Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

Experimental Parasitology xxx (2017) 1e7

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Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri Gi-Sang Seong a, 1, Hae-Jin Sohn a, 1, Heekyoung Kang a, Ga-Eun Seo a, Jong-Hyun Kim b, Ho-Joon Shin a, * a

Department of Microbiology, Ajou University School of Medicine, Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

We produced anti-NfCPB and antiNfCPB-L McAbs using a cell fusion technique. High antigenicity of 2C9(NfCPB) and 1C8(NfCPB-L) McAbs selected for use. 2C9 and 1C8 proteins were localized on cytoplasm, pseudopodia and amoebastomes. Two McAbs will be useful for the pathoimmunological studies of PAM.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 July 2017 Received in revised form 18 August 2017 Accepted 11 September 2017 Available online xxx

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced antiNfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. © 2017 Elsevier Inc. All rights reserved.

Keywords: Naegleria fowleri Cathepsin B protein Cathepsin B-Like protein Cell fusion Monoclonal antibody

* Corresponding author. E-mail address: [email protected] (H.-J. Shin). 1 Author contributions: Gi-Sang Seong and Hae-Jin Sohn contributed equally to this work. http://dx.doi.org/10.1016/j.exppara.2017.09.004 0014-4894/© 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: Seong, G.-S., et al., Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri, Experimental Parasitology (2017), http://dx.doi.org/10.1016/j.exppara.2017.09.004

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G.-S. Seong et al. / Experimental Parasitology xxx (2017) 1e7

1. Introduction Free-living pathogenic Naegleria fowleri, a causal agent of primary amoebic meningoencephalitis (PAM) in humans, inhabits natural environments such as ponds, rivers, hot springs and swimming pools, where they can occur in three different forms, trophozoite, cyst and occasionally flagellate cell (Culbertson, 1970; Ma et al., 1990; Visvesvara et al., 2007). Increasingly N. fowleri, the “brain-eating amoeba”, is becoming recognized as an important pathogenic microorganism of concern in aquatic recreation activities (Heggie, 2010; Sifuentes et al., 2014). In the United States, Australia and Europe, more than 300 cases of PAM have been reported (Budge et al., 2013; De Jonckheere, 2011; Yoder et al., 2010). Furthermore, death due to PAM, as a neglected tropical disease (NTD), has been increasing in Asian countries such as Pakistan, India, Thailand and Vietnam (Gupta et al., 2009; Shakoor et al., 2011; Siddiqui and Khan, 2014; Siripanth, 2005). The symptoms of PAM, due to infection of N. fowleri via the nasal cavity from warm water contaminated with amoebic trophozoites, are headache, nausea, vomiting, fever and meningitis (Schuster and Visvesvara, 2004; Shin and Im, 2004). Because the clinical symptoms of PAM are similar to acute purulent encephalitis and bacterial meningitis, early clinical diagnosis is very difficult. Thus, PAM diagnosis depends mainly on finding trophozoites in tissue biopsies or cultivation of CSF (central spinal fluid). But the practice is not easy. Recently developed diagnostic methods depend on primerbased PCR (polymerase chain reaction) analysis post tissue biopsy or cultivation of CSF (Kang et al., 2015; Ma et al., 1990; Qvarnstrom et al., 2006; Reveiller et al., 2002; Schuster and Visvesvara, 2004). Two possible pathogenic mechanisms of N. fowleri causing PAM are recognized. The contact-dependent mechanism is concerned with the formation of amoebastomes, or food-cups, activated by nfa1 and nf-action genes. A contact-independent mechanism, with the secretion of phospholipase A, neuraminidase, elastase, perforin-like protein and cysteine protease, also has been reported (Kang et al., 2005; Kim et al., 2008; Marciano-Cabral and Cabral, 2007; Schuster and Visvesvara, 2004; Shin et al., 2001; Sohn et al., 2010). Previously, Lee et al. (2014) cloned N. fowleri cathepsin B (NfCPB) and cathepsin B-Like (NfCPB-L) proteins consisting of 1.038bp (assumed molecular weight 38.4 kDa) and 939bp (34 kDa) fragments, respectively. The refolding recombinant cathepsin B (rNfCPB) and cathepsin B-like (rNfCPB-L) proteins play important roles in the proteolytic activities on immunoglobulin, collagen, fibronectin, hemoglobin and albumin (Lee et al., 2014). In the present study, in order to develop useful monoclonal antibodies essential for further patho-immunological studies, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (rNfCPB McAb and rNfCPB-L McAb) using the cell fusion technique and observed their immunological characteristics. 2. Materials and methods 2.1. Amoebae culture and preparation of lysate and excretorysecretory protein Pathogenic N. fowleri (Carter Nf69 strain, ATCC 30215) and nonpathogenic N. gruberi (Schardinger strain; ATCC 30960) were axenically cultured at 37 C in Nelson's medium and PYNFH medium, respectively, with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) according to methods previously described (Kang et al., 2015). Acanthamoeba castellanii and A. polyphaga were axenically cultured at 25 C in PYG medium according to previously reported methods (Sohn et al., 2017). Amoebic lysates (Nf-lysate, Ng-lysate, Ac-lysate and Ap-lysate) were prepared by the freezing-thawing

method, and after ultracentrifugation at 12,000 rpm at 4 C for 2hr, the aqueous supernatants were collected (Kim et al., 2016). Preparation of N. fowleri excretory-secretory protein (Nf-ESP) was performed using the previously reported method (Kim et al., 2009). Briefly, N. fowleri trophozoites were harvested in 50 ml tubes and incubated in PBS (pH 7.4) at 37 C for 1 h. After centrifugation at 800 g for 5min, the collected supernatant was used as the Nf-ESP. 2.2. Recombinat NfCPB and NfCPB-L protein preparation To obtain the rNfCPB and rNfCPB-L proteins, the nfcpb and nfcpbL genes were cloned into the pExp5-TOPO TA vector (Invitrogen, USA); pExp5-TOPO-nfcpb and pExp5-TOPO-nfcpb-L were then transformed into Escherichia coli strain BL21 (DE3) according to the method previously described (Lee et al., 2014). Following inoculation into Luria-Bertani medium containing 100 mg/ml of ampicillin, E. coli was incubated with 200 rpm rotation at 37 C until 0.4e0.6 OD600 was reached. After the addition of 1 mM isopropylb-D-thiogalactopyranoside (IPTG, Goldbio, USA), E. coli was incubated with 200 rpm rotation at 37 C for 4 h and pelleted down. The pellet was resuspended in 8M urea lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01M Tris-HCl) and pelleted down again. The collected supernatant was subjected to purification with nickel-nitrilotriacetic acid (Ni-NTA) chromatography (QIAGEN, USA) and concentration by Amicon® Ultradevice (cut-off 10 kDa; Millipore, USA). Purified rNfCPB and rNfCPB-L proteins were stored at 20 C until use. 2.3. rNfCPB and rNfCPB-L immunization Immunization methods previously described (Lee et al., 2007) were employed. Each 50 mg of rNfCPB and rNfCPB-L proteins was mixed with 100 ml of Freund's complete adjuvant (Sigma, USA) and intraperitoneally injected into 6-week-old female BALB/c mouse. Next, the mice were intraperitoneally boosted twice with 25 mg of each protein mixed with 100 ml of Freund's incomplete adjuvant (Sigma, USA) at two-week intervals. Polyclonal antibody formation was evaluated from caudal vein bloods by ELISA (antigen, 0.1 mg/ 100ml/96-well; secondary antibody, 1:10,000 diluted goat antimouse IgG conjugated alkaline phosphatase). At 4 days before cell fusion, a final boosting with 25 mg of each protein suspended in PBS (pH 7.4) was administered. 2.4. Cell fusion and monoclonal antibody production The cell fusion technique used was a slight modification of the methods of Lee and Kim (Kim et al., 2012; Lee et al., 2007). Briefly, the splenocytes isolated from immunized mouse under anesthesia with Ketamin-Rompun solution was fused with myeloma cells (F/o cell line) cultured in complete DMEM (Dulbeco's Modified Eagle Medium, Gibco, USA) medium by adding PEG (polyethylene glycol) solution. The mixture was cultured in 96-well plate with HAT (hypoxanthine-aminopterin-thymidine, Sigma) medium at 37 C, in a 5% CO2 incubator for 3 days. Next, the cultivating medium in each 96-well culture plate was replaced with HT (hypoxanthinethymidine, Sigma) medium. After the identification of hybridoma cells from each well, the secretion of antibody from hybridoma cells was evaluated by ELISA. The hybridoma cells producing antibodies were scaled up in 24-well plates and consequently 75-cm2 culture flask (Nunc, USA) by culturing at 37 C, in a 5% CO2 incubator. Selection of monoclones producing monoclonal antibodies was made using the limiting dilution method (Kim et al., 2012). Massive production of monoclonal antibodies was obtained from ascites of mice injected with monoclone cells. Then, antibody immunoglobulins were purified in a Protein A column (proA™, KOREA), concentrated by Amicon® Ultradevice, isotypes determined with



an isotyping kit (Roche, USA), and stored at

20 C until use.

3

produced (PBS, 0.091; normal mouse serum, 0.139; immunized mouse serum, 2.094) (Fig. 3B).

2.5. SDS-PAGE and western blotting 3.4. Selection of monoclone producing monoclonal antibody In order to observe the immunochemical characters of monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and western blotting were practiced according to previously reported methods (Lee et al., 2017). Briefly, 12% SDS-PAGE and polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) for western blotting were used. Blotted PVDF membranes presoaked with 5% skim milk (BD, USA) were reacted with anti-NfCPB McAb or anti-NfCPB-L McAb (each diluted 1:200) for 1 h and treated with the goat anti-mouse IgG-conjugated alkaline phosphatase (1:10,000 diluted; Sigma, USA). The color reaction was observed by adding 20 ml of BCIP/NBT (5-bromo-4-chloro-3indolylphos-phate/nitro blue tetrazolium tablet; Sigma, USA).

To obtain the monoclone producing monoclonal antibody, a limiting dilution with each of two polyclones, 2CF11 and 2DE9 for rNfCPB, and 1DH9 and 1DH10 for rNfCPB-L, was carried out. Finally, two monoclones, 2D6 (2.068) and 2D11 (1.075) for rNfCPB protein, were selected from 2CF11 cells, and five monoclones, 2A7 (1.35), 2B7 (1.538), 2E2 (1.755), 2E9 (1.541) and 2C9 (1.689), were selected from 2DE9 cells (PBS, 0.086; normal mouse serum, 0.134; immunized mouse serum, 2.086) (Fig. 4A). For rNfCPB-L protein, two monoclones, 1C8 (2.075) and 1D9 (2.114) and one monoclone, 1E5 (2.056), were selected from 1DH10 and 1DH9 cells, respectively (PBS, 0.086; normal mouse serum, 0.153; immunized mouse serum, 2.228) (Fig. 4B).

2.6. Immunocytochemistry

3.5. Isotypes of monoclonal antibodies

To observe the cellular locations of NfCPB or NfCPB-L proteins in N. fowleri trophozoites, immunocytochemistry (indirect fluorescent antibody test) was performed using a modification of previously reported methods (Kang et al., 2005). Briefly, N. fowleri trophozoites were placed on a slide clover-glass and fixed with 10% formaldehyde for 10 min. After washing with PBS (pH 7.4) twice, 1% ammonium hydroxide (for permeabilization) and 3% BSA solution (for reducing non-specific reactions) were reacted for 5 min at room-temperature. Next, NfCPB McAb or NfCPB-L McAb (1:200 diluted in 3% BSA solution) was added and reacted at 4 C for 18hr. After washing with PBS (pH 7.4) twice, anti-mouse antibody conjugated fluorescein isothiocyanate (Sigma, USA) (diluted 1:200 in 3% BSA solution) was added. After reaction and washing, the final slide cover-glass was inverted onto a glass microscope slide with mounting solution (VECTOR, USA) and examined under a confocal microscope (Zeiss, Germany).

Each two monoclonal antibodies, 2C9 and 2D6 for rNfCPB, and 1C8 and 1D9 for rNfCPB-L were subjected to isotyping analysis. The isotypes of 2C9, 2D6, 1C8 and 1D9 were IgG2a, IgG1, IgG3 and IgG1, respectively (Table 1).

3. Results 3.1. Recombinant NfCPB and NfCPB-L proteins Purified recombinant NfCPB and NfCPB-L proteins produced from E. coli expression and Ni-NTA purification system were electrophoresed on 12% SDS-PAGE gels and showed molecular weights of 38.4 and 34.3 kDa for rNfCPB and rNfCPB-L proteins, respectively (Fig. 1). Finally, total concentration of 10 mg/ml each for rNfCPB and rNfCPB-L proteins was produced. 3.2. Antibody formation After immunization of purified rNfCPB and rNfCPB-L proteins into each mouse, the formation of antibody was evaluated from mice sera by ELISA. The ELISA value for rNfCPB protein were 1.133e2.204s of optical density (O.D.) in three mice (Fig. 2A). For rNfCPB-L protein, the O.D. values were 2.242e2.251 in two mice (Fig. 2B). 3.3. Hybridoma cells producing monoclonal antibody After cell fusion, four hybridoma clones producing antibodies for rNfCPB protein were obtained: 2AB6 (0.991), 2CB1 (1.019), 2CF11 (2.123) and 2DE9 (0.93) (PBS, 0.089; normal mouse serum, 0.126; immunized mouse serum, 2.156) (ELISA O.D. values) (Fig. 3A). Five hybridoma clones, 1AH12 (2.081), 1CA6 (1.549), 1CB7 (2.085), 1DH9 (1.772), 1DH10 (1.907) for rNfCPB-L protein were

3.6. Antigenicity and species-specificity of 2C9 and 1C8 monoclonal antibodies To observe the antigenicity of produced monoclonal antibodies against various antigens of N. fowleri, the purified 2C9 and 1C8 McAbs were reacted with N. fowleri lysate (Nf-lysate), Nf-ESP and rNfCPB and rNfCPB-L proteins. As determined by western blotting, 2C9 McAb reacted with Nf-lysate and Nf-ESP (excretory-secretory protein) as the response showed a 28 kDa band, and 38.4 kDa band for rNfCPB protein (Fig. 5A). In addition, 1C8 McAb reacted with Nflysate and Nf-ESP, which showed a 24 kDa reacted band, and 34 kDa band for rNfCPB-L protein (Fig. 5B). To observe the speciesspecificity of the monoclonal antibodies, the purified 2C9 and 1C8 McAbs were reacted with Nf-lysate, Ng-lysate, Ac-lysate and Aplysate. As determined by western blotting, 2C9 (for rNfCPB) and 1C8 (for rNfCPB-L) McAbs only reacted with Nf-lysate (Fig. 6). 3.7. Intracellular localization of NfCPB and NfCPB-L proteins To observe the locations of NfCPB and NfCPB-L proteins in N. fowleri trophozoites, an immuno-cytochemistry assay was performed using 2C9 (for NfCPB) and 1C8 (for NfCPB-L) McAbs. As seen in the immuno-fluorescence reactions with 2C9 and 1C8 McAbs, the NfCPB and NfCPB-L proteins were observed on cell membranes, especially on psuedopodia and food-cup structures (Fig. 6). 4. Discussion Pathogenic N. fowleri, informally known as the “brain-eating amoeba”, has a worldwide distribution, and PAM has been recognized as an important disease associated with hot summer activities such as swimming and water-skiing (De Jonckheere, 2011; Sifuentes et al., 2014; Zaheer, 2013). In particular, PAM is an NTD has been reported in Pakistan in connection with religious ablution practices in which amoeba-contaminated water was used (Siddiqui and Khan, 2014). Trophozoites of N. fowleri enter the nasal cavity, penetrate the olfactory mucosa and migrate along the olfactory nerve, crossing the cribriform plate until reaching the olfactory bulbs of the central nerve system, where it causes acute PAM symptoms such as severe headache, nausea, vomiting and fever.


4


Fig. 1. SDS-PAGE band patterns of purified recombinant NfCPB (A) and NfCPB-L proteins (B). Lane 1 and 4, IPTG-uninduced E. coli lysate; lane 2 and 5, IPTG-induced E. coli lysate; lane 3, purified rNfCPB protein; lane 6, purified rNfCPB-L protein; M, protein size marker.

(A)

(B) NfCPB

2.5

2

2 Absorbance at 405nm

Absorbance at 405nm

NfCPB-L

2.5

1.5

1

0.5

1.5

1

0.5

0

0 PBS

Normal

M1

M2

M3

PBS

Normal

M1

M2

Fig. 2. Antibody formation in mice sera after 3rd immunization with rNfCPB (A) and rNfCPB-L (B) by ELISA analysis. PBS and Normal (normal mouse serum; 1:200 diluted) were used as negative controls, M; number of immunized mouse serum (1:200 diluted).

During the gradual process, severe haemorrhagic necrosis develops in the forebrain (Visvesvara et al., 2007). In previous studies, cysteine protease, cathepsin B and cathepsin B-like proteins, were considered necessary for the contactindependent pathological mechanisms of Paragonimus westermani (lung fluke), Schistosoma mansoni (blood fluke), Clonorchis sinensis (liver fluke), Angiostrongylus cantonensis (nematode) and Entamoeba histolytica (intestinal amoeba), in which they cause lysis of hosts' structural proteins (collagen and fibronectin), functional proteins (hemoglobin and albumin) and immune molecules (IgG, IgM and IgA) (Chappell and Dresden, 1986; Keene et al., 1986; Long et al., 2015; Na et al., 2008, Na et al., 2006). For the contactindependent pathological mechanism of N. fowleri, cysteine protease, cathepsin B and cathepsin B-Like proteins were concerned with invasion into host cells, evasion of host immune defenses and nutrition uptake (Kim et al., 2008, Kim et al., 2009; Lee et al., 2014). In order to develop useful monoclonal antibodies essential for patho-immunological studies, we produced in total seven kinds of anti-NfCPB McAbs and three of anti-NfCPB-L McAbs using the cell fusion technique. Among these, 2C9 and 2D6 monoclones

producing high titer of monoclonal antibodies against the rNfCPB protein were selected and injected intraperitoneally into mice to obtain large scale antibody production. Finally, purified 2C9 McAb was authentically reacted with Nf-lysate and rNfCPB protein, and yielded a 28 kDa band, but did not react with Ng-lysate, Ac-lysate or Ap-lysate (i.e., other free-living amoebae). In a previous study, DNA sequence of the N. gruberi cathepsin B gene had a 56% homology with the CPB gene of N. fowleri (Lee et al., 2014). This result suggests that 2C9 McAb has high antigenicity and species-specificity. For the NfCPB-L protein, 1C8 and 1D9 McAb were selected and subjected to massive production via mouse ascites. The final purified 1C8 McAb reacted with Nf-lysate and purified rNfCPB-L protein, yielding a 24 kDa band. IC8 did not react with Ng-lysate, Aclysate or Ap-lysate. The gene sequence homology between N. gruberi and N. fowleri NfCPB gene was 46% (Lee et al., 2014). This result also suggests that 1C8 McAb has a high antigenicity and species-specificity. In addition to the results of western blotting with two McAbs (2C9, 1C8), the reacting band sizes with Nf-lysate and Nf-ESP were smaller than those of rNfCPB and rNfCBP-L proteins. It has been



5

(A) NfCPB

2.5 Absorbance at 405nm

Absorbance at 405nm

2.5 2 1.5

1 0.5 0

NfCPB

2

1.5 1 0.5 0

(B) NfCPB-L

NfCPB-L 2.5 Absorbance at 405nm

Absorbance at 405nm

2.5 2 1.5 1 0.5

2 1.5 1 0.5 0

0

Fig. 3. Antibody formation in hybridoma cells from 75-cm2 culture flask by ELISA analysis. Hybridoma after cell fusion and monoclonal cells post liming dilution were examined by ELISA against rNfCPB (A) and rNfCPB-L (B) proteins. PBS and Normal (normal mouse serum; 1:200 diluted) were used as negative controls; immunized mouse serum (1:200 diluted) was used as positive control.

Fig. 4. SDS-PAGE band patterns of Nf-lysate (lane 1), Nf-ESP(lane 3), rNfCPB (lane 5A) and rNfCPB-L protein (lane 5B), and western blot analysis with 2C9 McAb (A) reacted with Nflysate (lane 2), Nf-ESP (lane 4) and rNfCPB (lane 6) and 1C8 (B) McAb reacted with Nf-lysate (lane 2), Nf-ESP (lane 4) and rNfCPB-L (lane 6). M; protein size marker.

Table 1 Isotypes of monoclonal antibodies. Monoclonal antibodies

Isotype

2C9 (anti-NfCPB) 2D6 (anti-NfCPB) 1C8 (anti-NfCPB-L) 1D9 (anti-NfCPB-L)

IgG2a/kappa IgG1/kappa IgG3/kappa IgG1/kappa

suggested that cathepsin B and cathepsin B-L protein are initially transcribed as longer precursor molecules, and then undergo a cleavage course in which some amino acids of precursor are deleted

during post-translation modification. Through the analysis of amino acids sequences of NfCPB and NfCPB-L proteins, the active forms of rNfCPB and rNfCBP-L proteins were evaluated as having molecular weights of 25 kDa and 24 kDa, respectively. In the results of immunocytochemistry assays with 2C9 and 1C8 McAbs, NfCPB and NfCPB-L proteins were mainly localized on cell membranes, pseudopodia and food-cup structures. We suggest that NfCPB and NfCPB-L proteins may be easily secreted from N. fowleri trophozoites. As an indirect finding, 2C9 and 1C8 McAbs reacted well with each Nf-ESP. To further evaluate this question, further studies of antibody-reactions using culture media supernatant and mRNA or gene expression of NfCPB proteins by real-time PCR


6


Fig. 5. Immunoblot analysis of monoclonal antibodies reacted with lysates of various amoebae. (A) 2C9 McAb showed 28 kDa reacting band with Nf-lysate. (B) 1C8 McAb showed 24 kDa reacting band with Nf lysate. M; protein size marker.

Fig. 6. Localization of NfCPB and NfCPB-L protein in N. fowleri trophozoites by immunocytochemistry assay using monoclonal antibody. Immuno-fluorecent reaction of 2C9 (A) and 1C8 (B) McAbs were predominantly showed on pseudopodia (arrow) and food-cup (arrow head). Scale bar; 10 mm.

analysis should be carried out in the amoebic culture system. Additionally, whether NfCPB and NfCPB-L proteins are concerned with amoebic movement and phagocytosis, which may affect infection mechanisms of N. fowleri, should be elucidated in further studies. Finally, two 2C9 and 1C8 McAbs will be useful for studying the patho-immunological mechanisms by which N. fowleri evades the immune response and causes PAM in its hosts. Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (Ministry of Science, ICT & Future Planning) (No. 2015R1C1A2A01054526). References Budge, P.J., Lazensky, B., Van Zile, K.W., Elliott, K.E., Dooyema, C.A., Visvesvara, G.S.,

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Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies.

Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization.

Immunization of mice against Naegleria fowleri infection.

Characterisation of cathepsin B and collagenolytic cathepsin from human placenta.

Production and characterization of monoclonal antibodies against aflatoxin B1.

Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Molecular characterization of the cathepsin B of turbot (Scophthalmus maximus).

Collagenolytic cysteine proteinases of bone tissue. Cathepsin B, (pro)cathepsin L and a cathepsin L-like 70 kDa proteinase.

N-formylpyrazolines and N-benzoylpyrazolines as novel inhibitors of mammalian cathepsin B and cathepsin H.

In vitro effects of amphotericin B on growth and ultrastructure of the amoeboflagellates Naegleria gruberi and Naegleria fowleri.

Naegleria fowleri: pathogenesis, diagnosis, and treatment options.

Monoclonal antibodies to gelonin: production and characterization.

Cathepsin B: Basis Sequence: Mouse.

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O.

Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins.

Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases.

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Gene structure of mouse cathepsin B.

Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and the myofibrillar structure.

Naegleria fowleri: fine structural localization of acid phosphatase and heme proteins.