STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

Sandra L.M. AVILA, Tania R. TOZETTO-MENDOZA, Viviana G. ARRUK & Antonio Walter FERREIRA

SUMMARY

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20^oC up to 10 days or at -70^oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

KEYWORDS: Malaria; Plasmodium falciparum; Antigen; ELISA; Western blotting; Serodiagnosis.

INTRODUCTION

The definition of an ideal antigen to be used as a support for diagnostic purposes and for the evaluation of the success of therapy is a goal to be reached. The antigen complexity of eukaryotic organisms involved in infectious processes is one of the most important limiting factor for attaining this aim. Also, the response to epitopes of low immunogenicity may be masked by the immunological background induced by multiple infectious processes in populations residing in areas with precarious living conditions. Indeed cross-reactions between antigens of several bacteria, fungi and parasites are very common. In malaria, many serologic tests using blood stage of Plasmodium falciparum as antigen for the detection of antibodies have been employed for diagnostic purposes, seroepidemiological surveys or therapeutic follow-up. P. falciparum presents different antigens during its evolutive stages as well as a high degree of genetic polymorphism and antigen variation, resulting in antigenic differences between isolates from different regions^4,14.

Indirect immunofluorescence (IIF)^1,24 and the enzyme linked immunosorbent assay (ELISA)^12,13,25 are the most widely used tests, mainly because of their satisfactory results in terms of sensitivity, specificity and reproducibility. ELISA for the detection of malarial antibodies has been introduced by Voller et al.³¹ At that time the test used Plasmodium knowlesi infected monkey erythrocytes as antigen, and since then different antigens and various techniques have been employed^8,15. ELISA has some advantages when compared to other tests such as the possibility of automation, is not time consuming and measures antibody levels at relative low cost.

Another method used to detect antibodies against plasmodia is Western blotting. This technique has been used to study the humoral response to different protein antigen components of the parasite by detecting bands defining the infecting species, phase of the disease, different levels of protection, therapeutic efficiency, etc²⁸.

In both ELISA and Western blotting, soluble components of plasmodia are adsorbed to inert supports. The variety of antigens used in different laboratories has led to conflicting results and to controversies. A strict standardization of all preparation steps of the antigen extract is therefore fundamental to obtain consistent results not only in diagnostic or epidemiological studies, but also in the serological follow-up of treated patients.

In a previous investigation, we studied six different methods for the extraction of soluble parasite components using three different detergents, sodium deoxycholate (ionic), Triton X-100 (non-ionic) and amphoteric Zwittergent; a chaotropic agent (urea); a saline solution (NaCl) and an alkaline solution (NaOH). The extraction with Zwittergent showed the best results in both ELISA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)^2,23.

In the present study, we determined the ideal conditions for the isolation of somatic antigens of P. falciparum obtained from in vitro culture and evaluated different storage conditions of the antigen extract to be used in ELISA and Western blotting.

MATERIALS AND METHODS

Sera

Sera from patients from a malaria-endemic area (Peixoto de Azevedo, Mato Grosso, Brazil) with a previous history of malaria and positive for the presence of anti-P. falciparum IgG antibodies in ELISA and IIF were used as positive controls in the standardization of the ELISA (6 sera) and of the Western blotting (12 sera). Sera from 6 blood donors from the Fundação Pró-Sangue, São Paulo Blood Center, Brazil, who have never had malaria or lived in a malaria-endemic area, were used as negative controls.

Antigen preparation

P. falciparum (ZwAg). P. falciparum (ADA isolate from Rondônia, 1982) parasites were grown in vitro according to the method of Trager & Jensen³⁰. Cultures consisted of 5% washed O+ human red blood cells in RPMI 1640 (Sigma Chem. Co.) supplemented with 25 mM Hepes, 10 mM glucose, 0.005% hypoxanthine, 0.25% sodium bicarbonate and 10% heat-inactivated human plasma serum (type A+). Red blood cells at 10% late-stage schizont parasitemia were washed three times with 0.01 M phosphate-buffered saline, pH 7.2 (PBS). The final pellet was resuspended in PBS containing 0.04% (w/v) saponin (Merck) in PBS and incubated for 20 min at room temperature, with occasional mixing. The lysate was centrifuged (3,000 x g, 4^oC, 15 min) and the pellet was washed three times with PBS. The Zwittergent detergent was chosen in the present study since it proved to be a good extracting agent of somatic antigens giving a preparation of parasites retained within permeable erythrocyte ghosts^2,23. It is an amphoteric detergent (critical micellar concentration: 3.6 mM) that presents the solubilization properties of proteins, including cellular membrane proteins, but without being denaturing^16,21.

The parasite pellet was stored under different conditions: up to 10 days at -20^oC (batch I), 4 months at -20^oC (batch II) and 2 months at -70^oC (batch III).

The parasite pellet was further processed with PBS containing 200 µl of 1% Zwittergent 3-14 (Calbiochem Co.) and incubated for 20 min at room temperature²³. After centrifugation (10,000 x g at 4^oC for 30 min) the supernatant was collected and the protein content was determined by the method of Lowry et al¹⁸.

Different variables of the P. falciparum antigen preparation were studied: the addition of a mixture of protease inhibitors (treatment named i) to the following final concentrations: 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 µg/ml antipain and 1 trypsin inhibitor unit (TIU) aprotinin, and dialysis against PBS for 18 h after Zwittergent extraction (treatment named d). Antigen extracts were stored for different periods of time (0,15,30, and 60 days) and at different temperatures (-20ºC, -70ºC, and in liquid N₂). The protein pattern of each aliquot was determined by SDS-PAGE and its immunoreactivity was analyzed by ELISA. The variable presenting the best results was used for standardization of the Western blotting conditions. Fig. 1 summarizes the variables used for P. falciparum antigen extraction.

We also studied different P. falciparum isolates (SA, JST and TAN, 1995) obtained from volunteers with high parasitemia, living in the city of Peixoto de Azevedo, Mato Grosso. For this, Glycerolyte® 57 solution (Fenwal) (1.7 ml) was added to each 1-ml blood sample and aliquots were maintained in liquid N₂ and transported to the Laboratory of Immunology, Instituto de Medicina Tropical de São Paulo. Antigen extracts from these isolates were obtained by treatment with Zwittergent and a mixture of protease inhibitors as described above and the reactivity of the different isolates was compared to that of the ADA isolate used as reference.

Normal red blood cells (O+). Non-infected red blood cells (~300 ml) were processed as described above and used as control antigen.

ELISA

Flat-bottom plastic plates (NUNC) were sensitized with 100 µl of the P. falciparum antigen extract or of control antigen (5 µg/ml) in PBS, for 2 h at 37^oC and for 18 h at 4^oC. Plates were then washed 3 times with PBS containing 0.05% Tween 20 (PBS-Tw20) and blocked with 5% skim milk in PBS-Tw20 (200 µl/well) for 1 h at 37^oC. Positive and negative standard sera diluted 1/100 in PBS-Tw20 containing 1% skim milk (serum diluent) (100 µl/well) were then added in duplicate. After a 40-min incubation at 37^oC, plates were washed again as above and incubated with a peroxidase-conjugated anti-human IgG (g-chain specific) (Biolab Mérieux S.A.) diluted in serum diluent (100 µl/well) for 40 min at 37^oC. After washing, the enzymatic reaction was developed with 0.0022 M o-phenylenediamine chromogen solution (Sigma Chemical Co.) containing 0.013 M 30 % hydrogen peroxide (Merck) in 0.05 M citrate-phosphate buffer, pH 5.0 (100 µl/well), for 30 min at 37^oC in the dark. The reaction was stopped by the addition of 2 N H₂SO₄ (50 µl/well). Absorbance was determined at 492 nm with a microtiterplate reader.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting

Proteins of P. falciparum and control antigens were separated by SDS-PAGE according to the method of Laemmli¹⁷. Aliquots of the extracts were diluted in sample buffer, boiled for 3 min and applied to an 8% polyacrylamide gel using a vertical and discontinuous system. Electrophoresis was performed at an initial voltage of 40 V followed by 120 V after penetration of the samples into the running gel until the indicator dye reached the end of the separating gel. Two types of sample buffer were compared: DTT buffer containing 50 mM dithiothreitol (DTT), 62 mM Tris-HCl, pH 6.8, 0.2% sodium dodecyl sulfate (SDS) and 10% glycerol, and urea buffer containing 1 M urea, 0.01 M Tris-HCl, 0.001% bromophenol blue, 10% glycerol, 1% SDS and ß-mercaptoethanol. The following proteins were used as standard molecular mass markers: ovalbumin (45 kDa), bovine albumin (66 kDa), phosphorylase B (97.4 kDa), and ß-galactosidase (116 kDa) (Sigma Chem. Co.). After electrophoresis, the gel was stained with silver according to the method of Merril et al.²⁰ or transferred onto nitrocellulose membranes.

Western blotting

Antigen fractions separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes (Millipore) for 18 h using a Mini-Protean II® apparatus (BioRad), as described by Towbin et al²⁹. To determine the transfer efficiency, the nitrocellulose membranes were stained with 0.1% Ponceau S (Sigma) in 10% acetic acid. The membranes were cut into 4-mm strips and immediately used for the immunoenzymatic assay or stored for analysis of stability. Different variables of the in situ immunoenzymatic assay were studied: a) blocking solution: 0.5%, 1% and 5% skim milk in PBS-Tw20; b) blocking time: 15 min, 30 min, 45 min, 1 h and 2 h at 37^oC; c) serum diluent: 0.5%, 2% and 5% skim milk in PBS-T; d) serum dilution: 1/20, 1/50 and 1/100; e) incubation time and serum temperature: 1 h, 2 h and 4 h at 37^oC and 18 h at 4^oC; f) conjugate diluent: 0.5%, 2% and 5% skim milk in PBS-T; g) time and temperature of incubation with the conjugate: 1 h and 2 h at room temperature and 30 min at 37^oC; h) chromogen solution: solution A (6 mg 4-chloro-a-naphthol in 2 ml methanol added to 10 ml PBS and 10 µl 30% H₂O₂) and solution B (5 mg diaminobenzidine and 150 µl 30% H₂O₂ in 30 ml PBS).

Statistical analysis

Multiple independent data were compared by the parametric analysis of variance test (ANOVA) and by the nonparametric test of Kruskal Wallis. Significance was defined at the 95% level.

RESULTS

P. falciparum antigen preparations were analyzed regarding their protein pattern by SDS-PAGE and regarding the immunoreactivity of anti-P. falciparum antibody-positive and -negative control sera by Western blotting and ELISA.

The best SDS-PAGE results were obtained with antigens extracted using protease inhibitors, from parasite pellets stored for 0-10 days at -20^oC (I) and up to 2 months at -70^oC (III). Under these conditions, clearly defined protein bands were obtained ranging in molecular mass from 17 to 170 kDa (Fig. 2a,b,d-j). SDS-PAGE of antigen extracts from parasite pellets stored at -20^oC for 4 months (batch II) did not show a satisfactory protein pattern, mainly in terms of high molecular mass bands (Fig. 2c).

Fig. 2.
Electrophoresis of different batches of P. falciparum (ADA isolate) antigen extracts on 8% polyacrylamide gel (SDS-PAGE). SDS gel electrophoresis was performed on the day of antigen extraction using P. falciparum pellets stored for up to 10 days at -20^oC, batch I (2A,B), 4 months at -20^oC, batch II (2C), and 2 months at -70^oC, batch III (2D). SDS gel electrophoresis of batch III was performed after 60 days of storage at -20^oC (E,H), and -70^oC (F,I), and in liquid N₂ (G,J). Two different sample buffers were studied: urea and ß-mercaptoethanol (A,C,D,E,F,G) and dithiothreitol (B,H,I,J). MW = Molecular weight x 10^-3.

The two types of sample buffer studied, urea buffer (Fig. 2e-g) and DTT buffer (Fig. 2h-j), did not show much significant differences in the quality of electrophoresis. Urea buffer showed a slightly better definition of bands and was therefore chosen for the subsequent study.

The different temperatures of storage employed did not interfere significantly with the gel pattern up to 30 days of storage. After this time, the extracts stored at -70^oC and in liquid N₂ presented a better band resolution than those stored at -20^oC, irrespective of storage time (Fig. 2e-j).

The addition of protease inhibitors led to the most reproducible protein patterns under the different storage conditions, concerning time (15, 30 and 60 days) and temperature (-20^oC, -70^oC and N₂) of storage, whereas dialysis of the product did not interfere with the resolution of the protein bands. Fig. 3 shows the gel of batch I extracted in the presence of inhibitors and without dialysis (a-c) and without inhibitors or dialysis (d-f) stored for 60 days at different temperatures.

Fig. 3.
Electrophoresis on 8% polyacrylamide gel (SDS-PAGE) of P. falciparum (ADA isolate) antigens obtained from parasite pellets stored for up to 10 days at -20^oC (batch I) and extracted with (A-C) or without (D-F) the addition of protease inhibitors to the extracting agent. The extract was stored for 60 days at -20^oC (A,D), -70^oC (B,E) and in liquid N₂ (C,F). MW = Molecular weight x 10^-3.

After definition of the best antigen extract by SDS-PAGE, standardization of Western blotting with control sera pool was performed. Different blocking solutions and different incubation times and temperatures for the sera and conjugate were studied. The best results were obtained under the following conditions: blocking of the strips for 15 min at room temperature with 5% skim milk in PBS-Tw20, incubation of the sera diluted in 2% skim milk in PBS-Tw20 at 4^oC overnight, incubation of the conjugate diluted in 2% skim milk in PBS-Tw20 for 1 h at room temperature and incubation with the 4-chloro-a-naphthol chromogen solution for 15 min. A significant loss of immunoreactivity of the antibodies was observed when the strips were blocked for 2 h at room temperature. Three serum dilutions were tested (1/10, 1/50 and 1/100); since no difference in reactivity was observed we chose the 1/50 dilution for the subsequent study (Fig. 4).

Fig. 4.
Western blotting of pooled sera positive (a-i) or negative (j-l) for anti-P. falciparum antibodies using the P. falciparum antigen extract. Different conditions were studied: blocking with 5% (a,d,g,j), 2% (b,e,h,k), and 0.5% (c,f,i,l) skim milk in PBS-Tw20, and different incubation times with the blocking solution 2 h (a-c), 1 h (d-f) and 15 min (g-l)). MW = Molecular weight x 10^-3.

Different incubation times and temperatures of the sera were tested in order to reduce the reaction time, and incubation at 4^oC overnight provided the best results (data not shown).

The control preparation of non-parasitized human O+ red blood cells showed various protein bands by SDS-PAGE, ranging from 15 to 160 kDa, with some of them having a molecular mass similar to that of the protein bands of the parasite antigen preparation. The same control preparation was transferred onto nitrocellulose membranes and Western blot analysis was performed with 12 anti-P. falciparum positive and 6 negative control sera. Nine of the positive sera presented reactivity against some of the following protein bands: 62 kDa (n = 7), 58 kDa (n = 5), 40 kDa (n = 4), 65 kDa (n = 3), 68 kDa (n = 2), 70 kDa (n = 2), 75 kDa (n = 1), and 43 kDa (n = 1). None of the negative control sera showed reactivity against the bands of the control preparation.

P. falciparum antigen extracts stored for different periods of time and at different temperatures were also analyzed by ELISA. When the tests were performed on days 0 and 15 after antigen extraction, no statistically significant difference in the absorbance values obtained for batches I, II and III were observed, independently of the presence or absence of treatment with protease inhibitors and/or dialysis (P> 0.05). On days 30 and 60 significant differences in absorbance were observed, independently of the addition of protease inhibitors or the storage temperature of the antigen extract, with batch B showing a significant reduction in absorbance from day 30 on (P < 0.05). The storage temperature of the extract did not alter these results (P > 0.05) (Fig. 5).

Fig. 5. Mean ELISA absorbance of six positive (+) and six negative (-) control sera for the erythrocytic forms of P. falciparum. The parasite pellets were stored under different conditions before extraction: up to 10 days at -20^oC (batch I), 4 months at -20^oC (batch II) and 2 months at -70^oC (batch III). Extraction was performed with Zwittergent or with Zwittergent added of protease inhibitors (treatment named i ), with Zwittergent and subsequent dialysis (treatment named d), and with Zwittergent added of protease inhibitors and subsequent dialysis (treatment named di ). The extract was stored at three different temperatures, -20^oC (batches I-20, II-20, III-20), -70^oC (batches I-70, II-70, III-70), and in liquid N₂ (batches I-N, II-N, III-N), for 0, 15, 30 and 60 days.

The influence of addition of protease inhibitors during extraction and subsequent dialysis of the extract for detergent removal was determined for the three batches (I, II and III) and the results obtained for batch I were analyzed by ANOVA. No significant differences in storage temperatures were observed between days 0, 15, 30 and 60 (P > 0.05), except for a higher decrease in absorbance in the tests performed with the dialyzed extracts (p<0,05).

The decrease in absorbance along time (days 0, 15, 30 and 60 after extraction) was not significant up to 30 days for all treatments within batch I (P > 0.05). The extract prepared without dialysis or inhibitors was stable at the studied temperatures, up to 60 days. On day 60, significant differences in the storage at -20^oC were observed between the d, di and i treatments (P < 0.05). Storage at -70^oC and in N₂ up to 60 days also led to excellent reproducibility of the results obtained for i and d.

To determine whether the maintenance of parasites in in vitro culture for a long period of time would interfere with their immunoreactivity and consequently with their use as antigen in serologic tests, we compared the ADA isolate of P. falciparum (Rondônia, 1982) with other isolates (JST, SA and TAN) obtained from blood samples of patients living in Peixoto de Azevedo in May, 1995. Fig. 6 shows the reactivity of a pool of positive sera with different parasite isolates in Western blotting. We observed that the ADA isolate, although maintained in in vitro culture for more than 10 years, preserved the protein band pattern present in more recent isolates. This result was confirmed by ELISA. Fig. 7 shows the mean reactivity index (RI) of positive or negative sera with antigens obtained from different P.falciparum isolates in ELISA. The different isolates were efficient in distinguishing reactive and non-reactive sera. No significant difference in the RI values of reactive sera was observed using different isolates (P = 0.0246, Kruskal-Wallis test). A significant difference (P < 0.0001) was observed among the non-reactive sera but no mean value was above the reactivity threshold (RI = 1.0).

Fig. 6.
Western blotting of a pool of positive sera with P. falciparum antigen extracts obtained from different isolates: ADA (Rondônia, 1982), and TAN, SA and JST (Peixoto de Azevedo, Mato Grosso, 1995). MW = Molecular weight x 10^-3.

DISCUSSION

In the present study we standardized the isolation of a somatic P. falciparum antigen and determined the best conditions for reproducibility of results in ELISA and Western blotting tests.

A wide variety of antigens of the erythrocytic phase of P. falciparum have been used in immunoenzymatic tests for malaria serology. The sensitivity and specificity of serologic tests such as ELISA and Western blotting depend on the adequate standardization of the solid phase, including isolation, purification and storage of the antigen and the type of blocking. If the test is not carefully standardized, these factors can markedly affect the reproducibility of results.

High parasitemias of mature forms, necessary for the isolation of sufficient amounts of parasites and good protein yield after extraction of somatic antigens, are difficult to get within a short period of time. This corresponds to a limiting factor for the use of large-scale serologic tests. We tested two temperatures to store the parasite pellet, -20ºC and -70ºC, commonly used in almost all laboratories. In this paper, we only presented the results of the storage up to 10 days and 4 months at -20ºC and 2 months at -70ºC. The antigen activity was been directly related with the conditions of storage of the whole parasite. Best results were obtained using parasites stored up to 10 days at -20^oC or for 2 months at -70^oC. The storage for 4 months at -20ºC highly reduced the quality of the extracted antigens. FRANCO¹³ recommended a storage time of up to 4 days at -20^oC. Our observation that the storage time can be extended presents advantages, especially when the production of a large antigen batch is necessary. Conversely, as far as storage temperatures of the antigen extract are concerned, storage at -70^oC or in liquid N₂ would be ideal, mainly when the time of storage is longer than 30 days.

We also studied the possibility that the detergent used for antigen extraction could interfere with the results of serologic tests. For this purpose, the final product was dialyzed using a membrane with a pore of 10,000 molecular weight cut-off, process that removes detergents with the characteristics of Zwittergent¹⁶. Since dialysis did not interfere with the results under some conditions, it was suppressed from the procedure and time for antigen preparation was reduced.

The addition of protease inhibitors during the process of antigen extraction is commonly used to reduce the degradation of proteins of interest^10,19. In general, a mixture of inhibitors is used affecting different classes of proteases in an empirical way. We tested the addition of a mixture of two cysteine proteinase (leupeptin and antipain) and two serine proteinase (PMSF and aprotinin) inhibitors and extracts obtained with and without inhibitors showed similar ELISA and SDS-PAGE results. If confirmed, these results indicate that the addition of inhibitors is unnecessary in antigen preparation to be only used in ELISA, therefore reducing its cost.

We also standardized the best conditions for Western blotting using the antigen extract that showed the best SDS-PAGE pattern. The incubation time with the blocking solution was the variable that most interfered with the results. DenHollander & Befus⁹ reported a loss of proteins adsorbed to the nitrocellulose membranes during blocking with skim milk depending on both the milk concentration and the time the membrane was in contact with the blocking solution. Our results are in agreement with these observations since we obtained good results with only 15 min of blocking. After 1 h of blocking, the loss of some bands was observed. The time of serum incubation also altered the results, with overnight incubation at 4^oC providing the best results.

Many proteins of the P. falciparum antigen extract recognized by positive control sera had a molecular weight identical or similar to that of previously described parasite antigens. These proteins include the 80-kDa protein present on the merozoite surface which is the target for antibodies inhibiting the invasion by merozoites²⁷; the 140/130/110-, 82-, 65/40- and 42-kDa proteins described as rhoptry antigens which are important in the invasion process of erythrocytes and are able to induce an immune response^6,11,22; the 126-kDa parasitophorus vacuole protein⁷; the hsp70 heat shock protein⁵; the 56-kDa glycoprotein present in young and mature trophozoites and schizonts and expressed on the merozoite surface at the end of schizogony²⁶ and the 55-kDa protein of the endocytic vacuole of the parasite with a cathepsin D-like protease activity³.

The SDS-PAGE protein pattern of the control preparation of non-parasitized erythrocytes revealed numerous bands corresponding to proteins present in the antigen extract of P. falciparum, which may be proteins comigrating in the electrophoretic field. A few bands were recognized by positive control sera in Western blotting, probably due to reactivity against normal blood proteins modified during infection. However, negative control sera recognized no protein band of the parasite antigen extract or the control preparation.

The present paper describes conditions for the production of a stable P. falciparum blood stage antigen to be used in the solid phase of ELISA and Western blotting tests, for the detection of antibodies in reliable and reproducible circumstances. Based on these results, a wide seroepidemiological study using serum samples of different populations from endemic and non-endemic areas in Brazil and the standard antigen is being conducted in order to evaluate and establish serologic profiles for human malaria in Brazil.

RESUMO

Padronização de procedimentos para a preparação de antígeno de Plasmodium falciparum a serem utilizados em testes sorológicos.

O objetivo deste estudo foi padronizar variáveis técnicas para o armazenamento de Plasmodium falciparum e de seus componentes antigênicos. Sedimentos de parasitas foram obtidos do cultivo in vitro de P.falciparum e estocados em diferentes temperaturas por diferentes períodos de tempo. De cada variável, foram extraídos os componentes antigênicos com detergente anfótero Zwittergent na presença e na ausência de inibidores de proteases e submetidos ou não a posterior diálise. Os produtos foram estocados por 15, 30 e 60 dias em diferentes temperaturas e caracterizados por SDS-PAGE. A atividade antigênica de cada extrato foi determinada por ELISA e Western blotting usando soros positivos e negativos para anticorpos IgG anti-formas eritrocitárias de P.falciparum. Os extratos antigênicos obtidos de parasitas estocados até 10 dias a -20ºC ou por 2 meses a -70ºC e tratados com inibidores de proteases, sob as diferentes condições de armazenamento, apresentaram melhor definição das bandas protéicas no SDS-PAGE e Western blot e melhores resultados no ELISA, permitindo diferenciação segura dos soros positivos e negativos.

ACKNOWLEDGEMENTS

The Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), process nº 95/5287-6 and Laboratório de Investigação Médica (LIM/48), School of Medicine, University of São Paulo supported this work. We thank Mr. Nei Rodrigues Carvalho Filho and Mr. Felix Bezerra Junior for the laboratory support.

Correspondence to: Dr. Sandra L. M. Avila, Instituto de Medicina Tropical, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar 470, 05403-140, São Paulo, SP, Brasil. Phone: [phone]; Fax: [phone], E-mail: [e-mail].

Received: 20 March 1998

Accepted: 20 October 1998

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Publication Dates

• Publication in this collection
  02 Mar 1999
• Date of issue
  Sept 1998

History

• Accepted
  20 Oct 1998
• Received
  20 Mar 1998