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Purification of the 67-kDa lectin-like glycoprotein of Trypanosoma cruzi, LLGP-67, and its evaluation as a relevant antigen for the diagnosis of human infection
FEMS Microbiology Letters 220 (2003) 149^154
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Puri¢cation of the 67-kDa lectin-like glycoprotein of Trypanosoma cruzi, LLGP-67, and its evaluation as a relevant antigen for the diagnosis of human infection Iva¤n S. Marcipar, Elina Welchen, Cintia Roodveldt, Alberto J. Marcipar, Ariel M. Silber INTEBIO, Facultad de Bioqu|¤mica y Ciencias Biolo¤gicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje ‘El Pozo’, C. C. 242 (3000) Santa Fe, Argentina Received 5 November 2002 ; received in revised form 20 January 2003 ; accepted 24 January 2003 First published online 16 February 2003
Abstract In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas’ disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively. < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diagnosis ; Chagasic infection ; 67-kDa lectin-like glycoprotein ; LLGP-67 ; Galactose; Trypanosoma cruzi
1. Introduction Chagas’ disease or American trypanosomiasis is caused by the protozoan parasite Trypanosoma cruzi. This infection a¡ects 16^18 million people in the Americas, with 90 million people at risk [1]. Infected reduviid bugs transmit the disease naturally to humans when broken skin or mucosal tissue comes into contact with insect feces containing metacyclic trypomastigotes. Clinical symptoms in the acute phase are non-speci¢c and often not recognized as being due to T. cruzi infection, and in the chronic phase, most of infection cases are asymptomatic. For this reason, diagnosis based on speci¢c antibody detection is a relevant tool to investigate the serologic status of people coming from endemic areas [2]. Most of antigens commonly used
* Corresponding author. Present address: Instituto de Qu|¤mica, Universidade de Sa‹o Paulo, Av. Lineu Prestes 748 Bloco 10 andar Te¤rreo (05508-900) Sa‹o Paulo, Brazil. Tel. : +55 (11) 3091-3810 R. 233. E-mail address : [email protected] (A.M. Silber).
for the diagnosis of T. cruzi infection are usually complex mixtures of molecules extracted from whole parasites or parasite fractions. Not surprisingly, these mixtures may give rise to false positive reactions and cross-react with sera from other pathogens like Leishmania spp. [3,4]. A variation in terms of reproducibility for diagnosis reagents based on whole or semi-puri¢ed parasite extracts was also reported [5,6]. A variety of recombinant antigens have been proposed in order to optimize the diagnosis of the infection, particularly in terms of speci¢city. However, most of them showed not to be as sensitive as native antigens (for a review see [7]). Attempts to replace parasite extracts by proteins puri¢ed from the parasite that present higher sensitivity and speci¢city scores have been made, and a variety of native molecules have been evaluated [8^15]. However, direct puri¢cation of proteins from total extracts of the parasite is generally di⁄cult because of their low relative quantities and multiple puri¢cation steps, which generally leads to low yields of puri¢ed proteins. In this context, simpli¢ed methods have been developed that can easily be scaled up for the obtainment of antigens for diagnosis.
0378-1097 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00090-9
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Recently, our group has described a 67-kDa galactose binding protein, de¢ned as a 67-kDa lectin-like glycoprotein or LLGP-67. LLGP-67 is present in epimastigote and tripomastigote stages of the parasite, and has been shown to be involved in the host cell invasion and host cell receptor recognition in the endothelial cells of the mammary artery, atrium and right ventricle [16]. The identi¢cation and puri¢cation of this protein have been previously achieved on the basis of its speci¢c interactions with galactose residues displayed on the surface of desialylated human erythrocytes. However, this method has no practical value to obtain the amounts of protein required to perform assays such as an evaluation of its antigenic pro¢le for human diagnosis of T. cruzi infection. A main disadvantage is the relatively low stability of erythrocyte columns. In the present work we describe a straightforward method for the puri¢cation of the LLGP-67 antigen on the basis of its galactose binding activity and demonstrate the relevance of LLGP-67 as antigen for diagnosis of human infection by T. cruzi.
2. Materials and methods 2.1. Parasites T. cruzi epimastigotes (Tulahuen 0 strain) were cultured in conditions allowing exponential growth in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum at 28‡C [17]. 2.2. Total homogenates (TH) T. cruzi epimastigotes were harvested, washed in phosphate-bu¡ered saline (PBS) and resuspended in 5 volumes of 1 mM tosyl-L-lysinechloromethylketone (TLCK) and 1 mM phenylmethylsulfonyl £uoride (PMSF) in distilled water. After frozen and thawed (four times), the suspension was subjected to sonication (20 kHz, 30 watt, 2 min). The lysate was cleared by centrifugation at 15 000Ug for 15 min and the supernatant was used for subsequent puri¢cation. Total protein determination was carried out according to Bradford [18]. 2.3. A⁄nity chromatography Chromatography on erythrocytes or agarose (Sigma) was performed (1U10 cm columns Bio-Rad). The columns were loaded with 1 mg of protein from epimastigote extracts in a total volume of 1 ml. The column was washed with PBS until no protein could be detected by absorbance at 280 nm. Speci¢cally bound protein was eluted by 0.15 M galactose in PBS. Fractions of 3 ml each were collected, and their protein concentration determined. Where indicated, fractions corresponding to the absorbance peak
were concentrated by tangential ¢ltration using a Millipore membrane (MWCO = 10 kDa, Millipore). 2.4. Immune serum Serum was obtained by using a modi¢cation of the procedure described by Tijsen [19]. A female, 3-month-old rabbit was subcutaneously inoculated with 0.6 ml of puri¢ed protein in 0.6 ml of Freund’s complete adjuvant (protein concentration 0.5 mg ml31 ) that was obtained as described above. Fifteen days later the animal was boostered with 0.6 ml puri¢ed protein (0.25 mg ml31 ) plus 0.6 ml of Freund’s incomplete adjuvant. Two weeks after the booster, blood was collected and sera were separated. Prior to immunizations, blood extractions for preimmune controls were performed. Serum against LLGP-67 that had been puri¢ed on erythrocyte membranes was obtained as previously described [16]. 2.5. Serum titration by enzyme-linked immunosorbent assay (ELISA) Polystyrene microplates were sensitized with 1 Wg of the 67-kDa lectin-like glycoprotein (LLGP-67). The microplates were incubated with di¡erent dilutions of immune rabbit serum in PBS^1% low fat milk. Following washing, microplates were incubated with the goat anti-rabbit IgG peroxidase conjugate (Sigma Chemical Company, USA) that was diluted 1/1000 in PBS^1% milk before use. All incubations were performed at 37‡C for 60 min. The reaction was developed using tetramethyl benzidine (TMB) in H2 O2 and the absorbance was read at 450 nm. Cuto¡ values for each dilution were calculated as the absorbance average of assays performed with negative sera plus two standard deviations. The serum titer was 4000. 2.6. Human sera Human chagasic sera were selected from discarded blood from blood banks. The T. cruzi infection status was determined by using two di¡erent conventional tests: commercial ELISA and indirect hemagglutination (IHA) based on total homogenate antigens. Sera were characterized as positive or negative based on concordant results from both reactions. 2.7. ELISA with human sera Polystyrene microplates (Costar, USA) were sensitized with 1 Wg per well of 67-kDa lectin-like glycoprotein (LLGP-67) obtained from the agarose column. Microplates were incubated with a 1/200 dilution of human sera in PBS^1% low fat milk. After washing, the microplates were incubated with the second antibody and developed as described above.
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2.8. Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE)
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3. Results
A 12% polyacrylamide gel electrophoresis under reducing conditions was carried out according to Laemmli [20]. The gels were stained with Coomassie brilliant blue. 2.9. Western blot Epimastigotes TH (50 Wg per lane) were subjected to 12% SDS^PAGE and electroblotted to a nitrocellulose membrane. Membranes were incubated with sera in PBS with 1% albumin (1/1000) and, subsequently, with anti-rabbit IgG peroxidase conjugate, diluted 1/1000 in PBS with 1% albumin. The reaction was developed with 0.05% diaminobenzidine and 0.04 volumes of H2 O2 in PBS. 2.10. Data analysis The data obtained as optical densities at 450 nm were analyzed by using computer graphics software (Micrococcal Origin 5.0). The cuto¡ value of ELISA was calculated as the mean OD450 of the true negative sera plus two standard deviations (S.D.). Positive and negative results in ELISA tests with LLGP-67 were compared with the serologic status obtained by the respective reference technique, as described above. U indices [21] (observed agreements against chance-expected agreement by chance) were calculated for the ELISA results according to the formula U = (Po 3Pe )/(13Pe ), were Po is the observed agreement and Pe is the expected agreement by chance.
The ability of a non-derivatized agarose matrix to speci¢cally retain a previously described galactose binding protein (LLGP-67) from a whole extract of epimastigotes of T. cruzi was evaluated. Epimastigote TH applied to the column and the eluate was fractionated. After washing, the retained material was speci¢cally eluted using 0.15 M galactose as competing ligand. Protein elution from the column was followed by absorbance at 280 nm. A single elution peak was observed, indicating that free galactose was able to compete with galactose groups displayed by non-derivatized agarose in order to liberate the galactose binding protein (Fig. 1A). Epimastigote TH, protein puri¢ed by agarose chromatography, and LLGP-67 puri¢ed on erythrocyte membranes were submitted to SDS^PAGE (Fig. 1B). A single 67-kDa band was obtained for both puri¢cation methods. The fact that the molecular mass and galactose binding properties are identical for both proteins suggests that proteins obtained using both puri¢cation methods are the same, namely LLGP-67. In order to further con¢rm the identity of the protein that has been puri¢ed on agarose and was previously described as LLGP-67 antigen, speci¢c sera against LLGP67 and the protein puri¢ed on agarose were used in a Western blot assay. Proteins from epimastigote TH, LLGP-67 and the agarose-puri¢ed protein were separated by SDS^PAGE, blotted to nitrocellulose membranes and then incubated with the respective speci¢c antibodies. As shown in Fig. 2, both speci¢c sera recognized the same
Fig. 1. A: Chromatogram corresponding to LLGP-67 puri¢cation on non-derivatized agarose. 1 ml of parasite protein extract was loaded at a concentration of 1 mg ml31 . The arrow indicates the addition of 0.15 mM competing galactose. B: SDS^PAGE: Parasite extracts £owed through the column (lane a) and LLGP-67 puri¢ed on non-derivatized agarose (lane b) or on erythrocyte membranes (lane c) were submitted to a polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue.
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Fig. 2. Identity assay: Sera against samples of LLGP-67 puri¢ed on non-derivatized agarose interaction and erythrocyte membranes were used in a Western blot assay. Lanes a and e, preimmune sera (as controls); lanes b, c and d, serum against LLGP-67 puri¢ed on non-derivatized agarose ; lanes f, g and h, serum against LLGP-67 puri¢ed on erythrocyte membranes. On lanes a, b, e and f, epimastigote total homogenate was electrophoresed as antigen. On lanes c and g, agarose-puri¢ed LLGP-67 was used. On lanes d and h, erythrocyte membrane-puri¢ed LLGP-67 was used.
single 67-kDa band. This result con¢rmed the identity of the puri¢ed protein as LLGP-67. The capacity of the chromatographic matrix for the puri¢cation of LLGP-67 was determined. The column was loaded with increasing concentrations of T. cruzi extract. Each puri¢cation fraction from the elution peak was collected, pooled, and concentrated to a ¢nal volume of 1 ml and total protein concentration was determined in each
case. By this method we calculated a column capacity of 10.38 Wg ml31 for LLGP-67. Finally, the agarose-puri¢ed LLGP-67 was evaluated for its suitability as an antigen for diagnosis against a human panel of T. cruzi infected sera (Fig. 3). Fortynine out of 50 chagasic sera were reactive against LLGP-67 (98.00% sensitivity) and 52 out of 53 non-chagasic sera showed no reaction against both puri¢ed molecules (98.11% speci¢city). When the U index was calculated from these data, the obtained value was 0.96 (P 6 0.001). To con¢rm that ELISA reactions were revealing a speci¢c reactivity against the agarose-puri¢ed LLGP-67, the same antigen preparation was used in Western blot assays. Pools containing sera from individuals that reacted positive or negative (as control) in the ELISA were subjected to Western blot analysis. A single 67-kDa protein was recognized by positive pools of sera (Fig. 4), showing that the reactivity of chagasic sera observed by ELISA was speci¢c for the agarose-puri¢ed LLGP-67.
4. Discussion In a previous work, we have established that the LLGP67 is a glycoprotein with galactose binding activity present in epimastigote and trypomastigote stages. It was originally obtained by puri¢cation from epimastigotes by its interaction with desialylated erythrocyte membranes on the basis of galactose binding activity [16]. Based on these results we evaluated di¡erent methods of puri¢cation in order to obtain material for studies of antigenic relevance for human diagnosis. In the present study, we propose a direct method to a⁄nity purify LLGP-67 from total parasite homogenates on the basis of its previously described
Fig. 3. Human sera that were previously characterized as positive or negative for T. cruzi infection were evaluated by ELISA using LLGP-67 as antigen. Cuto¡ (two standard deviations over the mean of negative values) is indicated.
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kDa
a
b
c
d
e
67 kDa
Fig. 4. Speci¢city of ELISA reactivity. The reactivity of pools of human sera de¢ned as negative (lane a) or positive (lanes b, c, d and f) for T. cruzi infection were tested by Western blot against LLGP-67. In each case, same blots of LLGP-67 were used for ELISA and Western blot in order to demonstrate that reactivity of sera observed by ELISA was due to recognition of LLGP-67.
galactose binding activity. The ability of this protein to speci¢cally recognize and bind galactose residues on non-derivatized agarose was useful in order to establish a simple one-step puri¢cation. A single a⁄nity peak corresponding to a 67-kDa protein was obtained when free galactose had been added to the column in order to compete with exposed galactose residues in the agarose matrix. The electrophoretic pattern of the puri¢ed protein agreed with that shown by LLGP-67 puri¢ed on the basis of its binding to erythrocyte membranes [16]. Indeed, the protein could be puri¢ed to apparent homogeneity. In order to con¢rm the identity of the obtained protein and LLGP67, a speci¢c immune serum was prepared and its reactivity pattern in a Western blot assay was compared with that obtained by a speci¢c serum against LLGP-67 puri¢ed by other methods. Cross-speci¢c recognition among sera and puri¢ed antigens also was evaluated, suggesting identity between both molecules. The capacity of non-derivatized agarose as a puri¢cation matrix was also characterized resulting in 10.38 Wg ml31 . It is remarkable that non-derivatized agarose is a relatively inexpensive and easily obtainable material. In view of this, increasing the column size was neither di⁄cult nor expensive in order to obtain the required amounts of protein for diagnostic purposes. Finally, enzyme immunoassays were performed using agarose-puri¢ed LLGP-67 as antigen. In these assays we showed that
153
LLGP-67 was a speci¢c and sensitive antigen for human diagnosis of T. cruzi infection (U index = 0.96). The U index indicates concordance between the ELISA test using LLGP-67 as antigen and the reference criterion that was used for characterization of sera. It is interesting to remark that U values between 0.91 and 1 are considered highly concordant [22]. The identi¢cation of T. cruzi proteins that can be used for detection of the human infection is a relevant topic in diagnosis research of Chagas’ disease. Major e¡orts are made to identify antigens that permit the replacement of complex extracts or unde¢ned fractions by de¢ned parasite components (mainly proteins and glycoproteins) and recombinant antigens in order to improve sensitivity, speci¢city and reproducibility of diagnosis [7,23,24]. Once cloned and established the expression conditions, recombinant antigens can be easily obtained in large scale. Interestingly, a variety of recombinant antigens displaying high sensitivity or speci¢city have been described in the last years. However it was di⁄cult to identify antigens showing both characteristics, sensitivity and speci¢city, simultaneously, and in view of that, a combination of recombinant antigens is used [25]. On the other hand, it should be taken into account that recombinant antigens expressed in heterologous systems will show di¡erences in post-translational modi¢cations with respect to native antigens. One of the major post-translational modi¢cations are glycosylations. The addition of glycosidic moieties in glycoproteins strongly a¡ects their antigenic pro¢le for the diagnosis of T. cruzi infection, since it was observed that glycoproteins might be immunodominant, in particular for the rise of lytic antibodies [26]. In this context, the use of these puri¢ed glycoproteins for diagnosis may be relevant. Most of recombinant antigens that have been evaluated up to now were expressed in Escherichia coli, and have no glycosylations at all. The use of glycosidic structures of native glycoprotein has provided high speci¢city antigens [14]. In view of the above-mentioned considerations, mixtures of recombinant antigens or recombinant and native puri¢ed antigens are being proposed in order to improve the speci¢city and reproducibility without loss of sensitivity. We propose that the agarose a⁄nity chromatography method can be used to obtain LLGP-67 as an antigen for the diagnosis of T. cruzi infection. Recently, our group has studied the evolution of serum avidity indices on the follow up of T. cruzi infection in rats that had been experimentally infected, as a model for the humoral response [27]. It was observed that no speci¢c pattern existed with respect to the rise of high avidity antibodies against speci¢c antigens: some antigens induce the development of a high avidity antibody response along time in contrast to others that induce a low avidity antibody response. Since LLGP-67 has been shown to be a relevant antigen it would be interesting to follow up the avidity of the antibody response against this protein along time. The possi-
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bility of obtaining antigens that rise high avidity antibody responses opens new perspectives in the setting up of diagnosis systems, since highly stringent conditions for antibody binding to antigens will provide a tool to regulate the speci¢city for the antigen recognition process by antibodies.
[12]
[13]
Acknowledgements [14]
The authors acknowledge to Dr. Maria Ju¤lia Manso Alves and Dr. Henning Ulrich for critical readings and suggestions. This work was partially funded by Universidad Nacional del Litoral, CAID No. 052/009.
References
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[1] Schmun‹is, G.A. (1991) Trypanosoma cruzi, the etiologic agent of Chagas’ disease: status in the blood supply in endemic and nonendemic countries. Transfusion 31, 547^557. [2] Tanowitz, H.B., Kirchho¡, L.V., Simon, D., Morris, S.A., Weiss, L.M. and Wittner, M. (1992) Chagas’ disease. Clin. Microbiol. Rev. 5, 400^419. [3] Frasch, A.C.C. and Reyes, M.B. (1990) Diagnosis of Chagas’ disease using recombinant DNA technology. Parasitol. Today 6, 137^139. [4] Chiaramonte, M.G., Zwirner, W.N., Caropresi, S.L., Taranto, N.J. and Malchiodi, E.L. (1996) Trypanosoma cruzi and Leishmania spp. Human mixed infection. Am. J. Trop. Med. Hyg. 54, 271^273. [5] Camargo, M.E., Segura, E.L., Kagan, I.G., Souza, J.M.P., Carvalheiro, J.R., Yanovsky, J.F. and Guimara‹es, M.C.S. (1986) Collaboration on the standardization of Chagas disease in Ame¤ricas : an appraisal. Bull. Pan. Am. Health Organ. 20, 233^244. [6] Saez-Alquezar, A., Luquetti, A.O., Borges-Pereira, J., Moreira, E.F., Gadahela, M., Garcia Zapata, M.T. and Strugo Arruda, A.H. (1997) Estudo multice¤ntrico: validacYa‹o do desempenho de conjuntos diagno¤sticos de hemaglutinacYa‹o indirecta dispon|¤veis no Brasil para o diagno¤stico serolo¤gico da infecYa‹o pelo Trypanosoma cruzi. Rev. Patol. Trop. 26, 343^374. [7] da Silveira, J.F., Umezawa, E.S. and Luquetti, A.O. (2001) Chagas disease : recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends Parasitol. 17, 286^291. [8] Kirccho¡, L.V., Gam, A.A., Gusmao, R.A., Goldsmith, R.S., Rezende, J.M. and Rassi, A. (1987) Increased speci¢city of serodiagnosis of Chagas’ disease by detection of antibody to the 72- and 90kilodalton glycoproteins of Trypanosoma cruzi. J. Infect. Dis. 155, 561^564. [9] Scharfstein, J., Schechter, M., Sena, M., Peralta, J.M., MendoncaPreviato, L. and Miles, M.A. (1988) Trypanosoma cruzi: characterization and isolation of a 57/51,000 m.w. surface glycoprotein (GP57/ 51) expressed by epimastigotes and bloodstream trypomastigotes. J. Immunol. 137, 1336^1341. [10] Martinez, J., Campetella, O., Frasch, A.C.C. and Cazzulo, J.J. (1991) The major cysteine proteinase (Cruzipain) from Trypanosoma cruzi is antigenic in human infections. Infect. Immun. 59, 4275^4277. [11] Solana, M.E., Katzin, A.M., Umezawa, E.S. and Miatello, C.S. (1995) High speci¢city of Trypanosoma cruzi epimastigote ribonucleo-
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Puri¢cation of the 67-kDa lectin-like glycoprotein of Trypanosoma cruzi, LLGP-67, and its evaluation as a relevant antigen for the diagnosis of human infection Iva¤n S. Marcipar, Elina Welchen, Cintia Roodveldt, Alberto J. Marcipar, Ariel M. Silber INTEBIO, Facultad de Bioqu|¤mica y Ciencias Biolo¤gicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje ‘El Pozo’, C. C. 242 (3000) Santa Fe, Argentina Received 5 November 2002 ; received in revised form 20 January 2003 ; accepted 24 January 2003 First published online 16 February 2003
Abstract In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas’ disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively. < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diagnosis ; Chagasic infection ; 67-kDa lectin-like glycoprotein ; LLGP-67 ; Galactose; Trypanosoma cruzi
1. Introduction Chagas’ disease or American trypanosomiasis is caused by the protozoan parasite Trypanosoma cruzi. This infection a¡ects 16^18 million people in the Americas, with 90 million people at risk [1]. Infected reduviid bugs transmit the disease naturally to humans when broken skin or mucosal tissue comes into contact with insect feces containing metacyclic trypomastigotes. Clinical symptoms in the acute phase are non-speci¢c and often not recognized as being due to T. cruzi infection, and in the chronic phase, most of infection cases are asymptomatic. For this reason, diagnosis based on speci¢c antibody detection is a relevant tool to investigate the serologic status of people coming from endemic areas [2]. Most of antigens commonly used
* Corresponding author. Present address: Instituto de Qu|¤mica, Universidade de Sa‹o Paulo, Av. Lineu Prestes 748 Bloco 10 andar Te¤rreo (05508-900) Sa‹o Paulo, Brazil. Tel. : +55 (11) 3091-3810 R. 233. E-mail address : [email protected] (A.M. Silber).
for the diagnosis of T. cruzi infection are usually complex mixtures of molecules extracted from whole parasites or parasite fractions. Not surprisingly, these mixtures may give rise to false positive reactions and cross-react with sera from other pathogens like Leishmania spp. [3,4]. A variation in terms of reproducibility for diagnosis reagents based on whole or semi-puri¢ed parasite extracts was also reported [5,6]. A variety of recombinant antigens have been proposed in order to optimize the diagnosis of the infection, particularly in terms of speci¢city. However, most of them showed not to be as sensitive as native antigens (for a review see [7]). Attempts to replace parasite extracts by proteins puri¢ed from the parasite that present higher sensitivity and speci¢city scores have been made, and a variety of native molecules have been evaluated [8^15]. However, direct puri¢cation of proteins from total extracts of the parasite is generally di⁄cult because of their low relative quantities and multiple puri¢cation steps, which generally leads to low yields of puri¢ed proteins. In this context, simpli¢ed methods have been developed that can easily be scaled up for the obtainment of antigens for diagnosis.
0378-1097 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00090-9
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Recently, our group has described a 67-kDa galactose binding protein, de¢ned as a 67-kDa lectin-like glycoprotein or LLGP-67. LLGP-67 is present in epimastigote and tripomastigote stages of the parasite, and has been shown to be involved in the host cell invasion and host cell receptor recognition in the endothelial cells of the mammary artery, atrium and right ventricle [16]. The identi¢cation and puri¢cation of this protein have been previously achieved on the basis of its speci¢c interactions with galactose residues displayed on the surface of desialylated human erythrocytes. However, this method has no practical value to obtain the amounts of protein required to perform assays such as an evaluation of its antigenic pro¢le for human diagnosis of T. cruzi infection. A main disadvantage is the relatively low stability of erythrocyte columns. In the present work we describe a straightforward method for the puri¢cation of the LLGP-67 antigen on the basis of its galactose binding activity and demonstrate the relevance of LLGP-67 as antigen for diagnosis of human infection by T. cruzi.
2. Materials and methods 2.1. Parasites T. cruzi epimastigotes (Tulahuen 0 strain) were cultured in conditions allowing exponential growth in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum at 28‡C [17]. 2.2. Total homogenates (TH) T. cruzi epimastigotes were harvested, washed in phosphate-bu¡ered saline (PBS) and resuspended in 5 volumes of 1 mM tosyl-L-lysinechloromethylketone (TLCK) and 1 mM phenylmethylsulfonyl £uoride (PMSF) in distilled water. After frozen and thawed (four times), the suspension was subjected to sonication (20 kHz, 30 watt, 2 min). The lysate was cleared by centrifugation at 15 000Ug for 15 min and the supernatant was used for subsequent puri¢cation. Total protein determination was carried out according to Bradford [18]. 2.3. A⁄nity chromatography Chromatography on erythrocytes or agarose (Sigma) was performed (1U10 cm columns Bio-Rad). The columns were loaded with 1 mg of protein from epimastigote extracts in a total volume of 1 ml. The column was washed with PBS until no protein could be detected by absorbance at 280 nm. Speci¢cally bound protein was eluted by 0.15 M galactose in PBS. Fractions of 3 ml each were collected, and their protein concentration determined. Where indicated, fractions corresponding to the absorbance peak
were concentrated by tangential ¢ltration using a Millipore membrane (MWCO = 10 kDa, Millipore). 2.4. Immune serum Serum was obtained by using a modi¢cation of the procedure described by Tijsen [19]. A female, 3-month-old rabbit was subcutaneously inoculated with 0.6 ml of puri¢ed protein in 0.6 ml of Freund’s complete adjuvant (protein concentration 0.5 mg ml31 ) that was obtained as described above. Fifteen days later the animal was boostered with 0.6 ml puri¢ed protein (0.25 mg ml31 ) plus 0.6 ml of Freund’s incomplete adjuvant. Two weeks after the booster, blood was collected and sera were separated. Prior to immunizations, blood extractions for preimmune controls were performed. Serum against LLGP-67 that had been puri¢ed on erythrocyte membranes was obtained as previously described [16]. 2.5. Serum titration by enzyme-linked immunosorbent assay (ELISA) Polystyrene microplates were sensitized with 1 Wg of the 67-kDa lectin-like glycoprotein (LLGP-67). The microplates were incubated with di¡erent dilutions of immune rabbit serum in PBS^1% low fat milk. Following washing, microplates were incubated with the goat anti-rabbit IgG peroxidase conjugate (Sigma Chemical Company, USA) that was diluted 1/1000 in PBS^1% milk before use. All incubations were performed at 37‡C for 60 min. The reaction was developed using tetramethyl benzidine (TMB) in H2 O2 and the absorbance was read at 450 nm. Cuto¡ values for each dilution were calculated as the absorbance average of assays performed with negative sera plus two standard deviations. The serum titer was 4000. 2.6. Human sera Human chagasic sera were selected from discarded blood from blood banks. The T. cruzi infection status was determined by using two di¡erent conventional tests: commercial ELISA and indirect hemagglutination (IHA) based on total homogenate antigens. Sera were characterized as positive or negative based on concordant results from both reactions. 2.7. ELISA with human sera Polystyrene microplates (Costar, USA) were sensitized with 1 Wg per well of 67-kDa lectin-like glycoprotein (LLGP-67) obtained from the agarose column. Microplates were incubated with a 1/200 dilution of human sera in PBS^1% low fat milk. After washing, the microplates were incubated with the second antibody and developed as described above.
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2.8. Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE)
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3. Results
A 12% polyacrylamide gel electrophoresis under reducing conditions was carried out according to Laemmli [20]. The gels were stained with Coomassie brilliant blue. 2.9. Western blot Epimastigotes TH (50 Wg per lane) were subjected to 12% SDS^PAGE and electroblotted to a nitrocellulose membrane. Membranes were incubated with sera in PBS with 1% albumin (1/1000) and, subsequently, with anti-rabbit IgG peroxidase conjugate, diluted 1/1000 in PBS with 1% albumin. The reaction was developed with 0.05% diaminobenzidine and 0.04 volumes of H2 O2 in PBS. 2.10. Data analysis The data obtained as optical densities at 450 nm were analyzed by using computer graphics software (Micrococcal Origin 5.0). The cuto¡ value of ELISA was calculated as the mean OD450 of the true negative sera plus two standard deviations (S.D.). Positive and negative results in ELISA tests with LLGP-67 were compared with the serologic status obtained by the respective reference technique, as described above. U indices [21] (observed agreements against chance-expected agreement by chance) were calculated for the ELISA results according to the formula U = (Po 3Pe )/(13Pe ), were Po is the observed agreement and Pe is the expected agreement by chance.
The ability of a non-derivatized agarose matrix to speci¢cally retain a previously described galactose binding protein (LLGP-67) from a whole extract of epimastigotes of T. cruzi was evaluated. Epimastigote TH applied to the column and the eluate was fractionated. After washing, the retained material was speci¢cally eluted using 0.15 M galactose as competing ligand. Protein elution from the column was followed by absorbance at 280 nm. A single elution peak was observed, indicating that free galactose was able to compete with galactose groups displayed by non-derivatized agarose in order to liberate the galactose binding protein (Fig. 1A). Epimastigote TH, protein puri¢ed by agarose chromatography, and LLGP-67 puri¢ed on erythrocyte membranes were submitted to SDS^PAGE (Fig. 1B). A single 67-kDa band was obtained for both puri¢cation methods. The fact that the molecular mass and galactose binding properties are identical for both proteins suggests that proteins obtained using both puri¢cation methods are the same, namely LLGP-67. In order to further con¢rm the identity of the protein that has been puri¢ed on agarose and was previously described as LLGP-67 antigen, speci¢c sera against LLGP67 and the protein puri¢ed on agarose were used in a Western blot assay. Proteins from epimastigote TH, LLGP-67 and the agarose-puri¢ed protein were separated by SDS^PAGE, blotted to nitrocellulose membranes and then incubated with the respective speci¢c antibodies. As shown in Fig. 2, both speci¢c sera recognized the same
Fig. 1. A: Chromatogram corresponding to LLGP-67 puri¢cation on non-derivatized agarose. 1 ml of parasite protein extract was loaded at a concentration of 1 mg ml31 . The arrow indicates the addition of 0.15 mM competing galactose. B: SDS^PAGE: Parasite extracts £owed through the column (lane a) and LLGP-67 puri¢ed on non-derivatized agarose (lane b) or on erythrocyte membranes (lane c) were submitted to a polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue.
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Fig. 2. Identity assay: Sera against samples of LLGP-67 puri¢ed on non-derivatized agarose interaction and erythrocyte membranes were used in a Western blot assay. Lanes a and e, preimmune sera (as controls); lanes b, c and d, serum against LLGP-67 puri¢ed on non-derivatized agarose ; lanes f, g and h, serum against LLGP-67 puri¢ed on erythrocyte membranes. On lanes a, b, e and f, epimastigote total homogenate was electrophoresed as antigen. On lanes c and g, agarose-puri¢ed LLGP-67 was used. On lanes d and h, erythrocyte membrane-puri¢ed LLGP-67 was used.
single 67-kDa band. This result con¢rmed the identity of the puri¢ed protein as LLGP-67. The capacity of the chromatographic matrix for the puri¢cation of LLGP-67 was determined. The column was loaded with increasing concentrations of T. cruzi extract. Each puri¢cation fraction from the elution peak was collected, pooled, and concentrated to a ¢nal volume of 1 ml and total protein concentration was determined in each
case. By this method we calculated a column capacity of 10.38 Wg ml31 for LLGP-67. Finally, the agarose-puri¢ed LLGP-67 was evaluated for its suitability as an antigen for diagnosis against a human panel of T. cruzi infected sera (Fig. 3). Fortynine out of 50 chagasic sera were reactive against LLGP-67 (98.00% sensitivity) and 52 out of 53 non-chagasic sera showed no reaction against both puri¢ed molecules (98.11% speci¢city). When the U index was calculated from these data, the obtained value was 0.96 (P 6 0.001). To con¢rm that ELISA reactions were revealing a speci¢c reactivity against the agarose-puri¢ed LLGP-67, the same antigen preparation was used in Western blot assays. Pools containing sera from individuals that reacted positive or negative (as control) in the ELISA were subjected to Western blot analysis. A single 67-kDa protein was recognized by positive pools of sera (Fig. 4), showing that the reactivity of chagasic sera observed by ELISA was speci¢c for the agarose-puri¢ed LLGP-67.
4. Discussion In a previous work, we have established that the LLGP67 is a glycoprotein with galactose binding activity present in epimastigote and trypomastigote stages. It was originally obtained by puri¢cation from epimastigotes by its interaction with desialylated erythrocyte membranes on the basis of galactose binding activity [16]. Based on these results we evaluated di¡erent methods of puri¢cation in order to obtain material for studies of antigenic relevance for human diagnosis. In the present study, we propose a direct method to a⁄nity purify LLGP-67 from total parasite homogenates on the basis of its previously described
Fig. 3. Human sera that were previously characterized as positive or negative for T. cruzi infection were evaluated by ELISA using LLGP-67 as antigen. Cuto¡ (two standard deviations over the mean of negative values) is indicated.
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kDa
a
b
c
d
e
67 kDa
Fig. 4. Speci¢city of ELISA reactivity. The reactivity of pools of human sera de¢ned as negative (lane a) or positive (lanes b, c, d and f) for T. cruzi infection were tested by Western blot against LLGP-67. In each case, same blots of LLGP-67 were used for ELISA and Western blot in order to demonstrate that reactivity of sera observed by ELISA was due to recognition of LLGP-67.
galactose binding activity. The ability of this protein to speci¢cally recognize and bind galactose residues on non-derivatized agarose was useful in order to establish a simple one-step puri¢cation. A single a⁄nity peak corresponding to a 67-kDa protein was obtained when free galactose had been added to the column in order to compete with exposed galactose residues in the agarose matrix. The electrophoretic pattern of the puri¢ed protein agreed with that shown by LLGP-67 puri¢ed on the basis of its binding to erythrocyte membranes [16]. Indeed, the protein could be puri¢ed to apparent homogeneity. In order to con¢rm the identity of the obtained protein and LLGP67, a speci¢c immune serum was prepared and its reactivity pattern in a Western blot assay was compared with that obtained by a speci¢c serum against LLGP-67 puri¢ed by other methods. Cross-speci¢c recognition among sera and puri¢ed antigens also was evaluated, suggesting identity between both molecules. The capacity of non-derivatized agarose as a puri¢cation matrix was also characterized resulting in 10.38 Wg ml31 . It is remarkable that non-derivatized agarose is a relatively inexpensive and easily obtainable material. In view of this, increasing the column size was neither di⁄cult nor expensive in order to obtain the required amounts of protein for diagnostic purposes. Finally, enzyme immunoassays were performed using agarose-puri¢ed LLGP-67 as antigen. In these assays we showed that
153
LLGP-67 was a speci¢c and sensitive antigen for human diagnosis of T. cruzi infection (U index = 0.96). The U index indicates concordance between the ELISA test using LLGP-67 as antigen and the reference criterion that was used for characterization of sera. It is interesting to remark that U values between 0.91 and 1 are considered highly concordant [22]. The identi¢cation of T. cruzi proteins that can be used for detection of the human infection is a relevant topic in diagnosis research of Chagas’ disease. Major e¡orts are made to identify antigens that permit the replacement of complex extracts or unde¢ned fractions by de¢ned parasite components (mainly proteins and glycoproteins) and recombinant antigens in order to improve sensitivity, speci¢city and reproducibility of diagnosis [7,23,24]. Once cloned and established the expression conditions, recombinant antigens can be easily obtained in large scale. Interestingly, a variety of recombinant antigens displaying high sensitivity or speci¢city have been described in the last years. However it was di⁄cult to identify antigens showing both characteristics, sensitivity and speci¢city, simultaneously, and in view of that, a combination of recombinant antigens is used [25]. On the other hand, it should be taken into account that recombinant antigens expressed in heterologous systems will show di¡erences in post-translational modi¢cations with respect to native antigens. One of the major post-translational modi¢cations are glycosylations. The addition of glycosidic moieties in glycoproteins strongly a¡ects their antigenic pro¢le for the diagnosis of T. cruzi infection, since it was observed that glycoproteins might be immunodominant, in particular for the rise of lytic antibodies [26]. In this context, the use of these puri¢ed glycoproteins for diagnosis may be relevant. Most of recombinant antigens that have been evaluated up to now were expressed in Escherichia coli, and have no glycosylations at all. The use of glycosidic structures of native glycoprotein has provided high speci¢city antigens [14]. In view of the above-mentioned considerations, mixtures of recombinant antigens or recombinant and native puri¢ed antigens are being proposed in order to improve the speci¢city and reproducibility without loss of sensitivity. We propose that the agarose a⁄nity chromatography method can be used to obtain LLGP-67 as an antigen for the diagnosis of T. cruzi infection. Recently, our group has studied the evolution of serum avidity indices on the follow up of T. cruzi infection in rats that had been experimentally infected, as a model for the humoral response [27]. It was observed that no speci¢c pattern existed with respect to the rise of high avidity antibodies against speci¢c antigens: some antigens induce the development of a high avidity antibody response along time in contrast to others that induce a low avidity antibody response. Since LLGP-67 has been shown to be a relevant antigen it would be interesting to follow up the avidity of the antibody response against this protein along time. The possi-
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bility of obtaining antigens that rise high avidity antibody responses opens new perspectives in the setting up of diagnosis systems, since highly stringent conditions for antibody binding to antigens will provide a tool to regulate the speci¢city for the antigen recognition process by antibodies.
[12]
[13]
Acknowledgements [14]
The authors acknowledge to Dr. Maria Ju¤lia Manso Alves and Dr. Henning Ulrich for critical readings and suggestions. This work was partially funded by Universidad Nacional del Litoral, CAID No. 052/009.
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