interfere with absorption in the maximum for peroxidase at 403 nm. Enzymatic activity of free and combined peroxidase was determined through the pyrogallol technique, standardized against a purpurogallin solution, as indicated by the producers of the enzyme. Relative enzymatic activity was taken as the percent ratio of the observed activity and the theoretical one corresponding to the molecules of enzyme in the conjugates. This ratio indicates the decay of enzymatic activity due to conjugation. characteristics of the Immunologica peroxidase conjugates were determined by immunoelectrophoretic and immunodiffusion analyses, as defined by Beutner et al. ( 1968)) for immunofluorescence conjugates. In this way conjugate C, for example, employed in the tests here included, had a titer of 1: 10 against human IgG, a molar
138
TRYPANOSOMIASIS
E/P ratio of 1.7 and a relative activity of 62%.
enzymatic
Serum samples Venous blood was collected from patients with well defined clinical and laboratory diagnoses. These included 90 patients with Chagas’ disease, 60 patients with other diseases, parasitic or not, and presenting antibodies against the etiological agent or autoantibodies, and 15 individuals considered as normal. Serum was kept at -20 C and for use diluted in PBS at 1:20 for qualitative tests. Doubling dilutions were done for the titration of reactive samples. Positive and negative controls samples were obtained from reagent and nonreagent sera previously tested against T. cruzi antigens by immunofluorescence, complement fixation ( Camargo 1966), and hemagglutination procedures (Camargo et al. 1973). Immunoperoxidase
tests
Tests were performed with epimastigotes culture forms of T. cruzi, fixed on small square areas drawn on microscope slides with the help of nail varnish as described for the immunofluorescence test (Camargo 1966). About 0.02 ml of serum dilution were pipetted per area and the slides incubated at 37 C for 45 min, washed in two changes of PBS, 10 min each, and carefully dried with bibulous paper. Areas were then covered with conjugate C5, diluted in PBS according to titer, and incubated for another 45 min, washed and dried in a similar way. Development of color through the catalytic activity of any peroxidase bound to the parasitic forms was done by the technique of Graham and Karnovsky ( 1966). For this purpose, slides were covered for 30 min at room temperature with a layer of a reactive solution. This was prepared by diluting 75 mg of diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in 100 ml of a Tris-WC1 buffer, 0.05 M,
134
FERREIRA,
FIG
2. Positive
(a)
and
negative
CAMARGO
(II)
AND
results
NAKAHARA
in
the peroxitlase
antiglobulin
test fob
TrypanoJoma cruzi antibodies.
pH 7.6, and adding to the solution hydrogen peroxide to the concentration of 0.1%. Slides were rapidly washed in distilled water and mounted with DPX (BDH Chemicals, Poole, England) and coverglass. Tests were observed with standard light microscope, under a 40x dry and a 100x oil immersion objective. Positive tests corresponded to a brown staining of the parasites, evaluated from + to 4+ according to intensity. In ncgative cases, parasites were barely seen through a very faint staining, observed also when saline was substituted for sera (Fig. 2). No parasite staining was demonstrated
when slides were treated exclusively the staining solution. Immtinojhorescence
with
tests
These were performed as described ( Camargo 1966). The antihuman IgG fluorescein conjugate was prepared from rabbit immune serum according to the technique of Clark and Shepard (1963), free fluorcscein being removed by chromatography of the reagent mixture in a Sephadex G-2.5 column. The conjugate presented a F/P = 10 weight ratio, corresponding to about four molecules of
SERO
DIAGNOSIS
OF
TABLE Delermination
I
of Maximal Reactivity Dilution of Peroxidase Conjugate C:, against a Positive Standard Serum against Tyrpanosoma cruxia
Dilutions of positive serum
Dilutions 1:lO
1:20 1:40 1:80 1:160 1:320 1:640 1: 1280 Negative serum 1:20 a 14:
Intensity
135
TRYPANOSOMIASIS
of positive
1:20
1
1:40
0
0
reactions-0:negative
of conjugate 1:80
I:160
3 3 3 2 1 0 0
3 2 2 1 1 0 0
0
0
1:320
0
results.
fluorescein per molecule of globulin. For use, it was diluted so as to give maximal reactivity ( Beutner 1968). Tests were read with the help of a binocular microscope provided with dark-field, 40x oil immersion objective, HBO 200 W mercury bulb, a K500 interference excitation filter and a SO (Zeiss) barrier filter.
tests employing different dilutions of the conjugate, as indicated in Table I. According to these results, the dilution of I:20 of conjugate was chosen for use in the routine tests. Comparative Results of Zmmunoperoxidase and Immunofluorescence Tests
RESULTS
Positive results for the immunoperoxidase test were observed in all 90 cases of T. cruzi infection, with titers ranging from 1: 20 to 1:640. There was a direct correspondence with the respective titers in the immunofluorescence test (Table II), which
Titration of the Immunoperoxidase Conjugate In order to determine the dilutions conjugates giving maximal reactivity, positive standard serum was titrated
of a in TABLE
Comparative
Results between the Immunoperoxidase and the ZvnmunoJluorescence Tests, in 90 Serum Samples from Patients with Chagas’ Disease
Immunofluorescence test
Immunoperoxidase Nonreagent (>1:20)
Nonreagent (>1:20) 1:20 1:40 1:80 I:160 1:320 1:640
II
1: 20
1:40
1:80
3 1
1 6
test 1:160
1:320
1:640
0 2
1
3 17 10
1 6 27 3
2 5 2
136
FERREIRA,
CAMARGO
TABLE Results oj the Immunoperoxidase
AND
NAKAHARA
III
Test in Sera from Palients with Other Diseases and LVormals.
Diagnosis
NO.
Schistosomiasis Acute lymphonodular Toxoplasmosis Malaria Mucocutaneous leishmaniasis Early syphilis South American blastomycosis Histoplasmosis Ricket,tsiosis (Maculous fever) With antinuclear antibodies With antimitochondrial antibodies With antismooth muscle antibodies With antiparietal cells antibodies Normals
1.5 II 12 3 7 2 I 2 2 I 2 2 1;,
Total
7.i
were the same or differed by only one dilution in 85 cases (correspondence = 0.94). Seven cases were also positive from the group of 53 patients with other parasitic of infectious diseases. However, five of these were also reagent in the immunofluorescence, complement fixation and hemagglutination test for antibodies reacting with T. cruzi antigens. Negative tests were seen in sera from normals and in sera with autoantibodies (antinucleus, smooth muscle, gastric parietal cells, mitochondria) as revealed by immunofluorescence tests with adequate tissue sections ( Table III). DISCUSSION
Very similar qualitative and quantitative results were obtained in the indirect antiglobulin test for T. cruzi antibodies employing either peroxidase or fluorescein This indicates comparative conjugates. levels of sensitivity for both procedures when performed as described. For immunofluorescence test such levels depend on several factors including reactivity of antigens and characteristics of the optical equipment and conjugates. In order to ensure a maxima1 response of the
React,ive
Nonreactive 14 11 9 2 7 2 0 2 2 2 2 1.i
7
6X
fluorochrome, an interference filter was employed to select from the mercury bulb emission the wavelengths corresponding to maximal absorption in the fluorescein spectrum. Also the barrier filter had a maximal transmission for the fluorescent emission. The fluorescein conjugate besides having a high labeling ratio, was employed in a dilution giving maximal reactivity. For the peroxidase test, conditions were also chosen to yield a high sensitivity. The determination of the labeling intensity of the enzyme conjugate indicated from one to two molecules of enzyme per globulin molecule, with a higher frequency of those more heavily labeled. The relative enzymatic activity for the conjugate showed that a decay of no more than 38%’ of the peroxidase activity had resulted from the tagging procedure. Also in the tests the conjugate was diluted so as to give a maximal reactivity and to result in the highest titers for reagent sera. For the development of color in the slides the diaminobenzidine reaction was preferred because it was easy to perform and gave stable preparations. It was observed that intensity of color depended on the concentration of hydrogen peroxidase and length of the reaction, both of which had to be carefully determined
SERO DIAGNOSIS OF TRYPANOSOMIASIS
for each new batch of diaminobenzidine hydrochloride. In order to study the specificity of the indirect antiglobulin immunoperoxidase test for T. cruzi antibodies, serum samples from patients presenting high levels of antibodies against other agents or selfcomponents were chosen. The few positive results found can be ascribed to mixed infections, since patients not only came from areas endemic for American trypanosomiasis but also positive results were obtained by a different serologic test for T. cruzi antibodies. In the two cases for which only the peroxidase test was positive, titers of 1:20 were found, suggesting the 1:40 dilution of sera is more appropriate for screening purposes. REFERENCES AVRAMEAS, S. 1969. Coupling of enzymes to protein with glutaraldehyde. Use of the conjugate for the detection of antigens and antibodies. Immunochemistry 6,43-55. BEUTNER, E. H., SEPULVEDA, M. R., AND BARNETT, E. U. 1968. Quantitative studies of immunofluorescent staining. Relationships of characteristics of unabsorbed antihuman IgG nonspecific staining properties in an indirect test for antinuclear factors. Bulletin of the World Health Organization 39, 587-606. CAMARGO, M. E. 1966. Fluorescent antibody test for the serodiagnosis of American Trypanosomiasis. Technical modifications employing preserved culture forms of Trypanosomu cruzi in a slide test. Revista do Institute de Medicina Tropkal de SBo Paul0 8, 227-234. CA~IARGO, M. E., HOSHINO, S., ANU SIQUEIRA, C. R. V. 1973. Hemagglutination with preserved, sensitized cells, a practical test for routine
137
serologic diagnosis of American trypanosomiasis. Rev&a do Institute de Medicina Tropical 15, 81-85. CLARK, H. W. AND SHEPARD, C. C. 1963. A dialysis technique for preparing fluorescent antibody. Virology 20, 642-644. FERREIRA, A. W., CAMARGO, M. E., AND NAKAHARA, 0. S. 1975. Labeling and evaluation of peroxydase conjugates for indirect antiglobulin antibody test. Revista do lnstituto de Medicina Tropical de Sa’o Paulo. In press. FERRI, R. G., AND COSSERMELLI, W. 1964. Analyse immunoelectrophorktique. Micro et macro methodes. Revue FranCake d’Btude Cliniques et Biologiques 9, 134-138. FIFE, E. H., JR., AND MUSCHELL 1959. Fluorescent antibody technique for serodiagnosis of Trypanosoma cruzi. Proceeding of the Society for Experimental Biology and Medicine 101,
540-543. GRAHAM, R. C., JR., AND KARNOVSKY, M. J. 1966. The early stages of absorption of injected horseradish-peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. The Journal Hbtochemistry and CytochemistTy 14, 291-302. NAKANE, P. K., AND PIERCE, G. B., JR. 1966. Enzyme-labeled antibodies: Preparation and application for the localization of antigens. The Journal of Histochembtry and Cytochemistry 14, 929-931. RAM, J. S., NAKANE, P. K., RAWLINSON, D. G., AND PIERCE, G. G. 1966. Enzyme labelled antibodies for ultrastructural studies. Federation Proceedings 25, 732. SOBER, H. A., GUTTER, F. J., WICKOFF, M. M., AND PETERSON, E. A. 1966. Chromatography of proteins. II. Fractionation of serum proteins on anion-exchange cellulose. The JownaZ of the American Chemical Society 78, 756. SOUZA, S. L., AND CAMARGO, M. E. 1962. The use of filter paper blood smears in a practical fluorescent test for American Trypanosomiasis. Rev&a do Znstituto de Medicina Tropical de %v Paul0 8,255-258.
37, 131-137
PARASITOLOGY
Trypanosoma
(19%)
cruzi: lmmunoperoxidase for Serologic Diagnosis
Antibody
Test
A. WALTER FERREIRA, M. E. CAMARGO, AND 0. S. NAKAHARA Institute
de Medicina
Tropical
de Sa’o Puulo, S&o Paulo, Brasil
(Submitted for publication
May 24, 1973)
FERHEIRA, A. WALTER, CAMARGO, M. E., AMI NAKAHARA, 0. S. 1975. Trypanosoma cruzi: Immunoperoxiclase antibody test for serologic diagnosis. Experimental Parasitology 37, 131-137. An indirect antiglobulin immunoperoxidase test was developed for the serological diagnosis of American Trypanosomiasis. Purified rabbit antihuman IgG was labeled with the enzyme and the conjugate so obtained was characterized according to its immune and enzymatic activities, with the help of such parameters as the authors have recently described. For a maximal sensitivity in the tests, high antibody levels and a high-labeling ratio were chosen, as well as dilutions of conjugate ensuring maximal reactivity. Positive tests were found in all 90 serum samples from patients with Chagas’ disease and titers did not differ significantly from those observed in immunofluorescence tests done in parallel. The specificity of both tests was also similar, as indicated by the results found for serum samples from 60 patients with other diseases, parasitic or not, showing high levels of antibodies against other infective agents or autoantibodies, and in 15 normals. INDEX DESCRIPTORS: F’eroxidase antiglobulin test; Trypanosoma crzlzi; Antibodies; Techniques; American trypanosomiasis; Serology; Immunity.
There is a great need of practical tests for the serological diagnosis of infections by Trypanosoma cruzi, in view of its widespread incidence in many areas of Latin America and the risks of contamination through blood transfusion from infected donors. Also, easily performed serologic tests are necessary for surveys, in order to determine the prevalence of the infection or to evaluate measures intended to eradicate transmission. In such a context, the immunofluorescence antibody test represented a significant contribution since it is at once sensitive, specific and practical for routine and Muschell 1959; purposes, (Fife Camargo 1966). As many as 20 tests can be performed on a single microscope slide and not only are serum samples satisfactory for the test but also eluates from fingertip
blood collected on filter paper, (Souza and Camargo 1966), making it favourable for seroepidemiology studies. However, the requirement of a fluorescence microscope certainly reduces the possibility of employing this test for routine purposes in small laboratories and blood banks. The recent development of immunoenzymatic tests (Ram 1966; Nakane and Pearce 1966), may represent a more practical solution since preparations can be observed through a standard microscope and are also more stable than the fluorescence test slides. In this paper we describe the antiglobulin immunoperoxidase test for T. cruzi antibodies and present results in parallel with immunofluorescence tests of the same samples. Serum samples were examined from patients with Chagas’ dis131
Copyright All rights
0 1975 by Academic of reproduction in any
Press, Inc. form reserved.
132
FERREJRA,
CAMARGO
AND NAKAHARA
FIG. 1. Sephadex G-100 chromatogram of the peroxidase in 280 nm and (----) 403 nm (. . * * .).
ease, other parasitic and nonparasitic eases, and from healthy individuals. MATERIAL
Enzymatic
dis-
AND METHODS
Conjugates
Immunoperoxidase conjugates were prepared from rabbit serum against human IgG. Animals were inoculated with a purified IgG fraction, isolated from a pool of human serum according to a modification of the technique of Sober et al. (1956). This technique includes precipitation with 1.56 M ammonium sulphate and chromatographic isolation through a DEAE Sephadex A-50 column. The IgG fraction was eluted with Tris-HCI buffer, pH 8, between 0.01 and 0.03 M. Purity of the concentrated eluate was ascertained by immunoelectrophoretic analysis (Ferri and Cossermelli 1964). Animals were injected intramuscularly with 5 mg protein in complete Freund’s adjuvant followed a week later by a similar dose in incomplete adjuvant. Intravenous inoculations were then given twice a week for two weeks with 2 mg globulins in saline solution. The rabbits’ immune serum from bleedings 10 days after the last injection was specific for IgG as shown by a immunoelectrophoretic precipitation line against total human
conjugation
mixture.
Densitometries
serum and against a purified IgG fraction. An immunodiffusion titer (Beutner et al. 1968), of 1:64 was found against this fraction. Horseradish peroxidase, (tipe II, Sigma Chemical Co.), was conjugated to the globulin fraction precipitated from the immune serum by 1.56 M ammonium sulphate. Glutaraldehyde was used as the conjugating agent, as described by Avrameas (1969). For this purpose, 12 mg peroxidase dissolved in 1.0 ml of phosphate buffer 0.1 M, pH 6.8, were added to a volume of globulin saline containing 6 mg protein. The mixture was stirred at room temperature and 0.05 ml of a 1% glutaraldehyde solution were added dropwise. Stirring was maintained for 2 hr and to remove any free enzyme from the conjugate, the mixture was precipitated with 1.56 M ammonium sulphate and dialysed for 24 hr at 4 C against PBS ( NaCl 0.15 M, phosphates 0.01 hl, pH 7.2). Better results could be obtained by chromatography in Sephadex G-100, which allowed not only to remove free peroxidase but also to separate labeled and unlabeled globulins. For this purpose, the mixture was pipetted on a 40 cm x 1.5 cm Sephadex G-100 column and eluted with PBS at a rate of eight drops a minute, 20 drops being collected per tube. Densi-
SERO
DIAGNOSIS
OF
tometries were evaluated for each tube at 280 nm and 403 nm in a Beckman-Due espectrophotometer (Fig. 1). With the help of immunodiffusion of eluates against human globulins and against a rabbit antiperoxidase serum, it was determined that conjugate was present in Tubes l-8, followed by unlabeled globulins in successive tubes. Most free enzyme eluted between Tubes 32 and 45. The conjugates were characterized by the intensity of labeling measured by the molar enzyme-protein ratio (E/P) and by the relative enzymatic activity as defined by Ferreira et al. (in press). For this purpose, protein concentration was determined by the biuret technique, standardizecl with a Kjehldahl analysed human globulin solution. From the protein values obtained for the conjugates, the respective amounts corresponding to the enzyme were discounted. For the peroxidase determination, a batophenanthroline reaction for Fe was employed as already described by us, (Ferreira et al., in press). This technique was preferred to the more simple one by absorbance determination, ( Avrameas, 1969) because proteins interfere with absorption in the maximum for peroxidase at 403 nm. Enzymatic activity of free and combined peroxidase was determined through the pyrogallol technique, standardized against a purpurogallin solution, as indicated by the producers of the enzyme. Relative enzymatic activity was taken as the percent ratio of the observed activity and the theoretical one corresponding to the molecules of enzyme in the conjugates. This ratio indicates the decay of enzymatic activity due to conjugation. characteristics of the Immunologica peroxidase conjugates were determined by immunoelectrophoretic and immunodiffusion analyses, as defined by Beutner et al. ( 1968)) for immunofluorescence conjugates. In this way conjugate C, for example, employed in the tests here included, had a titer of 1: 10 against human IgG, a molar
138
TRYPANOSOMIASIS
E/P ratio of 1.7 and a relative activity of 62%.
enzymatic
Serum samples Venous blood was collected from patients with well defined clinical and laboratory diagnoses. These included 90 patients with Chagas’ disease, 60 patients with other diseases, parasitic or not, and presenting antibodies against the etiological agent or autoantibodies, and 15 individuals considered as normal. Serum was kept at -20 C and for use diluted in PBS at 1:20 for qualitative tests. Doubling dilutions were done for the titration of reactive samples. Positive and negative controls samples were obtained from reagent and nonreagent sera previously tested against T. cruzi antigens by immunofluorescence, complement fixation ( Camargo 1966), and hemagglutination procedures (Camargo et al. 1973). Immunoperoxidase
tests
Tests were performed with epimastigotes culture forms of T. cruzi, fixed on small square areas drawn on microscope slides with the help of nail varnish as described for the immunofluorescence test (Camargo 1966). About 0.02 ml of serum dilution were pipetted per area and the slides incubated at 37 C for 45 min, washed in two changes of PBS, 10 min each, and carefully dried with bibulous paper. Areas were then covered with conjugate C5, diluted in PBS according to titer, and incubated for another 45 min, washed and dried in a similar way. Development of color through the catalytic activity of any peroxidase bound to the parasitic forms was done by the technique of Graham and Karnovsky ( 1966). For this purpose, slides were covered for 30 min at room temperature with a layer of a reactive solution. This was prepared by diluting 75 mg of diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in 100 ml of a Tris-WC1 buffer, 0.05 M,
134
FERREIRA,
FIG
2. Positive
(a)
and
negative
CAMARGO
(II)
AND
results
NAKAHARA
in
the peroxitlase
antiglobulin
test fob
TrypanoJoma cruzi antibodies.
pH 7.6, and adding to the solution hydrogen peroxide to the concentration of 0.1%. Slides were rapidly washed in distilled water and mounted with DPX (BDH Chemicals, Poole, England) and coverglass. Tests were observed with standard light microscope, under a 40x dry and a 100x oil immersion objective. Positive tests corresponded to a brown staining of the parasites, evaluated from + to 4+ according to intensity. In ncgative cases, parasites were barely seen through a very faint staining, observed also when saline was substituted for sera (Fig. 2). No parasite staining was demonstrated
when slides were treated exclusively the staining solution. Immtinojhorescence
with
tests
These were performed as described ( Camargo 1966). The antihuman IgG fluorescein conjugate was prepared from rabbit immune serum according to the technique of Clark and Shepard (1963), free fluorcscein being removed by chromatography of the reagent mixture in a Sephadex G-2.5 column. The conjugate presented a F/P = 10 weight ratio, corresponding to about four molecules of
SERO
DIAGNOSIS
OF
TABLE Delermination
I
of Maximal Reactivity Dilution of Peroxidase Conjugate C:, against a Positive Standard Serum against Tyrpanosoma cruxia
Dilutions of positive serum
Dilutions 1:lO
1:20 1:40 1:80 1:160 1:320 1:640 1: 1280 Negative serum 1:20 a 14:
Intensity
135
TRYPANOSOMIASIS
of positive
1:20
1
1:40
0
0
reactions-0:negative
of conjugate 1:80
I:160
3 3 3 2 1 0 0
3 2 2 1 1 0 0
0
0
1:320
0
results.
fluorescein per molecule of globulin. For use, it was diluted so as to give maximal reactivity ( Beutner 1968). Tests were read with the help of a binocular microscope provided with dark-field, 40x oil immersion objective, HBO 200 W mercury bulb, a K500 interference excitation filter and a SO (Zeiss) barrier filter.
tests employing different dilutions of the conjugate, as indicated in Table I. According to these results, the dilution of I:20 of conjugate was chosen for use in the routine tests. Comparative Results of Zmmunoperoxidase and Immunofluorescence Tests
RESULTS
Positive results for the immunoperoxidase test were observed in all 90 cases of T. cruzi infection, with titers ranging from 1: 20 to 1:640. There was a direct correspondence with the respective titers in the immunofluorescence test (Table II), which
Titration of the Immunoperoxidase Conjugate In order to determine the dilutions conjugates giving maximal reactivity, positive standard serum was titrated
of a in TABLE
Comparative
Results between the Immunoperoxidase and the ZvnmunoJluorescence Tests, in 90 Serum Samples from Patients with Chagas’ Disease
Immunofluorescence test
Immunoperoxidase Nonreagent (>1:20)
Nonreagent (>1:20) 1:20 1:40 1:80 I:160 1:320 1:640
II
1: 20
1:40
1:80
3 1
1 6
test 1:160
1:320
1:640
0 2
1
3 17 10
1 6 27 3
2 5 2
136
FERREIRA,
CAMARGO
TABLE Results oj the Immunoperoxidase
AND
NAKAHARA
III
Test in Sera from Palients with Other Diseases and LVormals.
Diagnosis
NO.
Schistosomiasis Acute lymphonodular Toxoplasmosis Malaria Mucocutaneous leishmaniasis Early syphilis South American blastomycosis Histoplasmosis Ricket,tsiosis (Maculous fever) With antinuclear antibodies With antimitochondrial antibodies With antismooth muscle antibodies With antiparietal cells antibodies Normals
1.5 II 12 3 7 2 I 2 2 I 2 2 1;,
Total
7.i
were the same or differed by only one dilution in 85 cases (correspondence = 0.94). Seven cases were also positive from the group of 53 patients with other parasitic of infectious diseases. However, five of these were also reagent in the immunofluorescence, complement fixation and hemagglutination test for antibodies reacting with T. cruzi antigens. Negative tests were seen in sera from normals and in sera with autoantibodies (antinucleus, smooth muscle, gastric parietal cells, mitochondria) as revealed by immunofluorescence tests with adequate tissue sections ( Table III). DISCUSSION
Very similar qualitative and quantitative results were obtained in the indirect antiglobulin test for T. cruzi antibodies employing either peroxidase or fluorescein This indicates comparative conjugates. levels of sensitivity for both procedures when performed as described. For immunofluorescence test such levels depend on several factors including reactivity of antigens and characteristics of the optical equipment and conjugates. In order to ensure a maxima1 response of the
React,ive
Nonreactive 14 11 9 2 7 2 0 2 2 2 2 1.i
7
6X
fluorochrome, an interference filter was employed to select from the mercury bulb emission the wavelengths corresponding to maximal absorption in the fluorescein spectrum. Also the barrier filter had a maximal transmission for the fluorescent emission. The fluorescein conjugate besides having a high labeling ratio, was employed in a dilution giving maximal reactivity. For the peroxidase test, conditions were also chosen to yield a high sensitivity. The determination of the labeling intensity of the enzyme conjugate indicated from one to two molecules of enzyme per globulin molecule, with a higher frequency of those more heavily labeled. The relative enzymatic activity for the conjugate showed that a decay of no more than 38%’ of the peroxidase activity had resulted from the tagging procedure. Also in the tests the conjugate was diluted so as to give a maximal reactivity and to result in the highest titers for reagent sera. For the development of color in the slides the diaminobenzidine reaction was preferred because it was easy to perform and gave stable preparations. It was observed that intensity of color depended on the concentration of hydrogen peroxidase and length of the reaction, both of which had to be carefully determined
SERO DIAGNOSIS OF TRYPANOSOMIASIS
for each new batch of diaminobenzidine hydrochloride. In order to study the specificity of the indirect antiglobulin immunoperoxidase test for T. cruzi antibodies, serum samples from patients presenting high levels of antibodies against other agents or selfcomponents were chosen. The few positive results found can be ascribed to mixed infections, since patients not only came from areas endemic for American trypanosomiasis but also positive results were obtained by a different serologic test for T. cruzi antibodies. In the two cases for which only the peroxidase test was positive, titers of 1:20 were found, suggesting the 1:40 dilution of sera is more appropriate for screening purposes. REFERENCES AVRAMEAS, S. 1969. Coupling of enzymes to protein with glutaraldehyde. Use of the conjugate for the detection of antigens and antibodies. Immunochemistry 6,43-55. BEUTNER, E. H., SEPULVEDA, M. R., AND BARNETT, E. U. 1968. Quantitative studies of immunofluorescent staining. Relationships of characteristics of unabsorbed antihuman IgG nonspecific staining properties in an indirect test for antinuclear factors. Bulletin of the World Health Organization 39, 587-606. CAMARGO, M. E. 1966. Fluorescent antibody test for the serodiagnosis of American Trypanosomiasis. Technical modifications employing preserved culture forms of Trypanosomu cruzi in a slide test. Revista do Institute de Medicina Tropkal de SBo Paul0 8, 227-234. CA~IARGO, M. E., HOSHINO, S., ANU SIQUEIRA, C. R. V. 1973. Hemagglutination with preserved, sensitized cells, a practical test for routine
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serologic diagnosis of American trypanosomiasis. Rev&a do Institute de Medicina Tropical 15, 81-85. CLARK, H. W. AND SHEPARD, C. C. 1963. A dialysis technique for preparing fluorescent antibody. Virology 20, 642-644. FERREIRA, A. W., CAMARGO, M. E., AND NAKAHARA, 0. S. 1975. Labeling and evaluation of peroxydase conjugates for indirect antiglobulin antibody test. Revista do lnstituto de Medicina Tropical de Sa’o Paulo. In press. FERRI, R. G., AND COSSERMELLI, W. 1964. Analyse immunoelectrophorktique. Micro et macro methodes. Revue FranCake d’Btude Cliniques et Biologiques 9, 134-138. FIFE, E. H., JR., AND MUSCHELL 1959. Fluorescent antibody technique for serodiagnosis of Trypanosoma cruzi. Proceeding of the Society for Experimental Biology and Medicine 101,
540-543. GRAHAM, R. C., JR., AND KARNOVSKY, M. J. 1966. The early stages of absorption of injected horseradish-peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. The Journal Hbtochemistry and CytochemistTy 14, 291-302. NAKANE, P. K., AND PIERCE, G. B., JR. 1966. Enzyme-labeled antibodies: Preparation and application for the localization of antigens. The Journal of Histochembtry and Cytochemistry 14, 929-931. RAM, J. S., NAKANE, P. K., RAWLINSON, D. G., AND PIERCE, G. G. 1966. Enzyme labelled antibodies for ultrastructural studies. Federation Proceedings 25, 732. SOBER, H. A., GUTTER, F. J., WICKOFF, M. M., AND PETERSON, E. A. 1966. Chromatography of proteins. II. Fractionation of serum proteins on anion-exchange cellulose. The JownaZ of the American Chemical Society 78, 756. SOUZA, S. L., AND CAMARGO, M. E. 1962. The use of filter paper blood smears in a practical fluorescent test for American Trypanosomiasis. Rev&a do Znstituto de Medicina Tropical de %v Paul0 8,255-258.
