Efficacy of Iron Oxide Nanoparticles in Diagnosis of Schistosomiasis

Document Type : Original Article

Author

• Ibrahim R Aly

Head of Parasitology Dep. Theodor Bilharz Research Inst.

Abstract

Highlights

INTRODUCTION

Schistosomiasis, which is also known as bilharziasis, is one of the most worldwide spread disease-causing the highest rates of morbidity and mortality after malaria. Schistosomiasis infects about 200 million people worldwide. The contamination takes place by getting in contact with fresh water contaminated with cercaria which penetrates the skin causing infection.¹ There are 3 main types of Schistosoma that cause human infections: S. mansoni, S. haematobium, or S.japonicum.² In Egypt, the urinary schistosomiasis is representing a serious health problem to deal with. Its complications add more burden on the national control programs and on the national economy.³ Due to controlling programs over the last decade, a decline in the prevalence of human schistosomiasis in Egypt has been reported, however the disease is still endemic in many foci.⁴ Schematically, there are three different known approaches for the diagnosis of parasitic infections, direct (detecting parasitic ova in stool or urine), indirect (relaying on biochemical assays) and immunological methods which are the most sensitive and specific methods in measure the immune response (antibodies) to certain parasitic antigens and/or detecting circulating parasitic antigens.⁶ Proteases and peptidases are proteolytic enzymes that operate in virtually every biological phenomenon. They function not only as individual enzymes but often in cascades or networks. They also provide essential functions in all life forms.⁷ Proteases operate at the host-parasite interface facilitating migration, digestion of host proteins and probably immune evasion.^8,9

This study aimed to determine the sensitivity and specificity of ELISA-based iron oxide nanoparticles, evaluate the efficacy of iron oxide nanoparticlesfor diagnosis of human schistosomiasis infections and to compare between ELISA-based iron oxide nanoparticle and traditional sandwich ELISA.

MATERIALAND METHODS

Animals: New Zealand white male rabbits were purchased from Agriculture Faculty, Cairo University (Giza, Egypt), weighing approximately 1.5 Kg and 2 months old. They were examined and confirmed to be free from parasitic infections. The rabbits were housed in the animal house at Theodore Bilharz Research Institute (TBRI) (Giza, Egypt), under standard laboratory care at 21°C, 16% moisture, the animals were supplied with filtered water with salts 1cm/5 liter and vitamins 1cm/10 liter, also their diet hold a minimum of 15% protein, 3% fat and 22% fiber. Internationally valid guidelines were applied to animal experiments.

Parasites: Adult worms of Schistosoma haematobium used for the preparation of purified tegumental antigen 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.¹⁰

Study population: A total of 128 individuals were enrolled in the present study. Thirty individuals were free from any parasitic infections served as normal control. Sixty patients infected with S. haematobium and 38 patients infected with other helminthic parasites (It included 10 patients infected with H. nana, 8 with Fasciolagigantica, 8 with Ascarislumbricoides and 12 with Ancylostomaduodenale).

Preparation and purification of tegumental from S. haematobium.

Homogenization:

Freshadult worms ofSchistosoma haematobiumweresuspended in 10 ml of phosphate buffer saline (PBS) anddialyzed against lysis buffer [8 M Urea, 2MThiourea,4%3,3,Cholamidopropyl,dimethylammonium, propanesulfonate (CHAPS),50 Mdithiothreitol (DTT),20 M MTris and complete Mini Protease Inhibitor Cocktail Tablets (Roche)].After dialysis, the adult worms were homogenizedunder continuous agitation for 2 hours at room temperature

witha glasshomogenizer,followed by 10 repeated

passagesthrough a 30-gauge hypodermicneedle.Thenthe homogenate was centrifuged at 20,000×g for 30 min at25°C and the supernatant was collected and stored at −70°C.

Purification of schistosomal antigen was done by DEAE Sepharose CL-6B and the protein content was estimated by the Bio-Rad kit. Characterization of schistosomal antigen was done using Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Harlow & Lane¹¹and Myers.¹²

Assessment of reactivity of the schistosomal antigen of S.haematobium by indirect ELISA: This method was performed, with some modifications from the original method of Engvall and Perlmann.¹³

Immunization of rabbit for production of polyclonal antibodies: Rabbit anti-serum was obtained by immunizing New Zealand white rabbit (approximately 1.5Kg weight) with 1mg of purified schistosomal antigen that was given to the rabbit in the entire course of immunization. The rabbit received priming dose intramuscular injection (i.m) at four sites (1mg purified schistosomal antigen mixed 1:1 incomplete Freund's adjuvant (CFA), (Sigma).

Three booster doses were given, each was 0.5 mg antigen emulsified in equal vol. of incomplete Freund's adjuvant (IFA), (Sigma). The first boosting was two wk. after the priming dose. The following boosting doses were given at weekly intervals, according to Fagbemi.¹⁴

The rabbit was bled for collection of serum one week later after preliminary testing of titer by indirect ELIZA.

Purification of rabbit anti- purified schistosomal antigen serum: Rabbit IgG purification steps were based on two different methods: Ammonium sulfate precipitation method¹⁵ and caprylic acid treatment.¹⁶

Protein content was measured after each purification step using the Bradford method.¹⁷ The efficiency of the purification steps was measured by 12% SDS-room temperature. The plates were washed 5 times with

washing buffer. Hundred μl/well of substrate solution [one tablet of OPD (Sigma) dissolved in 25 ml of 0.05 M phosphate citrate buffer, pH 5 with urea hydrogen peroxidase (Sigma)] was added to each well and the plates were incubated in the dark at room temperature for 30 min. Fifty μl/well of 8 N H₂S0₄ was added to stop the enzyme-substrate reaction. The absorbance was measured at 492 nm using ELISA reader (Bio-Rad microplate reader Richmond, Ca).

Statistical analysis: Data are expressed as mean (M) ±standard deviation (SD). Statistical analysis was performed with the aid of the SPSS computer program (version windows 16.0).

RESULTS

Characterization of S. haematobium membrane antigen by (SDS-PAGE)

The protein fractions resulted from the preparation methods were analyzed by 12.5% SDS-PAGE under reducing condition and showed 2 major at 65 and 95 KDa and many minor bands representing S. haematobium membrane antigen (Fig. 1).

Fig. 1: SDS-PAGE of S. haematobium membrane antigen

Lane 1: Molecular weight of the standard protein

Lane 2: Crude S. haematobium antigen

Lane 3: Purified S. haematobium membrane antigen

Assessment of reactivity and specificity of the prepared S. haematobium membrane antigen by indirect-ELISA

The antigenicity of the S. haematobium membrane antigen was tested by indirect ELISA technique. Serum samples from an infected human with S. haematobium gave a strong reaction against S. haematobium membrane antigen with mean OD reading equal to 0.929 and no cross-reactions were recorded with sera of patients infected with other parasites e.g., Fasciola, H. nana and Ascaris (Table 1).

Table 1: Reactivity of S. haematobium membrane antigen by indirect ELISA

pAb titers detection and testing for reactivity against S. haematobium membrane antigen by indirect ELISA

An increasing antibody level started 1 wk after the first booster dose. Three days after the 2^nd booster dose immune sera gave a high titer against S. haematobium membrane antigen with OD of 2.97 at 1/250 dilution (Fig. 2).

Fig. 2:  Reactivity of immunized rabbit anti- S. haematobium membrane antigen antisera (diluted 1/250) against S. haematobium membrane antigen by indirect ELISA

Protein content determination of the purified anti-S.haematobiumIgG-pAb

The total protein content of the rabbit’s crude serum-containing anti-S.haematobiumpAb was 7.2 mg/ml. Using the 50% ammonium sulfate precipitation method, the protein content was 5.1 mg/ml, while following a 7% caprylic acid precipitation method, the content dropped to 3.5 mg/ml. Finally, the protein content of the highly purified anti-S.haematobium IgG-pAb subjected to ion-exchange chromatography method (DEAE Sephadex A-50 ion exchange column chromatography) was 2.1 mg/ml.

Figure (3) shows the OD₄₉₂ profile of the IgG fractions obtained following purification by DEAE Sephadex A-50 ion-exchange column chromatography. The eluted IgG is represented by a single peak with a maximum OD value 0.48 at fraction number 17.

Fig. 3: OD₂₈₀ profile of fraction obtained following rabbit’s anti-S.haematobium IgG-pAb purification by DEAE ion-exchange chromatography

Characterization of anti-S.haematobium IgG-pAb by SDS-PAGE

The purity of IgG-pAb after each step of purification was assayed by 12% SDS-PAGE under reducing conditions. Analysis of 50% ammonium sulfate-precipitated proteins by 12% SDS-PAGE under reducing conditions showed that precipitated proteins appeared as several bands.

The purified IgGpAb after ion exchange chromatography was represented by only 2 bands, Land H-chain bands at 31 and 53 kDa, respectively. The pAb appears free from other proteins (Fig. 4).

Fig. (4): 12.5% SDS-PAGE of anti-S. haematobiumIgG-pAb before and after purification

Lane 1: Molecular weight of the standard protein

Lane 2: Crude anti- S. haematobium membrane antigen IgG-pAb (before purification)

Lane 3: Precipitated proteins after 50% ammonium sulfate treatment

Lane 4: Purified IgG-pAb after 7% caprylic acid treatment

Lane 5: Purified IgG-pAb after ion-exchange chromatography.

Assessment of specificity of the purified pAb by indirect ELISA

The produced anti- S. haematobium membrane antigenIgG-pAb diluted 1/250 in PBS/T buffer gave strong reactivity to S. haematobium membrane antigen. The OD means reading at 492 nm for S. haematobium membrane antigen was 2.84 compared to 0.24, 0.19, 0.31 and 0.41 for Fasciolia, H.nana, Ancylostoma and Ascaris infected sera, respectively (Table 2).

Table 2: Specificity of rabbit anti- S. haematobium membrane antigen IgG-pAb against different parasitic antigens by indirect ELISA

Detection of S. haematobium membrane antigen in serum samples by sandwich ELISA

Table 3: Results of S. haematobium membrane antigen detection in serum samples by sandwich ELISA

Detection of S. haematobium

 membrane antigen in serum samples conjugated with IO by sandwich IMB -ELISA

The calculated cut off OD value was 0.282. The presence of S. haematobium membrane antigen in serum samples of the different studied groups was evaluated by the nanomagnetic beads method. The mean OD value of the S. haematobium infected group (1.97±0.22) was significantly higher than that of other parasites group (group B).

Out of 60 schistosomiasis cases, 57 cases gave positive results, while 3 cases gave negative results, giving a sensitivity of 95%. All the 30 healthy controls (group C) were negative being below the cut off value for S. haematobium membrane antigen positivity giving a 100% specificity. In group B (patients with other parasitic infections), only 5 cases were detected as positive (2 with Fasciola, 1 with Ascarislumbricoides, 1 case with H. nana and 1 case withAncylostomaduodenale infection), while the other 33 cases were negative giving specificity of the procedure of 92.6% to group B. The P-value was < 0.001 which means that there is a statistical significance in positivity between S. haematobiuminfectedgroup and other tested groups (Table 4).

Table 4: Results of S. haematobium membrane antigen detection in serum samples conjugated with IO by sandwich IMB-ELISA

Table (4) shows the incidence of positivity for antigen detection in human sera by the two diagnostic techniques used in the study. The two techniques illustrated a high percentage of positivity reaching 100% in serum of the high infection subgroup. In the moderate infection subgroup, the incidence of positivity for antigen in serum was 80%, 92%, 84% and 96% by sandwich ELISA, sandwich IMB-ELISA, respectively. Whereas, in the light infection subgroup, the incidence of positivity for antigen in serum was 80%, 92%, 84% and 96% by sandwich ELISA, sandwich IMB-ELISA, respectively.

The other parasites-infected groups mainly with Fasciola gigantica showed some degree of cross-reactivity with antigens in human sera.

DISCUSSION

Schistosomiasis is one of the main occupational diseases, acquired by man through activities associated with freshwaters such as farming, washing, bathing, and recreation. It has been recognized as a disease of significant socio-economic and public health importance second to malaria ²⁰. Urinary schistosomiasis remains a major health burden in endemic areas of Africa and the Middle East, affecting more than 110 million people in rural, agricultural and peri-urban areas ²¹. Estimates show that at least 220.8 million people required preventive treatment in 2017, out of which more than 102.3 million people were reported to have been treated. Preventive treatment, which should be repeated over a number of years, will reduce and prevent morbidity. Schistosomiasis transmission has been reported from 78 countries. However, preventive chemotherapy for schistosomiasis, where people and communities are targeted for large-scale treatment, is only required in 52 endemic countries with moderate-to-high transmission.²⁰

In Egypt, schistosomiasis is still representing a serious health problem to deal with. Its complications add more burden to

the national control programs and on the national economy. Due to control programs over the last decade, a decline in the prevalence of human schistosomiasis in Egypt has been reported, however, the disease is still endemic in many foci. ^3,4 Schistosomiasis haematobium was highly prevalent (60%) both in the Nile Delta and Nile Valley South of Cairo in districts of perennial irrigation while it was low (6%) in districts of basin irrigation. Schistosoma mansoni infected 60% of the population in the Northern and Eastern parts of the Nile Delta and only 6% in the Southern part.^21,22

The present work aimed to evaluate the efficacy of iron oxide nanoparticles for diagnosis of human Schistosomiasis infections, determine the sensitivity and specificity of ELISA-based iron oxide nanoparticles and compare between ELISA-based iron oxide nanoparticles and traditional sandwich ELISA.

The two Schistosomiasis antigens were used in the production of anti-Schistosomiasis IgG antibodies. Their antigenicity was tested by indirect ELISA technique and purified Schistosomiasis antigens gave positive reaction against Schistosomiasis infected sera and no cross-reactions against other parasites sera. The stronger reactivity was in favor of purified antigen.

The recorded sensitivity of sandwich ELISA using anti-crude Schistosomiasis IgG alone or conjugated with IO-PAbs was 85% and 95%, respectively and their specificities were 88.3% and 93.3%, respectively. As noted, higher diagnostic indices (sensitivity and specificity) were recorded in IO conjugated IgGs than traditional IgGs ELISA models.

In this study, the use of IO conjugated with IgG led to increasing the number of their binding sites and adsorptive capacity. This was attributed to their small size. Many authors used novel nano-diagnostic assays in the diagnosis of many parasitic infections, e.g., Schistosoma japonicum ²³, Schistosoma haematobium.²⁴

Lei et al.^{. 23} developed ELISA assay using IgYpAb-coated with a magnetic bead as a capture Ab and IgGm Ab as antigen-detection Ab against S.haematobium. The recorded sensitivity was 100% and 91.5% (in acute and chronic infection, respectively) and specificity 96.7%-100% with clonorchiasis and paragonimiasis positive cases.

Ibrahim et al. ²⁴ also developed a novel IMB-ELISA based IgG for the detection of excretory/secretory antigens in rabbit sera infected with S. haematobium. The reported sensitivity and specificity of the assay were 95% and 93.7%.   They used magnetic microbeads based-sandwich ELISA for the detection of S. haematobium circulating microsomal fraction antigen. The reported sensitivity and specificity of the assay were 96.5% and 96.3% compared to 88.2% and 87.3% by traditional sandwich ELISA.

It was noted that the application of the nanomagnetic beads significantly increased the sensitivity, specificity, and incidence of positivity of the technique towards higher detection of the antigen in moderate and light infected patients. The sensitivity of the traditional sandwich ELISA was 85% in serum and it increased by using the sandwich IMB-ELISA to be 95% in serum. The specificity of sandwich ELISA was 88.3% in serum and it increased by using the sandwich IMB-ELISA to be 93.3% in serum.

CONCLUSION

The data obtained concluded that the IMB-ELISA appears to be a sufficiently sensitive and feasible assay for the detection of schistosomal antigenemia and the evaluation of its potential use in human schistosomiasis needs more research.

Keywords

• S. haematobium
• Diagnosis
• Immunomagnetic bead ELISA technique – Paramagnetic nanoparticles