Analysis of Toxoplasma gondii antigens with sera from toxoplasmosis patients

Some proteins of the Toxoplasma gondii are recognized by IgG, IgM and IgA antibodies in patients with acute and chronic toxoplasmosis, depending on the strain and stage of the Toxoplasma. Sixty-nine sera from immunocompetent individuals were studied through the WesternBlot Test: 20 has an acute infection, 29 has a chronic toxoplasmosis infection and 20 were healthy (seronegatives). The protein analysis revealed by IgG and IgM antibodies were performed through the Immunoplot method in order to know their recognition frequency (f) and be valued as infection markers. In the acute phase, the IgM antibodies showed a recognition frequency (f = 0.60) for the 60kDa protein, and in the chronic phase the IgG antibodies showed a recognition frequency (f = 0.68) for the 12kDa protein. Seronegatives revealed no type of band. The protein of 12kDa can be a diagnostic marker of the chronic phase while protein 60kDa of the acute phase of toxoplasmosis. Key-words: Toxoplasmosis. Acute. Chronic. Antigen. Toxoplasma gondii. 271 Analysis of Toxoplasma gondii antigens with sera from toxoplasmosis patients Address to: Dra. María de la Luz Galván Ramírez. Centro de Investigación en Enfermedades Tropicales de la Universidad de Guadalajara. Lago Camecuaro no 2398 Colonia Lagos del Country. C.P. 44177. Guadalajara, Jalisco, México. Telefax: (52) (3) 617-9934 y 617-9935. Recebido para publicação em 09/06/97. Revista da Sociedade Brasileira de Medicina Tropical 31(3):271-277, mai-jun, 1998. ARTIGO Resumo Demonstrou-se que algumas proteínas do Toxoplasma gondii são reconhecidas pelos anticorpos IgG, IgM e IgA em pacientes com toxoplasmose aguda e crônica. A cepa e o estágio do protozoário interferem na diferença. Sessenta e nove soros de indivíduos imunocompetentes foram submetidos à pesquisa de anticorpos pela técnica de Western-Blot, sendo 20 dos soros provenientes de pacientes com infecção aguda e 29 de infecção crônica de toxoplasmose e 20 sem doença (soronegativos). A análise das proteínas reveladas pelos anticorpos IgG e IgM foi feita pelo método de Inmunoplot com a finalidade de se conhecer a freqüência de reconhecimento (f), e serem valorizados como marcadores de infecção. Na fase aguda os anticorpos IgM apresentaram uma freqüência de reconhecimento (f = 0.60) para a proteína de 60kDa. Na fase crônica, os anticorpos IgG apresentaram (f = 0.68) para a proteína de 12kDa. Soronegativos não revelaram nenhuma informação. A proteína de 12kDa pode ser um marcador diagnóstico da fase crônica, e a proteína de 60kDa pode ser um marcador diagnóstico da fase aguda da toxoplasmose. Palavras-chaves: Toxoplasmose. Aguda. Crônica. Antígeno. Toxoplasma gondii. Análise de antígenos de Toxoplasma gondii em soros de pacientes com toxoplasmose Maria de la Luz Galván-Ramírez, Cecilia Guillén-Vargas, Rafael Saavedra-Durán and Alfonso Islas-Rodríguez Revista da Sociedade Brasileira de Medicina Tropical 31:271-277, mai-jun, 1998. 272 Toxoplasma gondii is an obligate intracellular protozoan that causes chronic and acute forms of toxoplasmosis. Epidemiological studies indicates that 20% to 90% of adult population worldwide have been in contact with the parasite. The congenital infection is the most severe form of the disease, causing irreversible damage to the nervous system of the fetus, or abortion in pregnant women8 16. There are several proteic antigens obtain from Toxoplasma gondii that are recognized by antibodies from patients with some of the forms of toxoplasmosis1 2 6 13 14 15 18 22 24, but none of the antigens, have been clearly identified which have the characteristics of a marker for acute or chronic toxoplasmosis. Recently, several methods have been developed in order to detect seropositive subjects, not only with epidemiologic, but with diagnostic and prognostic purposes. Western Blot technique have been used in order to discriminate subjects with the chronic infection from subjects with the acute form of the disease4 5 7 9 13 20 but without conclusive results5 13. Here we report, using the tachyzoite stage antigen extract from Toxoplasma gondii in polyacrilamyde gel electrophoresis (SDS-PAGE) and Western Blot technique, the presence of antigenic markers for acute and chronic forms of toxoplasmosis, using the frequency analysis described by Larralde10. MATERIAL AND METHODS Subjects (Sera). We study forty-nine female patients, between 18-42 years old, from the Hospital of Obstetrics and Gynecology of the Mexican Social Security Institute at Guadalajara; Jalisco, Mexico. Twenty of them, were adult patients with acute toxoplasmosis and twentynine were adult patients with chronic toxoplasmosis. 20 normal subjects were used as the control group. The three groups: chronic, acute and subjects normal were accurately classified by criteria universally accepted8; such as the presence of lymphadenopathy for acute toxoplasmosis patients, or retinochoroiditis for chronic patients. Also we consider the presence of IgG antibodies for chronic patients and high titer of IgM antibodies for acute patients, by indirect immunofluorescence technic. All sera were divided in three parts and frozen at -20oC until used. Preparation of Toxoplasma antigen. The strain of Toxoplasma gondii used in this study was the "RH" (Sabin, 1941); maintained by serial passage every 2 days in the peritoneal cavity of Balb/c mice (donated in the Inst i tu te o f Biomedical Research of the Autonomous University of México, A.P. 70228, 04510 México, D.F. by Doctor Saavedra). Tachyzoites were obtained from the peritoneal cavity of the mice and placed in phosphate-buffer saline, pH 7.2 (PBS), containing heparin 0.1%, finally the pellet was washed in PBS twice. Contaminated mouse cells were removed by differential centrifugation at 300g followed by 3 washes with 0.15M NaCl solution. The pellet was suspended in 1ml of PBS, and stored in 100μl aliquots at 70oC17. Protein concentration was determined following Lowry's method12. SDS-PAGE and Western-Blot. The antigen obtained (1mg protein/ml) was mixed in a 1:2 dilution with the Laemmli sample buffer (0.02M Tris-HCL pH 6.8, 20% glycerol, 2% 2-mercaptoethanol and 0.05% bromophenolblue), and boiled at 100o for 5 minutes. The antigen was separated on a vertical slab gel with 15% acrylamide by electrophoresis technic, at a constant current of 25mA, in buffer which contained 5ml 30/.88% acrylamide/bisacrylamide, 1.8ml of 2M 8.8 Tris-base, 200μl 10% sodium dodecilsulfate, 3.1ml distilled water, 500μl of 10% ammonia persulfate, 5μl of TEMED. Markers of molecular weight (Mw), were run in the same gel with bovine serum albumin (Mw = 69kDa), glyceraldehyde 3-phosphate dehydrogenase (Mw = 36kDa), carbonic anhydrase (Mw = 30kDa), trypsinogen inhibitor (Mw = 21.5kDa) and lactalbumin (Mw = 14kDa); Sigma Chemical House. The proteins already separated, were transferred on to a nitrocellulose paper (Transbolt Transfer Medium, Bio-Rad, Richmond CA) using 25mM Tris-HCL buffer pH 8.2 containing 192mM glycine and 20%(v/v) methanol in an electrophoretic transfer chamber (Bio-Rad) overnight at 0.25mAmps21. The nitrocellulose st r ips were soaked for 30min in 0 .01M phosphate-buffer saline; containing 0.1%(v/v) Tween-20 (PBST), washed and then exposed for 90min, to 1:50 diluted human sera in PBST containing 5% non-fat dried milk. After washing two times in PBST, the nitrocellulose strips were incubated for 90min with peroxidase-conjugated rabbit anti-human IgG or IgM (DAKO, Copenhagen), diluted 1:1000 in the same buffer. After washing, in the nitrocellulose strips, were developed the bands Galván-Ramírez ML et al 273 color, with 3,3 diaminobenzidin tetrahydrochloride (DAB, DAKO, Copenhagen), DAB 2.5mg made in 10ml PBST plus CaCl 1% with 0.03% (v/v) H2O2. All incubations were carried out at room temperature and the reaction was stopped with H2O19. Immunoplot. A graphic method to measure from 0 to 1, the frequency of recognition (f) of each band f rom an ind iv idual ant igen recognized by specific antibodies in sera of sick and normal subjects through Western Blot thecnic, identifying with these diagnostic markers of crhonic and acute forms. Where (f)= The number of t imes that a band were recognized was divided with the total number of samples. The proteins candidates for diagnostic markers were considered, when their recognition frequency were > than 0.5. The bands recognized in sera from control subjects, were made into graphics according with their frequency (f) on their ordinates (Y), and the bands recognized in sick subjects in the abscissa (X). Interpretation of the graphic were necessary to rely on the regression line or the quadrants lines with the frequency > 0.5. The proteins situated near the regression line or in quadrants II and III were considered immunodominate and immunogenic, but they were not useful as diagnostic markers, on the contrary, proteins situated near the X and Y axes, in quadrants I and IV are candidates for diagnostic markers11.

Análise de antígenos de Toxoplasma gondii em soros de pacientes com toxoplasmose Maria de la Luz Galván-Ramírez, Cecilia Guillén-Vargas, Rafael Saavedra-Durán and Alfonso Islas-Rodríguez Toxoplasma gondii is an obligate intracellular protozoan that causes chronic and acute forms of toxoplasmosis.Epidemiological studies indicates that 20% to 90% of adult population worldwide have been in contact with the parasite.The congenital infection is the most severe form of the disease, causing irreversible damage to the nervous system of the fetus, or abortion in pregnant women 8 16 .
There are several proteic antigens obtain from Toxoplasma gondii that are recognized by antibodies from patients with some of the forms of toxoplasmosis 1 2 6 13 14 15 18 22 24 , but none of the antigens, have been clearly identified which have the characteristics of a marker for acute or chronic toxoplasmosis.
Recently, several methods have been developed in order to detect seropositive subjects, not only with epidemiologic, but with diagnostic and prognostic purposes.Western Blot technique have been used in order to discriminate subjects with the chronic infection from subjects with the acute form of the disease 4 5 7 9 13 20 but without conclusive results 5 13 .
Here we report, using the tachyzoite stage antigen extract from Toxoplasma gondii in polyacrilamyde gel electrophoresis (SDS-PAGE) and Western Blot technique, the presence of antigenic markers for acute and chronic forms of toxoplasmosis, using the frequency analysis described by Larralde 10 .

Subjects (Sera).
We study forty-nine female patients, between 18-42 years old, from the Hospital of Obstetrics and Gynecology of the Mexican Social Security Institute at Guadalajara; Jalisco, Mexico.Twenty of them, were adult patients with acute toxoplasmosis and twentynine were adult patients with chronic toxoplasmosis.20 normal subjects were used as the control group.The three groups: chronic, acute and subjects normal were accurately classified by criteria universally accepted 8 ; such as the presence of lymphadenopathy for acute toxoplasmosis patients, or retinochoroiditis for chronic patients.Also we consider the presence of IgG antibodies for chronic patients and high titer of IgM antibodies for acute patients, by indirect immunofluorescence technic.All sera were divided in three parts and frozen at -20 o C until used.
Preparation of Toxoplasma antigen.The strain of Toxoplasma gondii used in this study was the "RH" (Sabin, 1941); maintained by serial passage every 2 days in the peritoneal cavity of Balb/c mice (donated in the Institute of Biomedical Research of the Autonomous University of México, A.P. 70228, 04510 México, D.F. by Doctor Saavedra).Tachyzoites were obtained from the peritoneal cavity of the mice and placed in phosphate-buffer saline, pH 7.2 (PBS), containing heparin 0.1%, finally the pellet was washed in PBS twice.
Contaminated mouse cells were removed by differential centrifugation at 300g followed by 3 washes with 0.15M NaCl solution.The pellet was suspended in 1ml of PBS, and stored in 100µl aliquots at 70 o C 17 .Protein concentration was determined following Lowry's method 12 .
After washing two times in PBST, the nitrocellulose strips were incubated for 90min with peroxidase-conjugated rabbit anti-human IgG or IgM (DAKO, Copenhagen), diluted 1:1000 in the same buffer.After washing, in the nitrocellulose strips, were developed the bands color, with 3,3 diaminobenzidin tetrahydrochloride (DAB, DAKO, Copenhagen), DAB 2.5mg made in 10ml PBST plus CaCl 1% with 0.03% (v/v) H 2 O 2 .All incubations were carried out at room temperature and the reaction was stopped with H 2 O 19 .
Immunoplot.A graphic method to measure from 0 to 1, the frequency of recognition (f) of each band from an individual antigen recognized by specific antibodies in sera of sick and normal subjects through Western Blot thecnic, identifying with these diagnostic markers of crhonic and acute forms.Where (f)= The number of times that a band were recognized was divided with the total number of samples.The proteins candidates for diagnostic markers were considered, when their recognition frequency were > than 0.5.
The bands recognized in sera from control subjects, were made into graphics according with their frequency (f) on their ordinates (Y), and the bands recognized in sick subjects in the abscissa (X).
Interpretation of the graphic were necessary to rely on the regression line or the quadrants lines with the frequency > 0.5.The proteins situated near the regression line or in quadrants II and III were considered immunodominate and immunogenic, but they were not useful as diagnostic markers, on the contrary, proteins situated near the X and Y axes, in quadrants I and IV are candidates for diagnostic markers 11 .

RESULTS
Sera from patients with chronic and acute form of toxoplasmosis were selected by their clinical characteristics plus the presence of specific antibodies against Toxoplasma gondii.This selection help us to investigate the presence of an specific protein in order to differenciate each form of the disease as well as to be used as a diagnostic marker.
Western Blots.Illustrative Western Blots of the Toxoplasma gondii antigens reacting with sera from patients with acute or chronic toxoplasmosis as well as with sera from normal subjects are shown in Figure 1.Twenty sera from acute toxoplasmosis patients recognized 8 bands of molecular weights ranging from 19 to 90kDa.Twenty-nine sera, from chronic toxoplasmosis subjects, recognized 20 bands from 12 to 90kDa and finally, sera from normal subjects did not recognize any bands (Table 1).Immunoplots.The immunoplots of frequencies showed that sera containing IgM from acute toxoplasmosis patients frequently recognized two proteins: one of approximately 60kDa (f = 0.60) and the other of approximately 66kDa (f = 0.50).Normal sera did not recognize any of the antigenic bands (Figure 2A).
The immunoplots of frequencies from sera containing IgG of chronic toxoplasmosis patients, recognized one protein of approximately 12kDa (f = 0.68) and other of apporoximately 66 kDa (f = 0.55) and normal sera did not recognized any protein, Figure 2B.In Figure 2C, the immunoplot results shows the frequencies of data obtained with sera from chronic against sera from acute toxoplasmosis.Sera containing IgG from chronic individuals recognized more often one protein of approximately 12kDa (f = 0.68); while sera containing IgM from acute patients recognized one protein of approximately 60kDa (f = 0.60); other bands were recognized by antibodies presents in both chronic and acute patients but could not be selected as a diagnostic markers since they did not have significant recognition and were less than 0.5 (Table 1).

DISCUSSION
It is difficult to observe through the Western-Blot method, bands recognized by antibodies present in the sera from patients with chronic and acute toxoplasmosis.Using the immunoplot, described by Larralde in 1989 11 , makes it p o s s i b l e t o o r d e r t h e r e s u l t s o f t h e electroimmunotransference in such a way that the complex mix of antigens in general and those of Toxoplasma gondii in particular, can be classified quantitatively and specifically.Thus, this method constitutes a potential tool for bands discrimination, that possess diagnostic and prognostic information.
According with this analysis, three proteins of approximately 12, 60 and 66kDa were often recognized by the antibodies present in the sera from patients with different states of toxoplasmosis.
Due to the highest frequency recognition of 60kDa protein by IgM antibodies in acute patients, we consider this protein a good marker for this form of infection.Different authors had reported proteins with similar molecular weith recognized by IgM antibodies in patients with the acute form 22 23 .
In chronic infections the protein of 12kDa was recognized by the IgG antibodies with a frequency greater than 0.5.Since this protein was not recognized neither by normal or acute patients, we consider this as a marker protein when Immunoplot method is used.
The presence of the 66kDa protein in chronic as well as in the acute patients point to this protein as a marker for every persons that have been in contact with Toxoplasma gondii; for the same reason it can not be used as a marker in order to differentiated between the two forms of disease 2 7 .
It's impor tant to mention that with the immunoplot analysis, the proteins recognized with frequencies lower than 0.5 it can't be consider as a diagnostic markers for toxoplasmosis because of their low specificity.Although there are other quantitative diagnostic methods for primary toxoplasmosis based in IgG antibodies avidity 3 ; the serologic diagnosis of acute toxoplasmosis can be fortify by the production of monoclonal antibodies, highly specific, against the 60kDa marker protein and employing simpler methods and less expensive as the ELISA.
Further research is needed to confirm if the proteins of 12 and 60kDa can be considered as diagnostic markers for the acute and chronic stages of Toxoplasmosis.

Figure 1 -
Figure 1 -Western Blot of sera from patients with acute (A) and chronic (C) toxoplasmosis, and normal subjects (N) reacting with Toxoplasma gondii.

FrequencyFigure 2 -
Figure 2 -Immunoplots of (A) acute Toxoplasmosis patients versus normal individuals; (B) chronic versus normal, and (C) acute versus chronic.In (A) it can be observed the approximately 60kDa and 66kDa frequently recognized antigens as markers of acute Toxoplasmosis.In (B) it can be observed the approximately 12kDa and 66kDa frequentlyrecognized antigens as markers of the chronic form of the disease.In (C) it can be observed the above mentioned specific markers for acute and chronic forms and 66kDa protein.

Table 1 -
Molecular weights and frequency of recognition (f) of Toxoplasma gondii antigens by Western Blot toxoplasmosis acute and chronic.