Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess.
BACKGROUND
Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.


METHODS
Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.


RESULTS
rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.


CONCLUSIONS
The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
Syazwan
S
Saidin
Muhammad Hafiznur
MH
Yunus
Nor Dyana
ND
Zakaria
Khairunisak Abdul
KA
Razak
Lim Boon
LB
Huat
Nurulhasanah
N
Othman
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia. rahmah8485@gmail.com.
Rahmah
R
Noordin
D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D004749:Entamoebiasis / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D011772:Pyruvate, Orthophosphate Dikinase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011934:Reagent Strips / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D011934:Reagent Strips; D011994:Recombinant Proteins; D011772:Pyruvate, Orthophosphate Dikinase