Human and camel cystic echinococcosis - a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification

Abstract

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22 kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22 kDa. The purified IgG PsAb had been resolved at two bands at 52 kDa and at 32 kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Keywords: Echinococcus granulosus; Indirect ELISA; PCR; Protein-A sepharose gel affinity chromatography; SDS-page; Sandwich ELISA.

Fig. 1

Phylogenetic analysis using the maximum likelihood method based on NAD1 gene for Echinococcus sp. Our obtained isolate is highlighted (red dot). There was a total of 459 positions in the final dataset. Our genotype clustered in a well-supported branch (bootstrap value 78) with other E. granulosus references. The scale bar represents a 5% nucleotide sequence divergence

Fig. 2

Electrophoresis profile of protoscoleces hydatid cyst antigens, 10% SDS-PAGE slab gel stained with Coomassie brilliant blue dye. A: Camel protoscoleces hydatid cyst antigens (CPsAg): Lane CPsAg and Human protoscoleces hydatid cyst antigen (HPsAg): Lane HPsAg. B: Purified IgG PsAb after precipitation with 50% ammonium sulfate and purified by Affinity chromatography on protein-A Sepharose gel column: Lane IgG PsAb. Molecular weight standard protein: Lane M

Fig. 3

Binding activities in purified IgG PsAb towards camel E. granulosus antigen (CPsAg) (A) and Human E. granulosus antigen (HPsAg) (B) by indirect ELISA, at two-fold serially dilutions of specific purified IgG PsAb.

Fig. 4

Detection of circulating E. granulosus antigen in sera of human (A) and camels (B) infected with E. granulosus or other parasites in comparison to negative controls sera by using Sandwich ELISA

Fig. 5

Detection of circulating E. granulosus antigen in random sera of human by Sandwich ELISA

Fig. 6

Detection of circulating E. granulosus antigen in random sera of camel by Sandwich ELISA