Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target

Accepted Manuscript Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target Robert G.E. Krause, Ramona Hurdayal, David Choveaux, Jude Przyborski, Theresa H.T. Coetzer, J.P. Dean Goldring PII:

S0014-4894(17)30122-4

DOI:

10.1016/j.exppara.2017.05.007

Reference:

YEXPR 7411

To appear in:

Experimental Parasitology

Received Date: 16 March 2017 Accepted Date: 18 May 2017

Please cite this article as: Krause, R.G.E., Hurdayal, R., Choveaux, D., Przyborski, J., Coetzer, T.H.T., Goldring, J.P.D., Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.05.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Plasmodium glyceraldehyde-3-phosphate dehydrogenase: a potential malaria

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diagnostic target

3 Robert G.E. Krausea, Ramona Hurdayala,1, David Choveauxa,2, Jude Przyborskib,

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Theresa H.T. Coetzera, J.P.Dean Goldringa*

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Author addresses

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a

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Africa

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Biochemistry, University of KwaZulu-Natal, P.B. X01, Carbis Road, Scottsville 3209, South

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b

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35043 Marburg, Germany

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Rondebosch, 7701

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Africa

Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town,

Corresponding author

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*

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J.P. Dean Goldring: e-mail: [email protected], Tel: +27 33 260 5466

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Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925, South

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Parasitology, Faculty of Biology, Philipps University Marburg, Karl von Frisch Str. 8, D-

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Abstract

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Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting

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Plasmodial Histidine rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2

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is only expressed by Plasmodium falciparum parasites and the protein is not expressed in

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several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This

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study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-

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phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P.

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falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) E. coli host

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cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV)

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specific to P. falciparum GAPDH and one common to all mammalian malaria species

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(CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two

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recombinant proteins and the three epitopes and affinity purified. The antibodies detected

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the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a

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parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold

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higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and

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ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting

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potential as a malaria diagnostic biomarker.

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Key words: malaria; diagnosis; antibody; GAPDH; epitope; biomarker.

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1. Introduction

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Malaria is an ancient disease and was identified in Egyptian remains dating back ~2800 BC

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(Bianucci et al., 2015). In 2015 nearly 214 million malaria cases were recorded and around

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438 thousand people died of the disease, 66% of which were children under the age of five

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(WHO, 2015). This equates to 407 cases/min and one fatality/min. Most deaths result from

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a failure to diagnose and treat malaria before the onset of severe disease, at which point,

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even with proper treatment, mortality rates exceed 20% (Antia et al., 2008).

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55 Rapid diagnostic tests (RDTs) are currently the most accessible and user friendly form of

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malaria diagnosis, although microscopy remains the gold standard (Moody, 2002). RDTs are

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antibody based tests that diagnose malaria by detecting Plasmodium proteins such as

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P. falciparum histidine rich protein 2 (PfHRP2), and Plasmodium lactate dehydrogenase

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(PLDH) or aldolase (Mouatcho and Goldring, 2013). PfHRP2, which is only expressed by

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P. falciparum parasites, is presently the most widely used RDT. Mutations and deletions

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identified within the PfHRP2 gene in isolates from South America, Africa and Asia have

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however been found to compromise RDT specificity (Baker et al., 2005; Gamboa et al.,

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2010; Kumar et al., 2013; Deme et al., 2014).

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To allow for differential diagnosis of P. falciparum and P. vivax infections, PfHRP2 based

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tests are combined with PLDH and/or aldolase, which are expressed by all Plasmodium

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species (Dzakah et al., 2014). PLDH based tests are however preferred as they are more

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sensitive (Lee et al., 2006). The first PLDH based RDTs were developed in 1999 (Piper et

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al., 1999) and RDTs targeting PLDH alone include OptiMAL® and OPTIMAL-IT®. Native

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PfHRP2 appears to be present at higher concentrations than PLDH, resulting in detection of

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lower blood parasitemia (Martin et al., 2009; Marquart et al., 2012). However, PLDH offers

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the possibility of differential diagnosis based on species specific epitopes (Hurdayal et al.,

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2010). A biomarker similar to PLDH, which allows differential diagnosis and is present at

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higher concentrations than PLDH, could improve current antibody based malaria diagnosis.

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As Plasmodia infect and develop within red blood cells they divide to form between 14-32

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daughter merozoites within 24-72 hours (Baldacci and Menard, 2004; Collins and Jeffery,

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2007; Antia et al., 2008). Developing parasites require considerable metabolic energy

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(Daubenberger et al., 2000; Mehta et al., 2006). PLDH and aldolase are both glycolytic

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enzymes present at relatively high concentrations but which are cleared from infected

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patients’ blood within two to seven days of effective drug treatment, which makes them

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good indicators of current infections (Iqbal et al., 2004; De et al., 2016). Here we evaluate

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the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential diagnostic

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biomarker of P. falciparum/malaria infection given its inherent properties. The amino acid

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sequences of glycolytic enzymes are generally highly conserved, as shown for LDH and

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aldolase (Lee et al., 2006; Penna-Coutinho et al., 2011; Shin et al., 2013) and the same

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holds true for GAPDH (Cha et al., 2016). Plasmodium GAPDH has been assessed as a drug

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target (Campanale et al., 2003; Bruno et al., 2014) as have aldolase and PLDH (Read et al.,

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1999; Nemetski et al., 2015). GAPDH is also used as a PCR and protein diagnostic marker in

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several other infections including Brucella, Mycoplasma, Schistosoma and cancer prognosis

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(Wang et al., 2013a; Wang et al., 2013b; Guimaraes et al., 2014; Wareth et al., 2016).

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Interestingly GAPDH was recently identified as a potential vaccine candidate to prevent

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Kupffer cell invasion by malaria sporozoites (Cha et al., 2016). This adds malaria to the set

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of parasitic and bacterial diseases in which GAPDH is being pursued as a vaccine target, as

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reviewed by Perez-Casal and Potter (2016).

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Identifying new diagnostic biomarkers and developing new diagnostic reagents to address

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the shortfalls and potentially improve the performance of current RDTs remains important.

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The approach used in our study was twofold. We demonstrated that PfGAPDH was present

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at significantly higher concentrations than its glycolytic counterpart PfLDH, which may

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improve the detectable level of parasitemia in tests. We raised antibodies in chickens

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against peptides chosen as Plasmodium-common and P. falciparum-specific, similar to the

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approach we demonstrated with LDH antibodies (Hurdayal et al., 2010). All anti-

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recombinant PfGAPDH and anti-peptide antibodies detected Plasmodium and not host

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GAPDH. The P. falciparum specific anti-peptide antibodies detected P. falciparum GAPDH

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alone. The epitopes identified in our work and the anti-peptide antibodies raised could be

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useful for general or differential diagnosis of malaria infections. This work also demonstrates

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GAPDH as a promising alternative to LDH for use in RDTs.

110 2. Materials and methods

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2.1 In silico and bioinformatics work to identify protein targets and peptides

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Possible protein targets for malaria diagnosis were identified from transcriptome and

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proteomics data [www.plasmodb.org/; (Le Roch et al., 2003; Foth et al., 2011)]. Target

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peptides were assessed using sequence alignment, Predict7 and BLASTp analyses (Hurdayal

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et al., 2010). Candidate peptides were located on the protein crystal structure model of

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PfGAPDH using Deep view Swiss pdb viewer (http://swissmodel.expasy.org/repository/ and

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www.expasy.org/spdbv/).

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2.2 Recombinant expression and affinity purification of P. falciparum GAPDH and LDH

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The coding region for PfLDH (Pf(K1) strain), PfGAPDH (Pf(3D7) strain) and PyGAPDH were

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cloned into pKK223-3, pET-15(b) and pET-28(a) vectors respectively and confirmed by

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sequencing. BL21(DE3) E. coli (Novagen, Damstadt, Germany) were used as the expression

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host. No culture volumes exceeded 20% of the flask volume and all proteins were expressed

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using either auto-inducing terrific broth (TB) medium [1.2% (w/v) tryptone, 2.4% (w/v)

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yeast extract, 0.4% (w/v) glycerol, 0.231% (w/v) KH2PO4, 1.254% (w/v) K2HPO4] (Studier

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W.F., 2005) or lysogeny broth (LB) medium [1% (w/v) tryptone; 0.5% (w/v) yeast extract;

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85 mM NaCl; 11 mM glucose] supplemented with 0.3 mM Isopropyl thioglucopyranoside

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(IPTG). Depending on expression vector, cultures were supplemented with ampicillin (50

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µg/ml) or kanamycin (25 µg/ml). Overnight expression cultures (GAPDH at 30°C and LDH at

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37°C) were pelleted by centrifugation (4000 x g, 30 min, 4°C) and the supernatant

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removed. His-tagged recombinant proteins were purified from E. coli lysate supernatants

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using TALON® Co2+ resin according to manufacturer’s instructions. Briefly, culture pellets

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were suspended to 5% of their original culture volume [50 mM NaH2PO4; 300 mM NaCl; 10

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mM imidazole; 0.02% (w/v) NaN3 at pH 8.0] and lysed using 4 x 30 sec sonication (0.8

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Watts), centrifuged (12000 x g, 4°C, 20 min) and incubated with the resin for 1 hour at

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room temperature. The resin was washed with the suspension buffer until A280 values

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dropped to ~0.02. Bound His-tagged proteins were eluted with 250 mM imidazole.

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2.3 Protein concentration determination

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Protein concentrations were determined using the Bradford assay (Bradford, 1976). IgY and

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human IgG concentrations were calculated using A280 values and the IgY (Ɛ = 1.25)

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(Goldring and Coetzer, 2003) or human IgG (Ɛ = 1.35) (Semenova et al., 2004) extinction

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coefficients.

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145 2.4 SDS-PAGE

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Discontinuous reducing [5% (v/v) β-mercaptoethanol] SDS-PAGE with 12.5% running and

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4% stacking gels was used throughout the study (Laemmli, 1970). Gels were stained with

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Coomassie Brilliant Blue R-250 (Choveaux et al., 2012).

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2.5 Molecular exclusion chromatography

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Four milligrams of purified recombinant PfGAPDH (rPfGAPDH) was subjected to molecular

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exclusion chromatography on a Sephacryl S-200 column (equilibrated with 50 mM NaH2PO4;

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150 mM NaCl at pH 8.0), connected to an ÄKTA Prime Plus system. Standard running

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conditions of 0.5 ml/min, collecting 4 ml eluents for a total of 300 ml were used. The

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column was calibrated using the following standards: 6 mg blue dextran (2000 kD), 15 mg

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each of sheep IgG (150 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), and

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myoglobin (18.8 kDa) in a final volume of 3 ml. All samples, including the standards, were

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centrifuged (100000 x g, 4°C, 20 min) prior to loading the supernatant onto the column.

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Degassed buffers were used throughout.

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2.6 Raising antibodies in chickens

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Ethical clearance for the use of experimental animals in this study was granted by the

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animal ethics research committee of the University of KwaZulu-Natal (004/15/Animal). IgY

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antibodies against whole recombinant proteins as well as peptide targets were raised and

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affinity purified as previously described (Hurdayal et al., 2010; Krause et al., 2015). A

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method described by Lateef et al. (2007) was used to couple the CADGFLLIGEKKVSVFA

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peptide to the resin due to low peptide solubility.

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2.7 Characterising antibody specificity by western blot

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Western blotting was essentially performed as described by Towbin et al. (1979). The

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following solutions were used at room temperature: 5% (w/v) low fat milk powder in TBS

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(20 mM Tris; 200 mM NaCl at pH 7.4) for blocking (1 hour); 1 µg/ml primary (2 hours) and

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1/12000 secondary antibody (1 hour) solutions prepared in 0.5% (w/v) BSA-TBS.

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Nitrocellulose strips were developed for 30 min using 3.4 mM 4-chloro-1-naphthol and

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0.04% (v/v) H2O2. All images were captured using the Syngene G:Box system.

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2.8 Parasite culture and immunofluorescence

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Plasmodium falciparum 3D7 parasites were cultured in human O+ erythrocytes according to

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standard protocols, except cultures were incubated in gassed flasks (Spork et al., 2009).

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Synchronized asexual parasites were obtained by sorbitol treatment (Lambros and

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Vanderberg, 1979). Immunofluorescence microscopy was performed as described previously

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(Choveaux et al., 2012). Briefly, cells were fixed with 4% (v/v)

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paraformaldehyde/0.00075% (w/v) glutaraldehyde at 37°C for 30 min and quenched with

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125 mM glycine/PBS. Chicken antibodies directed against P. falciparum LDH and GAPDH

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were diluted in 3% (w/v) BSA/PBS and detected with goat anti-chicken-Cy3 antibody

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(DAKO, Santa Cruz). Hoechst 33258 (Molecular probes) was used at a concentration of 50

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ng/ml to stain parasite DNA. All images were acquired at room temperature on a Zeiss Cell

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Observer using appropriate filter sets.

190 2.9 Immunoprecipitation

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All sample incubations and washes were done at room temperature with agitation. All

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centrifugations were at 3000 x g, room temperature, 2 min in a 50 mM TrisHCl biffer pH 8

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containing; 150 mM NaCl; 0.1% (v/v) Triton X-100 was used throughout. Protein G beads

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were centrifuged, suspended and washed for 30 min. Following another centrifugation they

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were suspended in their original volume. Parasite lysates (1 ml) were pre-cleared by

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incubating with 20 µl protein G slurry for 30 min. These supernatants were then incubated

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with 25 µg anti-rPfGAPDH or rPfLDH IgY for 2 hours, followed by 1 hour incubation with 10

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µg bridging antibody (rabbit anti-chicken IgY). Fifty microliters of the protein G slurry was

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then added and incubated for 1 hour. Samples were underlayed with 1 M sucrose (200 µl)

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and centrifuged. The supernatant samples were discarded, or used as the starting material

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for further immunoprecipitation experiments. The final precipitated pellets were washed

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twice (1 ml) and centrifuged, after which they were prepared for SDS-PAGE and western

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blotting followed by densitometry analysis.

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2.10 Coupling IgY to HRPO

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The periodate coupling method of Nakane and Kawaoi (1974) was adapted, where the

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fluorodinitrobenzene reaction was omitted. Eight milligrams of horse radish peroxidase

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(HRPO) (1360 Units) was conjugated to an equivalent concentration of IgY.

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2.11 Quantitation of native P. falciparum GAPDH and LDH by DAS-ELISA

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ELISA plates were coated with 150 µl of an anti-peptide antibody (either Plasmodium

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common or P. falciparum-specific) as the capture antibody at 1 µg/ml in PBS pH 7.2 and

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incubated overnight at 4°C. All other antibody solutions were prepared in 0.5% (w/v) BSA-

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PBS and all subsequent incubation steps were done at 37°C. All washes were repeated 3

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times with 0.1% (v/v) PBS-Tween 20. After coating, the plates were washed and blocked

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with 0.5% (w/v) BSA-PBS for 1 hour and washed. The antigen-containing sample (150 µl

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per well) was loaded at the desired concentration. Recombinant protein solutions were

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prepared in 0.5% (w/v) BSA-PBS and used to prepare antigen standard curves. Plates were

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incubated for 2 hours with recombinant protein, washed and incubated for 2 hours with 150

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µl of either anti-rPfGAPDH-HRPO or anti-rPfLDH-HRPO diluted 1/200. After washing, 150 µl

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substrate solution was added per well and the plates were incubated in the dark for 30 min

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at room temperature and read immediately in an ELISA-plate reader at 405 nm.

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Background controls included: no antigen, no capture and no detection antibody. All results

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were corrected for background. The ELISAs were repeated three times (n=3) and all sample

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concentrations were done in triplicate. Statistical analysis was done using the Student’s t-

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test, with p ≤ 0.05 indicated with “*”.

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2.12 Human IgG pool antibodies

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Three hundred milligrams of a human anti-malaria hyperimmune serum pool was used to

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affinity purify human IgG antibodies against rPfLDH and rPfGAPDH as described previously

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(Choveaux et al., 2015).

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3. Results

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3.1 Selection of Plasmodial glyceraldehyde-3-phosphate dehydrogenase as a diagnostic

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target

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P. falciparum GAPDH is indicated as one of the most abundantly expressed parasite

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proteins, along with two existing diagnostic markers LDH and aldolase [Table 1 (Le Roch et

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al., 2003; Foth et al., 2011)]. PfGAPDH is a soluble, glycolytic enzyme with malaria-specific

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and malaria-species-specific amino-acid sequences (see below) predicted to enable

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identification of malaria parasites per se and differentiate them by species. The crystal

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structure of the protein is available (Satchell et al., 2005), which assisted protein analysis in

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this study (Table 1).

244 GAPDH amino-acid sequences were obtained from Plasmodb for P. falciparum, P. vivax and

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P. yoelii and aligned with avian and mammalian sequences (Fig. 1). The human, avian and

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murine sequences share over 91% identity with each other and between 62 and 67%

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identity with the plasmodial sequences. The plasmodial sequences include, murine (P.

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yoelii), monkey [P. knowlesi – also found in humans (Singh and Daneshvar, 2013)] and

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human (P. ovale, P. vivax, P. malariae and P. falciparum) and share 83 to 94% identity.

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When the amino-acid sequences of the human, murine and avian (chicken) protein were

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compared with the three malaria sequences, three unique malaria peptides were identified

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(Fig. 1). One of the peptides shares 100% sequence identity across all malaria species and

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was predicted to be a pan-malarial epitope, and two peptides unique to the P. falciparum

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GAPDH protein were identified. All three peptide sequences differed by ~50% with the

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human, murine or avian (chicken) orthologues (Table 2). The peptides were predicted to

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have good immunogenic properties [Predict7 program, see Hurdayal et al. (2010);

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Choveaux et al. (2012); (2015)] and were located on the surface of the 3-dimensional

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tetrameric structure of P. falciparum GAPDH, where they would be accessible to antibodies

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(Fig. 2).

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3.2 Recombinant expression of PfGAPDH and PyGAPDH

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The genes encoding the P. falciparum and the P. yoelii GAPDH proteins were expressed in E.

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coli host cells and the recombinant proteins isolated from lysed host cell supernatants. The

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recombinant P. falciparum GAPDH (rPfGAPDH) His-tagged fusion protein eluted from a

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TALON®Co2+ affinity matrix with an observed molecular mass of 38 kDa (6.55 mg from a 50

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ml culture, Fig. 3A), similar to the 38.8 kDa predicted from the amino-acid sequence with

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the addition of the His6 tag. Likewise the recombinant P. yoelii GAPDH (rPyGAPDH) had a

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molecular mass of 40 kDa, which corresponded to the predicted value of 40,328 kDa, with

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the His tag (8.95 mg from a 50 ml culture, Fig. 3B). The 38 kDa rPfGAPDH and the 40 kDa

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rPyGAPDH proteins were detected with anti-His tag antibodies (Fig. 3A/B right side panels).

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On passage over a Sephacryl S-200 gel filtration chromatography column, the purified

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rPfGAPDH protein eluted in two prominent peaks, one corresponding to the 38 kDa

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monomer and one to a 148 kDa tetramer (Fig. 3C). The high and lower molecular mass

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protein peaks resolved by electrophoresis at the same molecular mass on a reducing

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polyacrylamide gel (Fig. 3C insert).

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3.3 Raising antibodies against PfGAPDH

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Antibodies against the peptides (see above) coupled to a rabbit albumin carrier, and

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antibodies against rPfGAPDH were raised in chickens and affinity purified using the

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corresponding peptide, or protein coupled to an affinity matrix. Affinity purified antibody

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yields ranged from 1.9 to 9.5 mg (Table 3). The antibodies against the whole recombinant

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protein detected a 38 kDa rPfGAPDH and a 40 kDa rPyGAPDH protein in a western blot (Fig.

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4A), corresponding with the protein molecular mass detected by anti-His tag antibodies

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(Fig. 3). Antibodies against the common CKDDTPIYVMGINH-peptide detected both

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rPfGAPDH and rPyGAPDH as predicted (Fig. 4A). The antibodies raised against the two P.

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falciparum specific peptides (anti-CAEKDPSQIPWGKCQV and CADGFLLIGEKKVSVFA)

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detected rPfGAPDH but not rPyGAPDH (Fig. 4A). When incubated with a western blot of

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proteins from uninfected and Plasmodium infected red blood cell lysates, antibodies against

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whole protein detected a 36 kDa protein in infected but not in naïve, uninfected red cells.

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This corresponds well with the 36.6 kDa size predicted from the PfGAPDH genome

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sequence.

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3.4 Immunofluorescence microscopy to detect PfGAPDH

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Antibodies against rPfGAPDH were used to probe samples of infected blood by

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immunofluorescence microscopy. The antibodies showed PfGAPDH is expressed during the

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ring, trophozoite and schizont stages of the intraerythrocytic cycle with a cellular

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distribution similar to that of the sister and glycolytic enzyme diagnostic target, PfLDH (Fig.

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5), and likely to be localized in the parasite cytosol. This expression profile corresponds to

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data from both proteomic and mRNA expression experiments (Le Roch et al., 2003; Foth et

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al., 2011).

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3.5 Measuring the relative concentrations of PfLDH and PfGAPDH in P. falciparum lysates

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The relative concentrations PfLDH and PfGAPDH were measured in the same parasite lysate

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sample. Three experimental approaches were taken. The first was to incubate different

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concentrations of parasite lysate (based on total protein concentration) with the respective

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chicken IgY anti-protein antibodies followed by a rabbit anti-IgY antibody and protein G. The

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rabbit antibody is included because IgY binds poorly to protein A or G (Schade et al., 2005).

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The protein G/antibody/protein complex was then immunoprecipitated and analyzed by

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western blot (Fig. 6A). A representative scan of the intensity of the protein bands from a

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single result is presented on the right of the diagram. The protein bands detected by the

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anti-rPfGAPDH antibodies are consistently darker than those detected by the anti-rPfLDH

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antibodies over the protein range of 5 to 0.0625 mg/ml parasite lysate (Fig. 6A). The

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scanned image shows the same concentration pattern. The experiment was performed 3

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times with similar results. These data suggest that PfGAPDH is found at a higher

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concentration than PfLDH in this parasite lysate.

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A second approach was to incubate each concentration of parasite lysate with the anti-

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PfGAPDH IgY and precipitate the protein and then incubate the same sample with the anti-

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PfLDH IgY and precipitate the protein. The protein precipitate was evaluated by western blot

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and suggests PfGAPDH to be present in higher concentrations than PfLDH in the parasite

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lysate (Fig. 6B). The two separate experiments suggest that there is a 1.6 fold higher

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concentration of PfGAPDH than PfLDH. The immunoprecipitated PfGAPDH was detected by

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the common as well as one of the P. falciparum specific anti-peptide antibodies, where the

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precipitated PfLDH was not detected (Fig. 6C).

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A third approach was to determine the concentration of each protein by ELISA. The ELISA

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result followed a similar trend to the immunoprecipitation data and depicted a higher

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concentration of PfGAPDH than PfLDH in parasite lysate (Fig. 7). We understand the

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different methods are not directly comparable, owing to the different reagents required to

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precipitate or capture and detect the respective proteins from lysate samples, however, our

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data suggest a higher concentration of PfGAPDH than PfLDH. This is in good agreement with

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a previous proteomic study that ranked the expression of PfGAPDH higher than PfLDH (Foth

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et al., 2011).

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3.6 Levels of antibodies against PfLDH and PfGAPDH in a human malaria antibody pool

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To evaluate the potential presence of antibodies in patient sera that may interfere and

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compete with antibodies used in a detection system, a pool of antibodies isolated from 800

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patients with malaria was used (Goldring, 2004). The pool of antibodies was passed over an

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affinity matrix conjugated with rPfLDH, followed by passing over a matrix with rPfGAPDH as

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the ligand. The concentration of antibodies eluted from the column show that in this

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antibody pool there were proportionately more antibodies to PfLDH than to PfGAPDH (Fig.

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8).

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4. Discussion

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Our search for novel malaria diagnostic protein biomarkers began in silico using transcript

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and semi-quantitative proteomics data to rank potential targets in order of abundance (Le

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Roch et al., 2003; Foth et al., 2011). The majority of the malaria genome is thought to

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follow a “just in time” translational strategy (Le Roch et al., 2003), however proteomics

350

analyses suggest that protein levels do not always mirror changes in their transcript levels,

351

most likely due to post-translational modifications (Foth et al., 2011; Miao et al., 2013).

352

Combining both sets of data, we narrowed the number of malarial proteins from 5554 (Foth

353

et al., 2011) to 35 potential diagnostic biomarker proteins. Only those proteins conserved

354

amongst Plasmodium species, which would allow for detection of all species infecting

355

humans, were studied further. By aligning the amino acid sequences of the respective

356

protein orthologues from all human infecting Plasmodium species, proteins which expressed

357

both conserved, Plasmodium specific (pan-malarial), and unique species specific peptide

358

epitopes were selected. This narrowed the number of potential protein biomarkers to seven,

359

of which GAPDH ranked the highest in terms of mRNA transcript and protein abundance (Le

360

Roch et al., 2003; Foth et al., 2011). We therefore aimed to experimentally quantify the

361

abundance of PfGAPDH relative to the current protein biomarker PfLDH and assess the

362

specificity of anti-peptide antibodies raised against the selected Plasmodium GAPDH

363

epitopes identified in this study.

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GAPDH is present in all Plasmodia and is between 80 to 95% conserved amongst the five

366

human infecting species. These slight amino acid sequence variations allowed the selection

367

of species-specific peptide epitopes (Fig. 1), similar to a previous study with PLDH (Hurdayal

368

et al., 2010). The PGAPDH species-specific peptide epitopes were selected from regions of

369

largest amino acid variation. All the peptides considered, including the common peptide, are

370

located within the N-terminal Rossmann domain (Akinyi et al., 2008). An epitope composes

371

approximately 15 amino acids, of which the main antibody binding energy is between as few

372

as five amino acids (Benjamin and Perdue, 1996). Amongst 204 P. falciparum isolates

373

PfGAPDH has seven nonsynonymous single nucleotide polymorphisms (Cha et al., 2016), of

374

which only a conservative mutation (E71D) falls within one of our selected peptides making

375

this polymorphism unlikely to affect antibody binding to this epitope.

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376 PfLDH has two, and aldolase, three nonsynonymous SNPs, whereas PfHRP2 has 144. Such

378

variation has resulted in variable performance of PfHRP2 based tests (Baker et al., 2005).

379

Attempts have been made to resolve this by raising antibodies against more conserved

380

repeat regions of PfHRP2 (Verma et al., 2015), with a similar polypeptide based approach as

381

we have demonstrated here. This does not circumvent the failure of tests to detect isolates

382

in which PfHRP2 is partially or completely deleted (Gamboa et al., 2010; Kumar et al.,

383

2013; Deme et al., 2014). The conserved and essential nature of glycolytic proteins across

384

Plasmodium species makes them ideal biomarkers for diagnosis. The PfGAPDH crystal

385

structure (Satchell et al., 2005) was used to map the location of peptide candidates and we

386

selected those that were surface located on PfGAPDH (Fig. 2) to facilitate antibody binding.

387

To demonstrate the diagnostic potential of PGAPDH, we raised polyclonal chicken antibodies

388

against the purified whole rPfGAPDH and the Plasmodium specific GAPDH peptides and

389

assessed their specificity.

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We expressed and purified P. falciparum and the murine malaria orthologue P. yoelii GAPDH

392

(rPfGAPH and rPyGAPDH respectively, Fig. 3). The proteins resolved close to their expected

393

monomeric sizes of ~ 38 kDa for rPfGAPDH and ~ 40 kDa for rPyGAPDH on SDS-PAGE,

394

which was similar to previous studies (Daubenberger et al., 2000; Satchell et al., 2005;

395

Sangolgi et al., 2016). Size exclusion chromatography (Fig. 3C) demonstrated that

396

rPfGAPDH associated as a tetramer of ~148 kDa (Sangolgi et al., 2016), although it was

397

larger than the purified tetramer observed by Satchell et al. (2005) who removed the His

398

tag for crystallisation. Since rPfGAPDH forms tetramers in solution, it suggests the

399

recombinant protein adopts a conformation similar to native PfGAPDH. The rPfLDH protein

400

also formed a tetramer of ~145 kDa [data not shown; (Berwal et al., 2008)].

401

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Polyclonal chicken antibodies were raised against whole rPfGAPDH and rPfLDH (Krause et

403

al., 2015) and formed part of our analysis. The choice and source of antibodies in a

404

diagnostic test should be considered, as the Fc portion of mammalian IgG antibodies can

405

cross react with human Rheumatoid factor resulting in false positives (Iqbal et al., 2000).

406

An alternative is the use of IgM isotypes (Naot et al., 1981), or avian antibodies (Carlander

407

et al., 1999) as was chosen here. The polyclonal anti-rPfGAPDH antibodies were shown to

408

be specific for PGAPDH, and identified both PfGAPDH and PyGAPDH, which share 83% amino

409

acid identity (Fig. 4). Similarly Sangolgi et al. (2016) detected PyGAPDH with antibodies

410

raised against rPfGAPDH. Our antibodies did not detect human red blood cell GAPDH, in

411

western blots and immunofluorescence (Fig. 4 and 5), despite human GAPDH sharing

412

64.67% identity with PfGAPDH [Daubenberger et al. (2003) recorded 63.5% identity].

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413

Antibodies were raised against selected peptides. The antibodies against the common

415

Plasmodium peptide detected both rPfGAPDH and rPyGAPDH. The P. falciparum specific

416

antibodies detected only the rPfGAPDH protein and not the rPyGAPDH protein (Fig. 4) as

417

well as native PfGAPDH (Fig. 6C). Targeting these epitopes therefore allows for differential

418

detection of PGAPDH orthologues. Such specificity would allow differential diagnosis, which

419

is an essential guide for effective treatment of malaria, especially in light of increasing drug

420

resistance in both P. falciparum and P. vivax, as well as dormant liver stages of P. vivax

421

(WHO, 2015). The majority of current RDTs differentiate between P. falciparum and non-

422

P. falciparum infections by combining PfHRP2 with PLDH (Dzakah et al., 2014), however

423

more specific diagnosis may be required in the case of mixed infections (Douglas et al.,

424

2011). Our strategy was thus to identify epitopes that could both diagnose a general

425

malaria infection, or differentially diagnose P. vivax and P. falciparum infections.

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426 427

Although we only demonstrated the specificity of antibodies raised against the P. falciparum

428

species-specific epitopes here (Fig. 4), we suggest that the corresponding P. vivax peptides

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(Fig. 1 and Table 2) could be used to detect P. vivax orthologues as we demonstrated with

430

PLDH (Hurdayal et al., 2010). Interestingly, the corresponding P. knowlesi LDH-specific

431

peptide sequence shares 92% identity with its P. vivax counterpart, suggesting that

432

antibodies against this epitope would be specific for both orthologues of LDH. There is the

433

potential for selection of P. knowlesi specific peptide epitopes from the corresponding

434

regions within the GAPDH orthologues, although the P. falciparum epitopes selected here

435

share 81 and 80% identity respectively (Table 2). The specificity of such antibodies would

436

have to be assessed. An additional epitope not assessed here, ranging from amino acid

437

number 20 to 33 in the P. falciparum sequence (RSAYERNDVEVVAV) may also allow

438

P. knowlesi specificity. For this sequence, the identity between the P. vivax and P. knowlesi

439

orthologues is 79%.

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440

Immunofluorescence analysis of PfGAPDH and PfLDH (Fig. 5) showed very similar

442

distribution throughout the cytoplasm of the parasites, however PfGAPDH seemed to localise

443

to the nucleus in schizont stages. This distribution corresponded well with what has been

444

previously reported (Akinyi et al., 2008; Alam et al., 2014; Sangolgi et al., 2016). PfGAPDH

445

is also expressed during sporozoite and gametocyte stages (Trenholme et al., 2014; Cha et

446

al., 2016). We assessed PfGAPDH and PfLDH levels during the erythrocytic stage using a

447

semi-quantitative immunoprecipitation approach (Fig. 6), and a quantitative double

448

antibody sandwich ELISA (Fig. 7). The different experiments are not directly comparable.

449

Immunoprecipitation used polyclonal IgY against the whole protein, whereas the ELISA

450

employed a specific anti-peptide IgY as the capture antibody and a HRPO conjugated IgY

451

(against the whole protein) for the detection step. The different reagents and efficiency of

452

conjugate coupling could account for the numerical differences between the two results. The

453

capture ELISA method was used as PfLDH from parasite lysates bound poorly to the ELISA

454

plate preventing a direct ELISA approach. We found PfGAPDH levels to be higher than PfLDH

455

levels, which is in agreement with mRNA transcript (Le Roch et al., 2003) and proteomics

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data (Foth et al., 2011). The data suggests there to be 17.3 ng/ml of PfLDH in a 1%

457

parasitemia lysate and approximately 138.5 ng/ml PfGAPDH. Martin et al. (2009) quantified

458

the levels of PfLDH and PfHRP2 within a 1% parasite culture of the P. falciparum Dd2 strain

459

at approximately 28.9 ng/ml and 164.5 ng/ml, respectively. Therefore, PfGAPDH is present

460

between four to eight times the concentration of PfLDH, and in a concentration approaching

461

that of PfHRP2. Importantly this demonstrated that PfGAPDH is expressed throughout the

462

asexual red blood cell stages, which is when malaria symptoms develop and patients

463

present for diagnosis (Gwer et al., 2007). The higher PGAPDH levels in comparison to PLDH

464

could allow for the detection of lower parasitemia infections than current PLDH based tests.

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In glycolysis GAPDH functions in cycling NAD+/NADH with its counterpart LDH. The

467

Rossmann fold motif common to both PLDH and PGAPDH imparts this nucleotide binding

468

ability; also suggesting possible functions in transcriptional regulation, similar to their

469

mammalian counterparts (Kim and Dang, 2005). Evidence is accumulating to suggest that

470

there are “moonlighting” roles for seven of the ten glycolytic proteins, with GAPDH being

471

amongst these (Alam et al., 2014; Gomez-Arreaza et al., 2014). GAPDH orthologues have

472

been associated with various moonlighting functions including: DNA repair, RNA binding,

473

telomere binding, cell cycle regulation, histone expression, membrane fusion,

474

phosphorylation, phosphatidyl serine binding, nitric oxide interaction, cytoskeletal binding

475

and apoptosis, as reviewed by Sirover (2012). In Plasmodial studies Daubenberger et al.

476

(2003) observed PfGAPDH localising to the apical end of merozoites in the late schizont

477

stages and demonstrated its N-terminal dependent association with the insoluble membrane

478

fraction of infected red cell lysates in the presence of the GTPase Rab2. Native PfGAPDH was

479

subsequently detected in cytoplasmic, nuclear, cytoskeletal and cell membrane fractions of

480

asexual red blood cell stages and several protein-protein interactions were also observed

481

(Sangolgi et al., 2016). PfGAPDH is surface exposed on sporozoites (Lindner et al., 2013)

482

and plays a role in binding the CD68 surface receptor on liver Kupffer cells allowing liver

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483

invasion (Cha et al., 2016). Together these findings may begin to explain the higher

484

abundance of PfGAPDH in comparison to PfLDH (Fig. 6 and 7), again highlighting PGAPDH’s

485

potential for diagnosis.

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486 PfGAPDH has 64.67% sequence identity with human GAPDH, while PfLDH has 29.45%

488

sequence identity with human LDH. This supports the idea that the protein with least shared

489

sequence identity is likely to be more immunogenic. The further inference is that if GAPDH

490

is used in a diagnostic test, there are fewer antibodies in patient sera to compete with test

491

antibodies. This was supported by our screen of a human IgG pool (Fig. 8). There have been

492

no reports of human antibodies interfering with PLDH based tests to our knowledge, and our

493

findings suggest that PfGAPDH tests are also likely to be unaffected. Antibodies in patients’

494

sera against PfHRP2 have however been shown to influence PfHRP2 detection (Ho et al.,

495

2014).

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496

In vivo half-lives for aldolase and LDH are reported to be approximately seven days (Ashley

498

et al., 2009; Aydin-Schmidt et al., 2013) and seven to 28 days for HRP2 (Aydin-Schmidt et

499

al., 2013). The serum half-life of GAPDH has not, to this point, been determined. RDTs

500

targeting metabolic enzymes also hold potential for the monitoring of treatment outcome,

501

as has been done for PLDH and aldolase (Iqbal et al., 2004; Ashley et al., 2009). This is a

502

likely property of PGAPDH.

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504

DNA extracted from RDTs can be used effectively for further molecular analysis of

505

Plasmodium strains prevalent in a region. This could include screening for the presence of

506

drug resistance markers or it could serve as a quality control measure of the RDTs

507

themselves (Morris et al., 2013). This places RDTs at a pivotal point in the patient treatment

508

time line. RDTs also have a marked advantage of already being in the field and familiar to

509

users. Implementing any improvements and innovations to these tests therefore has

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important practical advantages in comparison to introducing a totally new test strategy. The

511

findings presented here provide evidence to support PGAPDH as a potential new biomarker

512

for the diagnosis of malaria. Our anti-peptide antibodies could also allow for both general

513

and differential diagnosis and testing these in an RDT format is essential. Overall, this could

514

aid in improving current tests without having major implications for end user

515

implementation. Interestingly, Cha et al. (2016) suggested target peptides for a PfGAPDH-

516

based vaccine against the liver stage of the malaria infection. The common peptide

517

“KDDTPIYVMGINH” selected in our work was amongst these and the specificity of our

518

antibodies supports that targeting this region would be specific to PGAPDH. It would be

519

interesting to further assess these antibodies in their sporozoite neutralization assay.

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520 Acknowledgements

522

This research was funded by the South African Medical Research Council, National Research

523

Foundation and the University of KwaZulu-Natal Research Incentive fund.

524

We thank Prof. R.L. Brady and Prof. L. Tilley for vectors expressing Plasmodium falciparum

525

lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase respectively.

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gland sporozoites. Mol Cell Proteomics. 12, 1127-1143.

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45. Marquart, L., Butterworth, A., McCarthy, J.S., Gatton, M.L., 2012. Modelling the

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dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to

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better understand malaria rapid diagnostic test performance. Malar J. 11, 74. 46. Martin, S.K., Rajasekariah, G.H., Awinda, G., Waitumbi, J., Kifude, C., 2009. Unified

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parasite lactate dehydrogenase and histidine-rich protein ELISA for quantification of

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Plasmodium falciparum. Am J Trop Med Hyg. 80, 516-522.

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47. Mehta, M., Sonawat, H.M., Sharma, S., 2006. Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities. J Vector Borne Dis. 43, 95-103.

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48. Miao, J., Lawrence, M., Jeffers, V., Zhao, F., Parker, D., Ge, Y., Sullivan, W.J., Jr.,

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Cui, L., 2013. Extensive lysine acetylation occurs in evolutionarily conserved

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metabolic pathways and parasite-specific functions during Plasmodium falciparum

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49. Moody, A., 2002. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 15, 66-78.

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diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -

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assessment of DNA extraction methods and field applicability. Malar J. 12, 106.

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presence of both rheumatoid factor and antinuclear antibodies. J Clin Microbiol. 14,

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K.A., Buscaglia, C.A., Nussenzweig, V., Sinnis, P., Levitskaya, J., Bosch, J., 2015.

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Inhibition by stabilization: targeting the Plasmodium falciparum aldolase-TRAP

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complex. Malar J. 14, 324.

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55. Penna-Coutinho, J., Cortopassi, W.A., Oliveira, A.A., Franca, T.C., Krettli, A.U., 2011.

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Antimalarial activity of potential inhibitors of Plasmodium falciparum

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dehydrogenase enzyme selected by docking studies. PloS One 6, e21237.

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56. Perez-Casal, J., Potter, A.A., 2016. Glyceradehyde-3-phosphate dehydrogenase as a

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57. Piper, R., Lebras, J., Wentworth, L., Hunt-Cooke, A., Houze, S., Chiodini, P., Makler,

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dehydrogenase (pLDH). Am J Trop Med Hyg. 60, 109-118. 58. Read, J.A., Wilkinson, K.W., Tranter, R., Sessions, R.B., Brady, R.L., 1999.

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dehydrogenase. J Biol Chem. 274, 10213-10218.

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59. Sangolgi, P.B., Balaji, C., Dutta, S., Jindal, N., Jarori, G.K., 2016. Cloning,

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phosphate dehydrogenase. Prot Expr Purif. 117, 17-25.

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B.J., Tilley, L., Colman, P.M., 2005. Structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum. Acta crystallographica. Section D, Biological crystallography 61, 1213-1221.

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61. Schade, R., Calzado, E.G., Sarmiento, R., Chacana, P.A., Porankiewicz-Asplund, J.,

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Terzolo, H.R., 2005. Chicken egg yolk antibodies (IgY-technology): a review of

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progress in production and use in research and human and veterinary medicine.

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Alternatives to laboratory animals : ATLA 33, 129-154.

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62. Semenova, V.A., Steward-Clark, E., Stamey, K.L., Taylor, T.H., Jr., Schmidt, D.S.,

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Martin, S.K., Marano, N., Quinn, C.P., 2004. Mass value assignment of total and

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subclass immunoglobulin G in a human standard anthrax reference serum. Clin

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63. Shin, H.I., Kim, J.Y., Lee, W.J., Sohn, Y., Lee, S.W., Kang, Y.J., Lee, H.W., 2013.

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Polymorphism of the parasite lactate dehydrogenase gene from Plasmodium vivax

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Korean isolates. Malar J. 12, 166.

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64. Singh, B., Daneshvar, C., 2013. Human infections and detection of Plasmodium knowlesi. Clin Microbiol Rev. 26, 165-184.

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65. Sirover, M.A., 2012. Subcellular dynamics of multifunctional protein regulation:

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mechanisms of GAPDH intracellular translocation. J Cell Biochem. 113, 2193-2200.

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66. Spork, S., Hiss, J.A., Mandel, K., Sommer, M., Kooij, T.W., Chu, T., Schneider, G.,

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Maier, U.G., Przyborski, J.M., 2009. An unusual ERAD-like complex is targeted to the

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apicoplast of Plasmodium falciparum. Eukaryot cell. 8, 1134-1145.

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67. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from

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polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc

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68. Trenholme, K., Marek, L., Duffy, S., Pradel, G., Fisher, G., Hansen, F.K., Skinner-

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Adams, T.S., Butterworth, A., Ngwa, C.J., Moecking, J., Goodman, C.D., McFadden,

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Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules. Antimicrob Agents Chemother. 58, 3666-3678.

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69. Verma, R., Jayaprakash, N.S., Vijayalakshmi, M.A., Venkataraman, K., 2015. Novel

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monoclonal antibody against truncated C terminal region of Histidine Rich Protein2

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(PfHRP2) and its utility for the specific diagnosis of malaria caused by Plasmodium

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falciparum. Exp Parasitol. 150, 56-66.

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70. Wang, D., Moothart, D.R., Lowy, D.R., Qian, X., 2013a. The expression of

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glyceraldehyde-3-phosphate dehydrogenase associated cell cycle (GACC) genes

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correlates with cancer stage and poor survival in patients with solid tumors. PloS One

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8, e61262.

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71. Wang, J., Zhao, F., Yu, C.X., Xiao, D., Song, L.J., Yin, X.R., Shen, S., Hua, W.Q.,

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Zhang, J.Z., Zhang, H.F., He, L.H., Qian, C.Y., Zhang, W., Xu, Y.L., Yang, J., 2013b.

752

Identification

753

excreted/secretory

754

immunoproteomic analysis. J Proteomics. 87, 53-67.

proteins products

inducing of

short-lived

Schistosoma

antibody

japonicum

SC

of

responses

adult

worms

from by

72. Wareth, G., Eravci, M., Weise, C., Roesler, U., Melzer, F., Sprague, L.D., Neubauer,

756

H., Murugaiyan, J., 2016. Comprehensive Identification of Immunodominant Proteins

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of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from

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Naturally Infected Hosts. Int J Mol Sci. 17, 659.

762

763

764

765

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767

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73. WHO, 2015. Guidelines for the Treatment of Malaria, 3rd ed, Geneva.

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Legends to Figures

769

Fig. 1. ClustalΩ alignment of the Plasmodium (P) GAPDH sequences: P. falciparum (Pf,

770

accession #: PF3D7_1462800); P. vivax (Pv, PVX_117322); P. yoelii (Py, PY17X_1330200)

771

with human (Hu, P04406.3); mouse (Mouse, AAH83149.1) and chicken (Gallus,

772

NP_989636.1) GAPDH amino acid sequences showing peptide epitopes chosen for chicken

773

antibody production. Identity between the malaria sequences (P. spp) was annotated below

774

the first three alignments. The inter-sequence identity is indicated at the bottom of the

775

alignment (overall). The unique P. falciparum peptides are highlighted in blue and green,

776

and the peptide common to all Plasmodium species highlighted in red.

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Fig. 2. The P. falciparum specific and the common Plasmodium epitopes were located on the

779

surface of the 3D tetrameric structure of PfGAPDH [1YWG (Satchell et al., 2005)] using

780

Swiss-Pdb viewer 4.0.1 (http://www.expasy.org.spdbv/). It should be noted that the

781

synthesized peptides contained additional N-terminal cysteines to allow for coupling

782

reactions.

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Fig. 3. Expression, purification, detection on SDS-PAGE and Sephacryl S-200 gel filtration

785

chromatography of rPfGAPDH and rPyGAPDH. Recombinant PfGAPDH was expressed at

786

30°C, induced with 0.3 mM IPTG, and rPyGAPDH at 37°C in auto-inducing Terrific broth

787

media; both were affinity purified with TALON (Co2+) resin in phosphate buffer pH 8.0 and

788

evaluated on a 12.5% reducing SDS-PAGE gels (A and B respectively). Molecular weight

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marker (Mw); lane 1, untransformed E. coli BL21(DE3); lane 2, un-induced; lane 3, IPTG

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induced; lanes 4-11, eluents off the affinity matrix with lane 4 unbound samples; lanes 5-6,

791

washes (10 mM imidazole); lanes 7-11, eluents (250 mM imidazole). The samples loaded in

792

(B) were the same order as in (A), except the un-induced control was omitted and only a

793

single wash was required. The left panels in (A and B) are the Coomassie R-250 stained

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reference gels of the western blots on the right, probed with mouse anti-His6-HRPO

795

monoclonal antibody at 1/5000 dilution. Affinity purified rPfGAPDH was passed over a

796

HiPrep 16/60 Sephacryl S-200 chromatography matrix (120 ml), in a 50mM NaH2PO4; 300

797

mM NaCl pH 8.0 buffer (C). The profile was recorded in milli Absorbance units (mAu)

798

labelled on the primary and secondary axes. The calibration standards (see materials and

799

methods) were included as the dashed line (plotted on the left axis), with the solid line

800

depicting the rPfGAPDH elution profile (plotted on the right axis). The eluted fractions

801

comprising the ~148 kDa and ~ 36 kDa peaks were run on a 12.5% reducing SDS-PAGE gel

802

and Coomassie R-250 stained (insert). The samples were loaded as follows: molecular

803

weight marker (Mw), peak 1 (42-52 ml) and peak 2 (64-68 ml).

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Fig. 4. Assessment of affinity purified anti-whole protein and anti-peptide antibody GAPDH

806

specificity. SDS-PAGE and western blot of rPfGAPDH probed with chicken anti-whole protein

807

and anti-peptide IgY antibodies (A), and detection of P. falciparum GAPDH (B) and LDH (C)

808

from a (D10) lysate. The specificity of the affinity purified IgY against rPfGAPDH and the

809

respective peptides were assessed by western blotting. The Coomassie R250 stained

810

reference gel in the far left panel (A) was loaded as follows: Molecular weight marker (Mw);

811

uninfected (O+) red blood cell lysate (lane 1); untransformed E. coli BL21(DE3) lysate (lane

812

2); purified rPfGAPDH (lane 3) and purified rPyGAPDH (lane 4). The blots were probed with

813

the respective affinity purified IgY antibodies as labelled above the blots at 1 µg/ml.

814

Infected (lane 1) and uninfected (lane 2) human red blood cell lysates were run on 12.5%

815

reducing SDS-PAGE gels, transferred to nitrocellulose and stained with Ponceau S (B and C

816

SDS-PAGE) and probed with anti-rPfGAPDH (B) or anti-rPfLDH (C) IgY antibodies. Antigen-

817

antibody interaction in all blots was detected with a rabbit anti-chicken-HRPO antibody at

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1/12000 dilution. Molecular weight markers were run in the lanes marked (Mw).

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Fig. 5. Immunolocalization of P. falciparum LDH and GAPDH in asexual P. falciparum (3D7)

821

parasites grown in vitro, synchronized by sorbitol treatment and prepared at ring,

822

trophozoite and schizont stages as indicated alongside the panels. Fixed parasites were

823

probed with chicken anti-rPfLDH or anti-rPfGAPDH antibodies and the chicken antibodies

824

were detected with a Cy3-conjugated antibody. Parasite DNA was stained with Hoechst and

825

the scale bar represents 5 µm. DIC images are shown as reference.

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Fig. 6. Immunoprecipitation of P. falciparum GAPDH and LDH from two samples of the same

828

P. falciparum (D10) culture lysate diluted from 5 to 0.625 mg/ml total protein (lanes 1 to 4

829

respectively). Anti-rPfGAPDH or anti-rPfLDH IgY (25ug), secondary rabbit anti-IgY

830

antibodies (10ug) and protein G were used throughout. (A) shows immunoprecipitation data

831

for separate lysate stocks, where in (B) PfGAPDH and PfLDH were precipitated sequentially

832

from the same stock. Lane 5 in (A) contained un-precipitated parasite lysate(5 mg/ml),

833

while an uninfected red blood cell lysate control (5 mg/ml) was loaded in the final lane of

834

both (A) and (B). Densitometry values for both GAPDH and LDH were shown alongside (A)

835

and (B) and expressed as INT/mm2. Lanes 1 and 2 in (C) were loaded with the precipitated

836

PfLDH or PfGAPDH from the 5 mg/ml lysate samples respectively and probed with chicken

837

anti- CAEKDPSQIPWGKCQV or anti- CKDDTPIYVMGINH antibodies at 5 µg/ml as indicated

838

above the blots. A rabbit anti-IgY-HRPO secondary antibody at 1/12000 dilution was used in

839

all blots.

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Fig. 7. Detection of P. falciparum GAPDH and LDH in Pf(D10) lysates by ELISA. Separate

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ELISA plates were coated with the common-Plasmodium anti-peptide antibodies at 1 µg/ml

843

as the capture antibodies. The anti- APGKSDKEWNRDDL antibody was used to capture

844

PfLDH and the anti- CKDDTPIYVMGINH antibody PfGAPDH. Total parasite lysate was added

845

at an initial concentration of 750 µg and double diluted three times. The resulting A405

846

values were converted to protein concentration values using prepared standard curves and

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847

the average values of triplicate readings were plotted with standard deviations shown.

848

Statistical analysis was done using the Student’s t-test, with p ≤ 0.05 indicated with “*”.

849 Fig. 8. The yields of affinity purified human IgG, from an anti-malaria hyper immune serum

851

pool, against recombinant P. falciparum LDH and GAPDH proteins was compared. Three

852

hundred milligrams of a human anti-malaria hyper immune serum pool was passed

853

consecutively over the recombinant P. falciparum GAPDH and LDH AminoLink® affinity

854

resins and the bound human IgG antibodies were eluted. The specificity of the antibodies

855

was assessed by enhanced chemiluminescence with the Coomassie R250 stained reference

856

gel on the left. All primary antibodies were used at 1/500 dilution as labelled above the

857

blots and detected with a rabbit anti-human IgG-HRPO secondary antibody at 1/6000. The

858

samples resolved on the 12.5% reducing SDS-PAGE gels were E. coli BL21(DE3) host cell

859

lysate (Lane 1); uninfected red blood cell lysate (Lane 2); infected P. falciparum (D10)

860

parasite culture lysate (Lane 3); affinity purified rPfGAPDH (Lane 4) and rPfLDH (Lane 5).

861

The yields of the affinity purified human IgG antibodies are indicated in the table alongside

862

the figure.

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Table 1 Compilation of the 35 most abundant Plasmodium proteins, with proteins that were of interest shaded in grey

Conserved amongst human

Human

Pan-Plasmodial

Species-specific

Crystal structure

malaria species

orthologue

peptides

peptides

available

Early transcribed membrane protein 11.2 Early transcribed membrane protein 2, putative Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Heat shock 70 kDa protein Circumsporozoite-related antigen

yes yes yes yes yes

only P.f. only P.f. all 5 P.f., P.v., P.k. only P.f.

no no yes yes no

no no yes yes no

no no yes no no

yes

PF11_0508 PF10_0019 PF10_0159

Hypothetical protein Early transcribed membrane protein Glycophorin-binding protein 130 precursor

no yes yes

only P.f. only P.f.

no no

no no

no no

9 10 11

PF10_0372 PF14_0486 PF11_0043

Hypothetical protein Elongation factor 2 60S acidic ribosomal protein p1, putative

no yes yes

P.f., P.v., P.k. P.f., P.v., P.k.

yes no

yes yes

no no

12 13 14 15 16 17 18 19 20 21

PFA0420w PFL2515c MAL6P1.91 PF13_0346 PFI1090w PFE0070w PF11_0039 PF14_0678 PF14_0425 PFI0875w

no no yes yes yes yes yes yes yes yes

P.f., P.v., P.k. P.f., P.v. P.f., P.v., P.k. only P.f. only P.f. only P.f. P.f., P.k. only P.f.

yes yes yes no no no yes yes

yes no yes no no no no no

no no no no no no yes no

22

PF08_0019

yes

P.f., P.v., P.k.

yes

yes

no

23

PF10_0155

Hypothetical protein Hypothetical protein Ornithine aminotransferase Ubiquitin-60S ribosomal protein L40 S-adenosylmethionine synthetase, putative Interspersed repeat antigen, putative Early transcribed membrane protein 11.1 Exported protein 2 Fructose-bisphosphate aldolase Heat shock protein 70-2 (BiP) Receptor for activated C kinase (PfRACK) = Guanine nucleotide-BP Enolase

yes

P.f., P.v., P.k.

yes

yes

no

24

PF07_0029

Heat shock protein 86

yes

P.f., P.v., P.k.

yes

yes

yes

25 26

PFI1105w Phosphoglycerate kinase MAL13P1.214 Phosphoethanolamine N-methyltransferase, putative (PMT) Heat shock protein 90 (Endoplasmin homolog precursor, PFL1070c putative)

yes yes

P.f., P.v., P.k. P.f., P.v., P.k.

yes no

yes yes

no yes

yes

P.f., P.v., P.k.

yes

yes

yes

28

PFI1475w

Merozoite surface protein 1 (MSP1)

yes

a

all 5

no

yes

yes

29

PFL2215w

Actin I

a

P.f., P.v., P.k.

no

yes

yes

30 31 32 33 34 35

PFF1300w PF14_0655 MAL13P1.56 PF13_0141 MAL8P1.17 PF11_0208

Pyruvate kinase, putative RNA helicase 45 (H45, eIF4A) M1-family aminopeptidase L-lactate dehydrogenase (LDH) Protein disulfide isomerase (PDI-8) Phosphoglycerate mutase

yes yes yes yes yes yes yes

P.f., P.v., P.k. P.f., P.v., P.k. P.f., P.v., P.k. all 5 P.f., P.v., P.k. P.f., P.v., P.k.

yes yes no yes yes yes

yes yes yes yes yes yes

yes yes yes yes yes yes

1 2 3 4 5

PF11_0040 PFB0120w PF14_0598 PF08_0054 PF11_0224

6 7 8

27

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#

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Only the Nterminus yes Only the Nterminus

yes yes yes yes yes

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The table was compiled from mRNA transcript and proteomics data (Foth et al., 2011; Le Roch et al., 2003).

4

a

5

Pan-Plasmodium and species-specific peptides were chosen based on ClustalΩ alignments and BLASTp searches.

6

Available P. falciparum crystal structures were searched for on the RCSB PDB database.

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denotes proteins that are either membrane bound or associated with the insoluble cytoskeleton.

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Table 2 Alignment of the selected GAPDH and LDH peptides

LDH common

PfGAPDH 1

LISDAELEAIFD (100)

APGKSDKEWNRDDL (100)

ADGFLLIGEKKVSVFA (100)

vivax

KITDEEVEGIFD (58)

APGKSDKEWNRDDL (100)

GDGCFTVGNKKIFVHS (38)

malariae

KITDAELDAIFD (75)

VPGKSDKEWNRDDL (93)

GDGKIIVGNKTINIHN (25)

ovale

KITDAELDAIFD (75)

APGKSDKEWNRDDL (100)

GEGMFTVGDKKIYVHS (31)

(67)

(100)

(50)

knowlesi Overall Identity 100 % Identity

KITDEEVEAIFD

APGKSDKEWNRDDL

*:* *::.***

.*************

falciparum, gorilla clade G1 to G3, chimpanzee clade C2 and C3

GDGFFTIGNKKIFVHH .:* : :*:*.: :.

falciparum,vivax, knowlesi, ovale, falciparum yoelii nigeriensis, semiovale, fragile, cynomolgi, gorilla clade G1 to G3, chimpanzee clade C2 and C3, chabaudi chabaudi, berghei, yoelii yoelii, vinkei petteri

2

PfGAPDH 2

GAPDH common

AEKDPSQIPWGKCQV (100)

KDDTPIYVMGINH (100)

SEKDPAQIPWGKYEI (67)

KDDTPIYVMGINH (100)

NEKEPSQIPWGKYGI (67)

KDDTPIYVMGINH (100)

SEKDPAQIPWGKYAI (67)

KDDTPIYVMGINH (100)

(60)

KDDTPIYVMGINH (100)

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PfLDH

falciparum

HEKDPANIPWGKYGI

SC

Plasmodium species

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**:*::*****

:

falciparum, reichenowi

************* falciparum, vivax, ovale curtisi, chabaudi chabaudi, malariae, knowlesi, coatneyi, reichenowi, fragile, brasilianum, cynomolgi, berghei, vinkei vinkei, yoelii yoelii, inui, gallinaceum

Percent sequence identity of the chosen peptide sequence (underlined) were given in brackets. The malariae (LDH:

4

AAS77572.1; GAPDH: ABU50375.1) and ovale (LDH: AIU41758.1; GAPDH: AJG43655.1) sequences were obtained from NCBI.

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% identity with Target species Target description

GAPDH

falciparum

chicken sequence

Chickens

Yield (mg)

protein peptide peptide peptide

66 25 40 54

3 1 1 1

9.5 1.9 8.5 6.3

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common

rPfGAPDH C-ADGFLLIGEKKVSVFA C-AEKDPSQIPWGKCQV C-KDDTPIYVMGINH

Type

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Highlights:

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GAPDH shows promise as a malaria diagnostic target. Antibodies against rPfGAPDH and specific peptides were raised in chickens. Antibodies detected recombinant and native PfGAPDH. Native PfGAPDH protein levels were 4-6 fold higher than the current target PfLDH.

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1) 2) 3) 4)