Benha Medical Journal

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 35  |  Issue : 2  |  Page : 134--138

Evaluation of nanogold particles-based enzyme-linked immunosorbent assay for detection of hydatidosis


Samia M Rashed, Mona E Naser, Ibrahim R Bayoumi, Nagwa S.M. Aly, Waleed E Mohamed, Amira S ElGhannm 
 Parasitology Department, Benha University, Benha, Egypt

Correspondence Address:
Dr. Amira S ElGhannm
Banha, Qalubia Governorate, 13511
Egypt

Abstract

Background Use of nanotechnology in clinical diagnosis meets the demands for increased sensitivity and early detection in less time. Purpose The aim of this study was to evaluate the nanogold particles-based dot-enzyme-linked immunosorbent assay (ELISA) as a test for detection of protoscolices antigen in serum samples of infected animals in comparison with traditional dot-ELISA. Methods A total of 76 blood samples were collected and included in the study: 36 sample of hydatidosis confirmed cases, 20 samples infected with other parasitic infection except hydatidosis as positive controls, and 20 samples as negative controls. Dot-ELISA was applied using two polyclonal antibodies against protoscolices antigen, the purified immunoglobulin G (IgG) polyclonal antibodies, and peroxidase-conjugated IgG, whereas in the nanogold dot-ELISA, the purified IgG polyclonal antibodies were conjugated with nanogold particles. Results On detection of protoscolices antigen by dot-ELISA, 31 (86.1%) of 36 serum samples were found to be positive, whereas nanogold dot-ELISA gave 34 (94.4%) positive serum samples. Dot-ELISA with nanogold particles had higher values than dot-ELISA regarding sensitivity (94.4 vs. 86.1%), positive predictive value (94.4 vs. 93.9%), negative predictive value (78.3 vs. 90%), and accuracy (92.9 vs. 87.5%), but specificity (90%) was the same for both tests. Conclusion Nanoparticles-based dot-ELISA is superior over traditional dot-ELISA for the detection of protoscolices antigen in hydatidosis. Dot-ELISA is rapid and easy to perform and the results can be read with the naked eye, so it does not require expensive equipment.



How to cite this article:
Rashed SM, Naser ME, Bayoumi IR, Aly NS, Mohamed WE, ElGhannm AS. Evaluation of nanogold particles-based enzyme-linked immunosorbent assay for detection of hydatidosis.Benha Med J 2018;35:134-138


How to cite this URL:
Rashed SM, Naser ME, Bayoumi IR, Aly NS, Mohamed WE, ElGhannm AS. Evaluation of nanogold particles-based enzyme-linked immunosorbent assay for detection of hydatidosis. Benha Med J [serial online] 2018 [cited 2018 Oct 17 ];35:134-138
Available from: http://www.bmfj.eg.net/text.asp?2018/35/2/134/239181


Full Text



 Introduction



Hydatidosis or cystic echinococcosis is a zoonotic parasitic disease, caused by infection with larval stage of Echinococcus granulosus. Although the disease occurs worldwide, it is endemic in Africa, South America, Europe, and Asia [1]. Mortality from hydatidosis is usually owing to the development of complications and is reported to be 2–4% [2]. The disease course is typically slow, and the diagnosis is often incidental owing to nonspecific symptoms [3]. Diagnosis is achieved by a combination of serologic tests and imaging, usually in conjunction with a history of exposure or immigration from an endemic area [4]. Many immunological assays have been developed for detection of antihydatid cyst antibodies and also for detection of hydatid antigens in the serum [5]. These include indirect hemagglutination, indirect immunoelectrophoresis, and enzyme-linked immunosorbent assay (ELISA) [6]. One of the main problems of antibody detection is that ∼40% of the surgically confirmed patients fail to show antibodies by various techniques [7]. Moreover, the antibodies may persist for a long time, even after removal of hydatid cyst by surgery or after clinical cure by chemotherapy [8]. So, the antibody detection assay cannot discriminate between the past and present infections in hydatidosis [5].

The circulating hydatid antigen is present in the active or recent infection and is absent in patients treated with surgery or chemotherapy Therefore, demonstration of the circulating antigen in the serum may indicate recent and active infection and may help in monitoring the efficacy of chemotherapy [9]. Detection of circulating antigens for hydatidosis has been confirmed by previous studies [10]. Nanodiagnostics involve the use of nanotechnology in clinical diagnosis to meet the demands for increased sensitivity and early detection in less time [11]. Nanomaterials have a large surface area, which enables attachment of large number of target specific molecules of interest for ultrasensitive detection. Conventional methods are limited to achieve this ultrasensitivity. Nanotechnology enables detection of a few microorganisms or target molecular analytes specific to pathogens. Moreover, it could allow rapid and real-time detection of the pathogens with relatively small sample volumes [11].

 Methods



Study groups

This study was conducted on 76 camel serum samples as follows: group 1 included 36 blood samples collected from lungs of naturally infected camels with hydatidosis and slaughtered at the abattoir of Toukh, Qalyubia Governorate; group 2 included 20 blood samples collected from camels infected with other parasitic diseases such as fascioliasis (positive control); and group 3 included 20 blood samples collected from parasite-free camels served as a negative control. The study was approved by the Research Ethical Committee of Benha faculty of medicine.Blood samples collection: Five microliters of blood was collected from each case. They were allowed to clot at room temperature for 2 h. Sera were separated by centrifugation at 2000 rpm for 10 min and stored at −20°C until used.Preparation of protoscolices antigen: Hydatid cysts’ fluid was collected from hydatid cysts removed from lungs of infected slaughtered camels at the abattoir of Toukh, Qalyubia Governorate, and then centrifuged at 10 000 rpm at 4°C for 60 min. The supernatant was removed, and the deposit containing protoscolices was prepared according to the method of Rafiei and Craig [12].

Purification of protoscolices antigen by diethyl aminoethyl (DEAE) sephadex is an effective method for separating proteins based on their charge. The DEAE group maintains a constant positive charge that is neutralized by counter ions, usually chloride ions. Other anions are capable of competing for the positive DEAE group [13]. The protein content was estimated by Life Science (Research, Education, Process Separations, Food Science) Bio-Rad SNC. (Hercules, California, USA) protein assay [14].

Production of polyclonal antibodies (pAbs) by immunization of a New Zealand white male rabbit by 1 mg of purified protoscolex antigen mixed with equal volume of complete Freund’s adjuvant (Sigma-Aldrich, Inc., Taufkirchen, Germany) was prepared for intramuscular injection of the rabbit according to the following regimen: priming dose of 1-mg purified protoscolex antigen injected at two sites (at both legs). First booster dose (0.5 mg antigen) injected 2 weeks after priming dose. Two booster doses (0.5 mg antigen each) were injected at weekly intervals, according to Fagbemi et al. [15]. The rabbit was killed 3 days after the last dose to obtain the blood sample. Centrifugation of the blood was done at 4000 rpm for 15 min. Rabbit serum containing antiprotoscoleces immunoglobulin G (IgG)-pAb was separated and fractionated and kept at −20°C.

Purification of rabbit antiprotoscolices IgG-pAb was carried out by ammonium sulfate precipitation [16] followed by caprylic acid purification [17].

Assessment of reactivity of Echinococcus protoscolex antigen was done using indirect ELISA according to Engvall and Perlmann [18].

Conjugation of antiprotoscolices IgG-pAb with horseradish peroxidase was done using periodate method according to Tijssen and Kurstak [19].

Conjugation of the antiprotoscolices IgG-pAb with gold nanoparticles was done according to Hermanson [20].

Dot-ELISA (antigen detection assay) was performed according to Boctor et al. [21], for detection of circulating protoscolex antigens by double-antibody sandwich procedures. Dot-ELISA test depends on the binding of the purified anti-E. granulosus IgG-pAb to a sensitive surface of nitrocellulose (NC) membrane as a capture matrix. The antigen under test binds the antibody; the bound amount is estimated by binding of a second antibody coupled to an active enzyme (peroxidase-conjugated antibody). Upon incubation with a suitable chromogenic enzyme substrate, the amount of second (conjugated) antibody binding is directly proportional to the amount of substrate reaction, as measured by visual reading of the color intensity [21].

Optimization of the test: Bio-dot apparatus was used with dot format sample template connected to vacuum source. It has 96 wells, each with diameter of 3 mm arranged in eight rows and 12 columns. NC was used as a capture matrix, 9×12 cm sheet with a pore size of 0.2 µm (Bio-Rad).

Procedure

The prewetted NC membrane was transferred to the Bio-dot apparatus and washed once with coating buffer for 5 min. After removing the excess solution, by suction, the membrane coated with 10–50 μl/well of the purified anti-Echinococcus IgG-pAb was diluted in carbonate buffer (1/250, 500, and 1000), and then incubated for variable times. Excess solution was removed, and then membrane was washed three times with 100 μl PBS-T/well.Then blocking solution was applied (10–50 μl/well), and incubated at room temperature for 15–45 min. Positive and negative control reference samples were added, diluted 1/1–1/32 in the diluents-blocking buffer, and then incubated for variable times (15–45 min) and washed three times with 100 μl PBS-T/well.HRP conjugated pAb was used in three dilutions (1/100, 250, and 500) diluted in the dilutent-blocking solution and incubated for variable times, and then the NC membrane was removed from the Bio-dot apparatus and washed five times with 100 μl PBS-T/well each time, followed by two times washing with PBS only.3,3′-Diaminobenzidine substrate was applied by immersing NC membrane in substrate solution. The reaction was stopped, just after development of the color, with cold distilled H2O.

Detection of circulating protoscolex antigen in serum samples by nanogold dot-ELISA

The procedure is same as mentioned for dot-ELISA but with the use of nanogold particles-pAb as a coating capture antibodies and NGBs-HRP-pAb as the conjugate antibodies.

Statistical analysis

The statistical analysis was performed using statistical analysis system (statistical package for social science), version 16. Validity tests (diagnostic sensitivity, specificity, and positive and negative predictive values) were done for the obtained results [22].

 Results



On detection of protoscolices antigen by dot-ELISA, 31 (86.1%) of 36 serum samples were found to be positive, which is statistically significant, whereas nanogold dot-ELISA gave 34 (94.4%) positive serum samples, which is statistically significant. Nanogold dot-ELISA had higher values than dot-ELISA regarding sensitivity (94.4 vs. 86.1%), positive predictive value (94.4 vs. 93.9%), negative predictive value (78.3 vs. 90%), and accuracy (92.9 vs. 87.5%) but specificity (90%) is the same for both tests.

 Discussion



Hydatid disease is essentially a disease of poor people residing in rural areas; hence, there is a need for a simple, economic diagnostic immunoassay for use at the field level or in a rural health center with inadequate facilities [23].

The present study agreed with Abou-Elhakam et al. [24] who reported that E. granulosus protoscolex antigen was detected in patient sera using dot-ELISA, and 48 of 50 patients were positive to Echinococcus spp., with sensitivity and specificity of 96 and 94%, respectively. The positive predictive value was 94% and negative predictive value was 90%.

Diab et al. [25] reported that Fasciola antigen detection in serum of sheep by sandwich ELISA showed 94.8% sensitivity and 95% specificity, whereas dot-ELISA was found to have sensitivity of 98.9% and specificity of 98.3%. So dot-ELISA gives better sensitivity and specificity than sandwich ELISA and is an easy to perform method for the rapid diagnosis of fascioliasis.

Concerning cross-reaction in animal cases ([Table 1] and [Table 2]), on using both dot-ELISA and nano dot-ELISA for detection of hydatidosis in group 2 (positive control), two cases infected with Fasciola gigantica were recorded as false positive. Cross-reaction is not only restricted to species belonging to the same phylum but is also extended to helminthes of different phyla such as the high degree of cross-reactivity between sera of cases infected with Taenia solium, Hymenolepis nana, Toxoplasma gondii, and E. granulosus. Moreover, a cross-reaction between F. gigantica, Trichinella spiralis, and E. granulosus was recorded [26].{Table 1}{Table 2}

The superiority of nano-based ELISA over traditional ELISA in diagnosis of parasitic diseases may be explained by the ability of nanoparticles to interact with matter at the nanoscale; the development of nanotechnology materials could potentially extend subcellular and molecular detection beyond the limits of conventional diagnostic modalities [27]. The small size and shape of the nanobeads enable them to be uniformly disseminated within the sample, thus improving the effectiveness of the antibody conjugation, and consequently enhancing the sensitivity of antigen detection [28].

Dot-ELISA has several advantages over sandwich ELISA, that is, nitrocellulose papers spotted with antigen are stable for at least 3 months at −20°C, and the results can be read with the naked eye, thus an expensive spectrophotometer is not required. The test is applicable to diagnose in the field setting as well as in laboratories that are not well equipped [29].

Rogan et al. [30] stated that dot-ELISA was developed as a field test for the diagnosis of hydatidosis in Kenya with 94% sensitivity and 90.3% specificity for Echinococcus infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Mihmanli M, Idiz UO, Kaya C, Demir U, Bostanci O, Omeroglu S, Bozkurt E. Current status of diagnosis and treatment of hepatic echinococcosis. World J Hepatol 2016; 8:1169–1181.
2Junghanss T, da Silva AM, Horton J, Chiodini PL, Brunetti E. Clinical management of cystic echinococcosis: state of the art, problems, and perspectives. Am J Tropical Med Hygiene 2008; 79:301–311.
3Manzano-Román R, Sánchez-Ovejero C, Hernández-González A, Casulli A, Siles-Lucas M. Serological diagnosis and follow-up of human cystic echinococcosis: a new hope for the future? Biomed Res Int 2015; 2015:428205.
4Pakala T, Molina M, Wu GY. Hepatic echinococcal cysts: a review. J Clin Transl Hepatol 2016; 4:39–46.
5Sadjjadi SM, Sedaghat F, Hosseini SV, Sarkari B. Serum antigen and antibody detection in echinococcosis: application in serodiagnosis of human hydatidosis. Korean J Parasitol 2009; 47:153–157.
6Metwally DM, Al-Olayan EM. Serum antibody detection in Ecchinococcosis: specificity of hydatidosis enzyme-linked immunosorbent assay. Life Sci J 2013; 10:2107–2110.
7Parija SC. A review of some simple immunoassays in the serodiagnosis of cystic hydatid disease. Acta Trop 1998; 70:17–24.
8Ray R, De PK, Karak K. Combined role of Casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian J Med Microbiol 2002; 20:79–82.
9Sunita T, Khurana S, Malla N, Dubey ML. Immunodiagnosis of cystic echinocooccosis by antigen detection in serum, urine, and saliva samples. Trop Parasitol 2011; 1:33–38.
10Siracusano A, Loppolo S, Notargiacomo S, Ortona R, Teggi A, de Rosa F, Vicari G. Detection of antibodies against Echinococcus granulosus major antigens and their subunits by immunoblotting. Trans Roy Soc Trop Med Hyg 1991; 85:239–243.
11Aly I, Zalat R, El Aswad BW, Moharm IM, Masoud BM, Diab T. Novel nanomagnetic beads based − latex agglutination assay for rapid diagnosis of human Schistosomiasis haematobium. World Acad Sci Eng Technol 2013; 7:977–983.
12Rafiei A, Craig PS. The immunodiagnostic potential of protoscolex antigens in human cystic echinococcosis and the possibleinfluence of parasite strain. Ann Trop Med Parasitol 2002; 96:383–389.
13Smith AM, Dowd AJ, McGonigle S, Keegan PS, Brennan G, Trudgett A, Dalton JP. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol Biochem Parasitol 1993; 62:1–8.
14Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976; 72:248–254.
15Fagbemi BO, Obarisiagbon IO, Mbuh JV. Detection of circulating antigen in sera of Fasciola gigantica infected cattle with antibodies reactive with a Fasciola-specific 88-kDa antigen. Vet Parasitol 1995; 58:235–246.
16Nowotny A. Basic exercises in immunochemistry. New York, NY: Springer-Verlag Inc.; 1979. pp. 1-3.
17McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods 1987; 96:271–278.
18Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971; 8:871–874.
19Tijssen P, Kurstak E. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugate for enzyme immunoassay. Anal Biochem 1984; 136:451–457.
20Hermanson GT. Bioconjugate techniques, functional targets. 2nd ed. San Diego, CA: Academic Press; 2008. pp. 3-136.
21Boctor FN, Stek MJ Jr, Pitrt JB, Kamal R. Simplification and standardization of dot-elisa for human Schistosomiasis mansoni. J Parasitol 1987; 73:589–592.
22Gonzalez-Sapienza G, Lorenzo C, Nieto N. Improved immunodiagnosis of cystic hydatid disease by using a synthetic peptide with higher diagnostic value than that of its parent protein. J Clin Microbiol 2000; 38:397–398.
23Parija SC. Recent trends in the serodiagnosis of hydatid disease. Southeast Asian J Trop Med Public Health 1991; 22(Suppl):371–376.
24Abou-Elhakam HM, Farid AA, Mahana NA, Bauiomy IR, Elameer AM. Dot ELISA as a field test for hydatid diagnosis. J Egypt Soc Parasitol 2016; 46:441–452.
25Diab TM, Rabia I, Komy WEL, Amir AEL. Efficacy of sandwich and dot-ELISA in Diagnosis of fascioliasis using a pair of polyclonal antibodies against cathepsin L antigen in naturally infected sheep. Res J Parasitol 2011; 6:90–100.
26El-Moghazy FM, Abdel-Rahman EH. Cross-reaction as a common phenomenon among tissue parasites in farm animals. Glob Vet 2012; 8:367–373.
27Hu Y, Fine DH, Tasciotti E, Bouamrani A, Ferrari M. Nanodevices in diagnostics. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2011; 3:11–32.
28Chen L, Wei H, Guo Y, Cui Z, Zhang Z, Zhang XE. Gold nanoparticle enhanced immuno-PCR for ultrasensitive detection of Hantaan virus nucleocapsid protein. J Immunol Methods 2009; 346:64–70.
29Rokni MB, Samani A, Massoud J, Nasr MB. Evaluation of dot-ELISA method using excretory-secretary antigens of Fasciola hepatica in laboratory diagnosis of human fasciolosis. Iran J Parasitol 2006; 1:26–30.
30Rogan MT, Craig PS, Zeyhle E, Romig T, Lubano GM, Deshan L. Evaluation of a rapid dot-ELISA as a field test for the diagnosis of cystic hydatid disease. Trans R Soc Trop Med Hyg 1991; 85:773–777.