Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess
Authors: Khairunisak Abdul Razak
Nurulhasanah Othman
Nor Dyana Zakaria
Syazwan Saidin
Rahmah Noordin
Muhammad Hafiznur Yunus
Lim Boon Huat
Journal: BMC Infectious Diseases (2014)
Discipline: IMMUNOLOGY
Keywords: Amoebic liver abscess; Pyruvate phosphate dikinase; Diagnostic marker; Recombinant protein; Western blot
Affiliations: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia. .

Abstract:  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. Conclusion: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples. 

Retracted: No

PubPeer comments:
 November 2014 Erratum found here: https: bmcinfectdis.biomedcentral.com articles 10.1186 1471-2334-14-533 It reads:  After publication of this article [1], it was brought to our attention that we had failed to make it clear that Figures one (Figure 1 here), two (Figure 2 here) and three (Figure 3 here) were put together by grouping elements from different images. The revised figures, in which this grouping has been made explicit in the arrangement, and figure legends, in which the adjustments are clearly stated, are shown below. , 
 I reported this paper in April 2014, because of image concerns in Figure 2. Lanes 4 and 5 appear to look remarkably similar ![file] , 
 In addition, lanes 1 in Figures 1 and 2 looked remarkably similar, while lanes 2 also looked very similar. ![file] Here are the full figures again, marked in a different way. According to the legends, it appears that the lanes 2 in both figures did not represent the same sample. Of particular note, the fattest band in lanes 2 of each figure is shown at a different molecular weight (55 kDa in Figure 1 and 45 kDa in Figure 2, respectively). ![file] , 
 Retracted [8 September 2022] . ----------------------- The Editor has retracted this article. After publication, concerns were raised about unexpected similarities in Figs. 1 and 2. They were addressed in an Erratum, but additional concerns emerged, specifically: - Apparent similarities of the backgrounds in lanes 4 and 5 in Fig. 2 in the original article, as well as lanes 3u20134 in Image 1A in the correction [1]. - Apparent similarities of the backgrounds in lane 2 of Fig. 1 and lane 2 of Fig. 2 in the original article, as well as lanes 1 and 6 in Image 1A in the correction [1]. The authors have provided further explanation for the figure similarity, however the Editor found these to not be satisfactory. Furthermore, the authors were unable to provide evidence of the ethics approval for the study. None of the authors agree to this retraction. ,