Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)

Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries. Intestinal amoebiasis may produce symptoms such as colitis and dysentery; while extraintestinal amoebiasis commonly results in amoebic liver abscess (ALA). The commercia...

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Main Author:	Syazwan Saidin
Format:	thesis
Language:	eng
Published:	2017
Subjects:	RC Internal medicine
Online Access:	https://ir.upsi.edu.my/detailsg.php?det=4704
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spelling	oai:ir.upsi.edu.my:47042020-02-27 Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR) 2017 Syazwan Saidin RC Internal medicine Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries. Intestinal amoebiasis may produce symptoms such as colitis and dysentery; while extraintestinal amoebiasis commonly results in amoebic liver abscess (ALA). The commercial antigen detection tests for intestinal infection vary in their sensitivities and specificities; most of them use native parasite antigen extract which have specificity issues. Thus this study was aimed at improving the laboratory diagnosis of ALA and intestinal amoebiasis. Previously, pyruvate phosphate dikinase (PPDK) had been identified as a marker for detection of extraintestinal amoebiasis, thus the recombinant form of the protein (rPPDK) was used in this study to develop a rapid antibody dipstick test for ALA. Search for new anti-amoebic agents is important due to the side-effects of metronidazole and reports of resistance against it. PPDK is involved in a key pathway in the energy metabolism of the parasite, thus is a promising target for a new anti-amoebic agent. Therefore, the present study was also aimed to investigate in vitro anti-amoebic activity of potential compounds using E. histolytica HM-l: IMSS. The PPDK gene was custom cloned into pET28a(+) expression vector, transformed into E. coli BL21 (DE3), expressed and purified. Diagnostic sensitivity and specificity of the purified rPPDK protein were evaluated by Western blot using individual serum samples. For comparison, the above procedures were also performed to produce recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (rGal/GaINAc lectin). Western blots using patients and control serum samples probed with anti-human IgG4-HRP showed higher diagnostic sensitivity (93.3%) and specificity (100%) as compared to blots using IgG and IgGl as secondary antibodies. rPPDK was found to show better specificity when compared to rGal/GalNAc lectin. A rapid dipstick test using rPPDK was developed and evaluated for ALA serodiagnosis. The results showed 96.7% (n=29/30) diagnostic sensitivity and 100% (n=40/40) specificity. For the development of an antigen detection rapid test for intestinal amoebiasis, polyclonal antibody (PAb) against rPPDK, rGal/GalNAc lectin and E. histolytica excretory-secretory antigens (Eh ESA) were produced. The best detection of E. histolytica in stool samples was shown when anti-rPPDK PAb was lined on the dipstick and gold conjugated anti-Eh ESA PAb was used as the detector reagent. Performance of the developed dipstick test was compared with commercial TechLab E. histolytica II ELISA and real-time PCR, using 70 stool samples from patients and controls. Ten compounds from the National Cancer Institute (NCI) were selected from a previous virtual screening performed against ATP site of a three-dimensional PPDK model. Using an enzymatic assay, seven of the compounds showed inhibitory activities, they include NSC349156 and NSC228137 with MIC values of 25 11M and 50 11M respectively. Using NBT reduction assay, NSC349156 showed the lowest IC50 (14.04 11M), followed by NSC228137 (20.7 11M); while IC50 of metronidazole was 6.15 11M. After 24 hours of treatment, NSC228137 and NSC349156 exhibited 37.61% and 75.73% growth inhibition, respectively. In conclusion, rPPDK was successfully used in this study for development of rapid antibody and antigen detection tests for diagnosis of extraintestinal and intestinal amoebiasis respectively. In addition, two compounds showed potential to serve as anti-amoebic agents. 2017 thesis https://ir.upsi.edu.my/detailsg.php?det=4704 https://ir.upsi.edu.my/detailsg.php?det=4704 text eng closedAccess Doctoral Universiti Pendidikan Sultan Idris Fakulti Sains dan Matematik N/A
institution	Universiti Pendidikan Sultan Idris
collection	UPSI Digital Repository
language	eng
topic	RC Internal medicine
spellingShingle	RC Internal medicine Syazwan Saidin Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
description	Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries. Intestinal amoebiasis may produce symptoms such as colitis and dysentery; while extraintestinal amoebiasis commonly results in amoebic liver abscess (ALA). The commercial antigen detection tests for intestinal infection vary in their sensitivities and specificities; most of them use native parasite antigen extract which have specificity issues. Thus this study was aimed at improving the laboratory diagnosis of ALA and intestinal amoebiasis. Previously, pyruvate phosphate dikinase (PPDK) had been identified as a marker for detection of extraintestinal amoebiasis, thus the recombinant form of the protein (rPPDK) was used in this study to develop a rapid antibody dipstick test for ALA. Search for new anti-amoebic agents is important due to the side-effects of metronidazole and reports of resistance against it. PPDK is involved in a key pathway in the energy metabolism of the parasite, thus is a promising target for a new anti-amoebic agent. Therefore, the present study was also aimed to investigate in vitro anti-amoebic activity of potential compounds using E. histolytica HM-l: IMSS. The PPDK gene was custom cloned into pET28a(+) expression vector, transformed into E. coli BL21 (DE3), expressed and purified. Diagnostic sensitivity and specificity of the purified rPPDK protein were evaluated by Western blot using individual serum samples. For comparison, the above procedures were also performed to produce recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (rGal/GaINAc lectin). Western blots using patients and control serum samples probed with anti-human IgG4-HRP showed higher diagnostic sensitivity (93.3%) and specificity (100%) as compared to blots using IgG and IgGl as secondary antibodies. rPPDK was found to show better specificity when compared to rGal/GalNAc lectin. A rapid dipstick test using rPPDK was developed and evaluated for ALA serodiagnosis. The results showed 96.7% (n=29/30) diagnostic sensitivity and 100% (n=40/40) specificity. For the development of an antigen detection rapid test for intestinal amoebiasis, polyclonal antibody (PAb) against rPPDK, rGal/GalNAc lectin and E. histolytica excretory-secretory antigens (Eh ESA) were produced. The best detection of E. histolytica in stool samples was shown when anti-rPPDK PAb was lined on the dipstick and gold conjugated anti-Eh ESA PAb was used as the detector reagent. Performance of the developed dipstick test was compared with commercial TechLab E. histolytica II ELISA and real-time PCR, using 70 stool samples from patients and controls. Ten compounds from the National Cancer Institute (NCI) were selected from a previous virtual screening performed against ATP site of a three-dimensional PPDK model. Using an enzymatic assay, seven of the compounds showed inhibitory activities, they include NSC349156 and NSC228137 with MIC values of 25 11M and 50 11M respectively. Using NBT reduction assay, NSC349156 showed the lowest IC50 (14.04 11M), followed by NSC228137 (20.7 11M); while IC50 of metronidazole was 6.15 11M. After 24 hours of treatment, NSC228137 and NSC349156 exhibited 37.61% and 75.73% growth inhibition, respectively. In conclusion, rPPDK was successfully used in this study for development of rapid antibody and antigen detection tests for diagnosis of extraintestinal and intestinal amoebiasis respectively. In addition, two compounds showed potential to serve as anti-amoebic agents.
format	thesis
qualification_name
qualification_level	Doctorate
author	Syazwan Saidin
author_facet	Syazwan Saidin
author_sort	Syazwan Saidin
title	Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
title_short	Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
title_full	Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
title_fullStr	Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
title_full_unstemmed	Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)
title_sort	recombinant pyruvate phosphate dikinase (rppdk): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (ir)
granting_institution	Universiti Pendidikan Sultan Idris
granting_department	Fakulti Sains dan Matematik
publishDate	2017
url	https://ir.upsi.edu.my/detailsg.php?det=4704
_version_	1747833136923803648

Recombinant pyruvate phosphate dikinase (rPPDK): development of antibody and antigen rapid dipstick tests for amoebiasis and identification of potential antiamoebic compounds (IR)

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