- Email: [email protected]
Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library
Accepted Manuscript Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library Ruqaiyyah Siddiqui, Huma Kulsoom, Salima Lalani, Naveed Ahmed Khan PII:
S0014-4894(16)30063-7
DOI:
10.1016/j.exppara.2016.04.001
Reference:
YEXPR 7226
To appear in:
Experimental Parasitology
Received Date: 1 September 2015 Revised Date:
13 January 2016
Accepted Date: 2 April 2016
Please cite this article as: Siddiqui, R., Kulsoom, H., Lalani, S., Khan, N.A., Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.04.001. 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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Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage
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antibody display library
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Ruqaiyyah Siddiqui1, Huma Kulsoom2, Salima Lalani2, Naveed Ahmed Khan1*
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Malaysia; 2Department of Biological and Biomedical Sciences, Aga Khan University, Pakistan.
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Department of Biological Sciences, Faculty of Science and Technology, Sunway University,
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Short title: B. mandrillaris antibodies using phage display library
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*Corresponding address: Naveed Ahmed Khan, Department of Biological Sciences, Faculty of
15
Science and Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-
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8622. Ext: 7176. Fax: 60-(0)3-5635-8630. E-mail: [email protected]
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Abstract Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for
23
successful prognosis. Current methods for identifying this organism rely on culture and
24
microscopy, antibody-based methods using animals, or involve the use of molecular tools that
25
are expensive. Here, we describe the isolation of antibody fragments that can be used for the
26
unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were
27
isolated from a bacteriophage antibody display library. Individual clones were studied by
28
enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed
29
specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a
30
diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying
31
their biological role(s) is discussed further.
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Keywords: Phage display library; Balamuthia; ELISA; Immunocytochemistry; Diagnosis
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Balamuthia mandrillaris is a recently discovered protist pathogen that is a causative agent of granulomatous amoebic encephalitis. Once acquired, the infection results in death in
37
almost all reported cases. Under harsh conditions, the organism maintains itself in the cyst form.
38
Favorable conditions promote its conversion to a vegetative trophozoite stage, which in turn
39
causes the skin lesions and/or brain infection (Visvesvara et al., 1990; Martinez and Visvesvara,
40
1997). The brain infection is typically marked by the presence of granulomatous infiltrations in
41
infected tissues, thus named as granulomatous amoebic encephalitis (Visvesvara et al., 1990;
42
Martinez and Visvesvara, 1997; Siddiqui and Khan, 2008). B. mandrillaris infection can occur in
43
anyone, regardless of the immune status, as compared to other free living amoeba such as A.
44
castellanii, which generally targets immunocompromised patients. The successful treatment is
45
difficult due to poor and delayed diagnosis. In majority of cases, definitive analysis is made on
46
autopsy (Lobo et al., 2013). Current methods of detection include PCR-based methods,
47
immunofluorescence and culture techniques (Visvesvara et al., 1990; Kiderlen et al., 2008). The
48
use of immunofluorescence assays has become increasingly popular as microbial antigens are
49
easier to test as they are usually tested within the clinical specimen but the isolation of antibody
50
fragments is expensive, time consuming or may have ethical concern due to the use of animals
51
for the development of antibody-based methods. Bacteriophage antibody display libraries
52
expressing single-chain Fv antibody fragments have been developed as an alternative way of
53
isolating specific antibodies by random pairing of heavy and light chain fragments isolated from
54
naïve human lymphocytes to express single chain of variable fragment specific to the antigens of
55
B. mandrillaris (Khan et al., 2000). Specific antibodies can be readily isolated without the need
56
of animals. Here, we describe the isolation of B. mandrillaris-specific antibodies using
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3
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bacteriophage antibody library. These antibodies provide the reagents to establish a specific and
58
rapid detection assay for B. mandrillaris. Bacteriophage library, E. coli TG1 and helper phage KO7 were obtained from M.
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Yamabhai, Suranaree University of Technology, Thailand. The human brain microvascular
61
endothelial cells (HBMECs), B. mandrillaris and A. castellanii were obtained and cultured as
62
described previously (Kulsoom et al., 2014). For isolation of B. mandrillaris-specific antibody
63
fragments, library clones were amplified as described previously (Khan et al., 2000). Briefly, E.
64
coli TG1 were incubated at 37°C in the presence of TYE 1% glucose and 100 µg/mL ampicillin
65
until the absorbance at 600nm was approximately 0.5. Helper phage was then added to the
66
culture and incubated for a further 30 min at 37°C without shaking. Next, kanamycin was added
67
(to select for bacteriophage-containing clones), and cultures incubated at 30°C overnight.
68
Subsequently, the culture was centrifuged at 11,000 x g for 15 min and pellet resuspended in a
69
1/5 volume of 20% (w/vol) polyethylene glycol 6000 in 2.5 M NaCl for 1 h at 4°C. After
70
incubation and three washes, the pellet was resuspended in PBS with 15% glycerol and
71
centrifuged at 1,500 x g for 10 min. Finally, the supernatant containing the bacteriophage
72
particles was filtered (pore size, 0.45 mm) and titres were determined by serial dilutions prior to
73
storage at -20°C. Clones amplified were subjected to multiple rounds of positive bio-panning
74
using B. mandrillaris and negative bio-panning using A. castellanii as described previously
75
(Khan et al., 2000). A total of 90 clones that showed binding affinity to B. mandrillaris were
76
subjected to repeated rounds (i.e., four) of ELISA using anti-M13-HRP conjugated antibody
77
(Abcam) on iMark Bio-rad microplate reader. From these, 14 clones were selected that
78
reproducibly exhibited absorbance values of more than 0.5 at 450 nm, corresponding to higher
79
binding affinity to B. mandrillaris (data not shown). For immunofluorescence assays, B.
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mandrillaris, A. castellanii, E. coli and HBMEC were pelleted and re-suspended in RPMI. Cells
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were then treated with selected clones at a dilution of 1:100. After 3X washing with milk-PBS,
82
cells were incubated with anti-M13-FITC conjugated antibody and visualized under fluorescent
83
microscope. None of the 14 clones showed reactivity against A. castellanii, E. coli and HBMEC
84
(Fig. 1). The results are representative of three independent experiments. In each experiment, cell
85
cultures were obtained from original stocks and incubated with clones for ELISA and
86
immunofluorescence assays. All 14 clones assessed using immunofluorescence showed positive
87
reactivity against B. mandrillaris in all three independent experiments performed with more than
88
80% cells showing positive reactivity (Fig. 1). Moreover, clones were also tested against the cyst
89
stage of B. mandrillaris to determine whether reactivity is stage-specific. Out of 14, only 8
90
clones showed reactivity with cyst stage of B. mandrillaris as described above, suggesting that
91
specificity is stage-specific. The results showed that selected antibody clones isolated from
92
phage library can detect trophozoite as well as cyst stage of B. mandrillaris (e.g., clone #46)
93
while others (e.g., clone #88) exhibited binding affinity to cyst alone. These findings suggest
94
affinity of clones to different antigens on the surface of B. mandrillaris. These clones can
95
provide a useful tool to characterize surface antigens of B. mandrillaris trophozoites and cysts as
96
well as study transformation of B. mandrillaris into different stages of its life cycle. This is the
97
first report that bacteriophage antibody display technology has been used in the development of
98
an antibody for the identification of B. mandrillaris. Bacteriophage antibody display libraries are
99
potentially useful and powerful tools that allow the rapid generation of antibody reagents for use
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100
in diagnostic assays. This technology is effective in designing an antibody that recognises a
101
given protein epitope and studying its role in cellular differentiation, motility, cell division,
102
phagocytosis, chemotaxis and other biological processes. In summary, antibody fragments from
5
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a bacteriophage library can be a method of choice for generation of antibodies which are cost
104
effective, reproducible, do not require animals and can form the basis to develop clinical
105
applications.
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Competing interests: The authors declare that they have no competing interests.
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Acknowledgements: This work was partially supported by Sunway University, Malaysia; and
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the University Research Council, Aga Khan University, Pakistan.
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References
1. Khan, N.A., Greenman, J., Topping, K.P., Hough, V.C., Temple, G.S., Paget, T.A., 2000.
113
Isolation of Acanthamoeba-specific antibodies from a bacteriophage display library. J.
114
Clin. Microbiol. 38, 2374-2377.
115 116
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2. Kiderlen, A.F., Radam, E., Lewin, A., 2008. Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene. BMC Microbiol. 8, 210. 3. Kulsoom, H., Baig, A.M., Siddiqui, R., Khan, N.A., 2014. Combined drug therapy in the
118
management of granulomatous amoebic encephalitis due to Acanthamoeba spp., and
120 121 122 123 124
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Balamuthia mandrillaris. Exp. Parasitol. 145, S115-S120.
4. Lobo, S.A., Patil, K., Jain, S., Marks, S., Visvesvara, G.S., Tenner, M., El Khoury, M.Y., 2013. Diagnostic challenges in Balamuthia mandrillaris infections. Parasitol. Res. 112, 4015-4019. 5. Martinez, A.J., Visvesvara, G.S., 1997. Free‐living, amphizoic and opportunistic amebas. Brain Pathol. 7, 583-598. 6
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6. Siddiqui, R., Khan, N.A., 2008. Balamuthia amoebic encephalitis: an emerging disease with fatal consequences. Microb. Pathogen. 44, 89-97. 7. Visvesvara, G.S., Martinez, A.J., Schuster, F.L., Leitch, G.J., Wallace, S.V., Sawyer, T.K., Anderson, M., 1990. Leptomyxid ameba, a new agent of amebic
129
meningoencephalitis in humans and animals. J. Clin. Microbiol. 28, 2750-2756.
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Figure legends
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Figure 1. Representative images of bacteriophage antibody clone reactivity against B.
133
mandrillaris, HBMEC, E. coli, A. castellanii under bright field (left) and fluorescence (right). A
134
& B are B. mandrillaris incubated without clone but only anti-M13-FITC; C & D are B.
135
mandrillaris incubated with clone (#46) plus anti-M13-FITC; E & F are HBMEC (clone + anti-
136
M13-FITC); G & H are E. coli (clone + anti-M13-FITC); and I & J are A. castellanii (clone +
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anti-M13-FITC). Magnification, X200.
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Highlights
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Balamuthia mandrillaris is a protist pathogen that can cause fatal infection Early diagnosis and aggressive treatment is a pre-requisite for successful prognosis Phage antibody technology was used to anti-B. mandrillaris antibody fragments Antibody clones did not bind to closely related amoeba and host cells indicating specificity.
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• • • •
S0014-4894(16)30063-7
DOI:
10.1016/j.exppara.2016.04.001
Reference:
YEXPR 7226
To appear in:
Experimental Parasitology
Received Date: 1 September 2015 Revised Date:
13 January 2016
Accepted Date: 2 April 2016
Please cite this article as: Siddiqui, R., Kulsoom, H., Lalani, S., Khan, N.A., Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.04.001. 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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1
Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage
3
antibody display library
RI PT
2
4
SC
Ruqaiyyah Siddiqui1, Huma Kulsoom2, Salima Lalani2, Naveed Ahmed Khan1*
5
6
7
1
8
Malaysia; 2Department of Biological and Biomedical Sciences, Aga Khan University, Pakistan.
M AN U
Department of Biological Sciences, Faculty of Science and Technology, Sunway University,
9 10
13
TE D
12
Short title: B. mandrillaris antibodies using phage display library
EP
11
*Corresponding address: Naveed Ahmed Khan, Department of Biological Sciences, Faculty of
15
Science and Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-
16
8622. Ext: 7176. Fax: 60-(0)3-5635-8630. E-mail: [email protected]
17
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1
ACCEPTED MANUSCRIPT
20 21
Abstract Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for
23
successful prognosis. Current methods for identifying this organism rely on culture and
24
microscopy, antibody-based methods using animals, or involve the use of molecular tools that
25
are expensive. Here, we describe the isolation of antibody fragments that can be used for the
26
unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were
27
isolated from a bacteriophage antibody display library. Individual clones were studied by
28
enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed
29
specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a
30
diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying
31
their biological role(s) is discussed further.
SC
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Keywords: Phage display library; Balamuthia; ELISA; Immunocytochemistry; Diagnosis
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2
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Balamuthia mandrillaris is a recently discovered protist pathogen that is a causative agent of granulomatous amoebic encephalitis. Once acquired, the infection results in death in
37
almost all reported cases. Under harsh conditions, the organism maintains itself in the cyst form.
38
Favorable conditions promote its conversion to a vegetative trophozoite stage, which in turn
39
causes the skin lesions and/or brain infection (Visvesvara et al., 1990; Martinez and Visvesvara,
40
1997). The brain infection is typically marked by the presence of granulomatous infiltrations in
41
infected tissues, thus named as granulomatous amoebic encephalitis (Visvesvara et al., 1990;
42
Martinez and Visvesvara, 1997; Siddiqui and Khan, 2008). B. mandrillaris infection can occur in
43
anyone, regardless of the immune status, as compared to other free living amoeba such as A.
44
castellanii, which generally targets immunocompromised patients. The successful treatment is
45
difficult due to poor and delayed diagnosis. In majority of cases, definitive analysis is made on
46
autopsy (Lobo et al., 2013). Current methods of detection include PCR-based methods,
47
immunofluorescence and culture techniques (Visvesvara et al., 1990; Kiderlen et al., 2008). The
48
use of immunofluorescence assays has become increasingly popular as microbial antigens are
49
easier to test as they are usually tested within the clinical specimen but the isolation of antibody
50
fragments is expensive, time consuming or may have ethical concern due to the use of animals
51
for the development of antibody-based methods. Bacteriophage antibody display libraries
52
expressing single-chain Fv antibody fragments have been developed as an alternative way of
53
isolating specific antibodies by random pairing of heavy and light chain fragments isolated from
54
naïve human lymphocytes to express single chain of variable fragment specific to the antigens of
55
B. mandrillaris (Khan et al., 2000). Specific antibodies can be readily isolated without the need
56
of animals. Here, we describe the isolation of B. mandrillaris-specific antibodies using
AC C
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M AN U
SC
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36
3
ACCEPTED MANUSCRIPT
57
bacteriophage antibody library. These antibodies provide the reagents to establish a specific and
58
rapid detection assay for B. mandrillaris. Bacteriophage library, E. coli TG1 and helper phage KO7 were obtained from M.
RI PT
59
Yamabhai, Suranaree University of Technology, Thailand. The human brain microvascular
61
endothelial cells (HBMECs), B. mandrillaris and A. castellanii were obtained and cultured as
62
described previously (Kulsoom et al., 2014). For isolation of B. mandrillaris-specific antibody
63
fragments, library clones were amplified as described previously (Khan et al., 2000). Briefly, E.
64
coli TG1 were incubated at 37°C in the presence of TYE 1% glucose and 100 µg/mL ampicillin
65
until the absorbance at 600nm was approximately 0.5. Helper phage was then added to the
66
culture and incubated for a further 30 min at 37°C without shaking. Next, kanamycin was added
67
(to select for bacteriophage-containing clones), and cultures incubated at 30°C overnight.
68
Subsequently, the culture was centrifuged at 11,000 x g for 15 min and pellet resuspended in a
69
1/5 volume of 20% (w/vol) polyethylene glycol 6000 in 2.5 M NaCl for 1 h at 4°C. After
70
incubation and three washes, the pellet was resuspended in PBS with 15% glycerol and
71
centrifuged at 1,500 x g for 10 min. Finally, the supernatant containing the bacteriophage
72
particles was filtered (pore size, 0.45 mm) and titres were determined by serial dilutions prior to
73
storage at -20°C. Clones amplified were subjected to multiple rounds of positive bio-panning
74
using B. mandrillaris and negative bio-panning using A. castellanii as described previously
75
(Khan et al., 2000). A total of 90 clones that showed binding affinity to B. mandrillaris were
76
subjected to repeated rounds (i.e., four) of ELISA using anti-M13-HRP conjugated antibody
77
(Abcam) on iMark Bio-rad microplate reader. From these, 14 clones were selected that
78
reproducibly exhibited absorbance values of more than 0.5 at 450 nm, corresponding to higher
79
binding affinity to B. mandrillaris (data not shown). For immunofluorescence assays, B.
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4
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mandrillaris, A. castellanii, E. coli and HBMEC were pelleted and re-suspended in RPMI. Cells
81
were then treated with selected clones at a dilution of 1:100. After 3X washing with milk-PBS,
82
cells were incubated with anti-M13-FITC conjugated antibody and visualized under fluorescent
83
microscope. None of the 14 clones showed reactivity against A. castellanii, E. coli and HBMEC
84
(Fig. 1). The results are representative of three independent experiments. In each experiment, cell
85
cultures were obtained from original stocks and incubated with clones for ELISA and
86
immunofluorescence assays. All 14 clones assessed using immunofluorescence showed positive
87
reactivity against B. mandrillaris in all three independent experiments performed with more than
88
80% cells showing positive reactivity (Fig. 1). Moreover, clones were also tested against the cyst
89
stage of B. mandrillaris to determine whether reactivity is stage-specific. Out of 14, only 8
90
clones showed reactivity with cyst stage of B. mandrillaris as described above, suggesting that
91
specificity is stage-specific. The results showed that selected antibody clones isolated from
92
phage library can detect trophozoite as well as cyst stage of B. mandrillaris (e.g., clone #46)
93
while others (e.g., clone #88) exhibited binding affinity to cyst alone. These findings suggest
94
affinity of clones to different antigens on the surface of B. mandrillaris. These clones can
95
provide a useful tool to characterize surface antigens of B. mandrillaris trophozoites and cysts as
96
well as study transformation of B. mandrillaris into different stages of its life cycle. This is the
97
first report that bacteriophage antibody display technology has been used in the development of
98
an antibody for the identification of B. mandrillaris. Bacteriophage antibody display libraries are
99
potentially useful and powerful tools that allow the rapid generation of antibody reagents for use
AC C
EP
TE D
M AN U
SC
RI PT
80
100
in diagnostic assays. This technology is effective in designing an antibody that recognises a
101
given protein epitope and studying its role in cellular differentiation, motility, cell division,
102
phagocytosis, chemotaxis and other biological processes. In summary, antibody fragments from
5
ACCEPTED MANUSCRIPT
a bacteriophage library can be a method of choice for generation of antibodies which are cost
104
effective, reproducible, do not require animals and can form the basis to develop clinical
105
applications.
RI PT
103
106
Competing interests: The authors declare that they have no competing interests.
108
Acknowledgements: This work was partially supported by Sunway University, Malaysia; and
109
SC
107
the University Research Council, Aga Khan University, Pakistan.
111
M AN U
110
References
1. Khan, N.A., Greenman, J., Topping, K.P., Hough, V.C., Temple, G.S., Paget, T.A., 2000.
113
Isolation of Acanthamoeba-specific antibodies from a bacteriophage display library. J.
114
Clin. Microbiol. 38, 2374-2377.
115 116
TE D
112
2. Kiderlen, A.F., Radam, E., Lewin, A., 2008. Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene. BMC Microbiol. 8, 210. 3. Kulsoom, H., Baig, A.M., Siddiqui, R., Khan, N.A., 2014. Combined drug therapy in the
118
management of granulomatous amoebic encephalitis due to Acanthamoeba spp., and
120 121 122 123 124
AC C
119
EP
117
Balamuthia mandrillaris. Exp. Parasitol. 145, S115-S120.
4. Lobo, S.A., Patil, K., Jain, S., Marks, S., Visvesvara, G.S., Tenner, M., El Khoury, M.Y., 2013. Diagnostic challenges in Balamuthia mandrillaris infections. Parasitol. Res. 112, 4015-4019. 5. Martinez, A.J., Visvesvara, G.S., 1997. Free‐living, amphizoic and opportunistic amebas. Brain Pathol. 7, 583-598. 6
ACCEPTED MANUSCRIPT
125 126 127
6. Siddiqui, R., Khan, N.A., 2008. Balamuthia amoebic encephalitis: an emerging disease with fatal consequences. Microb. Pathogen. 44, 89-97. 7. Visvesvara, G.S., Martinez, A.J., Schuster, F.L., Leitch, G.J., Wallace, S.V., Sawyer, T.K., Anderson, M., 1990. Leptomyxid ameba, a new agent of amebic
129
meningoencephalitis in humans and animals. J. Clin. Microbiol. 28, 2750-2756.
RI PT
128
SC
130
Figure legends
132
Figure 1. Representative images of bacteriophage antibody clone reactivity against B.
133
mandrillaris, HBMEC, E. coli, A. castellanii under bright field (left) and fluorescence (right). A
134
& B are B. mandrillaris incubated without clone but only anti-M13-FITC; C & D are B.
135
mandrillaris incubated with clone (#46) plus anti-M13-FITC; E & F are HBMEC (clone + anti-
136
M13-FITC); G & H are E. coli (clone + anti-M13-FITC); and I & J are A. castellanii (clone +
137
anti-M13-FITC). Magnification, X200.
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Highlights
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Balamuthia mandrillaris is a protist pathogen that can cause fatal infection Early diagnosis and aggressive treatment is a pre-requisite for successful prognosis Phage antibody technology was used to anti-B. mandrillaris antibody fragments Antibody clones did not bind to closely related amoeba and host cells indicating specificity.
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