Evaluation of Recombinant Fragments of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit for Serodiagnosis of Amebiasis

Abstract

We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose-and N-acetyl-D-galactosamine-inhibitable lectin, which is a cysteine-rich protein consisting of 1,101 amino acids (aa) and containing multiple CXXC motifs in amino acid sequences. In the present study, full-length Igl except for the signal sequences (aa 14 to 1088) and three fragments of Igl-the N-terminal part (aa 14 to 382), the middle part (aa 294 to 753), and the C-terminal part (aa 603 to 1088)-were prepared in Escherichia coli, and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA). Sera from 57 symptomatic patients with amebic liver abscess or amebic colitis, sera from 15 asymptomatic cyst passers, sera from 40 individuals with other protozoan infections, and sera from 50 healthy controls were used. The sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90 and 94%, respectively. When three fragments were used as antigens in the ELISA, the sensitivities were 56% in the N terminus, 92% in the middle part, and 97% in the C terminus. The specificities of the three antigens were 96% in the N terminus and 99% in both the middle and C-terminal fragments. These results demonstrate that Igl is well recognized in not only symptomatic but also asymptomatic patients with E. histolytica infection and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis.

Prevalence of Entamoeba spp. in Stool Samples of Patients with Amebiasis Suspect by Native-Lugol and ELISA

“…Dışkıda spesifik antijen tespitine dayalı ELISA testleri ise, kolay uygulanabilen, deneyim gerektirmeyen, ucuz, özgüllüğü ve hassaslığı yüksek olan tanı yöntemlerindendir. Günümüzde Entamoeba türlerinin değişik antijenlerine spesifik ELISA testleri kullanılmaktadır (6,13,14). Bunlardan E. histolytica'ya özgü olarak, galaktoz ve N-asetil-galaktozamin (Gal/GalNAc) lektine ve serin-zengin antijene karşı geliştirilmiş monoklonal antikorlar en yaygın kullanılanlardandır (11,15 (24) ise bunun aksine hastalığın 41 ve üstü yaşlarda daha sık görüldüğünü bildirmişlerdir.…”

Seroprevalence of Entamoeba histolytica Infection in China

“…19 Another advantage of the recombinant protein is that the use of defined proteins in serodiagnosis facilitates standardization of the assays. Moreover, production of large quantities of recombinant C-Igl may be an economically effective alternative to ameba trophozoite cultivation.…”

“…The supernate was used as crude antigen to detect antibodies against E. histolytica. oligonucleotide primers used were 19 EhIgl-S603: 5 -CCCTCGA GGAAGGACCAAATGCAGAAGAT-3 and EhIgl-AS1088: 5 -CCCTCGAGTTAAATGCCTTTAGCTCCATT-3 . The DNA fragment coding C-Igl was ligated into the expression vector pET19-b.…”

“…The C-terminus fragment of Igl is valuable for serodiagnosis of amebiasis. 19 In the present study, antibodies against E. histolytica were examined by ELISA using crude E. histolytica antigen and the recombinant fragment of the C-terminus fragment of Igl (C-Igl) of E. histolytica. A total of 1,312 serum samples collected from seven provinces in China had been examined.…”

“…[17][18][19] One of these useful antigens is the glycosylphosphatidylinisotol-anchored 150-kD intermediate subunit (Igl) of galactose-and N-acetyl-D-galactosamineinhibitable lectin. Recombinant Igl is well recognized in serum samples from patients with amebiasis, but not from patients with other protozoan infections.…”