Preparation of Crude ES Antigen

Evaluation of Schistosoma haematobium 27-29 kDa antigen for immunodiagnosis of Schistosomiasis haematobium

Azza El Amir Zoology Department, Faculty of Science, Cairo University

Key words: S. haematobium, SAWA, E/S, CP, ELISA, IgG, Immunodiagnosis.

ABSTRACT Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of schistosomiasis. This work is the first to demonstrate the immunodiagnostic potential of different Schistosoma haematobium (S. haematobium) antigens in human in Egypt. An enzyme linked immunosorbent assay (ELISA) was used to measure the levels of total immunoglobulin G (IgG) and IgG4 antibodies (Abs) against S. haematobium adult worm antigens (Ags) (SAWA), excretory/secretory Ags (E/S) and cysteine proteinase Ag (27-29 kDa) for the field diagnosis of schistosomiasis in Egypt. SDS-PAGE profiles of S. haematobium Ags showed several bands for SAWA, E/S and 27-29 kDa Ags which are characteristic of infections with Schistosoma spp. Purified protein fraction showed a single homogenous band of 27-29 kDa. For summarizing the potency of S. haematobium Ags, sensitivity rate, negative predictive value and diagnostic efficacy were calculated between data of 40 human patients in ELISA. SAWA Ag recorded 85.0 %, 77%, 90.0% with total IgG and 90.0 %, 83% and 93.3% with IgG4 isotype, respectively. While, E/S recorded 87.5 %, 80%, 92.0% with total IgG and 92.5%, 87%, 95.0% with IgG4 isotype, respectively. Purified 27-29 kDa Ag presents the higher significant (P<0.01) results recording 90.0 %, 83%, 93.3% with total IgG and 97.5%, 95%, 98.3% with IgG4 isotype, respectively. Data of the present study indicate that combining the detection of IgG4 isotype using the 27-29 kDa Ag in sera of Schistomiasis haematobium patients in an ELISA test could represent an effective immunodiagnostic tool for the detection of the disease in low worm burden population. In addition, this test could be useful in sero-epidemiolocal studies in low endemic areas and in diagnosis of the disease in travelers from non endemic countries visiting schistosomiasis endemic areas.

INTRODUCTION

In spite of the numerous efforts made to control their transmission, Schistosome parasites still represent a serious public health concern and a major economic problem. Schistosomiasis is associated with a variety of clinical syndromes that may lead to severe morbidity in many developing countries (Caffrey, 2007; Dissous et al., 2007). Early intervention with effective chemotherapeutic agents is most efficacious for treatment of the disease and relies on the availability of sensitive and specific methods for diagnosis. Development of sensitive as well as low-cost immunodiagnostics for detection of infected individuals would be an important step towards reaching the goal in schistosomiasis. The gold standard procedure in the diagnosis of schistosomiasis is the microscopic demonstration of eggs in stool and urine samples of patients. Detection of S. haematobium ova in urine of infected individuals remains the leading routine method for direct diagnosis of the disease. However, a homogeneous distribution of S. haematobium ova in urine is difficult to achieve (Braun-Munzinger and Southgate, 1992), but due to many obstacles it is not of valuable sensitivity (Hillyer, 1998). Sensitivity of all fecal examination methods is found to be poor and immunodiagnosis is considered essential for correct diagnosis (Agrawal, 2004). Many serological methods, thus far, have been challenged to diagnose human and animal schistosomiasis; most of them vary in specificity and sensitivity due to differences in materials and methods, and of course, may owe to differences in the nature of the parasite, being utilized to prepare Ag (Hassan et al., 2002; Intapan et al., 2003). Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis to non-endemic countries (Tsang and Wilkins, 1997). Different methods have been used, with enzyme-linked immunosorbent assay (ELISA) being the most widely developed (Hamilton et al., 1998). ELISA showed satisfactory sensitivity and high specificity in a very early phase after infection and may constitute a potentially useful method for laboratory diagnosis of Schistosomiasis mansoni Abs, Ags or even peptides. Also, ELISA was considered as a useful serological test for epidemiological studies in low endemic areas (Kanamura et al., 2002; Pardo et al., 2007; de Oliveira et al., 2008). The use of recombinant and purified antigens does not appear to have important advantages in comparison with the use of complex antigens. These antigens from adult worms and eggs from different species of Schistosoma are still the most widely used for the diagnosis of schistosomiasis (Hamilton et al., 1998; Al-Sherbiny et al., 1999). The use of parasite extracts in assays has two major disadvantages: they show extensive Ab cross- reactivity with other helminthes and they are difficult to standardize (Mott and Dixon, 1982). By contrast, defined and partially purified Schistosome Ags, e.g. those prepared from Schistosomes' eggs, enhance the magnitude of serological tests (Doenhoff et al., 1985; 1993). However, progress using subfractions of egg material in large-scale field trials has been impeded because these Ags are available only in limited quantities.

CP is a major component of the E/S products and because it is immunogenic at all stages of liver fluke development in the definitive host it can be used to diagnose both the acute and chronic stages of infections. Nevertheless regarding the variations of prepared Ag batches and some cross-reactivity observed using the aforementioned Ag, preparing and evaluating of a purer antigen sounds a significant idea. Recently more highlighting on the antigenic product of CP, chiefly cathepsin L1 (CL1) is warranted (Rokni and Gharavi, 2002).

Immunodiagnostic methods based on the detection of Abs continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosomal Ab detection assays though are very sensitive particularly in individuals from endemic areas (Van Lieshout et al., 2000), do not, however, differentiate between active and past infection and do not correlate with intensity of infection (Mott and Dixon, 1982). El-Raziky et al. (1974) and Ghanem et al. (1977) detected increased IgG value in acute and chronic infections in humans with active Schistosomiasis mansoni. IgG was reported to rise along the course of schistosomiasis. It rises at the early stages of active S. mansoni infection. IgG4 production was found related to susceptibility, suggesting therefore that the production of IgG4 Ab might block the functions of IgE and delay the development of protective immunity (Hagan et al., 1991).

MATERIALS AND METHODS Human subjects: Patients admitted to the Parasitology and Urology Department, TBRI were subjected to clinical, ultrasonographical, radiological and parasitological examination. S. haematobium eggs were quantified in 10 ml urine samples on 3 consecutive days by nucleopore filtration method (Peter et al., 1979) and the mean egg count was calculated. Based on results of parasitological examination and findings of clinical investigations, 40 patients with active S. haematobium infection were included in the present study. Patients harboring S. mansoni eggs or other parasites in their excreta were excluded from the study. Negative control group consisting of 20 healthy laboratory staff individuals were also included in the study. Blood samples were withdrawn from each individual and sera were separated, aliquoted and stored at –20oC until being used. Preparation of crude SAWA antigen Adult S. haematobium worms (103 worms) were supplied from the Schistosome Biological Supply Program Unit at Theodore Bilharz Research Institute (SBSP, TBRI) Giza, Egypt. Adult worms were recovered from the portal mesenteric vasculatures of laboratory infected hamsters by perfusion with heparinized saline as previously described by Smithers and Terry (1965). According to Da Silva and Ferri (1968), fresh adult worms were suspended in 10 ml of phosphate buffer saline (PBS) and homogenized with a glass homogenizer for 5 min on ice. The homogenate was ultracentrifuged for two hours at 45,000 rpm at 4˚C. The supernatant was collected and protein content was determined. Preparation of crude E/S antigens S. haematobium worms were washed with sterile PBS. The E/S Ag was prepared according to Santiago de Weil and Hillyer (1986) from a spent culture medium (RPMI, 1640, pH 7.3) (Sigma) containing 0.1 mM phenylmethyl-sulfonyl fluoride, 0.1 mM tosylamide-2-phenylethyl-chloromethyl ketone, 1 mm of L–trans–3–carboxyoxiran–2–carbonyl–L-leucy-lagmatine, 100 units/ml of penicillin,

o and 100 mg/ml of streptomycin in which the worms had been maintained for 6 hr at 37 C under 5% CO2 in air. After incubation, collected spent medium was clarified by centrifugation at 12.000 rpm for 30 min at 4oC. The medium was concentrated by ultrafiltration using Amicon YM 3 membrane filter (Grace & CO., Danvers, MA), dialyzed against distilled water containing the same proteinase inhibitors, aliquoted, and stored at -20oC till use. Purification of 27-29 kDa antigen (CP) by continuous-elution SDS-PAGE The continuous-elution SDS-PAGE was performed using the Prep-Cell (Model 491; Bio-Rad, Hercules, CA) as recommended by the manufacturer. The discontinuous buffer system was used. A cylindrical column of polyacrylamide gel was prepared using a 28-mm internal diameter casting tube. It consisted of an upper 4% stacking gel and lower 12% resolving gel with heights of 2 and 5.5 cm, respectively. The casting tube was assembled with the upper and lower buffer chambers and the later was connected to an external recirculation pump (Bio-Rad). 5 mg of protein from SAWA were incubated for 5 min in a boiling water bath in 1 ml of sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl, pH 6.8 and 0.001% bromphenol blue) before loading in the gel individually. The recirculation pump speed was operated at 100 ml/min. Each sample was subjected to electrophoresis for 10 hr at 250 V, 40 mA, and 12 W. Resolved proteins were collected at the base of the gel column using a dialysis membrane with a 6-kDa cut-off. The purified proteins were continuously and individually eluted at a flow rate of 1 ml/min using an external peristaltic pump (Econo-Pump; Bio-Rad). The absorbance of elute at 280 nm was monitored using a UV monitor (Model EM-1; Bio-Rad). The cut-off absorbance was an optical density (OD) of 0.05 and fractionated proteins were retrieved (0.5 ml/tube) using a programmable fraction collector (Model 2128; Bio-Rad). The fractions containing the specific component with an approximate molecular weight (MW) of 27-29 kDa (CP Ag) were identified by SDS-PAGE. These fractions containing the component of interest were pooled, dialyzed overnight against 10 mM citrate buffer (pH 4.9) and lyophilized (Chapell and Dresden, 1987). SDS-PAGE profile of S. haematobium Ag preparations (Purity of 27-29 kDa Ag): The eluate was analyzed by SDS-PAGE using the Mini-Protean II Cell (Bio-Rad), under reducing conditions on a 12 % gel prepared by the method of Laemmli (1970). For molecular weight estimation, low range prestained MW markers (Bio-Rad) were ran in parallel. After electrophoresis, the resolved polypeptides were revealed by staining with Coomassie brilliant blue molecular weight standards. Assessment of anti- S. haematobium total IgG and IgG4 by ELISA Detection of anti- S. haematobium IgG and IgG4 was assayed against S. haematobium SAWA, E/S and 27-29 kDa Ags using indirect ELISA, according to the method of Engvall and Perlman (1971) with some modifications. Briefly, wells of microtitre plates (Costar, Cambridge, MA) were coated with 5 ng/ml of either crude SAWA, E/S or 27-29 kDa (100 µl/well) in coating buffer (0.05 M carbonate buffer, pH 9.6). The plates were covered and kept over night at room temperature, then blocked with 200 µl/well 1% bovine serum albumin in PBS/0.05% Tween 20 (PBS/T), pH 7.2 for 2 hr at room temperature. The plates were washed with PBS/T 3 times then 100 µl of subjects diluted sera (1:200), were added to each well. Plates were then incubated for one hr at room temperature. After incubation, plates were washed three times with PBS/T. For the detection of total IgG 100 µl of diluted (1:1000) anti-human IgG peroxidase-labeled conjugate, gamma chain-specific (Sigma) were added to each well. While for the detection of IgG4 goat specific conjugate anti-human IgG4 (subclass-specific binding) labeled horse- radish peroxidase (Sigma) were used at a dilution of and 1:400 in washing buffer. Plates were then incubated for 30 min at room temperature, then washed 5 times with PBS/T and incubated with 100 µl/well of ortho-phenylenediamine (OPD) (Sigma) substrate for 30 min. The reaction was stopped with

50 l/well 8 N H2SO4 and absorbance was measured at 492 nm using a microplate ELISA reader (Bio- Rad Microplate reader, Richmond, CA, USA). The cut off value was calculated as the mean (X) optical density (OD) of the healthy control sera 2SD. Samples showing OD values equal to or greater than the cut off value were considered positive. Sensitivity and diagnostic efficacy of ELISA were evaluated. Statistical analysis The data are presented as mean  standard deviation of mean (X  SD). The means of the different groups were compared globally using the analysis of variance ANOVA. The data were considered significant if P values were less than 0.05. The parameter of sensitivity and diagnostic efficacy were determined according to Galen (1980) using the formulae as: Sensitivity = Test (+ve) ______x 100 Total confirmed (+ve) Negative predictive value = __ True (-ve)______x 100 True (-ve) + Test (-ve) Diagnostic efficacy = Test (+ve) + Test (-ve)__ x 100 True (+ve) + True (–ve)

RESULTS 1. Purity of 27-29 kDa separated antigen: To confirm the purity of the 27-29 kDa separated fraction a sample was subjected to SDS-PAGE in parallel with SAWA Ags. SDS-PAGE profiles of S. haematobium Ags (SAWA, E/S and 27-29 kDa) were investigated by 12% SDS-PAGE. Stained gels showed that SAWA was separated in multiple bands of MW ranging from 12-117 kDa with major bands of 105, 79, 66, 58, 45, 34, 29-27 and 14 kDa and minor bands of 100, 38, 36, 32, 25 and 12 kDa. SDS-PAGE profile of the purified fraction showed a single homogenous band of 29-27 kDa (Fig. 1). 2. Reactivity of patient sera in ELISA: Data illustrated in Figure (1) show the reactivity of sera collected from a total of 40 schistosomiasis haematobium patients in ELISA using different Ag preparations (SAWA, E/S and 27-29 kDa) and anti- human IgG or IgG4 as secondary antibodies. ELISA reactivity of IgG in patient sera against SAWA resulted in an OD values ranged from 0.28 to 0.88 with X±SD of 0.58±0.30 (Figure 1A), while for IgG4 isotype the OD range was significantly (P<0.01) higher recording a range of 0.45 to 0.93 with X±SD of 0.69±0.24 (Figure 1B), while testing control sera resulted in OD ranging from 0.25 to 0.13 with X±SD of 0.19±0.058 and from 0.35 to 0.19 with X±SD of 0.27±0.078. On the other hand, using E/S for the detection of total IgG and IgG4 in patients sera resulted in OD ranged from 0.46 to 0.96 with X±SD 0.71±0.25 (Figure 1C) and 0.56 to 1 with X±SD 0.78±0.22 (Figure 1D), respectively. Also the control sera reactivity resulted in an OD ranged from 0.33 to 0.19 with X±SD 0.26 ±0.066 for IgG and from 0.36 to 0.02 with X±SD of 0.28±0.075. Capturing of anti-S. haematobium Abs using 27-29 kDa Ags in ELISA showed the strongest reactivity in comparison to other two Ags indicated by the OD ranging from 0.54 to 0.17 with X±SD of 0.85±0.31 for IgG (Figure 1E) and from 0.82 to 1.48 X±SD of 1.15±0.33 for IgG4 (Figure 1F). The control sera reactivity with CP resulted in an OD ranged from0.35 to 0.21 with X±SD of 0.28±0.069 for IgG and from 0.39 to 0.23 with X±SD of 0.31±0.079 for IgG4. In summary, the highest ELISA reactivity of patient sera was achieved by combining the detection of IgG4 against 27-29 kDa Ags. 3. Sensitivity and the diagnostic efficacy of the ELISA assay: According to the calculated cutoff value for the ELISA test using SAWA as Ag for the detection of IgG or IgG4, 34 and 35 patients were recorded positive, respectively with OD higher than the calculated cutoff, resulting in test sensitivities of 85% and 90%, respectively. Negative controls tested did not exceed the calculated cutoff values indicating specificities of 100% for both tests. Test negative predictive value and diagnostic efficacy were calculated for all Ags. From data depicted in Table (1), negative predictive value recorded 77% and 83%, while, diagnostic efficacy was 90% and 93.3% for SAWA detecting IgG and IgG4, respectively. On the other hand, testing patient sera in ELISA using E/S as Ag for detection of reactive IgG or IgG4 indicated that 35 and 37 patients were recorded positive, with sensitivities reaching 87.5% and 92.5% respectively. Again none of the negative control reactions exceeded the cutoff values indicating 100% specificities for both tests. Negative predictive and diagnostic efficacy values were increased with E/S Ag to be 80%, 87% and 92%, 95% with IgG and IgG4, respectively. Finally, testing patient sera in ELISA using 27-29 kDa as Ag for capture of IgG or IgG4 resulted in 36 and 39 positive OD exceeding the cutoff values for both tests with sensitivities of 90% and 97.5% respectively. Also, none of the negative control sera reacted with OD exceeding the cutoff values for both tests with specificities of 100% for both tests. Negative predictive and test efficacy values recorded the highest percentage with 27-29 kDa Ag proving its potency in detection of both total IgG and IgG4 isotype Abs recording 83%, 95% and 93.3%, 98.3%, respectively (Table 1). In conclusion, combining the detection of IgG4 against 27-29 kDa Ags in ELISA resulted in a reliable test for S. haematobium diagnosis indicated by the highest sensitivity, negative predictive value and diagnostic efficacy. DISCUSSION Schistosomiasis diagnosis is usually dependent on the detection of Schistosoma eggs in samples of feces and urine. Nevertheless, the erratic egg laying cycles of the parasite and the low and fluctuating excretion of eggs in stool or urine of the host (Hillyer, 1998) resulting the low sensitivity (do not exceed 30%) of the parasitological examination and accordingly in misdiagnosis of many infected patients, which finally lead to patient mistreatment and continuity of the transmission of the disease. Immunodiagnosis of the disease based on the detection of anti-parasite antibodies or circulating antigens of the parasite in patient’s body fluids is currently the best way to increase the chance for the detection of misdiagnosed cases by parasitological examination; moreover immunodiagnosis could be used more effectively than conventional diagnosis for epidemiologic studies. Up-to-date, ELISA is the technique of choice over several other techniques like hemagglutination, immunofluorescence, and the circumoval precipitin test concerning reliability, field applicability and need for sophisticated methodology. So, it is essential to establish a reliable ELISA test aiming at the diagnosis and seroepidemiology of the disease (Rokni et al., 2002; Hadighi et al., 2003). Hence, many ELISA tests have been developed by a lot of investigators around the world for the immunodiagnosis of parasitic diseases, especially for schistosomiasis. Ags used in such ELISAs ranged greatly from crude antigenic preparations, purified Ags and even recombinant Ags. Although, the use of crude Ags from adult worms and eggs have a high sensitivity and specificities (healthy non-endemic negative controls) for the diagnosis of infection (Noya et al., 1995); however, cross reactivity with other parasites resulting in false positives for infections with other trematodes (Fasciola), intestinal nematodes (especially hookworms) and cestodes (Tenia) (Noya et al., 2002). Such cross-reactivities result in the decrease of the specificities of such assays when used in endemic areas where polyparasitism is common in many resident individuals (Tchuem Tchuente et al., 2003). The main reason of using crude Ags from adult worms over using Ags from E/S or eggs is their greater facility and their high yield of antigenic material (Hamilton et al., 1998). The present study was performed as an attempt to compare the diagnostic efficacy of S. haematobium SAWA, E/S and CP Ags in Ab diagnosis of S. haematobium infection in human. Several authors investigated the potential diagnostic efficacy of tests using many purified schistosomal Ags, such as CEF6 (Ghandour et al., 1997), cathodic circulating Ags (Qian and Deelder, 1993), and the recombinant Ags rTEG of S. japonicum (Li et al., 1999), 34, 35 and 38 kDa (Mikhail et al., 1997) and r22 kDa of S. mansoni (Pardo et al., 2004) have demonstrated acceptable sensitivities, although they are less than those of tests using the crude Ags from adult worms and eggs. In the present study, we found the reactivity of parasitologically confirmed S. haematobium patient sera in ELISA for the detection of total IgG and IgG4 isotype using crude antigenic preparations (SAWA and E/S) as Ags showed that this assay is highly sensitive with a diagnostic efficacy of 90%, 93.3% and a negative predictive value of 77%, 83% for SAWA, and 92%, 95% and 80%, 87% for E/S respectively with total IgG and IgG4; these results indicated that detection of IgG4 isotype is more effective in immunodiagnosis of S. haematobium than the detection of IgG Abs using SAWA or E/S as Ag. Our data confirm and extend those of Feng et al. (2000), De Oliveira et al. (2003), Sorgho et al. (2005), Bahgat et al. (2006) and Planchart et al. (2007) who reported acceptable sensitivity of assays using crude SAWA and worm vomit Ags for the detection of Abs in S. mansoni patient sera and better sensitivities were reached (94%) for the detection of IgG4 isotype in individuals with low worm burden. Also, it has been reported that, detection of IgG4 isotype against Fasciola worm crude antigenic preparation is more specific in immunodiagnostic assays for fascioliasis than other IgG isotypes (Maher et al., 1999). Moreover, another advantage for the detection of IgG4 over total IgG has been reported recently by Mohanty et al. (2006) indicating that elevated Ag-specific IgG4 Abs in the blood of tested individuals were the indicators of circulating parasite in infected individuals who do not have apparent clinical symptoms of Schistosomiasis haematobium. In an attempt to improve the immunodiagnosis of S. haematobium we used the purified 27-29 kDa Ag in ELISA for the detection of total IgG and IgG4 isotype in patient sera. Our data indicated that, the use of the 27-29 kDa Ag was more efficient in the immunodiagnosis of schistosomiasis haematobium over other crude antigenic preparations as indicated by the highest sensitivity, negative predictive value and diagnostic efficacy reaching 90%, 83%, 93.3% for IgG detection and 97.5%, 95%, 98.3% for IgG4 detection. Again, combining the detection of IgG4 isotype with the purified Ag in ELISA resulted in the highest diagnostic efficacy for the disease. Our data confirm and extend those reported earlier using purified Ags for the improvement of the immunodiagnostic efficacy of ELISA assays for S. mansoni RP26 (Makarova et al., 2005; Gonçalves et al., 2006), cathepsin L1 (CL1) (Rokni et al., 2002), Sm 31/32 (Ruppel et al., 1990), Sm31 and Sm32 (El-Sayed et al., 1998), intestinal CPs Sm31/Sm32 (Planchart et al., 2007), 31/32 KDa (Li et al., 1997; Zhang et al., 1999) and Fasciola (26-28 kDa (Attallah et al., 2002), Fas2 (Espinoza et al., 2005; 2007), cathepsin-L CP (Raina et al., 2006), 27-kDa (Tantrawatpan et al., 2003) who reported that using those purified Ags for the detection of IgG4 resulted in better sensitivities for the detection of the parasitic infection even with the absence of eggs in patient excreta and clinical signs of the disease. The 27-29 kDa molecules have been reported earlier as the S. haematobium CP enzyme (ShCP3) based on its similarities to a 28 kDa CP from S. mansoni (Chapell and Dresden, 1987; Rege et al. (1992). Also, the CL of F. hepatica previously characterized by Wijffels et al. (1994) had a similar MW of our purified Ag in the range of 29 kDa. Accordingly, we suggested that our purified Ag might be the S. haematobium CP reported earlier. Moreover, we aim to further characterize the 27-29 kDa purified Ag by determining its enzymatic activity in future studies. The immunodiagnostic potential of different CP molecules from S. mansoni and Fasciola adult worms have been studied by many authors who indicated that these molecules had a significant sensitivities and specificities for the immunodiagnosis of schistosomiasis mansoni and fascioliasis (Rokni et al., 2003; Planchart et al., 2007) comparable to our results. From the data of the present study, we can conclude that combining the detection of IgG4 isotype using the 27-29 kDa Ag in sera of schistomiasis haematobium patients in an ELISA test could represent an effective immunodiagnostic tool for the detection of the disease in low worm burden population. In addition, this test could be useful in sero-epidemiolocal studies in low endemic areas and in diagnosis of the disease in travelers from non endemic countries visiting schistosomiasis endemic areas. Our future plans will concentrate on more characterization of the 27-29 kDa purified Ags and on the application of our developed test for a 10% survey in an endemic area and to study the specificity of the assay using sera from patients harboring parasite other than S. haematobium.

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Figure (1). Gel profile of adult S. haematobium worm antigens. SDS-PAGE (12% gel, under reducing conditions) of SAWA (5 mg), E/S and CP antigens. Migration position and molecular mass (m, in kDa) of prestained low M.w protein standards (Bio-Rad) are shown on left. Table 1: Sensitivity and diagnostic efficacy of anti-Schistosoma IgG and IgG4 isotype in ELISA using different S. haematobium antigens.

Total IgG (N=40) IgG4 (N=40) -ve -ve Schistosoma COV +ve* S DE COV +ve* S DE PV PV antigens

SAWA 0.31 34 85.0 % 77% 90.0% 0.43 36 90.0 % 83% 93.3%

E/S 0.40 35 87.5 % 80% 92.0% 0.44 37 92.5 % 87% 95.0%

CP 0.42 36 90.0 % 83% 93.3% 0.47 39 97.5 % 95% 98.3%

N : Number of S. haematobium parasitologically positive patients. COV : Cut off value +ve* : Number of true positive cases of OD above Cutoff values detected by ELISA. S : Sensitivity -ve PV : Negative predictive value DE : Diagnostic efficacy Total IgG IgG4 isotype

(A) (B)

(C) (D)

27-29 kDa

(E) (F)

Figure (1): Determination of total IgG and IgG4 isotype levels against different S. haematobium Ag preparations in ELISA against different S. haematobium Ags (SAWA, E/S and 27-29 kDa) in schistosomiasis patients. Absorbance values were determined at 492 nm. The ELISAs were performed in triplicate and the results represent averages of the results. Vertical bars represent the SD while, horizontal lines indicate the cutoff values= mean O.D obtained from healthy control+2SD.