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New developments in malaria diagnostics

New developments in malaria diagnostics REPORT mAbs 4:1, 120-126; January/February 2012; © 2012 Landes Bioscience Monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein 1,2, 1 1 3 3 1,2 Johanna H. Kattenberg, * Inge Versteeg, Stephanie J. Migchelsen, Iveth J. González, Mark D. Perkins, Petra F. Mens and Henk D.F.H. Schallig 1 2 Parasitology Unit; Royal Tropical Institute; Koninklijk Instituut voor de Tropen (KIT); KIT Biomedical Research; Amsterdam, The Netherlands; Academic Medical Centre; Centre Foundation for Innovative New Diagnostics (FIND); Geneva, Switzerland for Infection and Immunity; Amsterdam, The Netherlands; Key words: plasmodium, Plasmodium falciparum, malaria, diagnostics, rapid diagnostic test, monoclonal antibodies, glutamate rich protein, dihydrofolate reductase-thymidylate synthase, heme detoxification protein Abbreviations: RDTs, rapid diagnostic tests; GLURP, glutamate rich protein; DHFR-TS, dihydrofolate reductase-thymidylate synthase; HDP, heme detoxification protein; K , dissociation constant; HRP2, histidine rich protein; pLDH, parasite lactate dehydrogenase Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for © 2012 Landes Bioscience. novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity Do not distribute. against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, K ) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have K values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR- TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (K of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development. Unfortunately, it is not always widely available in many highly Introduction endemic areas or it is poorly executed, and for that reason an easy-to-use alternative is needed in situations where good quality Accurate early diagnosis and subsequent treatment are essential microscopy is unavailable. to reduce morbidity and mortality due to malaria. Diagnosis of malaria based on clinical signs and symptoms may lead to over- Over a decade ago, rapid diagnostic tests (RDTs) for malaria diagnosis even in highly endemic areas, as the clinical presenta- were introduced that have the advantages of being quick and 6,7 tions of the disease resemble those of a number of other common can be easily performed in remote settings. These lateral flow illnesses, such as pneumonia, equally associated with morbidity immuno-chromatographic tests are based on the detection of 1-3 and mortality. Introduction of effective but expensive, arte- malaria parasite antigens in patient blood by specific monoclo - misinin-based combination therapies, due to increasing parasite nal antibodies (mAbs) that bind the antigen and immobilize and resistance to older drugs such as chloroquine and sulfadoxine- mark it with a label on nitrocellulose. Commercially available pyrimethamine, increase the urgency for cheap and accurate RDTs for malaria all detect one or more of the following anti- diagnosis. gens: histidine rich protein 2 (HRP2), lactate dehydrogenase Microscopic examination of blood slides is, in principle, sen- (pLDH) or aldolase. Tests detecting HRP2 are most commonly sitive and specific for the parasitological detection of malaria, used because of their lower cost, better stability and lower detec- 8-11 if used correctly, and has been used for over a hundred years. tion threshold compared with pLDH-based tests. Moreover, *Correspondence to: Johanna H. Kattenberg; Email: [email protected] Submitted: 09/30/11; Revised: 10/25/11; Accepted: 10/25/11 http://dx.doi.org/10.4161/mabs.4.1.18529 120 mAbs Volume 4 Issue 1 REPORT REPORT Table 1. Antibody response of mouse sera to the recombinant antigen in ELISA at different days before each (booster) immunization during the immunization procedure Antigen Mouse Before immunization (Day 0) Day 29 Day 42 Day 48 #Cells * 10 harvested/ml GLURP R A 0 1:12,000 ≥1:15,36,000 1:3,20,000 60 B 0 1:3,000 ≥1:15,36,000 ≥1:102,40,000 60 GLURP R A 0 1:3,000 ≥1:15,36,000 1:40,000 30 B 0 1:24,000 ≥1:15,36,000 1:12,80,000 70 DHFR-TS A 0 1:24,000 ≥1:15,36,000 ≥1:102,40,000 50 B 0 1:24,000 ≥1:15,36,000 1:25,60,000 70 DHFR-1&2 A 0 1:12,000 ≥1:15,36,000 ≥1:102,40,000 20 B 0 1:24,000 ≥1:15,36,000 ≥1:102,40,000 50 HDP A 1:3,000 1:12,000* 1:48,000** ≥1:61,44,000*** 220 B 0 1:24,000* 1:96,000** 1:15,36,000*** 390 Mouse A of HDP already had a background titer at Day 0. This mouse was used for the fusion to myeloma cells; *day 28; **day 35; ***day 42. HRP2-based RDTs have better sensitivity than pLDH-based be found in the cytosol of the infected red blood cell and in later tests, although specificity is better for pLDH-based tests. stages it is trafficked to the food vacuole in transport vesicles HRP2-based tests, however, only detect Plasmodium falciparum together with heme. and the genetic diversity within the HRP2 antigen affects RDT Glutamate Rich Protein (GLURP) was chosen because it is a 13,14 sensitivity. Sensitivities for aldolase-detecting RDTs have highly antigenic exoantigen of P. falciparum that is expressed in often been reported to be poor, especially for non-falciparum par- all parasite life stages in the host, and its gene is conserved in geo- © 2012 Landes Bioscience. 15-19 25 asites. It is therefore not commonly used as a stand-alone test. graphically different isolates. The protein includes two repeat Concerns about test stability, performance (in terms of sensitivity regions, R1 and R2, of which the R2 repeat sequence is well con- and specificity) and antigen genetic diversity prompt the need for served between different isolates. Additionally, several major improvement of test performance characteristics, and therefore B-cell epitopes in the R0 (non-repetitive) region of GLURP have Do not distribute. 5,9,10,13,20 26 new antigens should be targeted by RDTs. been identified. This target is only suitable for P. falciparum In this study, novel antigens for detection by RDTs were diagnosis, because the gene is not present in the other species, but indentified through literature search. These antigen targets were it might be used in combination with other antibodies to make a chosen as suitable antigen targets according to the following three diagnostic test against multiple species. criteria. The first criterion is that the antigen should be unique for mAbs were raised against the P. falciparum specific antigen Plasmodia in some way, such as sequence or structure. Further, glutamate rich protein (GLURP) and the PAN-plasmodial anti- expression throughout the different life stages of the parasite is gens dihydrofolate reductase-thymidylate synthase (DHFR-TS) important, as well as having a conserved gene or regions within and heme detoxification protein (HDP). These antibodies were the gene, since antigenic variation can cause false negatives. screened for specificity against P. falciparum and Plasmodium Dihydrofolate reductase (DHFR) is an enzyme that functions vivax. Subsequently, affinity and detection limit were determined in the folate pathway in Plasmodium species, where it catalyzes and compared with commercially available HRP2 antibodies. the reduction of dihydrofolate to tetrahydrofolate. It was chosen because, unlike bacteria and higher eukaryotes, DHFR in pro- Results tozoa exists as a bi-functional protein with thymidylate synthase (TS). Moreover, the plasmodial DHFR domain contains two Generation and screening of mAbs. Antibody response to the 21,22 specific insert regions. All Plasmodia contain these inserts, antigens in mouse serum was measured during the immunization but the sequences vary slightly, making it potentially possible for process. High titers were observed for all antigens and 20 x 10 to 22 6 the antibodies to differentiate between the species. 390 x 10 cells/ml could be harvested from the spleens (Table 1). Heme detoxification protein (HDP) has been described as Half of the cells from one mouse for each antigen with the high- highly efficient in mediating the conversion of heme into hemo - est titer or cell count were fused with myeloma cells, except for zoin (malaria pigment). HDP is encoded by a single copy gene GLURP R2, where the cells might have been contaminated dur- containing 3 exons and is conserved within all (fully sequenced) ing harvest of the mouse with the highest titer. After selection Plasmodium species with a sequence identity of 60% and con- of hybridoma cells on selective medium with aminopterin, on 23,24 served within different P. falciparum isolates. HDP was cho- average 45% of the wells contained growing clones. These cells sen because of its unique function and although homologs have were screened for specific antibody production against P. falci- been found in other species, they only have a sequence identity parum NF54 and recombinant antigen in ELISA. Depending of less than 15% with plasmodium HDP. HDP is present in on the type of antigen, 10–25% of the growing hybridomas several life stages of Plasmodia and in the early ring stage it can (5–11% of the wells) were highly reactive with both NF54 and www.landesbioscience.com mAbs 121 Table 2. Overview and characteristics of hybridoma clones during immunization and selection process Percentage of clones highly reactive in ELISA Nr of wells #selected clones Antigen target With recombinant antigen With NF54 Proportion IgG Proportion IgM started with based on ELISA DHFR-TS insert 1 and 2 1008 8% 9% 16 1/16 15/16 TS 960 8% 6% 12 2/12 10/12 GLURP R 1504 5% 5% 4 1/4 3/4 GLURP R 576 9% 11% 10 7/10 3/10 HDP 2112 10% 5% 33 9/33 24/33 Table 3. Dissociation constants (K ) of monoclonal antibodies as determined by ELISA Target mAb Isotype K (nM) SE Target mAb Isotype K (nM) SE Target mAb Isotype K (nM) SE D D D DHFR D5 IgG3 0.10 0.014 HDP H1 IgG1 1.5 0.64 GLURP G2 IgG3 1.42 0.27 D6 IgM 0.068 0.015 H2 IgM 0.93 0.11 G4 IgG1 3.4 0.45 D7 IgM 0.36 0.039 H3 IgM 0.23 0.046 G12 IgM 0.54 0.089 D10 IgM 0.66 0.19 H4 IgG2b 0.23 0.057 G13 IgM 0.45 0.079 D13 IgM 0.41 0.20 H5 IgG3 2.1 0.82 G14 IgG2a 0.53 0.64 D15 0.80 0.49 H6 IgM 0.70 0.16 G15 IgG2a 0.54 0.11 D16 IgM 0.16 0.062 H7 IgM 0.32 0.030 G16 IgM 0.94 0.26 D19 IgG1 0.88 0.32 H8 IgM 0.56 0.081 G18 IgG1 0.70 0.12 D20 IgM ©0 .212012 Lande 0.10 H12 IgG2a s Bio 0.83 0.25scienc G19 e. IgG3 0.56 0.10 D24 IgM 2.4 1.3 H16 IgM 0.10 0.022 G22 IgG1 0.33 0.093 HRP2 PTL3 IgM 0.16 0.13 H18 IgM 0.21 0.022 G23 IgM 0.11 0.028 C1-13 IgG 1.0 0.049 G24 IgM 0.34 0.14 Do not distribute. G25 IgM 1.4 0.33 the corresponding recombinant antigen (Table 2). For each anti- antibodies (Table 4). The detection of antibody, expressed as gen the most reactive antibodies were selected and these cell lines absorbance at 450 nm, was linearly correlated with the antigen were further sub-cloned to derive monoclonal hybridoma cell concentration for all described mAbs (r ≥ 0.93 and p < 0.05) lines for further characterization. Isotyping of selected clones was (Fig. 1A–D). The strongest antibodies, D6, D15, H12 and H16 performed and, for the DHFR-TS antibodies, mostly IgM anti- are very comparable in absorbance pattern to the HRP2 antibod- bodies were selected, but for GLURP and HDP a larger propor- ies (Fig. 1A). tion of mAbs were IgG (Table 2). Detection of P. vivax. Although the antibodies were raised Dissociation constant of antibodies. Dissociation constants against P. falciparum recombinant antigens, five HDP (H2, were estimated by ELISA and are listed in Table 3 for selected H7, H10, H13 and H16) and four DHFR-TS (D7, D15, D16 DHFR-TS, HDP and GLURP antibodies. The DHFR-TS anti- and D28) of the selected antibodies were able to detect pooled bodies with the highest affinities are D5 and D6, for HDP H16 P. vivax patient samples in ELISA, besides P. falciparum. Positive and H18 and for GLURP are G22 and G23 (Table 3). The disso- absorbance at 450 nm in the ELISA ranged from 0.31–1.06 com- ciation constants of the commercially obtained HRP2 antibodies pared with 0.24 ± 0.03 of background absorption (no-antibody were not found in literature, but were measured in the present negative controls) and 0.22 ± 0.02 of GLURP (Pf specific) nega - study as 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13 tive controls. in the ELISA. This is in the same order of magnitude as the anti- sera against synthetic peptides of Pf HRP2 described by Tomar et Discussion al. Many of the developed antibodies for DHFR-TS, HDP and GLURP have a similar or higher affinity for their antigen than Concerns regarding the diagnostic accuracy and test stability of the HRP2 antibodies. malaria RDTs have prompted development of novel antibodies Crude parasite antigen detection. The ELISA with crude that could be incorporated in new diagnostic tests. In the pres- parasite antigen allowed the detection of native DHFR, HDP ent study, novel mAbs were developed and characterized against and GLURP antigens from culture at different concentrations the newly selected Plasmodium antigen targets dihydrofolate by selected mAbs. The lower detection limit of several developed reductase-thymidylate synthase, heme detoxification protein and antibodies was comparable to the detection limit of two HRP2 glutamate rich protein. 122 mAbs Volume 4 Issue 1 Table 4. Detection limit of crude parasite antigen in ELISA by selected In further test development, the antibodies should be com- antibodies compared with HRP2 antibodies bined to select good couples of capture- and detection-antibodies, mAb Detection limit of crude antigen and the detection limit of different P. falciparum culture isolates Antigen ng ng and patient samples should be evaluated next. The sandwich assay then needs to be transformed to a rapid test format for field HRP2 C1-13 250 8 application. Since stability of the reagents is a critical issue for HRP2 C1-13 25 8 the RDTs, the stability of the antibodies at ambient temperatures DHFR D6 250 25 needs to be investigated to choose the most stable antibodies. DHFR D15 25 25 Further research is needed if the DHFR-TS and HDP antibodies HDP H4 25 8 can also detect Plasmodium malariae and Plasmodium ovale para- HDP H12 25 8 sites. The mAbs presented in this study show much potential for further development in diagnostic assays. HDP H12 250 3 HDP H15 250 3 Material and Methods HDP H16 250 25 HRP2 PTL3 25 8 In vitro culture of P. falciparum. Plasmodium falciparum strains HRP2 PTL3 250 3 NF54 (UMC St. Radboud, Nijmegen, The Netherlands) and 3D7 (MRA-102, MR4, ATCC Manassas Virginia) parasites The present work generated several mAb-producing hybrid- were cultured in a candle jar on RPMI 1640 medium with oma cell lines, and specific clones were selected on the basis of L-Glutamine and 25 mM HEPES (Gibco, Invitrogen) with + + their reactivity with recombinant antigen and malaria cell cul- human A serum and O red blood cells according to the candle- 28,29 ture material. Affinities of the developed antibodies for their jar technique of Trager-Jensen. The parasitemia was deter- recombinant antigens are higher than, or comparable to, the two mined with Fields-stained microscopy slides and counted as tested commercially available HRP2 antibodies. High affinity percentage of infected red blood cells. The parasite strain used © 2012 Landes Bioscience. is important for sensitivity and the detection limit of diagnostic in the experiments depends on the isolate that was in culture assays, suggesting that the newly developed antibodies are good because the department changed the parasite strain to be used candidates for diagnostic test development. The antibodies can as a standard from NF54 to 3D7. NF54 is the parental isolate detect the native antigens from NF54 and 3D7 P. falciparum iso- of clone 3D7; therefore no difference is expected between the Do not distribute. lates in a concentration-dependent manner. Some of the antibod- clones concerning these antigens. The recombinant antigens were ies were able to give similar detection limit and absorbance as amplified from NF54 parasites (see section below) and the vec - the HRP2 antibodies, which is important for the sensitivity of tors were sequenced and compared with the published 3D7 genes a diagnostic test. Many antibodies had a lower absorbance than in PlasmoDB; the sequences were identical (data not shown). the HRP2 antibodies, but nevertheless many of them did have a Preparation of P. falciparum lysate. Parasite culture (~2–5%) similar detection limit. Higher absorbance does not necessarily was diluted 10 times in PBS and sonicated vigorously for 30 sec. mean a better antibody, but might also reflect the abundance of This suspension was used in the ELISA procedures described the antigen in the crude parasite extract. HRP2 is a very stable below. antigen and known to persist, and may therefore be more abun- P. vivax samples. Frozen samples from patients infected with dant in the extract, which seems like a plausible explanation, P. vivax parasites were provided by the Instituto de Medicina because the antibody affinities are very similar. The abundance Tropical “Alexander von Humboldt”—Universidad Peruana of the antigen in clinical samples is important for the detection Cayetano de Heredia in Peru. Samples were characterized with limit of a diagnostic assay, and should be kept into consideration microscopy. A pool of samples was used with a resulting parasit- for future test development. Some of the DHFR-TS and HDP emia of ~0.1% and used in the screening ELISA described below. antibodies were able to detect the homologous antigens in P. Production of recombinant antigens. The recombinant vivax, an important feature for malaria diagnostic tests, since the HDP protein was provided for the purpose of this study from available RDTs poorly detect this species (pLDH and aldolase), the Virginia Bioinformatics Institute, Virginia Tech. Primers 9,10 or not at all (HRP2). were developed to amplify gene fragments of the DHFR-TS and Mutations in the DHFR domain of Plasmodia are involved GLURP antigens and sequences are listed in Table 5. Genetic in anti-malarial resistance, and the presence of these mutations sequences were derived from PlasmoDB, with the follow- might inufl ence antibody binding. One of these mutations, ing accession numbers: DHFR-TS: PFD0830w and GLURP: Ser108, is a residue that is present in one of the recombinant anti- PF10_0344. Amplification was performed with Accuprime gens used in the present study, which is the penultimate amino Taq High Fidelity (Invitrogen) and the supplied PCR buffer acid at the C-terminal side of DHFR-TS insert 1 and 2. Whether II with the addition of 0.05 mM dNTPs and 0.2 μM each of the developed antibodies against DHFR-TS are affected by this forward and reverse primer. Amplification was performed on a mutation should be further determined by screening several Peltier Thermal cycler 100 with initial hot start of 1 min at 94°C genotypically different P. falciparum strains. and subsequently 40 cycles of 30 sec at 94°C, 30 sec at 58°C www.landesbioscience.com mAbs 123 © 2012 Landes Bioscience. Do not distribute. Figure 1. Detection of native antigens in a crude parasite extract dilution from P. falciparum culture with selected monoclonal antibodies. (A) HRP2 antibodies (C1–13 and PTL3, triangles) compared with strongest HDP (marked with H, solid lines) and DHFR-TS (marked with D, open squares) antibodies, (B) DHFR-TS antibodies, (C) HDP antibodies and (D) GLURP antibodies. Table 5. Primer sequences for recombinant antigens and encoding nucleotide range Fragment name Nucleotide range Primer Forward primer GGT GGT GCT AGC GCA TGT TGT AAG GTT GAA AGC DHFR-TS insert 1 and 2 46–327 in PFD0830w Reverse primer GGT GGT TGC TCT TCC GCA CCA GCT TGT TCT TCC CAT AAC Forward primer GGT GGT GCT AGC CAA AGT GAT CGA ACG GGA GTA TS 1024–1824 in PFD0830w Reverse primer GGT GGT TGC TCT TCC GCA AGC AGC CAT ATC CAT TGA AAT Forward primer GGT GGA CAT ATG AAT AAA CGA ATC GGG GGT CCT GLURP R 9–1281 in PF10_0344 Reverse primer GGT GGT TGC TCT TCC GCA TTT AGG GGA AGG CAC ATC GLURP R 2497–3204 in PF10_0344 Forward primer GGT GGT CAT ATG GAA GTG GCT CAT CCA GAA Reverse primer GGT GGT TGC TCT TCC GCA TTC ATT GTT ATT TGT TTG TGA TGG (DHFR1 and 2, TS, GLURP R ) or 60°C (GLURP R ) and E. coli High Efficiency (New England Biolabs) with heat shock 0 2 1.10 min at 68°C, and a final elongation step of 10 min at 68°C. at 42°C for 10 sec. Recombinant antigens were expressed and The PCR amplicons were precipitated with ethanol and mixed purified with Chitine beads as described in the IMPACT KIT with pTXB1 vector (IMPACT KIT, New England Biolabs) and (New England Biolabs) and confirmed on SDS-PAGE gel. This digested with BmtI (DHFR1&2 and TS) or NdeI (GLURP R work was performed in a controlled environment under GMO and GLURP R ) and SapI. All restriction enzymes were from license nr 02–080 of the Dutch ministry of Infrastructure and New England Biolabs and used as described by the manufac- Environment. turer. The digested vector and amplicons were ligated with T4 Immunization protocol and hybridoma culture. Ten to DNA ligase (Roche) and transformed to T7 Express Competent twelve week old specific pathogen free Balb/c mice (two for 124 mAbs Volume 4 Issue 1 each antigen) were immunized twice (with a 28-d interval) with Ultra-4 Centrifugal Filter Units (Millipore) until a concentration 100 μg recombinant antigen in PBS with 50% Stimune adjuvant of approximately 1 mg/ml was reached. (Prionics) intra peritoneal (i.p.) and subcutaneous (s.c.) and with Measurement of true affinity ( K ) by ELISA. The bind- a final booster after 11 d with 100 μg recombinant antigen in PBS ing affinities of the antibodies were determined by measuring intravenous (i.v.). All animal experiments were approved by the the dissociation constant (K ). Briefly, antigen at various con - -7 -11 local animal ethics committee. Antibody response in mice serum centrations ranging from 1 x 10 M to 5 x 10 M was mixed was determined with ELISA as described below. Splenocytes were with a constant amount of antibody in 0.1 M potassium phos- harvested and frozen according to the protocol from Marusich et phate, 2 mM EDTA, 10 mg/ml BSA, pH 7.8 and incubated for al. The cells of half a spleen were fused to myeloma cells accord- 15 h at room temperature. The antigen-antibody mix was then ing to a standardized protocol. Cell lines were selected based on transferred to micro-titer plates previously coated with capture ELISA results described below, and subsequently sub-cloned to antigens at 150 ng/well in 50 mM sodium carbonate pH 9.6 obtain single clones. Hybridoma cells were grown in Optimem (DHFR1&2, DHFR-TS, GLURP R , GLURP R or HDP) and 0 2 I with Glutamax (Invitrogen) with 10% FCS (Fetalclone I, incubated for 60 min at room temperature. Washing of plates and Hyclone) or Hybridoma Serum Free medium (Invitrogen). detection of the presence of antibody was performed as described Screening ELISA. Mice serum and supernatant of hybrid- for the screening ELISA. Dissociation constants were calculated oma cell lines were screened with ELISA for specific reactivity using regression analysis and the Scatchard-Klotz equation. In with recombinant antigen and NF54 and/or 3D7 culture lysate. comparison, two HRPII antibodies (C1–13 and PTL3, National ELISA plates (medium binding, Greiner) were coated over night Bioproducts Institute) were tested as well with recombinant at 4°C with recombinant antigen (20 μg/ml) in PBS or P. fal- HRP2 (ReaMatrix). ciparum lysate or a P. vivax patient sample (5,630 parasites/μl Crude parasite antigen ELISA. A crude parasite antigen diluted 1:5 in PBS) and subsequently washed with PBS + 0.1% extract was prepared from a late stage 3D7 culture (~3–4% 29,34 Tween 20 (PBST) and blocked with 5% BSA (Sigma) in PBS schizonts) with Percoll as described by Troye-Blomberg et al. for 1 h at 37°C. Plates were washed and incubated 1 h at 37°C This antigen extract was coated overnight on ELISA plates in a with 50 μl of supernatant of the hybridoma clones or mouse dilution series from 20-0.03 μg/ml in 1% BSA in PBST. 1% BSA © 2012 Landes Bioscience. 3 6 serum diluted in 1% BSA in PBST (1:3 x 10 –1:10 x 10 ). After in PBST was used as negative control and the wells were subse- washing, plates were incubated 1 h at 37°C with peroxidase quently blocked with 5% BSA in PBS for 30 min to 1 h at 37°C. conjugated goat-anti-mouse (Jackson Immunoresearch) 1:5,000 After washing 3 times with PBST, purified antibodies (5 μg/ml) diluted in 1% BSA in PBST. The presence of antibody was mea- were added to each dilution series. In comparison two HRP2 Do not distribute. sured by adding 0.04% 3,3',5,5'-Tetramethylbenzidine (Sigma), antibodies (C1–13 and PTL3, National Bioproducts Institute) 0.04% urea peroxide (Sigma) in 0.1 M Sodium acetate citrate were taken along on each ELISA plate in the same concentration. pH 4 and the reaction was stopped with 0.5 M Sulfuric acid. Statistical analysis. The dissociation constant (K ) was deter- Color development was measured at 450 nm in a SUNRISE mined by calculating the slope of the lines by regression analy- ELISA reader (Tecan). sis. The correlation between absorbance in the ELISA and Purification of mAbs. Antibodies were isotyped (Pierce the antigen concentration was determined by Pearson product Rapid ELISA Mouse Antibody Isotyping Kit, Thermo Scientific) moment correlation coefficient (PMCC). and HDP and DHFR-TS antibodies were purified directly from Acknowledgments hybridoma supernatant (filtered through 0.2 μm) with 1 ml HiTrap protein G HP columns (GE Healthcare) in case of This project is part of a collaborative effort for the improve- IgG and with 1 ml HiTrap IgM purification HP columns (GE ment of malaria diagnosis with the Foundation for Innovative Healthcare) in case of IgM, according to manufacturer’s instruc- New Diagnostics (FIND). Support for this project was provided tions. Purifications were performed on an ÄKTA FPLC system through funding from FIND. HRP2 recombinant protein was (Amersham Biosciences). Antibody containing fractions were provided by ReaMetrix India Pvt., Ltd.; anti-HRP2 antibodies pooled and concentrated with 50 kD (IgG) or 100 kD (IgM) by National Bioproducts Institute in South Africa; and the puri- Amicon Ultra-4 Centrifugal Filter Units (Millipore) until a fied recombinant heme-detoxification protein (HDP) by Virginia concentration of approximately 1 mg/ml was reached. Antibody Polytechnic Institute and State University (Virginia Tech.,) in concentration was determined by adsorption at 280 nm with a the USA. We thank Dionicia Gamboa from the Instituto de Nanodrop 1000 (Thermo Scientific). Medicina Tropical “Alexander von Humboldt”—Universidad The GLURP antibodies used in the experiments were pre- Peruana Cayetano de Heredia in Peru for sending samples from cipitated with ammonium sulfate. Hybridoma supernatant was patients infected with P. vivax parasites. Those samples were clarified by a 0.2 μm syringe filter (Millipore) and saturated collected in the frame of collaborative projects with FIND with ammonium sulfate pH 7.4 was slowly added till the final concen - the required approval of a local ethical committee. We thank tration of ammonium sulfate was 45% and left on a rotator for MR4 for providing us with 3D7 malaria parasites contributed by 45 min. Subsequently, the mixture was incubated O/N at 4°C Daniel Carucci and Alister Craig. We thank the Centre for clini- and then centrifuged at 3,893 g for 40 min. The pellet was resus- cal malaria studies of the University Medical Centre St. Radboud pended in 8 ml cold PBS and concentrated with 50 kD Amicon in Nijmegen for providing us with the NF54 malaria parasites. www.landesbioscience.com mAbs 125 13. Baker J, McCarthy J, Gatton M, Kyle DE, Belizario V, 24. Vinayak S, Rathore D, Kariuki S, Slutsker L, Shi References Luchavez J, et al. 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Tomar D, Biswas S, Tripathi V, Rao DN. Development 5. Bell D, Wongsrichanalai C, Barnwell JW. Ensuring specificity of a dipstick test (ICT Malaria P.f./P.v.) of diagnostic reagents: raising antibodies against syn- quality and access for malaria diagnosis: how can it compared with expert microscopy. Ann Trop Med thetic peptides of PfHRP-2 and LDH using micro- be achieved? Nat Rev Microbiol 2006; 4:682-95; Parasitol 2002; 96:15-8; PMID:11989528; http:// sphere delivery. Immunobiology 2006; 211:797- PMID:16912713; http://dx.doi.org/10.1038/nrmi- dx.doi.org/10.1179/000349802125000457. 805; PMID:17113917; http://dx.doi.org/10.1016/j. cro1474. imbio.2006.05.003. 17. Huong NM, Davis TM, Hewitt S, Huong NV, Uyen 6. Shiff CJ, Premji Z, Minjas JN. The rapid manual TT, Nhan DH, et al. Comparison of three antigen 28. Trager W, Jensen JB. Human malaria parasites in ParaSight-F test. A new diagnostic tool for Plasmodium detection methods for diagnosis and therapeutic moni- continuous culture. Science 1976; 193:673-5; falciparum infection. Trans R Soc Trop Med Hyg toring of malaria: a field study from Southern Vietnam. PMID:781840; http://dx.doi.org/10.1126/sci- 1993; 87:646-8; PMID:8296363; http://dx.doi. Trop Med Int Health 2002; 7:304-8; PMID:11952945; ence.781840. org/10.1016/0035-9203(93)90273-S. http://dx.doi.org/10.1046/j.1365-3156.2002.00869.x. 29. Moll K, Ljungström I, Perlmann H, Scherf A, Wahlgren 7. Piper R, Lebras J, Wentworth L, Hunt-Cooke A, 18. Mason DP, Kawamoto F, Lin K, Laoboonchai A, A. Methods in malaria research 2008; 5. Houzé S, Chiodini P, et al. Immunocapture diagnostic Wongsrichanalai C. A comparison of two rapid field 30. Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, © 2012 Landes Bioscience. assays for malaria using Plasmodium lactate dehydroge- immunochromatographic tests to expert microscopy Fischer S, Gajria B, et al. PlasmoDB: a functional nase (pLDH). Am J Trop Med Hyg 1999; 60:109-18; in the diagnosis of malaria. 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PMID:21735422. 126 mAbs Volume 4 Issue 1 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png mAbs Taylor & Francis

New developments in malaria diagnostics

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REPORT mAbs 4:1, 120-126; January/February 2012; © 2012 Landes Bioscience Monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein 1,2, 1 1 3 3 1,2 Johanna H. Kattenberg, * Inge Versteeg, Stephanie J. Migchelsen, Iveth J. González, Mark D. Perkins, Petra F. Mens and Henk D.F.H. Schallig 1 2 Parasitology Unit; Royal Tropical Institute; Koninklijk Instituut voor de Tropen (KIT); KIT Biomedical Research; Amsterdam, The Netherlands; Academic Medical Centre; Centre Foundation for Innovative New Diagnostics (FIND); Geneva, Switzerland for Infection and Immunity; Amsterdam, The Netherlands; Key words: plasmodium, Plasmodium falciparum, malaria, diagnostics, rapid diagnostic test, monoclonal antibodies, glutamate rich protein, dihydrofolate reductase-thymidylate synthase, heme detoxification protein Abbreviations: RDTs, rapid diagnostic tests; GLURP, glutamate rich protein; DHFR-TS, dihydrofolate reductase-thymidylate synthase; HDP, heme detoxification protein; K , dissociation constant; HRP2, histidine rich protein; pLDH, parasite lactate dehydrogenase Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for © 2012 Landes Bioscience. novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity Do not distribute. against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, K ) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have K values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR- TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (K of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development. Unfortunately, it is not always widely available in many highly Introduction endemic areas or it is poorly executed, and for that reason an easy-to-use alternative is needed in situations where good quality Accurate early diagnosis and subsequent treatment are essential microscopy is unavailable. to reduce morbidity and mortality due to malaria. Diagnosis of malaria based on clinical signs and symptoms may lead to over- Over a decade ago, rapid diagnostic tests (RDTs) for malaria diagnosis even in highly endemic areas, as the clinical presenta- were introduced that have the advantages of being quick and 6,7 tions of the disease resemble those of a number of other common can be easily performed in remote settings. These lateral flow illnesses, such as pneumonia, equally associated with morbidity immuno-chromatographic tests are based on the detection of 1-3 and mortality. Introduction of effective but expensive, arte- malaria parasite antigens in patient blood by specific monoclo - misinin-based combination therapies, due to increasing parasite nal antibodies (mAbs) that bind the antigen and immobilize and resistance to older drugs such as chloroquine and sulfadoxine- mark it with a label on nitrocellulose. Commercially available pyrimethamine, increase the urgency for cheap and accurate RDTs for malaria all detect one or more of the following anti- diagnosis. gens: histidine rich protein 2 (HRP2), lactate dehydrogenase Microscopic examination of blood slides is, in principle, sen- (pLDH) or aldolase. Tests detecting HRP2 are most commonly sitive and specific for the parasitological detection of malaria, used because of their lower cost, better stability and lower detec- 8-11 if used correctly, and has been used for over a hundred years. tion threshold compared with pLDH-based tests. Moreover, *Correspondence to: Johanna H. Kattenberg; Email: [email protected] Submitted: 09/30/11; Revised: 10/25/11; Accepted: 10/25/11 http://dx.doi.org/10.4161/mabs.4.1.18529 120 mAbs Volume 4 Issue 1 REPORT REPORT Table 1. Antibody response of mouse sera to the recombinant antigen in ELISA at different days before each (booster) immunization during the immunization procedure Antigen Mouse Before immunization (Day 0) Day 29 Day 42 Day 48 #Cells * 10 harvested/ml GLURP R A 0 1:12,000 ≥1:15,36,000 1:3,20,000 60 B 0 1:3,000 ≥1:15,36,000 ≥1:102,40,000 60 GLURP R A 0 1:3,000 ≥1:15,36,000 1:40,000 30 B 0 1:24,000 ≥1:15,36,000 1:12,80,000 70 DHFR-TS A 0 1:24,000 ≥1:15,36,000 ≥1:102,40,000 50 B 0 1:24,000 ≥1:15,36,000 1:25,60,000 70 DHFR-1&2 A 0 1:12,000 ≥1:15,36,000 ≥1:102,40,000 20 B 0 1:24,000 ≥1:15,36,000 ≥1:102,40,000 50 HDP A 1:3,000 1:12,000* 1:48,000** ≥1:61,44,000*** 220 B 0 1:24,000* 1:96,000** 1:15,36,000*** 390 Mouse A of HDP already had a background titer at Day 0. This mouse was used for the fusion to myeloma cells; *day 28; **day 35; ***day 42. HRP2-based RDTs have better sensitivity than pLDH-based be found in the cytosol of the infected red blood cell and in later tests, although specificity is better for pLDH-based tests. stages it is trafficked to the food vacuole in transport vesicles HRP2-based tests, however, only detect Plasmodium falciparum together with heme. and the genetic diversity within the HRP2 antigen affects RDT Glutamate Rich Protein (GLURP) was chosen because it is a 13,14 sensitivity. Sensitivities for aldolase-detecting RDTs have highly antigenic exoantigen of P. falciparum that is expressed in often been reported to be poor, especially for non-falciparum par- all parasite life stages in the host, and its gene is conserved in geo- © 2012 Landes Bioscience. 15-19 25 asites. It is therefore not commonly used as a stand-alone test. graphically different isolates. The protein includes two repeat Concerns about test stability, performance (in terms of sensitivity regions, R1 and R2, of which the R2 repeat sequence is well con- and specificity) and antigen genetic diversity prompt the need for served between different isolates. Additionally, several major improvement of test performance characteristics, and therefore B-cell epitopes in the R0 (non-repetitive) region of GLURP have Do not distribute. 5,9,10,13,20 26 new antigens should be targeted by RDTs. been identified. This target is only suitable for P. falciparum In this study, novel antigens for detection by RDTs were diagnosis, because the gene is not present in the other species, but indentified through literature search. These antigen targets were it might be used in combination with other antibodies to make a chosen as suitable antigen targets according to the following three diagnostic test against multiple species. criteria. The first criterion is that the antigen should be unique for mAbs were raised against the P. falciparum specific antigen Plasmodia in some way, such as sequence or structure. Further, glutamate rich protein (GLURP) and the PAN-plasmodial anti- expression throughout the different life stages of the parasite is gens dihydrofolate reductase-thymidylate synthase (DHFR-TS) important, as well as having a conserved gene or regions within and heme detoxification protein (HDP). These antibodies were the gene, since antigenic variation can cause false negatives. screened for specificity against P. falciparum and Plasmodium Dihydrofolate reductase (DHFR) is an enzyme that functions vivax. Subsequently, affinity and detection limit were determined in the folate pathway in Plasmodium species, where it catalyzes and compared with commercially available HRP2 antibodies. the reduction of dihydrofolate to tetrahydrofolate. It was chosen because, unlike bacteria and higher eukaryotes, DHFR in pro- Results tozoa exists as a bi-functional protein with thymidylate synthase (TS). Moreover, the plasmodial DHFR domain contains two Generation and screening of mAbs. Antibody response to the 21,22 specific insert regions. All Plasmodia contain these inserts, antigens in mouse serum was measured during the immunization but the sequences vary slightly, making it potentially possible for process. High titers were observed for all antigens and 20 x 10 to 22 6 the antibodies to differentiate between the species. 390 x 10 cells/ml could be harvested from the spleens (Table 1). Heme detoxification protein (HDP) has been described as Half of the cells from one mouse for each antigen with the high- highly efficient in mediating the conversion of heme into hemo - est titer or cell count were fused with myeloma cells, except for zoin (malaria pigment). HDP is encoded by a single copy gene GLURP R2, where the cells might have been contaminated dur- containing 3 exons and is conserved within all (fully sequenced) ing harvest of the mouse with the highest titer. After selection Plasmodium species with a sequence identity of 60% and con- of hybridoma cells on selective medium with aminopterin, on 23,24 served within different P. falciparum isolates. HDP was cho- average 45% of the wells contained growing clones. These cells sen because of its unique function and although homologs have were screened for specific antibody production against P. falci- been found in other species, they only have a sequence identity parum NF54 and recombinant antigen in ELISA. Depending of less than 15% with plasmodium HDP. HDP is present in on the type of antigen, 10–25% of the growing hybridomas several life stages of Plasmodia and in the early ring stage it can (5–11% of the wells) were highly reactive with both NF54 and www.landesbioscience.com mAbs 121 Table 2. Overview and characteristics of hybridoma clones during immunization and selection process Percentage of clones highly reactive in ELISA Nr of wells #selected clones Antigen target With recombinant antigen With NF54 Proportion IgG Proportion IgM started with based on ELISA DHFR-TS insert 1 and 2 1008 8% 9% 16 1/16 15/16 TS 960 8% 6% 12 2/12 10/12 GLURP R 1504 5% 5% 4 1/4 3/4 GLURP R 576 9% 11% 10 7/10 3/10 HDP 2112 10% 5% 33 9/33 24/33 Table 3. Dissociation constants (K ) of monoclonal antibodies as determined by ELISA Target mAb Isotype K (nM) SE Target mAb Isotype K (nM) SE Target mAb Isotype K (nM) SE D D D DHFR D5 IgG3 0.10 0.014 HDP H1 IgG1 1.5 0.64 GLURP G2 IgG3 1.42 0.27 D6 IgM 0.068 0.015 H2 IgM 0.93 0.11 G4 IgG1 3.4 0.45 D7 IgM 0.36 0.039 H3 IgM 0.23 0.046 G12 IgM 0.54 0.089 D10 IgM 0.66 0.19 H4 IgG2b 0.23 0.057 G13 IgM 0.45 0.079 D13 IgM 0.41 0.20 H5 IgG3 2.1 0.82 G14 IgG2a 0.53 0.64 D15 0.80 0.49 H6 IgM 0.70 0.16 G15 IgG2a 0.54 0.11 D16 IgM 0.16 0.062 H7 IgM 0.32 0.030 G16 IgM 0.94 0.26 D19 IgG1 0.88 0.32 H8 IgM 0.56 0.081 G18 IgG1 0.70 0.12 D20 IgM ©0 .212012 Lande 0.10 H12 IgG2a s Bio 0.83 0.25scienc G19 e. IgG3 0.56 0.10 D24 IgM 2.4 1.3 H16 IgM 0.10 0.022 G22 IgG1 0.33 0.093 HRP2 PTL3 IgM 0.16 0.13 H18 IgM 0.21 0.022 G23 IgM 0.11 0.028 C1-13 IgG 1.0 0.049 G24 IgM 0.34 0.14 Do not distribute. G25 IgM 1.4 0.33 the corresponding recombinant antigen (Table 2). For each anti- antibodies (Table 4). The detection of antibody, expressed as gen the most reactive antibodies were selected and these cell lines absorbance at 450 nm, was linearly correlated with the antigen were further sub-cloned to derive monoclonal hybridoma cell concentration for all described mAbs (r ≥ 0.93 and p < 0.05) lines for further characterization. Isotyping of selected clones was (Fig. 1A–D). The strongest antibodies, D6, D15, H12 and H16 performed and, for the DHFR-TS antibodies, mostly IgM anti- are very comparable in absorbance pattern to the HRP2 antibod- bodies were selected, but for GLURP and HDP a larger propor- ies (Fig. 1A). tion of mAbs were IgG (Table 2). Detection of P. vivax. Although the antibodies were raised Dissociation constant of antibodies. Dissociation constants against P. falciparum recombinant antigens, five HDP (H2, were estimated by ELISA and are listed in Table 3 for selected H7, H10, H13 and H16) and four DHFR-TS (D7, D15, D16 DHFR-TS, HDP and GLURP antibodies. The DHFR-TS anti- and D28) of the selected antibodies were able to detect pooled bodies with the highest affinities are D5 and D6, for HDP H16 P. vivax patient samples in ELISA, besides P. falciparum. Positive and H18 and for GLURP are G22 and G23 (Table 3). The disso- absorbance at 450 nm in the ELISA ranged from 0.31–1.06 com- ciation constants of the commercially obtained HRP2 antibodies pared with 0.24 ± 0.03 of background absorption (no-antibody were not found in literature, but were measured in the present negative controls) and 0.22 ± 0.02 of GLURP (Pf specific) nega - study as 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13 tive controls. in the ELISA. This is in the same order of magnitude as the anti- sera against synthetic peptides of Pf HRP2 described by Tomar et Discussion al. Many of the developed antibodies for DHFR-TS, HDP and GLURP have a similar or higher affinity for their antigen than Concerns regarding the diagnostic accuracy and test stability of the HRP2 antibodies. malaria RDTs have prompted development of novel antibodies Crude parasite antigen detection. The ELISA with crude that could be incorporated in new diagnostic tests. In the pres- parasite antigen allowed the detection of native DHFR, HDP ent study, novel mAbs were developed and characterized against and GLURP antigens from culture at different concentrations the newly selected Plasmodium antigen targets dihydrofolate by selected mAbs. The lower detection limit of several developed reductase-thymidylate synthase, heme detoxification protein and antibodies was comparable to the detection limit of two HRP2 glutamate rich protein. 122 mAbs Volume 4 Issue 1 Table 4. Detection limit of crude parasite antigen in ELISA by selected In further test development, the antibodies should be com- antibodies compared with HRP2 antibodies bined to select good couples of capture- and detection-antibodies, mAb Detection limit of crude antigen and the detection limit of different P. falciparum culture isolates Antigen ng ng and patient samples should be evaluated next. The sandwich assay then needs to be transformed to a rapid test format for field HRP2 C1-13 250 8 application. Since stability of the reagents is a critical issue for HRP2 C1-13 25 8 the RDTs, the stability of the antibodies at ambient temperatures DHFR D6 250 25 needs to be investigated to choose the most stable antibodies. DHFR D15 25 25 Further research is needed if the DHFR-TS and HDP antibodies HDP H4 25 8 can also detect Plasmodium malariae and Plasmodium ovale para- HDP H12 25 8 sites. The mAbs presented in this study show much potential for further development in diagnostic assays. HDP H12 250 3 HDP H15 250 3 Material and Methods HDP H16 250 25 HRP2 PTL3 25 8 In vitro culture of P. falciparum. Plasmodium falciparum strains HRP2 PTL3 250 3 NF54 (UMC St. Radboud, Nijmegen, The Netherlands) and 3D7 (MRA-102, MR4, ATCC Manassas Virginia) parasites The present work generated several mAb-producing hybrid- were cultured in a candle jar on RPMI 1640 medium with oma cell lines, and specific clones were selected on the basis of L-Glutamine and 25 mM HEPES (Gibco, Invitrogen) with + + their reactivity with recombinant antigen and malaria cell cul- human A serum and O red blood cells according to the candle- 28,29 ture material. Affinities of the developed antibodies for their jar technique of Trager-Jensen. The parasitemia was deter- recombinant antigens are higher than, or comparable to, the two mined with Fields-stained microscopy slides and counted as tested commercially available HRP2 antibodies. High affinity percentage of infected red blood cells. The parasite strain used © 2012 Landes Bioscience. is important for sensitivity and the detection limit of diagnostic in the experiments depends on the isolate that was in culture assays, suggesting that the newly developed antibodies are good because the department changed the parasite strain to be used candidates for diagnostic test development. The antibodies can as a standard from NF54 to 3D7. NF54 is the parental isolate detect the native antigens from NF54 and 3D7 P. falciparum iso- of clone 3D7; therefore no difference is expected between the Do not distribute. lates in a concentration-dependent manner. Some of the antibod- clones concerning these antigens. The recombinant antigens were ies were able to give similar detection limit and absorbance as amplified from NF54 parasites (see section below) and the vec - the HRP2 antibodies, which is important for the sensitivity of tors were sequenced and compared with the published 3D7 genes a diagnostic test. Many antibodies had a lower absorbance than in PlasmoDB; the sequences were identical (data not shown). the HRP2 antibodies, but nevertheless many of them did have a Preparation of P. falciparum lysate. Parasite culture (~2–5%) similar detection limit. Higher absorbance does not necessarily was diluted 10 times in PBS and sonicated vigorously for 30 sec. mean a better antibody, but might also reflect the abundance of This suspension was used in the ELISA procedures described the antigen in the crude parasite extract. HRP2 is a very stable below. antigen and known to persist, and may therefore be more abun- P. vivax samples. Frozen samples from patients infected with dant in the extract, which seems like a plausible explanation, P. vivax parasites were provided by the Instituto de Medicina because the antibody affinities are very similar. The abundance Tropical “Alexander von Humboldt”—Universidad Peruana of the antigen in clinical samples is important for the detection Cayetano de Heredia in Peru. Samples were characterized with limit of a diagnostic assay, and should be kept into consideration microscopy. A pool of samples was used with a resulting parasit- for future test development. Some of the DHFR-TS and HDP emia of ~0.1% and used in the screening ELISA described below. antibodies were able to detect the homologous antigens in P. Production of recombinant antigens. The recombinant vivax, an important feature for malaria diagnostic tests, since the HDP protein was provided for the purpose of this study from available RDTs poorly detect this species (pLDH and aldolase), the Virginia Bioinformatics Institute, Virginia Tech. Primers 9,10 or not at all (HRP2). were developed to amplify gene fragments of the DHFR-TS and Mutations in the DHFR domain of Plasmodia are involved GLURP antigens and sequences are listed in Table 5. Genetic in anti-malarial resistance, and the presence of these mutations sequences were derived from PlasmoDB, with the follow- might inufl ence antibody binding. One of these mutations, ing accession numbers: DHFR-TS: PFD0830w and GLURP: Ser108, is a residue that is present in one of the recombinant anti- PF10_0344. Amplification was performed with Accuprime gens used in the present study, which is the penultimate amino Taq High Fidelity (Invitrogen) and the supplied PCR buffer acid at the C-terminal side of DHFR-TS insert 1 and 2. Whether II with the addition of 0.05 mM dNTPs and 0.2 μM each of the developed antibodies against DHFR-TS are affected by this forward and reverse primer. Amplification was performed on a mutation should be further determined by screening several Peltier Thermal cycler 100 with initial hot start of 1 min at 94°C genotypically different P. falciparum strains. and subsequently 40 cycles of 30 sec at 94°C, 30 sec at 58°C www.landesbioscience.com mAbs 123 © 2012 Landes Bioscience. Do not distribute. Figure 1. Detection of native antigens in a crude parasite extract dilution from P. falciparum culture with selected monoclonal antibodies. (A) HRP2 antibodies (C1–13 and PTL3, triangles) compared with strongest HDP (marked with H, solid lines) and DHFR-TS (marked with D, open squares) antibodies, (B) DHFR-TS antibodies, (C) HDP antibodies and (D) GLURP antibodies. Table 5. Primer sequences for recombinant antigens and encoding nucleotide range Fragment name Nucleotide range Primer Forward primer GGT GGT GCT AGC GCA TGT TGT AAG GTT GAA AGC DHFR-TS insert 1 and 2 46–327 in PFD0830w Reverse primer GGT GGT TGC TCT TCC GCA CCA GCT TGT TCT TCC CAT AAC Forward primer GGT GGT GCT AGC CAA AGT GAT CGA ACG GGA GTA TS 1024–1824 in PFD0830w Reverse primer GGT GGT TGC TCT TCC GCA AGC AGC CAT ATC CAT TGA AAT Forward primer GGT GGA CAT ATG AAT AAA CGA ATC GGG GGT CCT GLURP R 9–1281 in PF10_0344 Reverse primer GGT GGT TGC TCT TCC GCA TTT AGG GGA AGG CAC ATC GLURP R 2497–3204 in PF10_0344 Forward primer GGT GGT CAT ATG GAA GTG GCT CAT CCA GAA Reverse primer GGT GGT TGC TCT TCC GCA TTC ATT GTT ATT TGT TTG TGA TGG (DHFR1 and 2, TS, GLURP R ) or 60°C (GLURP R ) and E. coli High Efficiency (New England Biolabs) with heat shock 0 2 1.10 min at 68°C, and a final elongation step of 10 min at 68°C. at 42°C for 10 sec. Recombinant antigens were expressed and The PCR amplicons were precipitated with ethanol and mixed purified with Chitine beads as described in the IMPACT KIT with pTXB1 vector (IMPACT KIT, New England Biolabs) and (New England Biolabs) and confirmed on SDS-PAGE gel. This digested with BmtI (DHFR1&2 and TS) or NdeI (GLURP R work was performed in a controlled environment under GMO and GLURP R ) and SapI. All restriction enzymes were from license nr 02–080 of the Dutch ministry of Infrastructure and New England Biolabs and used as described by the manufac- Environment. turer. The digested vector and amplicons were ligated with T4 Immunization protocol and hybridoma culture. Ten to DNA ligase (Roche) and transformed to T7 Express Competent twelve week old specific pathogen free Balb/c mice (two for 124 mAbs Volume 4 Issue 1 each antigen) were immunized twice (with a 28-d interval) with Ultra-4 Centrifugal Filter Units (Millipore) until a concentration 100 μg recombinant antigen in PBS with 50% Stimune adjuvant of approximately 1 mg/ml was reached. (Prionics) intra peritoneal (i.p.) and subcutaneous (s.c.) and with Measurement of true affinity ( K ) by ELISA. The bind- a final booster after 11 d with 100 μg recombinant antigen in PBS ing affinities of the antibodies were determined by measuring intravenous (i.v.). All animal experiments were approved by the the dissociation constant (K ). Briefly, antigen at various con - -7 -11 local animal ethics committee. Antibody response in mice serum centrations ranging from 1 x 10 M to 5 x 10 M was mixed was determined with ELISA as described below. Splenocytes were with a constant amount of antibody in 0.1 M potassium phos- harvested and frozen according to the protocol from Marusich et phate, 2 mM EDTA, 10 mg/ml BSA, pH 7.8 and incubated for al. The cells of half a spleen were fused to myeloma cells accord- 15 h at room temperature. The antigen-antibody mix was then ing to a standardized protocol. Cell lines were selected based on transferred to micro-titer plates previously coated with capture ELISA results described below, and subsequently sub-cloned to antigens at 150 ng/well in 50 mM sodium carbonate pH 9.6 obtain single clones. Hybridoma cells were grown in Optimem (DHFR1&2, DHFR-TS, GLURP R , GLURP R or HDP) and 0 2 I with Glutamax (Invitrogen) with 10% FCS (Fetalclone I, incubated for 60 min at room temperature. Washing of plates and Hyclone) or Hybridoma Serum Free medium (Invitrogen). detection of the presence of antibody was performed as described Screening ELISA. Mice serum and supernatant of hybrid- for the screening ELISA. Dissociation constants were calculated oma cell lines were screened with ELISA for specific reactivity using regression analysis and the Scatchard-Klotz equation. In with recombinant antigen and NF54 and/or 3D7 culture lysate. comparison, two HRPII antibodies (C1–13 and PTL3, National ELISA plates (medium binding, Greiner) were coated over night Bioproducts Institute) were tested as well with recombinant at 4°C with recombinant antigen (20 μg/ml) in PBS or P. fal- HRP2 (ReaMatrix). ciparum lysate or a P. vivax patient sample (5,630 parasites/μl Crude parasite antigen ELISA. A crude parasite antigen diluted 1:5 in PBS) and subsequently washed with PBS + 0.1% extract was prepared from a late stage 3D7 culture (~3–4% 29,34 Tween 20 (PBST) and blocked with 5% BSA (Sigma) in PBS schizonts) with Percoll as described by Troye-Blomberg et al. for 1 h at 37°C. Plates were washed and incubated 1 h at 37°C This antigen extract was coated overnight on ELISA plates in a with 50 μl of supernatant of the hybridoma clones or mouse dilution series from 20-0.03 μg/ml in 1% BSA in PBST. 1% BSA © 2012 Landes Bioscience. 3 6 serum diluted in 1% BSA in PBST (1:3 x 10 –1:10 x 10 ). After in PBST was used as negative control and the wells were subse- washing, plates were incubated 1 h at 37°C with peroxidase quently blocked with 5% BSA in PBS for 30 min to 1 h at 37°C. conjugated goat-anti-mouse (Jackson Immunoresearch) 1:5,000 After washing 3 times with PBST, purified antibodies (5 μg/ml) diluted in 1% BSA in PBST. The presence of antibody was mea- were added to each dilution series. In comparison two HRP2 Do not distribute. sured by adding 0.04% 3,3',5,5'-Tetramethylbenzidine (Sigma), antibodies (C1–13 and PTL3, National Bioproducts Institute) 0.04% urea peroxide (Sigma) in 0.1 M Sodium acetate citrate were taken along on each ELISA plate in the same concentration. pH 4 and the reaction was stopped with 0.5 M Sulfuric acid. Statistical analysis. The dissociation constant (K ) was deter- Color development was measured at 450 nm in a SUNRISE mined by calculating the slope of the lines by regression analy- ELISA reader (Tecan). sis. The correlation between absorbance in the ELISA and Purification of mAbs. Antibodies were isotyped (Pierce the antigen concentration was determined by Pearson product Rapid ELISA Mouse Antibody Isotyping Kit, Thermo Scientific) moment correlation coefficient (PMCC). and HDP and DHFR-TS antibodies were purified directly from Acknowledgments hybridoma supernatant (filtered through 0.2 μm) with 1 ml HiTrap protein G HP columns (GE Healthcare) in case of This project is part of a collaborative effort for the improve- IgG and with 1 ml HiTrap IgM purification HP columns (GE ment of malaria diagnosis with the Foundation for Innovative Healthcare) in case of IgM, according to manufacturer’s instruc- New Diagnostics (FIND). Support for this project was provided tions. Purifications were performed on an ÄKTA FPLC system through funding from FIND. HRP2 recombinant protein was (Amersham Biosciences). Antibody containing fractions were provided by ReaMetrix India Pvt., Ltd.; anti-HRP2 antibodies pooled and concentrated with 50 kD (IgG) or 100 kD (IgM) by National Bioproducts Institute in South Africa; and the puri- Amicon Ultra-4 Centrifugal Filter Units (Millipore) until a fied recombinant heme-detoxification protein (HDP) by Virginia concentration of approximately 1 mg/ml was reached. Antibody Polytechnic Institute and State University (Virginia Tech.,) in concentration was determined by adsorption at 280 nm with a the USA. We thank Dionicia Gamboa from the Instituto de Nanodrop 1000 (Thermo Scientific). Medicina Tropical “Alexander von Humboldt”—Universidad The GLURP antibodies used in the experiments were pre- Peruana Cayetano de Heredia in Peru for sending samples from cipitated with ammonium sulfate. Hybridoma supernatant was patients infected with P. vivax parasites. Those samples were clarified by a 0.2 μm syringe filter (Millipore) and saturated collected in the frame of collaborative projects with FIND with ammonium sulfate pH 7.4 was slowly added till the final concen - the required approval of a local ethical committee. We thank tration of ammonium sulfate was 45% and left on a rotator for MR4 for providing us with 3D7 malaria parasites contributed by 45 min. Subsequently, the mixture was incubated O/N at 4°C Daniel Carucci and Alister Craig. We thank the Centre for clini- and then centrifuged at 3,893 g for 40 min. The pellet was resus- cal malaria studies of the University Medical Centre St. Radboud pended in 8 ml cold PBS and concentrated with 50 kD Amicon in Nijmegen for providing us with the NF54 malaria parasites. www.landesbioscience.com mAbs 125 13. Baker J, McCarthy J, Gatton M, Kyle DE, Belizario V, 24. Vinayak S, Rathore D, Kariuki S, Slutsker L, Shi References Luchavez J, et al. 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Journal

mAbs – Taylor & Francis

Published: Jan 1, 2012

Keywords: Plasmodium falciparum; Dihydrofolate reductase-thymidylate synthase; Plasmodium; diagnostics; glutamate rich protein; heme detoxification protein; malaria; monoclonal antibodies; rapid diagnostic test

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