Effective diagnosis of schistosomiasis haematobium by Silver beads Sandwich ELISA

Schistosomiasis is a major public health problem. Diagnosis by simple and rapid immunoassays is a priority. The magnetic bead immunoassay using magnetic nanoparticles conjugated with anti-schistosomal antibody was evaluated for diagnosing human schistosomiasis infection. The present study was to evaluate the sandwich ELISA as a simple test for the detection of schistosomal antigen (CSA) in serum and urine samples of S. haematobium patients and compare it with ELISA. Investigation conducted on eighty six cases divided to three groups, 34 were positively for Schistosoma haematobium , 32 were positively for intestinal parasites ova and negative for S. haematobium ova in urine and 20 were negative urine and stool examination (Control). Immuno-magnetical bead based Enzyme-linked immunosorbent assay (ELISA) using for detected for antigen in sera and urine infected by S. haematobium . Sandwich ELISA sensitivities was 79.4% (serum) and 73.5% (urine) and which increase by used nano-sandwich ELISA to 88.2% (serum) and 82.4% (urine), respectively. Sandwich ELISA speci(cid:976)icities was 86.4% (serum) and 80.8% (urine) and which increase by used nano-sandwich ELISA to 93.3% (serum) and 88.5% (urine). We found that, nano-sandwich ELISA assay had highly sensitive and speci(cid:976)ically and technical method was applicably, fast, cheaper, accurate and promising diagnostic method for schistosomiasis.


INTRODUCTION
Schistosomiasis considered as one of the fundamental word related ailments obtained by man through exercises related with freshwater, for example, cultivating, washing, washing and amusement. It has been additionally perceived as an ailment of critical inancial and general wellbeing signi icance second just to jungle fever. Urinary schistosomiasis stays signi icant wellbeing trouble in ailment endemic zones of Africa and the Middle East particularly Egypt, in luencing in excess of 110 million individuals in provincial, agrarian, and periurban zones (Abdel-Wahab et al., 2000). In Egypt, urinary schistosomiasis is nevertheless representing a serious itness problem to deal with. Due to manage programs over the remaining decade, a decline in the occurrence of human schistosomiasis in Egypt has been reported, however the disease is still endemic in many foci (Contis and David, 1996;Al-Sherbiny et al., 1999). Detection of schis-tosomal circulating antigens looks to be an effective method for discriminate among preceding exposure and current infections. Schistosome antigens detecting as alternative to egg counting in urine (El-Shafei et al., 2002;Wilson et al., 2007). S. haematobium infections, egg antigens might need to more establishing in full-size amounts even when eggs counts degrees have been very low, Live eggs exercise of trapped in bladder wall (Bottieau et al., 2006;Demerdash et al., 1995). Sandwich ELISA technique are among the most commonly used assays in detection of schistosomal antigens proved to more sensitively and speci ically for antigen detection either in serum or in urine (Waugh et al., 2007). Detection of schistosomal circulating antigens appears to be an high quality method for discriminating among pre-exposure and contemporary infections. Schistosome antigens detection of taken distinguished area and may additionally be regarded as an alternative to egg counts in urine (Kahama et al., 1999). S. haematobium infecting, eggs antigens ought to be validated in quantity even egg rely stage was very low, might be related to live eggs activities trapped in bladders wall (Doenhoff et al., 2004) . Immunomagnetic beads were uniform, polymer particles coated by polystyrene shell which provided smooth hydrophobic surface to facilitate physical absorption of molecules (Gessler et al., 2006;Gundersen et al., 1992;Conlan et al., 2009). Our target is evaluating prepared S. haematobium antigen roles in detecting active schistosomal infections throughout raising anti-S. haematobium pAb using sandwich and nano-ELISA.

Study population
Our infestation conduct on S. haematobium infected cases from many endemic places in Egypt (El Fayoum, El-Sharkia and Kafr-EL-Shekh). The study was held in the period from April 2016 to June 2017. In the present study, a number of 86 individuals were enrolled. All cases and healthy volunteers subjected to medical and repeated parasitological stool examinations using Kato-Katz approach (Kongs et al., 2001), Merthiolate-Iodine-Formaldhyde concentration (MIFC) method and formol ether attention strategies (Knight et al., 1976). Urine evaluating perform for all groups and using sedimentation approach (Lier et al., 2009), nucleopore iltration techniques (Lengeler et al., 1993). Ethical problems handled in accordance to International Ethical Guidelines for Biomedical Research. Group A, S. haematobium infected (34), based on presences of Schistosoma egg in urine. Group B, Other para-sites than Schistosoma (32). Group C medical staff at Theodor Bilharz Research Institute (TBRI) served as parasite free-healthy negative control (20).

Preparation of antischistosomal worm antigen preparation polyclonal antibodies (Anti-SWAP pAbs)
Two rabbits were injected intramuscularly (i.m.), with 100µg of schistosomal worm antigen preparation (SWAP) with equal volumes from Freund's adjuvant. Then, 3 booster doses 0.5 mg SWAP with equal vol. of incomplete Freund's adjuvant give at 1 week. 1 week later, last booster dose, rabbit's sera obtained and polyclonal antibody (pAb) fractions were purify using 50% ammonium sulfate precipitation (McKinney and Parkinson, 1987). More puri ications from pAb by 4% caprilic acid.

Preparation and characterization of puri ied SWAP.
Adult clean schistosoma worms homogenized in 2 vol. from 20 mM Tris-Hcl buffer and centrifuged at 30.000 rpm / 30 min. Entire homogenization and centrifugation process was performed at 4 • C. Supernatant fraction were decant and assay to determination protein and store later on −20 • C till using. SWAP purify from the CPE by combination of ion-exchange chromatography on diethyl aminoethyl (DEAE)-Sephadex A-50 and gel iltration by Sephacryl high resolution (HR)-200. Absorbances of every fractions were assessment at 280 nm and purit ied of production protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis u der reducing condition (Laemmli, 1970).

Sandwich ELISA
The reactivity of anti-SWAP was explored using Sandwich ELISA. S-ELISA test based on the original method (Engvall and Perlmann, 1971) was used with microplate modi ication (Elkawaz and Ghaffarifar, 2009). Plates coat by 100 µl/well from pAb anti-SWAP (20 µg/ml) in carbonate buffer (0.06M, pH 9.6) and incubating overnight. Plates wash thrice by 0.1 M phosphate buffered saline/ Tween-20 (PBS/ T), and wells block by 200 µl of 2.5% FCS and incubating on 37 o C / 2 hr. Washing 3lates tribal times and 100µl (serum) adding to every well and incubating (2 h /37 • C) then 100 µl of peroxidaseconjugated anti-SWAP pAb dilute 1/250 add and incubation (1 hr). 100 µl from freshly prepared substrate solution dispense in every well / 5 min., then 50 µl from stopping buffer add for stoping enzymesubstrate over reaction. Absorbance measure at 492 nm by ELISA reader. Cut off values calculated as average OD reading of negative control ± SD.

Statistics
Data analyses by use student's t-test. Standard formulas used to estimating sensitivities, speci icities, positive and negative predictive ratio, also, false positive and negative rate, P< 0.05 (Snedecor and Cochran, 1991).

Preparation of S. haematobium SWAP antigen
Puri ication of S. haematobium SWAP antigen was shown in Figure 1. The eluted antigen following puri ication by DEAE sephacryl S-200 gel iltration column chromatography is single peak with max. Optical Density (OD) equal to 1.25 at fraction number 10 (representing the fraction with the highest protein content).  Lane 3: Partially puri ied after homogenization.

Detection of S. haematobium SWAP antigen in serum samples
The anti-S. haematobium SWAP IgG-pAb were using to detecting S. haematobium SWAP in human samples serum and urine and employed as antigen capture and HRP pAb as conjugate in sandwich ELISA. The sandwich ELISA technique was used for preliminary standardization and optimization of different materials concentrations and dilutions before the application of the technique on humans' s era.
Cut off values were 0.302 through S. haematobium SWAP delectation antigen in serum, the results showing that 27 of 34 were positive cases (79.4 %) of group infected with S. haematobium, while 7 of 34 were negative cases (false negative result) (20.6%). In cases with other parasitic infection only three 3 had positively by Fasciola, 2 by H. nana, 2 by Ascaris and no positive result 1 by Hookworms, 25 cases were negatively. Healthy control are negatively (Table 1).

Detection of S. haematobium SWAP antigen in serum samples by Nano-sandwich ELISA
The calculated cut off OD value was 0.225. The presence of S. haematobium SWAP antigen in serum samples was evaluated using nano-silver sandwich ELISA method and OD value of S. haematobium infected group (1.02±0.18) signi icant higher comparing to parasites group. 30 given positive results from 34 cases and only 4 gave false negative. Assay sensitivities was 88.2%. 20 healthy control negatively being below cut off values of SWAP positivity give 100% speci icity of procedures, comparing to infected group. In group B, 4 cases were positively (2 with Fasciola, 1 with Ascaris and 1 case with hookworms infection), 56 were negative giving speci icity of the procedure of 93.3% ( Table 2).

The incidence of positivity
The sensitivity and speci icity, of the two techniques using to detected S. haematobium SWAP in human sera, where use nano sandwich ELISA increase the sensitivity and speci icity of the techniques. The sensitivity of sandwich ELISA was 79.4% in serum and it increases by used nano sandwich ELISA to become 88.2% in serum. The speci icity of sandwich ELISA was 86.6% in serum and it increases through use nano-sandwich ELISA to become 92.3% in serum (Table 3).

DISCUSSION
In order to evaluate the different immunodiagnostic antigen detection assays, selection of a proper antigen and its puri ication followed by propagation of its speci ic antibodies and puri ication were mandatory. Comparative evaluation of S. haematobium SWAP antigen detection with sandwich ELISA relation to nanodiagnostic assays sandwich enzymelinked immunosorbent assay based on immunomagnetic beads (IMB-ELISA) techniques) was performed. SWAP antigen was used in the present study because using soluble egg antigens (SEA) and crude worm antigen have many drawbacks. The crude worm antigen shows weak immunogenicity due to few T cell epitopes and also its inability to induce lymphocytic proliferation of humans in early stage schistosomiasis (Parizade et al., 1994;Ondigo et al., 2018). Also, SEA shows immunoregulation and suppression as the disease persists and progresses, so it may not be reliable also in late stages (Ridi et al., 1997;Chand et al., 2010) . Standardization of sand-wich ELISA used for detection of SWAP antigen was carried out before the application of the technique on human samples.
The puri ied IgG fraction of the rabbit sera was employed as both antigen capture and peroxidase conjugated detecting antibody. On detection of S. haematobium SWAP antigen in human serum samples by sandwich ELISA; 7 out of 34 schistosomiasis haematobium cases (group A) gave positive results with 79.4% sensitivity. In healthy control group (group C), no positive results were obtained giving a speci icity of 100% compared to this group. While 7 cases out of the 30 patients with other parasitic infection (group B) showed positive results giving a speci icity of 86.0%. Correlation was positively among S. haematobium eggs number in 10 ml urine and SWAP levels in sera of S. haematobium infect using sandwich ELISA.
Similarly, Salah et al. (2006) detected SEA in serum samples of S. haematobium infected patients. They reported that the procedure gave a sensitivity of 89% and a speci icity of 100% .also, in agreement with the current study, Elkawaz and Ghaffarifar (2009)

CONCLUSION
Conclusion use of nano bead immunoassay offered the potential advantage of sensitivity and speci icity of the assay that may be due to the use of magnetic beads which can utilize larger surface area with highly binding capacities and rapidly reactions kinetics of solution with simples separation of bound and unbound materials on solid phases and provide good chance of enhance sensitivity of antigen detecting. The IMB-ELISA appears to be a suficiently sensitive and feasible assay for detection of schistosomal antigenemia.

Funding Support
The authors declare that they have no funding support for this study.