Jpn. J. Infect. Dis., 68, 474–480, 2015
Original Article
Novel IMB-ELISA Assay for Rapid Diagnosis of Human Toxoplasmosis Using SAG1 Antigen Shereen Hegazy2, Alyaa Farid1*, Ibrahim Rabae2, and Azza El-Amir1 1Zoology
Department, Science Faculty, Cairo University, Giza; and 2Parasitology Department, TBRI, Giza, Egypt
SUMMARY: Nanotechnology is important for medical diagnosis. Various nanoparticles have presented tremendous potential for diagnosing disease markers, pre-cancerous cells, fragment of viruses, specific proteins, antibodies, and other disease indicators. In general, nanoparticles are smaller than 1,000 nm and produced from different materials in different shapes such as spheres, rods, wires, and tubes. Our study aimed to develop a novel antigen-capture immunoassay based on IgG polyclonal antibody-coated magnetic microbead nanoparticles for the rapid detection of circulating surface antigen 1 of Toxoplasma gondii in human serum samples. Sandwich ELISA elicited a sensitivity of 92z, a specificity of 92.7z, a positive predictive value (PPV) of 92z, and a negative predictive value (NPV) of 92.7z. Immunomagnetic bead-ELISA showed sensitivity (98z), specificity (96.4z), PPV (96z), and NPV (98.1z) higher than that of sandwich ELISA. It is obvious that the use of magnetic microbead nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis.
antigens of the parasite, which are recognized as major study targets. There are 5 proteins in the superfamily of the surface antigens (SAG) of T. gondii, SAG1, SAG2 (22 kDa), p23, p35, and SAG3 (43 kDa). SAG1 is a 30 kDa glycoprotein designated as SAG1/p30 (6); it is stage specific, detected only in the tachyzoite stage, and absent in the bradyzoite stage (6,7). This antigen is abundant on the surface of both extracellular and intracellular tachyzoites (6). It has also been reported that SAG1 can elicit a lethal inflammatory process in a mouse model of pathogen-driven ileitis (8); however, the biological role of this surface protein remains unclear. SAG1 is considered as an important candidate antigen in diagnostic tests, with no known cross-reactivity with proteins from other microorganisms (6). This work aimed to evaluate SAG1 antigen as an early diagnostic marker of toxoplasmosis. A novel nanodiagnostic assay, immunomagnetic bead (IMB)-ELISA, based on IgG polyclonal antibody (pAb) was developed to detect circulating SAG1 antigens in human sera. Exploring an effective early diagnosis might be a key step towards prevention and eradication of this disease.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan parasite with an extremely wide geographic and host range (1). The course, symptoms, and consequences of T. gondii infection depend on the virulence and inoculum size of the parasite as well as on the genetic background and immune status of the infected host (2). It is estimated that one third of the world's population has been infected (3). High prevalence of the infection has been reported among pregnant women and women of childbearing age in Latin America, Eastern/ Central Europe, Middle East, south-east Asia, and Africa (4). Diagnosis of T. gondii infection relies mainly on serological tests enabling the detection of specific IgG, IgM, and IgA anti-Toxoplasma antibodies. Detection of IgM and IgA suggests an active T. gondii infection, whereas IgG alone leads to the diagnosis of a chronic infection. However, conflicting results may arise since anti-T. gondii IgM antibodies can be detected for more than a year after the initial infection (5). A major emphasis of medical technology is to improve diagnostic techniques to screen for infectious diseases. Such screening is required to identify illnesses, assess risk factors for disease onset, and determine progression or improvement of disease state. Especially, nanotechnology may improve the sensitivity, selectivity, speed, cost, and convenience of diagnostic tests. Furthermore, nanotechnology has also opened up the possibility of other screening strategies. Attachment of T. gondii to host cells involves surface
MATERIALS AND METHODS Participants: This study enrolled outpatients of the Theodor Bilharz Research Institute (TBRI) and ELkasrELainy Hospital. All patients and healthy volunteers were subjected to a panel of tests (according to the Toxoplasma Serological Profile [TSP]), consisting of the latex agglutination test, an ELISA based on crude T. gondii tachyzoite lysate (9), the Sabin-Feldman dye test (10), a double sandwich IgM ELISA, and the Differential agglutination test (Cofacetone [AC]-fixed versus that of formalin [HS]-fixed tachyzoites) (AC/HS test). These tests have been used successfully to determine if infections were acquired in the recent or more distant past. Ethical issues were strictly handled according to the International Ethical Guideline for Biomedical
Received October 14, 2014. Accepted January 5, 2015. J-STAGE Advance Publication April 10, 2015. DOI: 10.7883/yoken.JJID.2014.444 *Corresponding Author: Mailing address: Zoology Department, Science Faculty, Cairo University, Giza, Egypt. Tel: ＋201001089969, E-mail: alyaafarid＠yahoo.com 474
Novel IMB-ELISA Assay
finally by gel-filtration. The anti-T. gondii IgG pAbs produced had a very high degree of purity, except for few serum protein contaminants. Partially purified pAb was further adsorbed with fetal calf serum (FCS) to eliminate any non-specific binding with bovine antigens. The molecular weight was determined by SDS-PAGE, as previously described (20); the protein content was measured according to Bradford (21). The reactivity of anti-T. gondii IgG pAb against SAG1 antigen was measured by indirect ELISA with some modifications from the original method of Engvall and Perlman (15). Labeling of anti-SAG1 IgG pAb with horseradish peroxidase with the periodate method was performed as previously described (22). Sandwich ELISA: Polystyrene microtiter plates were coated with 100 ml/well of purified IgG pAb diluled in 8 ml carbonate buffer, pH 9.6. The plates were washed 3 times with a washing buffer (0.1 M PBS/Tween, PH 7.4/Tween). The free sites were blocked with 200 ml/well of 0.1z BSA/0.1 M PBS/T, pH 7.4 and incubated for 1 h at 379 C.The plates were washed 5 times with 0.1 M PBS/T. Sera (100 ml/well) were added and incubated for 2 h at 379 C, and the plates were washed 3 times. Peroxidase-conjugate IgG pAbs (100 ml/well) diluted in PBS were dispensed, and plates were incubated for 2 h at 379 C. The reaction was visualized by the addition of 100 ml/well of O-phenylene diamine substrate solution for 30 min in the dark at room temperature. The reaction was stopped by adding 50 ml/well of 8 N H2SO4, and the plates were read at 492 nm with an ELISA microplate reader (Bio Rad). IMB-ELISA: Microtitration plates were coated with 100 ml/well of anti-T. gondii pAbs coupled with magnetic microbead nanoparticles (200 mg/ml for IgG in 8 ml carbonate buffer, pH 9.6) and incubated overnight at room temperature. Plates were washed 3 times with 0.1 M PBS/T, pH 7.4. The remaining sites were blocked by 100 ml/well of 2.5z FCS/PBS/T and incubated for 2 h at 379 C. The plates were washed 3 times with PBS/T. Serum samples (100 ml) were pipetted into the wells in duplicate and incubated for 2 h at 379 C. The wells were washed 3 times, and 100 ml/well of peroxidase-conjugated pAbs were added and incubated for 1 h at room temperature. The reaction was visualized as described above. Statistical analysis: Data are expressed as mean ± standard deviation (SD) or percentages (z). The SPSS software (version 13 Windows) was used for data analysis.
Research. Prior to blood collection, the purpose of the study was explained individually to all participants and informed consents obtained. According to TSP, participants were divided into 3 main groups: (i) Fifty T. gondii-infected patients selected based on the presence of antibodies in their serum. (ii) Thirty-five patients harboring other parasites (E. histolytica, n ＝ 15; Giardia, n ＝ 5; and Cryptosporidium, n ＝ 15). (iii) Twenty parasite-free healthy individuals from the TBRI medical staff served as negative control. Serum samples from all groups were tested to detect circulating toxoplasmal antigens. Sera were separated and stored at －709 C until required. Animals: New Zealand white rabbits, weighting approximately 1.5 kg and 6 weeks old, were obtained and housed in TBRI. They were kept for 4 weeks (experiment duration) under standard laboratory care, i.e., 219 C, 16z moisture, filtered drinking water, and 15z protein, 3z fat, and 22z fiber diet. Blood samples were collected from healthy rabbits before inoculation and examined by ELISA for T. gondii antibodies and cross reactivity with other parasites, as previously described (16). Animal experiments were carried out according to the international guidelines and TBRI animal ethics committee. Preparation of tachyzoite proteins (SAG1 antigen) of T. gondii: T. gondii antigens were prepared from peritoneal exudates of BALB/c mice infected 3 days earlier with tachyzoites of T. gondii RH strain, as previously described (11). Mice peritoneal exudates were centrifuged at 2,000 g for 20 min, washed 3 times with phosphate buffer saline (PBS), subjected to sonication periods, and centrifuged at 12,000 g for 1 h, and supernatants were collected as soluble antigens. Protein content was determined by the Bradford method, and soluble antigens were stored at －209 C until use, as previously described (11,12). Purification of SAG1 antigen was carried out in 2 steps: diethylaminoethyl (DEAE)-Sephadex G-50 and G-200 ion exchange chromatography, as previously described (13), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12z slab gels (1 mm thick) under reducing conditions (2-mercaptoethanol) and stained with Coomassie blue (Bio-Rad, Hercules, CA, USA), as previously described (14). Following purification, the reactivity and specificity of T. gondii SAG1 antigen were tested by indirect ELISA (15). Preparation of IgG pAbs against SAG1 antigen: Rabbits free from T. gondii and other parasitic infections were inoculated for the production of antibodies against T. gondii SAG1 antigens. An intramuscular priming dose was injected at 4 sites, comprising 1 mg SAG1 antigen mixed with an equal volume of complete Freund's adjuvant (CFA; Sigma, St. Louis, MO, USA). Three booster doses were given, each containing 0.5 mg antigen emulsified in an equal volume of incomplete Freund's adjuvant (IFA, Sigma); the first boosting was given 2 weeks after the priming dose, and the following boosting doses were given at weekly intervals, as previous described (17). One week after the last booster dose, rabbit sera were harvested and the pAb fraction was purified by the 50z ammonium sulfate precipitation method (18). Further purification of pAb was performed with the 7z caprilic acid method (19) and
RESULTS Purification of 30 kDa SAG1 of T. gondii: (i) DEAESephadex G-50- ion exchange chromatography: Fig. 1 shows the OD280 profile of the antigen fractions obtained following purification by DEAE Sephadex G-50 ion exchange chromatography. The eluted antigens were represented by 4 peaks at 12, 22, 25, and 34 with a maximum OD value of 0.068 for fraction 25. The eluted protein fractions resulted from this purification method were analyzed by 12.5z SDS-PAGE under reducing condition and showed 3 major bands at 80, 30, and 27 kDa and many minor bands ranging from 110 kDa down to 17 kDa. (ii) DEAE-Sephadex G-200- ion ex475
Fig. 1. Elute profile for chromatography of 30 kDa SAG1 on DEAE sephadex G-50 ion exchange chromatography. Fig. 3. (Color online) SDS-PAGE of target antigens eluted from ion exchange chromatography column. Lane M: Low molecular weight standard, Lane 1: Purified antigen after DEAE sephadex G-200, Lane 2: Purified antigen after DEAE sephadex G-50, and Lane 3: Crude T. gondii antigen.
Table 1. The reactivity of SAG1 antigen by indirect ELISA Parasitic antigen
OD reading Mean ± SD
T. gondii E. histolytica Giardia Cryptosporidium
1.841 ± 0.231 0.294 ± 0.221 0.126 ± 0.049 0.142 ± 0.082
Fig. 2. Elute profile for chromatography of 30 kDa SAG1 on DEAE sephadex G-200 ion exchange chromatography.
after the second booster dose, immune sera displayed high titers against 30 kDa SAG1 with maximal OD of 1.5 at 1/100 dilution. The total protein content in the crude rabbit serum containing anti-T. gondii pAbs was 12.5 mg/ml. After 50z ammonium sulfate precipitation, the protein content was 8 mg/ml, while it dropped to 4 mg/ml following 7z caprylic acid precipitation. The purity of IgG pAbs after each purification steps was assayed by 12.5z SDS-PAGE under reducing condition. The purified IgG pAbs were represented by H- and L-chain bands at 53 and 31 kDa, respectively. The pAbs appear free from other proteins (Fig. 4). The reactivity of anti-T. gondii IgG pAbs against SAG1 and other parasite antigens was determined by indirect ELISA. The produced anti-T. gondii pAbs diluted 1/100 in PBS/T buffer elicited a strong reactivity towards SAG1. The OD reading at 492 nm for T. gondii antigen was 2.79 compared to 0.532, 0.326, and 0.243 for E. histolytica, Giardia, and Cryptosporidium, respectively. Standardization of sandwich ELISA: The optimum dilution of anti-T. gondii IgG pAb for the coating step was 1/80, while it was only 1/40 for peroxidase-conjugated anti-T. gondii IgG pAb. Detection of circulating T. gondii SAG antigen in human serum samples: (i) Sandwich ELISA: To measure the incidence of positivity for T. gondii, it was necessary at first to determine the cut off value for posi-
change chromatography: Fig. 2 shows the OD280 profile of the antigen fractions obtained following purification by DEAE Sephadex G-200 ion exchange chromatography. The eluted antigen was represented by a single peak with a maximum OD value of 0.15 for fraction 23. The eluted protein fraction was analyzed by 12.5z SDS-PAGE under reducing condition and showed a unique band at 30 kDa, which represents 30 kDa SAG1 (Fig. 3). Estimation of total protein content of T. gondii antigens: The protein content was 7 mg/ml of crude antigens and 3.5 and 1.5 mg/ml of purified antigens after DEAE-Sephadex G-50 and G-200 gel filtration chromatography, respectively. (i) Reactivity of SAG1 antigen by indirect ELISA: The antigenicity of the purified target antigen was tested by an indirect ELISA technique. Blood samples from human-infected with T. gondii elicited a strong reaction against 30 kDa SAG1 with a mean OD reading of 1.841, and no cross reactions were detected in samples of patients infected with other parasites (Table 1). Production of pAbs against SAG1: Test blood samples were withdrawn from New Zealand white rabbits before the injection of each immunizing dose. They were tested for the presence of specific anti-T. gondii antibodies by indirect ELISA, and antibody levels increased 1 week after the first booster dose. Three days 476
Novel IMB-ELISA Assay Table 3. Detection of 30 kDa SAG1 in human sera samples using IMB-ELISA technique Positive case
Negative case
Group (no. of animals) No. Healthy control (n ＝ 20) T. gondii (n ＝ 50) E. Histolytica (n ＝ 15) Giardia (n ＝ 5) Cryptosporidium (n ＝ 15) 1),2):
X ± SD
— 49 1.94 ± 0.32 11) 0.49 ± 0.21 0 — 11) 0.82 ± 0.14
No.
X ± SD
20 12) 14 5 14
0.12 ± 0.07 0.22 ± 0.032 0.24 ± 0.16 0.20 ± 0.09 0.14 ± 0.10
See Table 2, footnote.
Table 4. The sensitivity, specificity, PPV and NPV of sandwich and IMB-ELISA results used for detection of 30 kDa SAG1 in human sera samples
Fig. 4. (Color online) SDS-PAGE of IgG pAb (stained with coomassie blue), Lane M: Molecular weight standard protein, Lane 1: Crude anti-T. gondii IgG pAb, Lane 2: Purified IgG pAb after 50z ammonium sulfate treatment, and Lane 3: Purified IgG pAb after 7z caprylic acid treatment.
Negative case
Group (no. of animals) No. Healthy control (n ＝ 20) T. gondii (n ＝ 50) E. Histolytica (n ＝ 15) Giardia (n ＝ 5) Cryptosporidium (n ＝ 15) 1): 2):
X ± SD
46 1.61 ± 0.35 21) 0.58 ± 0.22 0 21) 0.63 ± 0.13
No.
X ± SD
20 42) 13 5 13
0.21 ± 0.07 0.28 ± 0.03 0.21 ± 0.15 0.15 ± 0.08 0.23 ± 0.10
Sensitivity (z )
Specificity (z )
PPV (z )
NPV (z)
ELISA IMB ELISA
92 98
92.7 96.4
92 96
92.7 98.1
bead method. The mean OD value of T. gondii-infected group (1.94 ± 0.32) was significantly higher than that of healthy control and other parasite groups (Table 3). One out of 50 T. gondii infected samples showed a false negative result, and the sensitivity of the assay was calculated to be 98z. All 20 negative controls were below the cut off value, while 2 out of 35 samples from individuals infected with other parasites (1 with E. histolytica and 1 with Cryptosporidium) were borderline, giving 96.4z specificity. PPV and NPV were 96z and 98.1z, respectively (Table 4).
Table 2. Detection of 30 kDa SAG1 in human sera samples using sandwich ELISA Positive case
Technique
DISCUSSION Infections by T. gondii are widely prevalent in humans and animals, especially in food animals throughout the world (23). Early diagnosis is important in patients who present clinical symptoms of toxoplasmosis and in pregnant women to prevent severe congenital infection in the developing fetus (24,25). Toxoplasmosis diagnosis depends on direct and indirect methods. The classical diagnosis of toxoplasmosis relies on serological methods for the detection of specific immunoglobulin antibodies. However, these methods have a poor efficiency, especially in neonates and immunodeficient patients (26). Owing to IgM long half-life in many patients and high percentages of non-specific results, toxoplasma-specific IgM assay is no longer of benefit in diagnosing acute toxoplasma infection (27–29). In some studies years ago, T. gondii antigens present in serum samples of experimentally infected animals suggests that antigens are detectable during the acute phase of toxoplasmosis (30,31). For instance, Raizman and Neva (32) showed the presence of circulating antigens in mouse sera by counter-current electrophoresis and agar gel diffusion on days 2–4 of infection. Huskinson et al. (33) could demonstrate the presence of T. gondii antigens in serum and urine of mice as early as 5 days post infection by western blot. Recently, Shojaee et al. (34) detected antigenemia from the fourth day after infection in mouse sera by immunoblotting. Moreover, to
False positive results. False negative results.
tivity or the line of demarcation between positive and negative samples. The cut off value for positivity was measured as the mean OD reading of the negative controls ＋ 2 SD of the mean. Tested samples showing OD values higher than the cut off value were considered positive for Toxoplasma. Table 2 shows the results of SAG1 antigen detection among the different groups. The cut off value for positivity was 0.428. The mean OD value of the T. gondii-infected group (1.61 ± 0.351) was significantly higher than that of both the healthy control (0.212 ± 0.067) and other parasite groups. Four out of 50 T. gondii-infected samples showed false negative results and the sensitivity of the assay was calculated to be 92z. All 20 negative controls were below the cut off value while 4 out of 35 samples from individuals infected with other parasites were border line, giving 92.7z specificity. The positive predictive value (PPV) and negative predictive value (NPV) were 92z and 92.7z, respectively. (ii) IMB-ELISA: The presence of T. gondii SAG1 antigen in human serum samples of the different groups was also evaluated by a nanomagnetic 477
body population; thus they offer a statistically relevant idea for the overall picture of an immune response. Our anti-SAG1 IgG pAb was represented by H- and L- chain bands at 53 and 31 kDa, respectively, and a 4 mg/ml protein concentration. These results are comparable with the yield of purified immunoglobulins from any biological fluid following similar purification procedures (58,59). The purified rabbit IgG fraction served as both antigen capture (1/80 mg/ml) and peroxidaseconjugated (1/40 mg/ml) detecting antibody in sandwich ELISA. The cut-off OD value for positivity in sandwich ELISA for SAG1 was 0.428 in serum samples. Many immunologic tests based on antigen detection (60,61) and molecular or proteomic diagnostic techniques (62,63) have been well studied and described. However, an affordable, easy-to handle, sensitive, and specific method is not yet available (64). An immunomagnetic bead-based immunoassay is a popular approach in diagnosis of many food-borne and infectious diseases. This innovative technique involves antibodycoated magnetic microbead nanoparticles, which trap antigens from liquid media. Furthermore, the small size and shape of the microbeads enable them to be evenly dispersed within the sample, thus improving the effectiveness of the antibody conjugation, and consequently enhancing the sensitivity of antigen detection (65,66). In this study, we developed a novel antigen-capture immunoassay based on IgG pAb-coated magnetic microbead nanoparticles for the detection of circulating SAG1 antigens of T. gondii in serum samples. The study was conducted on T. gondii-infected patients, patients infected with other parasites, and healthy controls. Sandwich ELISA revealed a sensitivity of 92z, specificity of 92.7z, PPV of 92z, and NPV of 92.7z. While IMB-ELISA sensitivity (98z), specificity (96.4z), PPV (96z), and NPV (98.1z) were higher than that of sandwich ELISA. Our results were comparable to those of Conlan et al. (67), who described the use of IMB as the solid phase for the portable detection of the CSF antigen in spleen samples and for the reliable detection of antibodies against CSF in animals vaccinated with a lapinized Cstrain. This IMB-ELISA is 100z sensitive and 91z specific in comparison to polyclonal antibody-based antigen-capture ELISA (AC-ELISA). Moreover, IMBantibody-ELISA is 97z sensitive and 95z specific. Lei et al. (68) developed a novel egg yolk antibody (IgY)coated magnetic bead antigen-capture immunoassay for the detection of a circulating antigen of S. japonica in serum samples of patients in endemic schistosomiasis areas of China. This IgY-IMB-ELISA comprises IgY pAb-coated magnetic beads as a capture antibody and a monoclonal IgG as a detection antibody. The sensitivity of this magnetic immunoassay is 100z in cases of acute infection and 91.5z in chronic cases of schistosomiasis, and no positive reaction was found in 49 healthy individuals. Cross-reactivity was 3.3z with clonorchiasis and 0z with paragonimiasis. There was a significant correlation between ELISA absorbance values and egg counts (eggs per gram feces) and a correlation coefficient of 0.88 in a small sample of 14 patients. The results demonstrated that IgY-IMB-ELISA is a sensitive and specific assay for detection of S. japonica. Ahmed et al. (69) also developed a novel IMB-ELISA based on IgG
increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful (35). Tachyzoites are thought to be responsible for acute phase of the infection and express immunodominant antigens that induce strong immune responses (36). Abdel-Rahman et al. (37) used T. gondii crude and affinity-purified tachyzoite antigens isolated from slaughtered sheep in the diagnosis of toxoplasmosis in horses. Conde de Felipe et al. (38) proved that 29–35 kDa T. gondii fractions can detect specific IgG in goats 2 weeks earlier than crude extract. The 30 kDa surface protein of T. gondii tachyzoite represents 3z to 5z of the parasite total proteins (39) (Kasper, 1987) and has been proposed for the diagnosis of acute acquired toxoplasmosis. Cesbron et al. (40) and Santaro et al. (41) have developed a double-sandwich ELISA based on this protein for detection of IgM antibodies. SAG1 is the main surface antigen of T. gondii that induces high titers of IgG, IgM, and IgA despite only accounting for 3–5z of total cellular proteins (42). In addition, this protein is also a tachyzoite-specific antigen and is highly conserved (43); thus, it is important for diagnostic purposes (44,45). Tachyzoites are the key disease-causing forms. During the acute stage of infection, tachyzoites activate a potent host immune response, which eliminates most of the parasites. The surface of the tachyzoite is the main target of the host immune response. SAG1 is a major surface antigen involved in this process. Although it only accounts for 3–5z of T. gondii total proteins, the majority of the antibodies are reactive against SAG1 during infection (46). Various SAG1 preparations purified from the parasite (47,48), produced by recombinant systems in Escherichia coli (49) or yeast (50), and its constituent peptides (51–53) stimulate the host humoral and cellular immunity, thereby providing protection against toxoplasmosis (54). As such, SAG1 features excellent antigenicity and immunogenicity and is valuable for both diagnosis and immunization. In 1980, Handman et al. (55) first identified SAG1 from T. gondii surface membrane antigens using a monoclonal antibody technique. Subsequently, Kasper et al. (56) applied mAb-affinity chromatography to isolate the protein. In 1988, Burg et al. (43) cloned the complete T. gondii sag1 gene, which has a length of 1.1 kb, encodes 336 amino acids, and yields a 30 kDa protein. In this study, we aimed to purify and characterize SAG1 antigen from T. gondii tachyzoites and evaluate its early diagnostic potential in human toxoplasmosis with sandwich ELISA and IMB-ELISA techniques, which may aid in the prevention of the infection and eradication of its transmission. When T. gondii antigens were analyzed by 12.5z SDS-PAGE under reducing condition, SAG1 antigen was detected as a 30 kDa protein with a concentration of 1.5 mg/ml. These results were similar to that of Villavedra et al. (42,57) who purified and isolated a fraction of 30–33 kDa from crude tachyzoite antigens, which was used successfully in the diagnosis of human toxoplasmosis by ELISA. The reactivity of SAG1 antigen was tested by indirect ELISA and elicited a strong reaction with a maximal mean OD reading of 1.841. This purified antigen was utilized for immunization of rabbits for the production of pAbs. Which contain the entire antigen-specific anti478
Novel IMB-ELISA Assay
for the detection of excretory/secretory (E/S) antigens in rabbit sera infected with S. haematobium. They detected E/S antigens in serum with a sensitivity of 95z a specificity of 93.7z compared to other parasitic infections and of 100z compared to healthy controls. On the other hand, detection of E/S antigens in urine elicited a sensitivity of 91z, and specificity of 93.7z compared to other parasitic infections and of 100z compared to healthy individuals. Furthermore, Aly et al. (70) evaluated a novel magnetic microbead based-latex agglutination assay (MMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardizing a novel magnetic microbead based-sandwich ELISA (MMB-S ELISA). They reported that the sensitivity of novel MMB-LAT was 82.4z versus MMB-S ELISA 96.5z and currently used sandwich ELISA 88.2z. The specificity of MMB-LAT was 83.6z versus MMB-S ELISA 96.3z and sandwich ELISA 87.3z. With our IMB-ELISA results showing a higher sensitivity and specificity than that of sandwich ELISA, it is obvious that the use of magnetic microbead nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis. The use of magnetic nanoparticles in immunoassay (nanomagnetic assay) combines magnetic nanoparticle high binding capacity and simple separation of bound and unbound materials as a solid phase with the rapid reaction kinetics of solutions, thus providing the chance of enhancing the sensitivity of antigen detection (71,72). The surface of the beads enables substantially more antibodies to be involved in the reaction with the antigen than in sandwich ELISA (73–75).
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
Conflict of interest None to declare.
26.
REFERENCES
27.
1. Current WL, Upton SJ, Long PL. Chapter 1. Taxonomy and life cycles. In: Long PL. editor. Coccidiosis of Man and Domestic Animals. Boca Raton, FL: CRC Press; 1990. p.1-16. 2. Dlugonska H. Toxoplasma rhoptries: unique secretory organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis. J Biomed Biotechnol. 2008;2008:632424. 3. Zhou P, Chen Z, Li HL, et al. Toxoplasma gondii infection in humans in China. Parasit Vectors. 2011;4:165-74. 4. Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol. 2009;39:1385-94. 5. Wong SY, Remington JS. Toxoplasmosis in pregnancy. Clin Infect Dis. 1994;18:853-61. 6. Handman E, Goding JW, Remington JS. Detection and characterization of membrane antigens of Toxoplama gondii. J Immunol. 1980;124:2578-83. 7. Kasper LH, Bradley MS, Pfefferkorn ER. Identification of stage specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies. J Immunol. 1984;132:443-9. 8. Rachinel N, Buzoni-Gatel D, Dutta C, et al. The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii. J Immunol. 2004;173:2725-35. 9. Choi WY, Nam HW, Youn JH, et al. Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme linked immunosorbent assay. Kor J Parasitol. 1992;30:83-90. 10. Remington JS, Efron B, Cavanaugh E, et al. Studies on toxoplasmosis in El Savador prevalence and incidence of toxoplasmosis as measured by the Sabin-Feldman dye test. Trans R Soc Trop Med Hyg. 1970;64:252-67. 11. Brooks RG, Sharma SD, Remington JS. Detection of Toxoplas-
28.
29.
30. 31. 32. 33. 34. 35.
36. 37.
479
ma gondii antigen by a dot-immunobiding technique. J Clin Microbiol. 1985;21:113-6. Foroghi parvar F, Keshavarz H, Shojaee S. Detection of Toxoplasma gondii antigens in sera and urine of experimentally infected mice by capture–ELISA. Iranian J Parasitol. 2008;3:1-5. Timanova A, M äuller S, Marti T, et al. Ascaridia galli fatty acidbinding protein, a member of the nematode polyprotein allergens family. Eur J Biochem. 1999;261:569-76. Takacs B. Electrophoresis of proteins in polyacrylamide slab gels. In: Lefkovitz I, Pernis B.editors. Immunological Methods. New York: Academic Press; 1997. p.81-105. Engvall E, Perlmanm P. Enzyme-linked immunosorbent assay. Quantitative assay of IgG. Immunochem. 1971;8:871-4. Fagbemi BO, Guobadia EE. Immunodiagnosis of fascioliasis in ruminants using a 28-kDa cysteine protease of F. gigantica adult worm. Vet Parasitol. 1995;57:309-18. Fagbemi BO. Development and characterization of a monoclonal antibody reactive with 128 kDa protease of F. gigantica. Vet Parasitol. 1995;57:351-6. Nowotny A. A basic exercises in immunochemistry. In: Nowotny A. editor. A Basic Exercises in Immunochemistry. 2nd ed. BerlinHeidelberg-New York: Springer-Verlag; 1979. p.7-20. Mckinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from ascites fluid. J Immunol Methods. 1987;96:271-8. Harlow E, Lane D. editors. Antibodies: A Laboratory Manual. 1st ed. New York: Cold Spring Harbor Laboratory Press; 1988. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. Tijssen P, Kurstak P. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugate for enzyme immunoassays. Anal Biochem. 1984;136: 451-7. Jones JL, Dubey JP. Foodborne toxoplasmosis. Clin Infect Dis. 2012;55:845-51. Desmonts G, Daffos F, Forestier F, et al. Prenatal diagnosis of congenital toxoplasmosis. Lancet 1985;1:500-4. Doehring E, Reiter-Owona I, Bauer O, et al. Toxoplasma gondii antibodies pregnant women and their newborns in Dar-esSalaam. Am J Trop Med Hyg. 1995;52:546-8. Hafid J, Flori P, Raberin H, et al. Comparison of PCR, captureELISA and immunoblotting for detection of Toxoplasma gondii in infected mice. J Med Microbiol. 2001;50:1100-4. Beghetto E, Buffolano W, Spandoni A, et al. Use of an immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary Toxoplasma gondii infection during pregnancy. J Clin Microbiol. 2003;41:5414-8. Gras L, Gibert RE, Wallon M, et al. Duration of IgM response in women acquiring Toxoplasma gondii during pregnancy: Implication for clinical practices and cross sectional incidence studies. Epidemiol Infect. 2004;132:541-8. Kaul R, Chen P, Binder SR. Detection of Immunoglobulin M antibodies specific for Toxoplasma gondii with increased selectivity for recently acquired infections. J Clin Microbiol. 2004;42: 5705-9. Lindenschmidt EG. Enzyme-linked immunosorbent assay for detection of Toxoplasma gondii antigen in acute-phase toxoplasmosis. Eur J Clin Microbiol. 1985;4:488-92. Luft BJ, Remington JS. Toxoplasma encephalitis. J Infect Dis. 1998;157:1-6. Raizman RE, Neva FA. Detection of circulating antigen in acute experimental infections with T. gondii. J Infect Dis. 1975;132: 44-8. Huskinson J, Stepick-Biek P, Remington JS. Detection of antigens in urine during toxoplasmosis. J Clin Microbiol. 1989;27: 1099-101. Shojaee S, Keshavarz H, Rezaeian M, et al. Detection of Toxoplasma gondii antigens in sera from experimentally infected mice. Pak J Med Sci. 2007;23:100-2. Luo Q, Upadhya R, Zhang H, et al. Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry. Microbes Infect. 2011;13:1199-210. Gross U, Holpert M, Goebel S. Impact of stage differentiation on diagnosis of toxopla smosis. Ann Ist Super Sanita. 2004;40:65-70. Abdel-Rahman EH, Ghazy AA, Shaapan RM. Prevalence of antibodies to Toxoplasma gondii in farm horses in Egypt. J Egypt
Vet Med Assoc. 2005;65:99-112. 38. Conde de Felipe MM, Molina JM, Rodriguez-Ponce E, et al. IgM and IgG response to 29-35-kDa Toxoplasma gondii protein fractions in experimentally infected goats. J Parasitol. 2007;93:701-3. 39. Kasper LH. Isolation and characterization of a monoclonal antiP30 antibody resistant mutant of Toxoplasma gondii. Parasite Immunol. 1987;9:433-5. 40. Cesbron JY, Capron A, Ovlaqe G, et al. Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30). J Immunol Methods. 1985;83:151-8. 41. Santaro F, Afchain D, Pierce R, et al. Serodiagnosis of Toxoplasma infection using a purified parasite protein (P30). Clin Exp Immunol. 1985;62:262-9. 42. Khan IA, Ely KH, Kasper LH. A purified parasite antigen (P30) mediates CD8 ＋ T cell immunity against fatal Toxoplasma gondii infection in mice. J Immunol. 1991;147:3501-6. 43. Burg JL, Perelman D, Kasper LH, et al. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. Immunol. 1988;141:3584-91. 44. Siachoque H, Guzman F, Burgos J, et al. Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model. Exp Parasitol. 2006;114:62-5. 45. Harning D, Spenter J, Metsis A, et al. Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies. Clin Diagn Lab Immunol. 1996;3: 355-7. 46. Velge-Roussel F, Chard àes T, M áev áelec P, et al. Epitopic analysis of the Toxoplasma gondii major surface antigen SAG1. Mol Biochem Parasitol. 1994;66:31-8. 47. Debard N, Buzoni-Gatel D, Bout D. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect Immun. 1996;64:2158-66. 48. B äulow R, Boothroyd JC. Protection of mice from fatal Toxoplasma gondii infection by immunization with P30 antigen in liposomes. J Immunol. 1991;147:3496-500. 49. Petersen E, Nielsen HV, Christiansen L, et al. Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii. Vaccine. 1998;16:1283-9. 50. Biemans R, Gr áegoire D, Haumont M, et al. The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation. J Biotechnol. 1998;66:137-46. 51. Darcy F, Maes P, Gras-Masse H, et al. Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen. J Immunol. 1992;149:3636-41. 52. Godard I, Estaquier J, Zenner L, et al. Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis. Mol Immunol. 1994;31:1353-63. 53. Velge-Roussel F, Dimier-Poisson I, Buzoni-Gatel D, et al. AntiSAG1 peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines. Parasitology. 2001; 123:225-33. 54. Wang Y, Yin H. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii. Parasit Vectors. 2014;7:180. 55. Handman E, Goding JW, Remington JS. Detection and characterization of membrane antigen of Toxoplasma gondii. J Immunol. 1980;124:2578-83. 56. Kasper LH, Crabb JH, Pfefferkorn ER. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol. 1983;130:2407-12. 57. Villavedra M, Battistoni JJ, Rossi S, et al. Avidity analysis of the human immune response to a chitin binding protein of Toxoplasma gondii. Int J Parasitol. 2001;31:1087-92. 58. Bird P, Jones P, Allen D, et al. Analysis of the expression and secretion of isotypes of sheep B cell immunoglobulins with a panel of isotype-specific monoclonal antibodies. Res Vet Sci. 1995;59:
189-94. 59. Yang YB, Harrison K. Influence of column type and chromatographic conditions on the ion exchange chromatography of immunoglobulins. J Chromatogr A. 1996;743:171-80. 60. Robijn ML, Koeleman CA, Wuhrer M, et al. Targeted identification of a unique glycan epitope of Schistosoma mansoni egg antigens using a diagnostic antibody. Mol Biochem Parasitol. 2007; 151:148-61. 61. Sulbar áan GS, Ballen DE, Berm áudez H, et al. Detection of the Sm31 antigen in sera of Schistosoma mansoni-infected patients from a low endemic area. Parasite Immunol. 2010;32:20-8. 62. Lier T, Simonsen GS, Wang T, et al. Real-time polymerase chain reaction for detection of low-intensity Schistosoma japonicum infections in China. Am J Trop Med Hyg. 2009;81:428-32. 63. Robijn ML, Planken J, Kornelis D, et al. Mass spectrometric detection of urinary oligosaccharides as markers of Schistosoma mansoni infection. Trans R Soc Trop Med Hyg. 2008;102:79-83. 64. Engels D, Chitsulo L, Montresor A, et al. The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop. 2002;82:139-46. 65. Chen L, Deng L, Liu L, et al. Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureusand protein A. Biosens Bioelectron. 2007;22:1487-92. 66. Nagasaki Y, Kobayashi H, Katsuyama Y, et al. Enhanced immunoresponse of antibody/mixed-PEG coimmobilized surface construction of high-performance immunomagnetic ELISA system. J Colloid Interface Sci. 2007;309:524-30. 67. Conlan J, Khounsy S, Phruaravanh M, et al. Application of immunomagnetic bead technology for improved diagnosis of classical swine fever virus. In: Conlan J, Blacksell S, Morrissy C, et al. editors. Management of Classical Swine Fever and Foot-andMouth Disease in Lao PDR: proceedings of an international workshop held in Vientiane, Lao PDR, 20–21 November 2006. ACIAR Proceedings No. 128. Canberra, Australia: ACIAR, 2008;8:p.74-9. 68. Lei JH, Su BT, Xu H, et al. Evaluation of an IgY-based immunomagnetic enzyme-linked immunosorbent assay system for detection of circulating Schistosoma japonicum antigen in serum samples from patients in china. Am J Trop Med Hyg. 2011;85: 1054-9. 69. Ahmed A, El Amir A, Rabee I, et al. Effective Diagnosis of Schistosomiasis haematobium by Immunomagnetic Bead ELISA technique using Super-Paramagnetic Nanoparticles. J Am Sci. 2012;8:833-41. 70. Aly I, Zalat R, El Aswad BE, et al. Novel nanomagnetic beads based–latex agglutination assay for rapid diagnosis of human Schistosomiasis haematobium. Int J Med Pharmaceut Sci Engin. 2013;7:641-7. 71. Gundersen SG, Haagensen I, Jonassen TO, et al. Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen. J Immunol Methods. 1992;148:1-8. 72. Ndhlovu P, Cadman H, Gundersen SG, et al. Optimization of the magnetic bead antigen capture enzyme immunoassay for the detection of circulating anodic antigens in mixed Schistosoma infections. Acta Trop. 1995;59:223-35. 73. Cudjoe KS, Thorsen LI, Sorensen T, et al. Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA. Int J Food Microbiol. 1991;12:313-21. 74. Nustad K, Paus E, Bomer OP. Magnetic particles as solid phase in immunoassays. In: Kemshead JT. editor. Magnetic Separation Techniques Applied to Cellular and Molecular Biology. Somerset, England: Wordsmiths' Conference Publications; 1991. p.39-46. 75. Patel PD, Blackburn C. Detection of food poisoning agents using immunomagnetic particles. In: Kemshead JT. editor. Magnetic Separation Techniques Applied to Cellular and Molecular Biology. Somerset, England :Wordsmiths' Conference Publications; 1991. p.93-105.
480
Original Article
Novel IMB-ELISA Assay for Rapid Diagnosis of Human Toxoplasmosis Using SAG1 Antigen Shereen Hegazy2, Alyaa Farid1*, Ibrahim Rabae2, and Azza El-Amir1 1Zoology
Department, Science Faculty, Cairo University, Giza; and 2Parasitology Department, TBRI, Giza, Egypt
SUMMARY: Nanotechnology is important for medical diagnosis. Various nanoparticles have presented tremendous potential for diagnosing disease markers, pre-cancerous cells, fragment of viruses, specific proteins, antibodies, and other disease indicators. In general, nanoparticles are smaller than 1,000 nm and produced from different materials in different shapes such as spheres, rods, wires, and tubes. Our study aimed to develop a novel antigen-capture immunoassay based on IgG polyclonal antibody-coated magnetic microbead nanoparticles for the rapid detection of circulating surface antigen 1 of Toxoplasma gondii in human serum samples. Sandwich ELISA elicited a sensitivity of 92z, a specificity of 92.7z, a positive predictive value (PPV) of 92z, and a negative predictive value (NPV) of 92.7z. Immunomagnetic bead-ELISA showed sensitivity (98z), specificity (96.4z), PPV (96z), and NPV (98.1z) higher than that of sandwich ELISA. It is obvious that the use of magnetic microbead nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis.
antigens of the parasite, which are recognized as major study targets. There are 5 proteins in the superfamily of the surface antigens (SAG) of T. gondii, SAG1, SAG2 (22 kDa), p23, p35, and SAG3 (43 kDa). SAG1 is a 30 kDa glycoprotein designated as SAG1/p30 (6); it is stage specific, detected only in the tachyzoite stage, and absent in the bradyzoite stage (6,7). This antigen is abundant on the surface of both extracellular and intracellular tachyzoites (6). It has also been reported that SAG1 can elicit a lethal inflammatory process in a mouse model of pathogen-driven ileitis (8); however, the biological role of this surface protein remains unclear. SAG1 is considered as an important candidate antigen in diagnostic tests, with no known cross-reactivity with proteins from other microorganisms (6). This work aimed to evaluate SAG1 antigen as an early diagnostic marker of toxoplasmosis. A novel nanodiagnostic assay, immunomagnetic bead (IMB)-ELISA, based on IgG polyclonal antibody (pAb) was developed to detect circulating SAG1 antigens in human sera. Exploring an effective early diagnosis might be a key step towards prevention and eradication of this disease.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan parasite with an extremely wide geographic and host range (1). The course, symptoms, and consequences of T. gondii infection depend on the virulence and inoculum size of the parasite as well as on the genetic background and immune status of the infected host (2). It is estimated that one third of the world's population has been infected (3). High prevalence of the infection has been reported among pregnant women and women of childbearing age in Latin America, Eastern/ Central Europe, Middle East, south-east Asia, and Africa (4). Diagnosis of T. gondii infection relies mainly on serological tests enabling the detection of specific IgG, IgM, and IgA anti-Toxoplasma antibodies. Detection of IgM and IgA suggests an active T. gondii infection, whereas IgG alone leads to the diagnosis of a chronic infection. However, conflicting results may arise since anti-T. gondii IgM antibodies can be detected for more than a year after the initial infection (5). A major emphasis of medical technology is to improve diagnostic techniques to screen for infectious diseases. Such screening is required to identify illnesses, assess risk factors for disease onset, and determine progression or improvement of disease state. Especially, nanotechnology may improve the sensitivity, selectivity, speed, cost, and convenience of diagnostic tests. Furthermore, nanotechnology has also opened up the possibility of other screening strategies. Attachment of T. gondii to host cells involves surface
MATERIALS AND METHODS Participants: This study enrolled outpatients of the Theodor Bilharz Research Institute (TBRI) and ELkasrELainy Hospital. All patients and healthy volunteers were subjected to a panel of tests (according to the Toxoplasma Serological Profile [TSP]), consisting of the latex agglutination test, an ELISA based on crude T. gondii tachyzoite lysate (9), the Sabin-Feldman dye test (10), a double sandwich IgM ELISA, and the Differential agglutination test (Cofacetone [AC]-fixed versus that of formalin [HS]-fixed tachyzoites) (AC/HS test). These tests have been used successfully to determine if infections were acquired in the recent or more distant past. Ethical issues were strictly handled according to the International Ethical Guideline for Biomedical
Received October 14, 2014. Accepted January 5, 2015. J-STAGE Advance Publication April 10, 2015. DOI: 10.7883/yoken.JJID.2014.444 *Corresponding Author: Mailing address: Zoology Department, Science Faculty, Cairo University, Giza, Egypt. Tel: ＋201001089969, E-mail: alyaafarid＠yahoo.com 474
Novel IMB-ELISA Assay
finally by gel-filtration. The anti-T. gondii IgG pAbs produced had a very high degree of purity, except for few serum protein contaminants. Partially purified pAb was further adsorbed with fetal calf serum (FCS) to eliminate any non-specific binding with bovine antigens. The molecular weight was determined by SDS-PAGE, as previously described (20); the protein content was measured according to Bradford (21). The reactivity of anti-T. gondii IgG pAb against SAG1 antigen was measured by indirect ELISA with some modifications from the original method of Engvall and Perlman (15). Labeling of anti-SAG1 IgG pAb with horseradish peroxidase with the periodate method was performed as previously described (22). Sandwich ELISA: Polystyrene microtiter plates were coated with 100 ml/well of purified IgG pAb diluled in 8 ml carbonate buffer, pH 9.6. The plates were washed 3 times with a washing buffer (0.1 M PBS/Tween, PH 7.4/Tween). The free sites were blocked with 200 ml/well of 0.1z BSA/0.1 M PBS/T, pH 7.4 and incubated for 1 h at 379 C.The plates were washed 5 times with 0.1 M PBS/T. Sera (100 ml/well) were added and incubated for 2 h at 379 C, and the plates were washed 3 times. Peroxidase-conjugate IgG pAbs (100 ml/well) diluted in PBS were dispensed, and plates were incubated for 2 h at 379 C. The reaction was visualized by the addition of 100 ml/well of O-phenylene diamine substrate solution for 30 min in the dark at room temperature. The reaction was stopped by adding 50 ml/well of 8 N H2SO4, and the plates were read at 492 nm with an ELISA microplate reader (Bio Rad). IMB-ELISA: Microtitration plates were coated with 100 ml/well of anti-T. gondii pAbs coupled with magnetic microbead nanoparticles (200 mg/ml for IgG in 8 ml carbonate buffer, pH 9.6) and incubated overnight at room temperature. Plates were washed 3 times with 0.1 M PBS/T, pH 7.4. The remaining sites were blocked by 100 ml/well of 2.5z FCS/PBS/T and incubated for 2 h at 379 C. The plates were washed 3 times with PBS/T. Serum samples (100 ml) were pipetted into the wells in duplicate and incubated for 2 h at 379 C. The wells were washed 3 times, and 100 ml/well of peroxidase-conjugated pAbs were added and incubated for 1 h at room temperature. The reaction was visualized as described above. Statistical analysis: Data are expressed as mean ± standard deviation (SD) or percentages (z). The SPSS software (version 13 Windows) was used for data analysis.
Research. Prior to blood collection, the purpose of the study was explained individually to all participants and informed consents obtained. According to TSP, participants were divided into 3 main groups: (i) Fifty T. gondii-infected patients selected based on the presence of antibodies in their serum. (ii) Thirty-five patients harboring other parasites (E. histolytica, n ＝ 15; Giardia, n ＝ 5; and Cryptosporidium, n ＝ 15). (iii) Twenty parasite-free healthy individuals from the TBRI medical staff served as negative control. Serum samples from all groups were tested to detect circulating toxoplasmal antigens. Sera were separated and stored at －709 C until required. Animals: New Zealand white rabbits, weighting approximately 1.5 kg and 6 weeks old, were obtained and housed in TBRI. They were kept for 4 weeks (experiment duration) under standard laboratory care, i.e., 219 C, 16z moisture, filtered drinking water, and 15z protein, 3z fat, and 22z fiber diet. Blood samples were collected from healthy rabbits before inoculation and examined by ELISA for T. gondii antibodies and cross reactivity with other parasites, as previously described (16). Animal experiments were carried out according to the international guidelines and TBRI animal ethics committee. Preparation of tachyzoite proteins (SAG1 antigen) of T. gondii: T. gondii antigens were prepared from peritoneal exudates of BALB/c mice infected 3 days earlier with tachyzoites of T. gondii RH strain, as previously described (11). Mice peritoneal exudates were centrifuged at 2,000 g for 20 min, washed 3 times with phosphate buffer saline (PBS), subjected to sonication periods, and centrifuged at 12,000 g for 1 h, and supernatants were collected as soluble antigens. Protein content was determined by the Bradford method, and soluble antigens were stored at －209 C until use, as previously described (11,12). Purification of SAG1 antigen was carried out in 2 steps: diethylaminoethyl (DEAE)-Sephadex G-50 and G-200 ion exchange chromatography, as previously described (13), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12z slab gels (1 mm thick) under reducing conditions (2-mercaptoethanol) and stained with Coomassie blue (Bio-Rad, Hercules, CA, USA), as previously described (14). Following purification, the reactivity and specificity of T. gondii SAG1 antigen were tested by indirect ELISA (15). Preparation of IgG pAbs against SAG1 antigen: Rabbits free from T. gondii and other parasitic infections were inoculated for the production of antibodies against T. gondii SAG1 antigens. An intramuscular priming dose was injected at 4 sites, comprising 1 mg SAG1 antigen mixed with an equal volume of complete Freund's adjuvant (CFA; Sigma, St. Louis, MO, USA). Three booster doses were given, each containing 0.5 mg antigen emulsified in an equal volume of incomplete Freund's adjuvant (IFA, Sigma); the first boosting was given 2 weeks after the priming dose, and the following boosting doses were given at weekly intervals, as previous described (17). One week after the last booster dose, rabbit sera were harvested and the pAb fraction was purified by the 50z ammonium sulfate precipitation method (18). Further purification of pAb was performed with the 7z caprilic acid method (19) and
RESULTS Purification of 30 kDa SAG1 of T. gondii: (i) DEAESephadex G-50- ion exchange chromatography: Fig. 1 shows the OD280 profile of the antigen fractions obtained following purification by DEAE Sephadex G-50 ion exchange chromatography. The eluted antigens were represented by 4 peaks at 12, 22, 25, and 34 with a maximum OD value of 0.068 for fraction 25. The eluted protein fractions resulted from this purification method were analyzed by 12.5z SDS-PAGE under reducing condition and showed 3 major bands at 80, 30, and 27 kDa and many minor bands ranging from 110 kDa down to 17 kDa. (ii) DEAE-Sephadex G-200- ion ex475
Fig. 1. Elute profile for chromatography of 30 kDa SAG1 on DEAE sephadex G-50 ion exchange chromatography. Fig. 3. (Color online) SDS-PAGE of target antigens eluted from ion exchange chromatography column. Lane M: Low molecular weight standard, Lane 1: Purified antigen after DEAE sephadex G-200, Lane 2: Purified antigen after DEAE sephadex G-50, and Lane 3: Crude T. gondii antigen.
Table 1. The reactivity of SAG1 antigen by indirect ELISA Parasitic antigen
OD reading Mean ± SD
T. gondii E. histolytica Giardia Cryptosporidium
1.841 ± 0.231 0.294 ± 0.221 0.126 ± 0.049 0.142 ± 0.082
Fig. 2. Elute profile for chromatography of 30 kDa SAG1 on DEAE sephadex G-200 ion exchange chromatography.
after the second booster dose, immune sera displayed high titers against 30 kDa SAG1 with maximal OD of 1.5 at 1/100 dilution. The total protein content in the crude rabbit serum containing anti-T. gondii pAbs was 12.5 mg/ml. After 50z ammonium sulfate precipitation, the protein content was 8 mg/ml, while it dropped to 4 mg/ml following 7z caprylic acid precipitation. The purity of IgG pAbs after each purification steps was assayed by 12.5z SDS-PAGE under reducing condition. The purified IgG pAbs were represented by H- and L-chain bands at 53 and 31 kDa, respectively. The pAbs appear free from other proteins (Fig. 4). The reactivity of anti-T. gondii IgG pAbs against SAG1 and other parasite antigens was determined by indirect ELISA. The produced anti-T. gondii pAbs diluted 1/100 in PBS/T buffer elicited a strong reactivity towards SAG1. The OD reading at 492 nm for T. gondii antigen was 2.79 compared to 0.532, 0.326, and 0.243 for E. histolytica, Giardia, and Cryptosporidium, respectively. Standardization of sandwich ELISA: The optimum dilution of anti-T. gondii IgG pAb for the coating step was 1/80, while it was only 1/40 for peroxidase-conjugated anti-T. gondii IgG pAb. Detection of circulating T. gondii SAG antigen in human serum samples: (i) Sandwich ELISA: To measure the incidence of positivity for T. gondii, it was necessary at first to determine the cut off value for posi-
change chromatography: Fig. 2 shows the OD280 profile of the antigen fractions obtained following purification by DEAE Sephadex G-200 ion exchange chromatography. The eluted antigen was represented by a single peak with a maximum OD value of 0.15 for fraction 23. The eluted protein fraction was analyzed by 12.5z SDS-PAGE under reducing condition and showed a unique band at 30 kDa, which represents 30 kDa SAG1 (Fig. 3). Estimation of total protein content of T. gondii antigens: The protein content was 7 mg/ml of crude antigens and 3.5 and 1.5 mg/ml of purified antigens after DEAE-Sephadex G-50 and G-200 gel filtration chromatography, respectively. (i) Reactivity of SAG1 antigen by indirect ELISA: The antigenicity of the purified target antigen was tested by an indirect ELISA technique. Blood samples from human-infected with T. gondii elicited a strong reaction against 30 kDa SAG1 with a mean OD reading of 1.841, and no cross reactions were detected in samples of patients infected with other parasites (Table 1). Production of pAbs against SAG1: Test blood samples were withdrawn from New Zealand white rabbits before the injection of each immunizing dose. They were tested for the presence of specific anti-T. gondii antibodies by indirect ELISA, and antibody levels increased 1 week after the first booster dose. Three days 476
Novel IMB-ELISA Assay Table 3. Detection of 30 kDa SAG1 in human sera samples using IMB-ELISA technique Positive case
Negative case
Group (no. of animals) No. Healthy control (n ＝ 20) T. gondii (n ＝ 50) E. Histolytica (n ＝ 15) Giardia (n ＝ 5) Cryptosporidium (n ＝ 15) 1),2):
X ± SD
— 49 1.94 ± 0.32 11) 0.49 ± 0.21 0 — 11) 0.82 ± 0.14
No.
X ± SD
20 12) 14 5 14
0.12 ± 0.07 0.22 ± 0.032 0.24 ± 0.16 0.20 ± 0.09 0.14 ± 0.10
See Table 2, footnote.
Table 4. The sensitivity, specificity, PPV and NPV of sandwich and IMB-ELISA results used for detection of 30 kDa SAG1 in human sera samples
Fig. 4. (Color online) SDS-PAGE of IgG pAb (stained with coomassie blue), Lane M: Molecular weight standard protein, Lane 1: Crude anti-T. gondii IgG pAb, Lane 2: Purified IgG pAb after 50z ammonium sulfate treatment, and Lane 3: Purified IgG pAb after 7z caprylic acid treatment.
Negative case
Group (no. of animals) No. Healthy control (n ＝ 20) T. gondii (n ＝ 50) E. Histolytica (n ＝ 15) Giardia (n ＝ 5) Cryptosporidium (n ＝ 15) 1): 2):
X ± SD
46 1.61 ± 0.35 21) 0.58 ± 0.22 0 21) 0.63 ± 0.13
No.
X ± SD
20 42) 13 5 13
0.21 ± 0.07 0.28 ± 0.03 0.21 ± 0.15 0.15 ± 0.08 0.23 ± 0.10
Sensitivity (z )
Specificity (z )
PPV (z )
NPV (z)
ELISA IMB ELISA
92 98
92.7 96.4
92 96
92.7 98.1
bead method. The mean OD value of T. gondii-infected group (1.94 ± 0.32) was significantly higher than that of healthy control and other parasite groups (Table 3). One out of 50 T. gondii infected samples showed a false negative result, and the sensitivity of the assay was calculated to be 98z. All 20 negative controls were below the cut off value, while 2 out of 35 samples from individuals infected with other parasites (1 with E. histolytica and 1 with Cryptosporidium) were borderline, giving 96.4z specificity. PPV and NPV were 96z and 98.1z, respectively (Table 4).
Table 2. Detection of 30 kDa SAG1 in human sera samples using sandwich ELISA Positive case
Technique
DISCUSSION Infections by T. gondii are widely prevalent in humans and animals, especially in food animals throughout the world (23). Early diagnosis is important in patients who present clinical symptoms of toxoplasmosis and in pregnant women to prevent severe congenital infection in the developing fetus (24,25). Toxoplasmosis diagnosis depends on direct and indirect methods. The classical diagnosis of toxoplasmosis relies on serological methods for the detection of specific immunoglobulin antibodies. However, these methods have a poor efficiency, especially in neonates and immunodeficient patients (26). Owing to IgM long half-life in many patients and high percentages of non-specific results, toxoplasma-specific IgM assay is no longer of benefit in diagnosing acute toxoplasma infection (27–29). In some studies years ago, T. gondii antigens present in serum samples of experimentally infected animals suggests that antigens are detectable during the acute phase of toxoplasmosis (30,31). For instance, Raizman and Neva (32) showed the presence of circulating antigens in mouse sera by counter-current electrophoresis and agar gel diffusion on days 2–4 of infection. Huskinson et al. (33) could demonstrate the presence of T. gondii antigens in serum and urine of mice as early as 5 days post infection by western blot. Recently, Shojaee et al. (34) detected antigenemia from the fourth day after infection in mouse sera by immunoblotting. Moreover, to
False positive results. False negative results.
tivity or the line of demarcation between positive and negative samples. The cut off value for positivity was measured as the mean OD reading of the negative controls ＋ 2 SD of the mean. Tested samples showing OD values higher than the cut off value were considered positive for Toxoplasma. Table 2 shows the results of SAG1 antigen detection among the different groups. The cut off value for positivity was 0.428. The mean OD value of the T. gondii-infected group (1.61 ± 0.351) was significantly higher than that of both the healthy control (0.212 ± 0.067) and other parasite groups. Four out of 50 T. gondii-infected samples showed false negative results and the sensitivity of the assay was calculated to be 92z. All 20 negative controls were below the cut off value while 4 out of 35 samples from individuals infected with other parasites were border line, giving 92.7z specificity. The positive predictive value (PPV) and negative predictive value (NPV) were 92z and 92.7z, respectively. (ii) IMB-ELISA: The presence of T. gondii SAG1 antigen in human serum samples of the different groups was also evaluated by a nanomagnetic 477
body population; thus they offer a statistically relevant idea for the overall picture of an immune response. Our anti-SAG1 IgG pAb was represented by H- and L- chain bands at 53 and 31 kDa, respectively, and a 4 mg/ml protein concentration. These results are comparable with the yield of purified immunoglobulins from any biological fluid following similar purification procedures (58,59). The purified rabbit IgG fraction served as both antigen capture (1/80 mg/ml) and peroxidaseconjugated (1/40 mg/ml) detecting antibody in sandwich ELISA. The cut-off OD value for positivity in sandwich ELISA for SAG1 was 0.428 in serum samples. Many immunologic tests based on antigen detection (60,61) and molecular or proteomic diagnostic techniques (62,63) have been well studied and described. However, an affordable, easy-to handle, sensitive, and specific method is not yet available (64). An immunomagnetic bead-based immunoassay is a popular approach in diagnosis of many food-borne and infectious diseases. This innovative technique involves antibodycoated magnetic microbead nanoparticles, which trap antigens from liquid media. Furthermore, the small size and shape of the microbeads enable them to be evenly dispersed within the sample, thus improving the effectiveness of the antibody conjugation, and consequently enhancing the sensitivity of antigen detection (65,66). In this study, we developed a novel antigen-capture immunoassay based on IgG pAb-coated magnetic microbead nanoparticles for the detection of circulating SAG1 antigens of T. gondii in serum samples. The study was conducted on T. gondii-infected patients, patients infected with other parasites, and healthy controls. Sandwich ELISA revealed a sensitivity of 92z, specificity of 92.7z, PPV of 92z, and NPV of 92.7z. While IMB-ELISA sensitivity (98z), specificity (96.4z), PPV (96z), and NPV (98.1z) were higher than that of sandwich ELISA. Our results were comparable to those of Conlan et al. (67), who described the use of IMB as the solid phase for the portable detection of the CSF antigen in spleen samples and for the reliable detection of antibodies against CSF in animals vaccinated with a lapinized Cstrain. This IMB-ELISA is 100z sensitive and 91z specific in comparison to polyclonal antibody-based antigen-capture ELISA (AC-ELISA). Moreover, IMBantibody-ELISA is 97z sensitive and 95z specific. Lei et al. (68) developed a novel egg yolk antibody (IgY)coated magnetic bead antigen-capture immunoassay for the detection of a circulating antigen of S. japonica in serum samples of patients in endemic schistosomiasis areas of China. This IgY-IMB-ELISA comprises IgY pAb-coated magnetic beads as a capture antibody and a monoclonal IgG as a detection antibody. The sensitivity of this magnetic immunoassay is 100z in cases of acute infection and 91.5z in chronic cases of schistosomiasis, and no positive reaction was found in 49 healthy individuals. Cross-reactivity was 3.3z with clonorchiasis and 0z with paragonimiasis. There was a significant correlation between ELISA absorbance values and egg counts (eggs per gram feces) and a correlation coefficient of 0.88 in a small sample of 14 patients. The results demonstrated that IgY-IMB-ELISA is a sensitive and specific assay for detection of S. japonica. Ahmed et al. (69) also developed a novel IMB-ELISA based on IgG
increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful (35). Tachyzoites are thought to be responsible for acute phase of the infection and express immunodominant antigens that induce strong immune responses (36). Abdel-Rahman et al. (37) used T. gondii crude and affinity-purified tachyzoite antigens isolated from slaughtered sheep in the diagnosis of toxoplasmosis in horses. Conde de Felipe et al. (38) proved that 29–35 kDa T. gondii fractions can detect specific IgG in goats 2 weeks earlier than crude extract. The 30 kDa surface protein of T. gondii tachyzoite represents 3z to 5z of the parasite total proteins (39) (Kasper, 1987) and has been proposed for the diagnosis of acute acquired toxoplasmosis. Cesbron et al. (40) and Santaro et al. (41) have developed a double-sandwich ELISA based on this protein for detection of IgM antibodies. SAG1 is the main surface antigen of T. gondii that induces high titers of IgG, IgM, and IgA despite only accounting for 3–5z of total cellular proteins (42). In addition, this protein is also a tachyzoite-specific antigen and is highly conserved (43); thus, it is important for diagnostic purposes (44,45). Tachyzoites are the key disease-causing forms. During the acute stage of infection, tachyzoites activate a potent host immune response, which eliminates most of the parasites. The surface of the tachyzoite is the main target of the host immune response. SAG1 is a major surface antigen involved in this process. Although it only accounts for 3–5z of T. gondii total proteins, the majority of the antibodies are reactive against SAG1 during infection (46). Various SAG1 preparations purified from the parasite (47,48), produced by recombinant systems in Escherichia coli (49) or yeast (50), and its constituent peptides (51–53) stimulate the host humoral and cellular immunity, thereby providing protection against toxoplasmosis (54). As such, SAG1 features excellent antigenicity and immunogenicity and is valuable for both diagnosis and immunization. In 1980, Handman et al. (55) first identified SAG1 from T. gondii surface membrane antigens using a monoclonal antibody technique. Subsequently, Kasper et al. (56) applied mAb-affinity chromatography to isolate the protein. In 1988, Burg et al. (43) cloned the complete T. gondii sag1 gene, which has a length of 1.1 kb, encodes 336 amino acids, and yields a 30 kDa protein. In this study, we aimed to purify and characterize SAG1 antigen from T. gondii tachyzoites and evaluate its early diagnostic potential in human toxoplasmosis with sandwich ELISA and IMB-ELISA techniques, which may aid in the prevention of the infection and eradication of its transmission. When T. gondii antigens were analyzed by 12.5z SDS-PAGE under reducing condition, SAG1 antigen was detected as a 30 kDa protein with a concentration of 1.5 mg/ml. These results were similar to that of Villavedra et al. (42,57) who purified and isolated a fraction of 30–33 kDa from crude tachyzoite antigens, which was used successfully in the diagnosis of human toxoplasmosis by ELISA. The reactivity of SAG1 antigen was tested by indirect ELISA and elicited a strong reaction with a maximal mean OD reading of 1.841. This purified antigen was utilized for immunization of rabbits for the production of pAbs. Which contain the entire antigen-specific anti478
Novel IMB-ELISA Assay
for the detection of excretory/secretory (E/S) antigens in rabbit sera infected with S. haematobium. They detected E/S antigens in serum with a sensitivity of 95z a specificity of 93.7z compared to other parasitic infections and of 100z compared to healthy controls. On the other hand, detection of E/S antigens in urine elicited a sensitivity of 91z, and specificity of 93.7z compared to other parasitic infections and of 100z compared to healthy individuals. Furthermore, Aly et al. (70) evaluated a novel magnetic microbead based-latex agglutination assay (MMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardizing a novel magnetic microbead based-sandwich ELISA (MMB-S ELISA). They reported that the sensitivity of novel MMB-LAT was 82.4z versus MMB-S ELISA 96.5z and currently used sandwich ELISA 88.2z. The specificity of MMB-LAT was 83.6z versus MMB-S ELISA 96.3z and sandwich ELISA 87.3z. With our IMB-ELISA results showing a higher sensitivity and specificity than that of sandwich ELISA, it is obvious that the use of magnetic microbead nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis. The use of magnetic nanoparticles in immunoassay (nanomagnetic assay) combines magnetic nanoparticle high binding capacity and simple separation of bound and unbound materials as a solid phase with the rapid reaction kinetics of solutions, thus providing the chance of enhancing the sensitivity of antigen detection (71,72). The surface of the beads enables substantially more antibodies to be involved in the reaction with the antigen than in sandwich ELISA (73–75).
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
Conflict of interest None to declare.
26.
REFERENCES
27.
1. Current WL, Upton SJ, Long PL. Chapter 1. Taxonomy and life cycles. In: Long PL. editor. Coccidiosis of Man and Domestic Animals. Boca Raton, FL: CRC Press; 1990. p.1-16. 2. Dlugonska H. Toxoplasma rhoptries: unique secretory organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis. J Biomed Biotechnol. 2008;2008:632424. 3. Zhou P, Chen Z, Li HL, et al. Toxoplasma gondii infection in humans in China. Parasit Vectors. 2011;4:165-74. 4. Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol. 2009;39:1385-94. 5. Wong SY, Remington JS. Toxoplasmosis in pregnancy. Clin Infect Dis. 1994;18:853-61. 6. Handman E, Goding JW, Remington JS. Detection and characterization of membrane antigens of Toxoplama gondii. J Immunol. 1980;124:2578-83. 7. Kasper LH, Bradley MS, Pfefferkorn ER. Identification of stage specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies. J Immunol. 1984;132:443-9. 8. Rachinel N, Buzoni-Gatel D, Dutta C, et al. The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii. J Immunol. 2004;173:2725-35. 9. Choi WY, Nam HW, Youn JH, et al. Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme linked immunosorbent assay. Kor J Parasitol. 1992;30:83-90. 10. Remington JS, Efron B, Cavanaugh E, et al. Studies on toxoplasmosis in El Savador prevalence and incidence of toxoplasmosis as measured by the Sabin-Feldman dye test. Trans R Soc Trop Med Hyg. 1970;64:252-67. 11. Brooks RG, Sharma SD, Remington JS. Detection of Toxoplas-
28.
29.
30. 31. 32. 33. 34. 35.
36. 37.
479
ma gondii antigen by a dot-immunobiding technique. J Clin Microbiol. 1985;21:113-6. Foroghi parvar F, Keshavarz H, Shojaee S. Detection of Toxoplasma gondii antigens in sera and urine of experimentally infected mice by capture–ELISA. Iranian J Parasitol. 2008;3:1-5. Timanova A, M äuller S, Marti T, et al. Ascaridia galli fatty acidbinding protein, a member of the nematode polyprotein allergens family. Eur J Biochem. 1999;261:569-76. Takacs B. Electrophoresis of proteins in polyacrylamide slab gels. In: Lefkovitz I, Pernis B.editors. Immunological Methods. New York: Academic Press; 1997. p.81-105. Engvall E, Perlmanm P. Enzyme-linked immunosorbent assay. Quantitative assay of IgG. Immunochem. 1971;8:871-4. Fagbemi BO, Guobadia EE. Immunodiagnosis of fascioliasis in ruminants using a 28-kDa cysteine protease of F. gigantica adult worm. Vet Parasitol. 1995;57:309-18. Fagbemi BO. Development and characterization of a monoclonal antibody reactive with 128 kDa protease of F. gigantica. Vet Parasitol. 1995;57:351-6. Nowotny A. A basic exercises in immunochemistry. In: Nowotny A. editor. A Basic Exercises in Immunochemistry. 2nd ed. BerlinHeidelberg-New York: Springer-Verlag; 1979. p.7-20. Mckinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from ascites fluid. J Immunol Methods. 1987;96:271-8. Harlow E, Lane D. editors. Antibodies: A Laboratory Manual. 1st ed. New York: Cold Spring Harbor Laboratory Press; 1988. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. Tijssen P, Kurstak P. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugate for enzyme immunoassays. Anal Biochem. 1984;136: 451-7. Jones JL, Dubey JP. Foodborne toxoplasmosis. Clin Infect Dis. 2012;55:845-51. Desmonts G, Daffos F, Forestier F, et al. Prenatal diagnosis of congenital toxoplasmosis. Lancet 1985;1:500-4. Doehring E, Reiter-Owona I, Bauer O, et al. Toxoplasma gondii antibodies pregnant women and their newborns in Dar-esSalaam. Am J Trop Med Hyg. 1995;52:546-8. Hafid J, Flori P, Raberin H, et al. Comparison of PCR, captureELISA and immunoblotting for detection of Toxoplasma gondii in infected mice. J Med Microbiol. 2001;50:1100-4. Beghetto E, Buffolano W, Spandoni A, et al. Use of an immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary Toxoplasma gondii infection during pregnancy. J Clin Microbiol. 2003;41:5414-8. Gras L, Gibert RE, Wallon M, et al. Duration of IgM response in women acquiring Toxoplasma gondii during pregnancy: Implication for clinical practices and cross sectional incidence studies. Epidemiol Infect. 2004;132:541-8. Kaul R, Chen P, Binder SR. Detection of Immunoglobulin M antibodies specific for Toxoplasma gondii with increased selectivity for recently acquired infections. J Clin Microbiol. 2004;42: 5705-9. Lindenschmidt EG. Enzyme-linked immunosorbent assay for detection of Toxoplasma gondii antigen in acute-phase toxoplasmosis. Eur J Clin Microbiol. 1985;4:488-92. Luft BJ, Remington JS. Toxoplasma encephalitis. J Infect Dis. 1998;157:1-6. Raizman RE, Neva FA. Detection of circulating antigen in acute experimental infections with T. gondii. J Infect Dis. 1975;132: 44-8. Huskinson J, Stepick-Biek P, Remington JS. Detection of antigens in urine during toxoplasmosis. J Clin Microbiol. 1989;27: 1099-101. Shojaee S, Keshavarz H, Rezaeian M, et al. Detection of Toxoplasma gondii antigens in sera from experimentally infected mice. Pak J Med Sci. 2007;23:100-2. Luo Q, Upadhya R, Zhang H, et al. Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry. Microbes Infect. 2011;13:1199-210. Gross U, Holpert M, Goebel S. Impact of stage differentiation on diagnosis of toxopla smosis. Ann Ist Super Sanita. 2004;40:65-70. Abdel-Rahman EH, Ghazy AA, Shaapan RM. Prevalence of antibodies to Toxoplasma gondii in farm horses in Egypt. J Egypt
Vet Med Assoc. 2005;65:99-112. 38. Conde de Felipe MM, Molina JM, Rodriguez-Ponce E, et al. IgM and IgG response to 29-35-kDa Toxoplasma gondii protein fractions in experimentally infected goats. J Parasitol. 2007;93:701-3. 39. Kasper LH. Isolation and characterization of a monoclonal antiP30 antibody resistant mutant of Toxoplasma gondii. Parasite Immunol. 1987;9:433-5. 40. Cesbron JY, Capron A, Ovlaqe G, et al. Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30). J Immunol Methods. 1985;83:151-8. 41. Santaro F, Afchain D, Pierce R, et al. Serodiagnosis of Toxoplasma infection using a purified parasite protein (P30). Clin Exp Immunol. 1985;62:262-9. 42. Khan IA, Ely KH, Kasper LH. A purified parasite antigen (P30) mediates CD8 ＋ T cell immunity against fatal Toxoplasma gondii infection in mice. J Immunol. 1991;147:3501-6. 43. Burg JL, Perelman D, Kasper LH, et al. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. Immunol. 1988;141:3584-91. 44. Siachoque H, Guzman F, Burgos J, et al. Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model. Exp Parasitol. 2006;114:62-5. 45. Harning D, Spenter J, Metsis A, et al. Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies. Clin Diagn Lab Immunol. 1996;3: 355-7. 46. Velge-Roussel F, Chard àes T, M áev áelec P, et al. Epitopic analysis of the Toxoplasma gondii major surface antigen SAG1. Mol Biochem Parasitol. 1994;66:31-8. 47. Debard N, Buzoni-Gatel D, Bout D. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect Immun. 1996;64:2158-66. 48. B äulow R, Boothroyd JC. Protection of mice from fatal Toxoplasma gondii infection by immunization with P30 antigen in liposomes. J Immunol. 1991;147:3496-500. 49. Petersen E, Nielsen HV, Christiansen L, et al. Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii. Vaccine. 1998;16:1283-9. 50. Biemans R, Gr áegoire D, Haumont M, et al. The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation. J Biotechnol. 1998;66:137-46. 51. Darcy F, Maes P, Gras-Masse H, et al. Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen. J Immunol. 1992;149:3636-41. 52. Godard I, Estaquier J, Zenner L, et al. Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis. Mol Immunol. 1994;31:1353-63. 53. Velge-Roussel F, Dimier-Poisson I, Buzoni-Gatel D, et al. AntiSAG1 peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines. Parasitology. 2001; 123:225-33. 54. Wang Y, Yin H. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii. Parasit Vectors. 2014;7:180. 55. Handman E, Goding JW, Remington JS. Detection and characterization of membrane antigen of Toxoplasma gondii. J Immunol. 1980;124:2578-83. 56. Kasper LH, Crabb JH, Pfefferkorn ER. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol. 1983;130:2407-12. 57. Villavedra M, Battistoni JJ, Rossi S, et al. Avidity analysis of the human immune response to a chitin binding protein of Toxoplasma gondii. Int J Parasitol. 2001;31:1087-92. 58. Bird P, Jones P, Allen D, et al. Analysis of the expression and secretion of isotypes of sheep B cell immunoglobulins with a panel of isotype-specific monoclonal antibodies. Res Vet Sci. 1995;59:
189-94. 59. Yang YB, Harrison K. Influence of column type and chromatographic conditions on the ion exchange chromatography of immunoglobulins. J Chromatogr A. 1996;743:171-80. 60. Robijn ML, Koeleman CA, Wuhrer M, et al. Targeted identification of a unique glycan epitope of Schistosoma mansoni egg antigens using a diagnostic antibody. Mol Biochem Parasitol. 2007; 151:148-61. 61. Sulbar áan GS, Ballen DE, Berm áudez H, et al. Detection of the Sm31 antigen in sera of Schistosoma mansoni-infected patients from a low endemic area. Parasite Immunol. 2010;32:20-8. 62. Lier T, Simonsen GS, Wang T, et al. Real-time polymerase chain reaction for detection of low-intensity Schistosoma japonicum infections in China. Am J Trop Med Hyg. 2009;81:428-32. 63. Robijn ML, Planken J, Kornelis D, et al. Mass spectrometric detection of urinary oligosaccharides as markers of Schistosoma mansoni infection. Trans R Soc Trop Med Hyg. 2008;102:79-83. 64. Engels D, Chitsulo L, Montresor A, et al. The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop. 2002;82:139-46. 65. Chen L, Deng L, Liu L, et al. Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureusand protein A. Biosens Bioelectron. 2007;22:1487-92. 66. Nagasaki Y, Kobayashi H, Katsuyama Y, et al. Enhanced immunoresponse of antibody/mixed-PEG coimmobilized surface construction of high-performance immunomagnetic ELISA system. J Colloid Interface Sci. 2007;309:524-30. 67. Conlan J, Khounsy S, Phruaravanh M, et al. Application of immunomagnetic bead technology for improved diagnosis of classical swine fever virus. In: Conlan J, Blacksell S, Morrissy C, et al. editors. Management of Classical Swine Fever and Foot-andMouth Disease in Lao PDR: proceedings of an international workshop held in Vientiane, Lao PDR, 20–21 November 2006. ACIAR Proceedings No. 128. Canberra, Australia: ACIAR, 2008;8:p.74-9. 68. Lei JH, Su BT, Xu H, et al. Evaluation of an IgY-based immunomagnetic enzyme-linked immunosorbent assay system for detection of circulating Schistosoma japonicum antigen in serum samples from patients in china. Am J Trop Med Hyg. 2011;85: 1054-9. 69. Ahmed A, El Amir A, Rabee I, et al. Effective Diagnosis of Schistosomiasis haematobium by Immunomagnetic Bead ELISA technique using Super-Paramagnetic Nanoparticles. J Am Sci. 2012;8:833-41. 70. Aly I, Zalat R, El Aswad BE, et al. Novel nanomagnetic beads based–latex agglutination assay for rapid diagnosis of human Schistosomiasis haematobium. Int J Med Pharmaceut Sci Engin. 2013;7:641-7. 71. Gundersen SG, Haagensen I, Jonassen TO, et al. Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen. J Immunol Methods. 1992;148:1-8. 72. Ndhlovu P, Cadman H, Gundersen SG, et al. Optimization of the magnetic bead antigen capture enzyme immunoassay for the detection of circulating anodic antigens in mixed Schistosoma infections. Acta Trop. 1995;59:223-35. 73. Cudjoe KS, Thorsen LI, Sorensen T, et al. Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA. Int J Food Microbiol. 1991;12:313-21. 74. Nustad K, Paus E, Bomer OP. Magnetic particles as solid phase in immunoassays. In: Kemshead JT. editor. Magnetic Separation Techniques Applied to Cellular and Molecular Biology. Somerset, England: Wordsmiths' Conference Publications; 1991. p.39-46. 75. Patel PD, Blackburn C. Detection of food poisoning agents using immunomagnetic particles. In: Kemshead JT. editor. Magnetic Separation Techniques Applied to Cellular and Molecular Biology. Somerset, England :Wordsmiths' Conference Publications; 1991. p.93-105.
480
