- Email: [email protected]
Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus
Accepted Manuscript Title: Application of Monoclonal Antibodies Generated Against Panton Valentine Leucocidin (PVL-S) Toxin for specific identification of Community Acquired Methicillin Resistance Staphylococcus aureus Author: Niveditha Sundar Poojary Shylaja Ramlal Radhika Madan Urs Murali Harishchandra Sripathy Harsh Vardhan Batra PII: DOI: Reference:
S0944-5013(14)00060-3 http://dx.doi.org/doi:10.1016/j.micres.2014.05.002 MICRES 25677
To appear in: Received date: Revised date: Accepted date:
26-11-2013 5-5-2014 9-5-2014
Please cite this article as: Poojary NS, Application of Monoclonal Antibodies Generated Against Panton Valentine Leucocidin (PVL-S) Toxin for specific identification of Community Acquired Methicillin Resistance Staphylococcus aureus, Microbiological Research (2014), http://dx.doi.org/10.1016/j.micres.2014.05.002 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.
Application of Monoclonal Antibodies Generated Against Panton Valentine Leucocidin
2
(PVL-S) Toxin for specific identification of Community Acquired Methicillin Resistance
3
Staphylococcus aureus
4
Niveditha Sundar Poojary1, Shylaja Ramlal1, Radhika Madan Urs1, Murali Harishchandra
5
Sripathy1, Harsh Vardhan Batra*1
Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore-
8
*Author for Correspondence
9
Dr. Harsh Vardhan Batra
us
570011. Karnataka, India.
7
cr
1
an
6
ip t
1
Director, outstanding scientist ‘H’
11
Defence Food Research Laboratory,
12
Siddartha Nagar, Mysore-570011. Karnataka, India.
13
E-mail: [email protected]
14
Phone: +91-821-2473671
15
Fax: +91-821-2473468
16
Co-authors:
17
Niveditha Sundar Poojary
18
E-mail: [email protected]
Ac ce p
te
d
M
10
19
1 Page 1 of 25
20
Dr. Shylaja Ramlal
21
e-mail: [email protected]
23
Radhika Madan Urs
24
e-mail: [email protected]
ip t
22
Dr. Murali Harishchandra Sripathy
27
e-mail: [email protected]
us
26
cr
25
Running title : Detection of CA-MRSA
M
29
an
28
30
Abstract:
32
Panton-Valentine
33
Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing
34
pneumonia comprised of two fractions namely PVL S and PVL F. In the present study, three
35
monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against
36
recombinant PVL-S (35 kDa) protein of Staphylococcus aureus. All the three MAbs specifically
37
reacted to confirm PVL- S positive strains of S. aureus recovered from clinical samples in
38
Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14
39
different pathogenic bacteria belonging to other genera and species in Western blot analysis.
40
Furthermore, a simple dot-ELISA method was standardized for the identification of PVL–S toxin
41
containing S. aureus strains. Initially in dot ELISA, Protein A (Spa) of S. aureus posed
42
background noise problems due to the non-specific binding of antibodies resulting in false
(PVL)
produced
by
Community
Acquired
Methicillin
Ac ce p
te
Leukocidin
d
31
2 Page 2 of 25
positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5%
44
milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL
45
monoclonal antibodies in dot- ELISA. Once standardized, this simple dot ELISA was evaluated
46
with nine PVL positive strains recovered from food, environmental and clinical samples and the
47
results were compared with PCR assay for the presence of PVL toxin genes and also with
48
Western blot analysis.
49
Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA
50
system can be of immense help in providing, rapid detection of the PVL toxin containing S.
51
aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of
52
CA-MRSA.
53
Key words: Staphylococcus aureus, Panton-Valentine leukocidin (PVL), Dot-ELISA,
54
Diethylpyrocarbonate
55
INTRODUCTION
ip t
43
te
d
M
an
us
cr
A 100% correlation was found between dot-ELISA, PCR assay and
Community associated Methicillin resistant Staphylococcus aureus (CA-MRSA) has
57
emerged as a major public health problem worldwide and epidemiologic data suggest that the
58
Panton–Valentine leukocidin (PVL) toxin is expressed by a large majority of CA-MRSA strain
59
(over 90%) and this could contribute to severe human infections, particularly in young and
60
immunocompetent hosts (Ching Wen Tseng et al. 2009). Rising antibiotic resistance is the
61
looming problem associated with clinical management of S. aureus. PVL is a pore forming toxin,
62
consists of two subunits, LukS-PV and LukF-PV, which in an octamer structure that is essential
63
for pore formation on host cells. PVL presumably breaches the body’s defense system by lysing
64
human polymorphonuclear cells (PMNs). CA-MRSA proliferates within elderly persons,
Ac ce p
56
3 Page 3 of 25
65
newborn infants and cancer patients and causes pneumonia, sepsis and proven to be a cause of
66
high mortality with the CA-MRSA strains bearing PVL (Jayasinghe and Francis JS 2005). Previously CA-MRSA was confined to hospitals and nursing homes and now it has
68
encroached upon immunocompetent populations and poses a growing threat to public health
69
worldwide (CDC). In India, probably due to overcrowding and poor hygiene, CA-MRSA has
70
spread and pronounced in bacteremias affecting neonates, especially from lower economic
71
sections, and breast abscesses in lactating mothers, becoming increasingly common in urban
72
areas (Namitha Desouza et al. 2010).
us
cr
ip t
67
To date, the diagnosis of PVL producing strains is mainly based on Polymerase Chain
74
Reactions (PCRs), Real-Time PCR assays and nucleic acid-hybridization kits (AdvanDx® PVL
75
Evigene™) for the detection of lukF-PV and lukS-PV genes (Yi-Wei Tang et al. 2007; Carrol and
76
Karen 2008). PCR systems require prior isolation of bacterial DNA, preparation of enzyme
77
reaction mix, and expensive instruments for nucleic acid amplification and sophisticated
78
laboratory facility. Moreover, quantitative measurement of toxin may be a better prognostic
79
marker than checking for the presence of PVL genes. The conventional method of identifying
80
Staphylococcus aureus is labour-intensive and time-consuming procedure (4 to 5 days) and the
81
detection of MRSA strains by antibiotic sensitivity test is also labour-intensive and time-
82
consuming procedure which is inadequate for making timely assessments on the microbiological
83
safety of foods and also in clinical laboratories (Samia et al. 2007).
Ac ce p
te
d
M
an
73
84
ELISA could be a better alternative in terms of simplicity, low cost and reliability but 99
85
% of S. aureus are capable of producing soluble and secretory Staphylococcal protein A (SpA)
86
which normally would interferes with the ELISA as Fc region of IgG antibodies would be
87
reacting to the SpA present in S. aureus resulting in false positive results (Goding et al. 1978).
4 Page 4 of 25
To prevent protein A interference specifically in ELISA systems several techniques have been
89
attempted (Hein et al. 2010). All these approaches are relatively expensive and most of them are
90
not fully adequate to remove SpA interference. DEPC has been suggested to inhibit non-specific
91
binding of Spa to IgG (Haake et al. 1982).
ip t
88
In the present study, we describe the generation of mouse monoclonal antibodies against
93
r-PVL-S fraction of S. aureus and also we report the development of a simple and novel dot
94
ELISA method employing these MAb for the specific detection of PVL-S toxin containing S.
95
aureus strains. The described dot-ELISA method does not require sophisticated instruments and
96
can be easily integrated into any diagnostic laboratories and might contribute towards timely
97
therapy of PVL associated infections.
d
100
MATERIALS AND METHODS
2.1 Bacterial cultures, chemicals and reagents:
te
99
M
98
an
us
cr
92
Bacterial strains used in this study are listed in Table 1. All the media used in this study
102
were purchased from Hi-Media labs (Mumbai, India). The S. aureus strains were grown in Brain
103
Heart Infusion broth (BHI) and plated on Baird Parker agar (BPA) supplemented with Egg yolk
104
tellurite emulsion. The plates were incubated at 37 OC for 24 hours. The isolates were identified
105
morphologically and biochemically by standard laboratory procedures (Murray et al. 2007) and
106
maintained in 15% glycerol stocks in -80 OC. Cloning host E. coli BL21 (DE3) pLysS was
107
grown and maintained in Luria Bertani broth/agar.
108
2.2 Construction of LukS-PV recombinant protein:
Ac ce p
101
109
The primers (Table 2) used in the construction of recombinant luk-S gene was designed
110
using the Generunner software. The gene sequences were retrieved from NCBI database. Each
5 Page 5 of 25
PCR reaction was performed with 1x pfu buffer with 2 mM MgSO4, 200 μM of dNTPs, 10
112
picomole each of forward and reverse primers, 50 ng of DNA template, one unit of pfu DNA
113
polymerase and the final volume was adjusted to 20 µl with nuclease free water. The PCR was
114
performed as follows: initial denaturation of 4 minutes at 94 OC followed by 30 cycles of
115
denaturation for 30 seconds at 94 OC, annealing at 57 OC for 30 seconds, extension of one minute
116
at 72 OC followed by a final extension of ten minutes at 72 OC. PCR products were analysed in
117
2% agarose with ethidium bromide. The PCR product was digested purified and digested with
118
BamHI and HindIII and inserted into pRSET A vector pre-digested with BamHI and HindIII.
119
The ligated product was transformed in to BL21(DE3)-pLysS E. coli host strains. The resultant
120
colonies were screened by colony PCR for the presence of insert by flanking T7 sequencing
121
primers. The plasmid was extracted from transformed strain and sequenced with T7 primers.
M
an
us
cr
ip t
111
The E. coli host cells harboring recombinant plasmids were grown overnight at 37 OC in
123
LB broth with 100 µg/ml ampicillin. One ml of this culture was further inoculated in to flasks
124
containing 25 ml LB broth containing 100 µg/ml ampicillin and grown at 37 OC with vigorous
125
shaking until the OD600 reached 0.7. One mM IPTG was added to the cells and allowed to grow
126
at 37 OC with vigorous shaking. One ml of cells were collected after 5 hrs of induction, lysed,
127
subjected to 12 % SDS-PAGE and stained with Coomassie brilliant blue to determine the
128
expression of the proteins. The proteins separated on SDS-PAGE was transferred on to a
129
nitrocellulose membrane by Western blot procedure, blocked with phosphate buffered saline
130
(PBS) containing 5% skim milk for 1 h followed by incubation with mouse monoclonal anti-
131
histidine antibodies. After washing with PBST (PBS + 0.05% Tween 20), the membrane was
132
incubated for 30 minutes with anti-mouse HRP labelled antibodies raised in goat. The membrane
133
was thoroughly washed with PBST and developed with 3,3`, 5, 5` -d Diaminobenzidine tetra
Ac ce p
te
d
122
6 Page 6 of 25
chloride and 0.004 % H2O2 in PBS. Later the reaction was stopped by washing the membrane
135
with distilled water. For bulk production, the culture was inoculated in 100 ml LB broth with
136
antibiotics. The recombinant protein was purified under denaturation conditions using urea as
137
denaturant by immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose
138
(Qiagen, Germany) as chelating resin and amino terminal hexa-histidine residues as affinity tag
139
according to manufacturer’s recommendations. The purity of the purified protein was analyzed
140
by 12% SDS-PAGE. The elutions were pooled and the protein concentration was quantified by
141
Lowry’s calorimetric assay using BSA as standard (Niveditha et al. 2013).
142
2.3
an
Primary and secondary antibody preparation:
us
cr
ip t
134
Four different groups of 8 female BALB/c mice aged between 6–8 weeks were
144
immunized intramuscularly in hind legs with 50 g of purified r-PVL-S proteins at an interval of
145
14 days (0, 14, 28 and 42 days). The first immunization was with Freund’s complete adjuvant
146
(FCA) and subsequent doses were provided with incomplete Freund’s adjuvant (IFA)
147
intramuscularly.
148
2.4
te
d
M
143
Ac ce p
Generation of monoclonal antibodies:
149
Approximately 108 SP 2/0 myeloma cells were fused with homogenized spleen cell
150
suspensions prepared from the spleens of immunized mice. Stable hybrid cell lines were selected
151
with Littlefield hypoxanthine-aminopterin thymidine (HAT) medium. The fused cells were
152
plated in 96 well tissue culture plates at several dilutions to ensure single clones per well. The
153
colonies were fed every fifth day with freshly prepared HAT medium. Approximately 2 weeks
154
after fusion, wells containing single and double macroscopic clones were tested for antibody
155
production and toxin specificity by indirect ELISA. Wells containing positive cells were cloned
156
by the limiting dilution method into 96-well tissue culture plates at least three times to ensure 7 Page 7 of 25
monoclonality. Of the 192 wells examined, only three clones gave strong positive signals in the
158
indirect ELISA with the highest affinity. Therefore, the supernatant of these three clones were
159
aspirated from the fusion well and subjected to limiting dilution for hybridoma selection. After
160
limiting dilution and ELISA screening, clones were used for the production of culture
161
supernatant for further large scale production of antibody.
162
2.5
cr
ip t
157
us
Isotyping of monoclonal antibodies:
Monoclonal antibodies obtained from immunized mice were used for detection of
164
specific antibody and antibody isotypes by enzyme-linked immunosorbent assay (ELISA) by
165
using a mouse MAb Isotyping kit Roche. Briefly, microtitre plates were coated with 100µl of
166
purified proteins (10 µg ml-1) in carbonate-bicarbonate buffer and the unbound sites were
167
blocked with skimmed milk powder (5 %) in PBS. Serially diluted test sera were added to the
168
wells and maintained at 37°C for 1 h, followed by similar incubation with Horse Radish
169
Peroxidase (HRPO)-conjugated goat anti-mouse IgG, IgM, IgG2a, IgG2b and IgG3 antibodies
170
(sigma aldrich India). Colour was developed with the substrate O-phenylenediamine
171
dihydrochloride (OPD) in presence of 0.4% H2O2 and absorbance was measured in ELISA plate
172
reader (Infinite M200 pro; Tecan, Grodig, Austria) at 470nm (Leary SE 1995; Uppada SB 2011).
173 174
2.6
Ac ce p
te
d
M
an
163
Dot immunobinding assay:
175
The crude protein preparations and purified recombinant proteins were coated on to
176
nitrocellulose membrane. The blots were blocked with phosphate buffered saline (PBS)
177
containing 5% skim milk along with different concentration of diethylpyrocarbonate (DEPC) for
178
1 h followed by incubation with 1:1000 dilution of anti-rPVL-S, mouse serum followed by 1 h
179
incubation with goat anti-mouse IgG-HRP conjugate (Sigma chemicals, USA). The membrane 8 Page 8 of 25
was thoroughly washed with PBST and chromogenic reaction was observed by incubating with
181
3,3`, 5, 5` -d Diaminobenzidine tetra chloride and 0.004 % H2O2 in PBS. Later the reaction was
182
stopped by washing the membrane with distilled water. For the assessment of sensitivity,
183
overnight culture of PVL positive S. aureus was serially diluted by using saline (0.9% of NaCl).
184
100 microlitre of sample drawn from each dilution and further processed for DNA extraction by
185
boiling lysis method. The DNA (1.0 μl) was used as template in PCR assay. 500 microlitre of
186
sample drawn from each dilution were pelleted in carbonate-bicarbonate buffer and Dot-ELISA,
187
was performed.
188
2.7
an
us
cr
ip t
180
Western blot for LukS-PV:
The crude protein preparations and purified recombinant proteins were resolved on 12%
190
polyacrylamide gel and were transferred on to nitrocellulose membrane (Towbin et al. 1979) and
191
blocked with phosphate buffered saline (PBS) containing 5% skim milk for 1 h. Incubation with
192
anti PVL-S MAbs followed by 1 h incubation with goat anti-mouse IgG-HRP conjugate (Sigma
193
chemicals, USA). Chromogenic reaction was observed by incubating with Diaminobenzidine
194
(DAB)-H2O2 substrate.
d
te
Ac ce p
195
M
189
196
RESULTS
197
3.1 Construction and Cloning of luk-S gene of PVL:
198
The individual gene luk-S was amplified from S. aureus genomic DNA (Fig.1a). After
199
cloning these genes into pRSETA vector, they were sequenced to determine if any mutations
200
have appeared. Positive clones were induced with 1mM IPTG for 5 hrs at 37 OC and the
201
expression of recombinant protein was detected in 12% SDS-PAGE gel stained with Coomassie
202
blue. The relative sizes of the proteins r-luk-S was in agreement with the predicted size i.e. 35 9 Page 9 of 25
203
kDa (Fig.1b). Subsequently, the expression or the proteins was confirmed by Western blot
204
analysis with anti-Histidine antibodies.
205
3.2 Purification of the recombinant protein: The recombinant proteins were purified from 100 ml induced culture by using
207
immobilized metal affinity chromatography on Ni-NTA agarose column (Qiagen). The cells
208
were induced for 5 h with 1 mM IPTG and they were collected and purified by urea denaturation
209
according to manufacturer’s protocol (Qiagen) (Fig.1b). The purified proteins were pooled and
210
dialysed into PBS + 10 mM Urea for 2 h at 4OC. After buffer exchange, the protein concentration
211
of luk-S protein was quantified to be 4.0 mg/ mL by Lowry’s calorimetric assay using bovine
212
serum albumin as a standard.
213
3.3 Monoclonal antibody to PVL-S toxin secreting clones:
M
an
us
cr
ip t
206
Stable hybrid cell lines were selected with Littlefield hypoxanthine-aminopterin thymidine
215
(HAT) medium. The fused cells were plated in 96 well tissue culture plates, from the 192 wells
216
examined, twenty hybridoma clones were obtained. From that only three clones, MAb1, MAb9
217
(B2, C1), MAb10 (C11, D12) gave strong positive signals in the indirect ELISA with the highest
218
affinity (Fig.2). Therefore, the supernatant of these three clones were aspirated from the fusion
219
well and subjected to limiting dilution for hybridoma selection. After limiting dilution and
220
ELISA screening, clones were used for the production of culture supernatant for further large
221
scale production of antibody.
222
Isotyping of monoclonal antibodies:
Ac ce p
te
d
214
223
To assess the specific antibody isotype in all three monoclonal antibodies, isotyping was
224
carried out by Indirect plate ELISA. MAb1 was found to be significantly higher in producing
225
IgG1 antibody isotype. MAb10 and MAb9 were biased towards the IgG2b and IgG3 isotype
10 Page 10 of 25
226
respectively, but they were statistically insignificant (p < 0.01) (Fig.3). Results were presented as
227
the means value ± standard error (SE) and the graph was created with the help of Graph Pad
228
Prism6 software. Statistical difference were analyzed and assumed with p value (p < 0.01).
230
ip t
229
3.4 Specificity:
The monoclonal antibodies raised against PVL-S protein were tested for their reactivity
232
by Indirect ELISA, Dot-ELISA and Western Blot analysis. MAb 1 reacted very strongly with
233
native toxin, which was shown by Indirect ELISA, Dot-ELISA and Western Blot analysis. The
234
MAb1 did not exhibit any cross-reactions with other organisms tested when compared with other
235
two monoclonal antibodies (Table 3).
236
3.6 Sensitivity:
M
an
us
cr
231
Sensitivity was determined by the comparative analysis between PCR and Dot-ELISA
238
assays through serial dilution method. PCR could detect upto 109 to 101 CFU/ml and Dot-ELISA
239
is also sensitive upto detect low cell concentration of 101 with all 3 monoclonal antibodies.
240
Hence the newly standardised Dot-ELISA based method is giving 100% correlation with PCR
241
(supplementary data). Reproducibility also checked with repeated testing of a newly developed
242
Dot-ELISA assay.
243
3.7 Simple Dot- ELISA assay with DEPC treatment:
Ac ce p
te
d
237
244
The MAb1 raised against recombinant luk-S protein was tested for their reactivity by
245
simple Dot ELISA assay by the addition of 10mM concentration of DEPC in blocking step has
246
reduced the interference of protein A in assay when compared with different concentrations like
247
1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM and15.0 mM. Assay showed that MAb1 reacted strongly
248
with native toxin of PVL positive Staphylococcus aureus strains which were compared with PVL
11 Page 11 of 25
249
negative Staphylococcus aureus strains (Fig.4). This assay correlates with the PCR method of
250
identification of 87 S. aureus isolates.
251
DISCUSSION A strong association of Panton-Valentine Leukocidin both in CA-MRSA related
253
outbreaks as well as CA-MRSA involved in chronic diseases has implications both for
254
epidemiological and clinical studies. Until recently, the CA-MRSA strains were susceptible to
255
many antibiotics other than the beta-lactams; however, resistance seems to be increasing and
256
multiple antibiotic resistant strains have started to emerge. Rapid identification of PVL in
257
methicillin-resistance S. aureus isolates causing severe infections is necessary in order to prevent
258
their potentially devastating consequences. PVL, a bi-component exotoxin consists of two
259
separately secreted and non-associated proteins (class S and class F components). Both the
260
components
261
permeabilization of target cells and leading to their lysis (König et al. 1995; Siqueira et al. 1997).
262
Employing a specific antibody and targeting the PVL molecule through a simple
263
immunoassay system would be a workable strategy for the quick identification of CA-MRSA
264
strains. Monoclonal antibodies overall have been utilized in large number of immunoassays for
265
the detection of toxins and pathogens.
266
generated against PVL-S fraction of S. aureus by using a recombinant PVL-S protein as the
267
immunogen. The spleen from a single immunized BALB/c mouse resulted in 19 antibody
268
producing hybrids which after cloning by limiting dilution, yielded 3 stable clones. MAb 1
269
belonged to IgG1 class and MAb 10 was IgG2b in nature. It implies that produced monoclonal
270
antibodies were directed towards Th2 and Th1 mediated antibody response against PVL-S toxin.
271
All the three MAbs generated were tested for specificity by dot ELISA and by Western blot
sequentially
and
synergistically,
inducing
the
activation
and
Ac ce p
te
d
interact
M
an
us
cr
ip t
252
In the present study, monoclonal antibodies were
12 Page 12 of 25
analysis. No reaction was seen against E. coli, S. flexneri, S. boydii, Klebsiella, S. typhi, S.
273
typhimurium, Proteus sp, Citrobacter, B. cereus, A. hydrophila, Y. enterocolitica and other
274
relevant pathogenic bacteria. All the three MAbs generated were also tested for sensitivity by
275
comparing with PCR and Dot-ELISA. However, non PVL producing S. aureus strains showed
276
false positive signal due to the presence of SpA. Different methods have been tried by several
277
workers to prevent SpA interference and there are addition of porcine IgG coupled to insoluble
278
matrix has been used to remove protein A from culture supernatants (Berdal et al. 1981; Fey and
279
Burkhard 1981). Revealing rabbit antibodies have been biotinylated so that the binding site of
280
Spa on the IgG molecule is masked (Hahn et al. 1986). Using IgG antibodies raised in rats and
281
sheep as a capturing antibody may contribute towards improved positive signal because of their
282
weak binding to SpA (Rogemond et al. 1991; Freed et al. 1982). In addition, chicken anti-protein
283
A Ig Y has been used to sequester protein A, since chicken IgY does not bind protein A in the Fc
284
region (Hoffman et al. 1996; Reddy et al. 2012). DEPC has also been suggested to inhibit non-
285
specific binding of Spa to IgG (Haake et al. 1982; Hein et al. 2010). Hence an attempt was made
286
to standardize a dot ELISA method by the incorporation of DEPC at concentrations ranging from
287
1 mM to 15 mM. Incorporation of 10 mM DEPC along with 5% milk in PBS in the blocking
288
step completely prevented non-specific binding of Spa to mouse anti-PVL monoclonal
289
antibodies in simple dot- ELISA. Once standardized, this simple dot ELISA was evaluated with
290
nine PVL positive and large numbers of PVL negative strains of S. aureus. False signals were
291
not observed in the DEPC treated dot ELISA. Similar results were also observed in Western blot
292
analysis (Table 3). The results are obtained within 3 hours. The isolates which were identified as
293
PVL producing strains by this newly described dot ELISA method were unequivocally detected
Ac ce p
te
d
M
an
us
cr
ip t
272
13 Page 13 of 25
294
positive by the in-house developed PCR format. Newly developed detection system is sensitive
295
upto to detect as low as 101cells. The importance of PVL detection from community-acquired MRSA has been gaining
297
attention. A method for amplifying and detecting the PVL gene by the PCR method, which is
298
currently used as a PVL detection method, requires expensive laboratory infrastructure and well-
299
experienced technicians and time-consuming. Moreover, at times the presence of genes might
300
not correlate with the expression of toxin. Specific and highly sensitive detection of the toxin
301
protein is difficult with the use of conventional techniques and also limited to specialized
302
laboratories with equipment and experience to perform such assays (Kalyan et al. 2008). To
303
facilitate the rapid detection of PVL toxin by routine clinical microbiology laboratories, the
304
newly described monoclonal antibody based dot-ELISA method reported here, can be of
305
immense help in providing, rapid and specific detection of the PVL toxin containing MRSA
306
strains at a relatively low cost which are added advantages over existing methods. This kit would
307
serve as a valuable tool for the reliable identification of PVL producing CA-MRSA strains from
308
clinical, food and environmental sources to arrest their dissemination and further expansion.
310
cr
us
an
M
d
te
Ac ce p
309
ip t
296
Acknowledgments:
311
Authors are thankful to the Director, DFRL for providing necessary facilities to conduct the
312
study. Niveditha. S is a Senior Research fellow funded by the Indian Council of Medical
313
Research (ICMR), India.
314 315
References:
14 Page 14 of 25
316
Berdal BP, Olsvik O, Omland T. (1981). A sandwich ELISA method for detection of
317
Staphylococcus aureus enterotoxins. Acta Pathol Microbiol Scand B89, 411–415.
318
Carrol and Karen C. (2008). Rapid Diagnostics for MRSA: current status, Molecular diagnosis
322
(1999). Four pediatric deaths from community-acquired methicillin resistant
cr
321
CDC.
Staphylococcus aureus-Minnesota and North Dakota. JAMA 282, 1123–1125. Ching Wen Tseng, Pierre Kyme,Jennifer Low,Miguel A. Rocha,Randa Alsabeh,Loren G. Miller,Michael
Arditi,Binh
An
Diep,
Victor
Nizet,Terence
M.
324
Doherty,David O. Beenhouwer, George Y. Liu. (2009). Staphylococcus aureus Panton
325
Valentine Lecocidin contributes to inflammation and Muscle Tissue injury, PLoS
326
one4(7), e6387.
M
an
323
Fey H, Burkhard G. (1981). Measurement of staphylococcal protein A and detection of protein
328
A-carrying
strains
329
ofImmnological Methods47, 99–107.
by
a
competitive
ELISA
method. Journal
te
staphylococcus
d
327
Otto,Moshe
us
320
ip t
and Therapy, Infectious diseases. 12, 1, 15-24.
319
Francis JS, Doherry MC, Johnston C, Sinha G, Ross T, Cai M S. (2005). Severe Community-
331
onset pneumonia in healthy adults caused by Methicillin resistant Staphylococcus aureus
332
carrying the Panton Valentine Leukocidin genes. Clin Infect Dis40, 100-7.
333 334
335 336
Ac ce p
330
Goding JW.(1978). Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 20, 241–53.
Haake DA, Franklin EC, Frangione B. (1982). The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate. J Immunol 129,190–2.
15 Page 15 of 25
337
Hahn IF, Pickenhahn P, Lenz W, Brandis H. (1986). An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. J Immunol Methods92, 25–9.
338
Hien M. Nguyen1, Migue A. Rocha, Koteswara R. Chintalacharuvu, and David O. Beenhouwer.
340
(2010). Detection and quantification of Panton-Valentine leukocidin in Staphylococcus
341
aureus cultures by ELISA and Western blotting: diethylpyrocarbonate inhibits binding of
342
protein A to IgG, J Immunol Methods 356(1-2), 1–5.
cr
ip t
339
Hoffman WL, Ruggles AO, Tabarya D. (1996). Chicken anti-protein A prevents Staphylococcus
344
aureus protein A from binding to human and rabbit IgG in immunoassays and eliminates
345
most false positive results. J Immunol Methods198, 67–77.
an
us
343
Jayasinghe, L and H. Bayley. (2005). The leukocidin pore: evidence for an octamer with four
347
LukF subunits and four LukS subunits alternating around a central axis. Protein Sci.14,
348
2550–2561.
d
M
346
Kalyan T.D,Balakrishna K, Murali H.S and Batra H.V. (2008). Construction of a recombinant
350
intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL
351
of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of
352
Staphylococcus aureus. 2008. Journal of Medical Microbiology 58, 577-583. Konig
Ac ce p
353
te
349
B,PrevostG,
PiemontY,
and
KonigW.(1995).
Effects
of
Staphylococcus
354
aureusleukocidins on inflammatory mediator release from human granulocytes. Journal
355
of Infectious Diseases171, 607–613.
356
Leary SE, Williamson ED, Griffin KF, Russell P, Eley SM, Titball RW. (1995). Active
357
immunization with recombinant V antigen from Yersinia pestis protects mice against
358
plague. Infect Immun63, 2854-2858.
16 Page 16 of 25
359
Namita D'Souza, Camilla Rodrigues and Ajita Mehta (2010). Molecular Characterization of
360
Methicillin-Resistant Staphylococcus aureus with Emergence of Epidemic Clones of
361
Sequence Type (ST) 22 and ST 772 in Mumbai, India. J. Clin. Microbiol 48(5), 1806-09. Niveditha Sundar Poojary*, Shylaja Ramlal, Murali Harishchandra Sripathy, Harsh Vardhan
363
Batra (2013). Application of Recombinant LUK-SF Protein of Panton-Valentine Toxin
364
of Community Acquired Methicillin Resistant Staphylococcus aureus for Toxin
365
Detection and Neutralization Properties. International Journal of Advanced Research 1,
366
1(10), 129-138.
us
cr
ip t
362
Reddy PK, Aravind S, Joseph Jeyabalaji Kingston, Murali HS, HV Batra (2013).Evaluation of
368
IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus
369
aureus without staphylococcal protein A interference. J Immunol Methods391,31–38.
M
an
367
Rogemond V, Da Costa Castro JM, Delaunay T, Cazaux C, Guinet RM. (1991). Protein A
371
insensitive ELISA detection of staphylococcal enterotoxin B. Lett Appl Microbiol13,
372
175–8.
te
d
370
Samia Perwaiz, Qamaruddin Barakzi, BadarJehan Farooqi, Nazia Khursheed, Nasim Sabir
374
(2007). Antimicrobial susceptibility pattern of clinical isolates of Methicillin Resistant
375
Staphylococcus aureus, J Pak Med Assoc57(1), 1-4.
Ac ce p
373
376
Siqueira J. A., Speeg-schatz c., Freitas f. I. S., Sahel j., Monteil'f H. and G. Prevost. (1997).
377
Channel-forming leucotoxins from Staphylococcus aureus cause severe inflammatory
378
reactions in a rabbit eye model. J. Med. Microbiol 46, 486-494.
379
Towbin H, Staehelin T, Gordon J. (1979). Electrophoretic transfer of proteins from
380
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl
381
Acad Sci U S A76(9), 4350-4.
17 Page 17 of 25
Uppada SB, Bhat AA, SahA, Donthamshetty RN. (2011). Enhanced humoral and mucosal
383
immune responses after intranasal immunization with chimeric multiple antigen peptide
384
of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice. Vaccine
385
29,9352-9360.
ip t
382
Yi-Wei Tang, Abdullah Kilic, Qunying Yang, Sigrid K. McAllister, Haijing Li, Rebecca S.
387
Miller, Melinda McCormac, Karen D. Tracy, Charles W. Stratton, Jian Han, and Brandi
388
Limbago. (2007). StaphPlex System for Rapid and Simultaneous Identification of
389
Antibiotic Resistance Determinants and Panton-Valentine Leukocidin Detection of
390
Staphylococci from Positive Blood Cultures, J Clin Microbiol 45(6), 1867–1873.
an
us
cr
386
Figure Legends:
392
Fig. 1
393
A. Agarose gel showing amplification of luk-S gene: Lane 1, PCR amplification of luk-S gene
394
(798 bp); Lane 2, 1 kb DNA ladder
395
B. Coomassie blue stained SDS-PAGE gel showing expression and purification of r-luk-S
396
protein by using immobilized metal affinity chromatography: Lane1, Prestained protein
397
ladder; lane 2, Induced E. coli BL21 (DE3) carrying pRSET A-luk-S vector (35kDa); lane 3,
398
Unstained protein ladder; last lanes 4, 5 & 6 are the elutions of Purified r-luk-S.
d
te
Ac ce p
399
M
391
400
Fig. 2 Plate ELISA showing reactivity of three Monoclonal antibody (MAb1, MAb9,
401
MAb10) against r-luk-S protein (10 µg ml-1) in duplicate wells: Lane A1, B1, C1, E1,
402
Negative control (PBS); Lane D1, positive control (PoAb as primary antibody); Lane A2 & A3,
18 Page 18 of 25
403
MAb 9; Lane B2 & B3, MAb 9; Lane C2 & C3, MAb 10; Lane D2 & D3, MAb 10; Lane E2 &
404
E3, MAb 1.
405
Fig. 3 Isotyping of Monoclonal antibodies:
407
Antibody isotypes specific to r-luk-S was evaluated by ELISA using isotype-specific monoclonal
408
antibodies for IgG1, IgG2a, IgG2b, IgG3 and IgM. The level of isotypes was expressed as
409
absorbance (A470 nm) of the colored complex developed in the Immunosorbent assay. Data are
410
represented as the mean value ± standard error (SE) and graph was created with the help of
411
Graph Pad Prism6 software. A statistical difference was assumed with p value (p < 0.01).
an
us
cr
ip t
406
412
Fig. 4 Dot ELISA showing reactivity of MAb1 against PVL positive and PVL negative S.
414
aureus strains
415
Lane A1-A6 are PVL positive and Lane B1-B6 are PVL negative S. aureus strains.
416
Lane A1, S. aureus E2147; Lane A2, S. aureus FRI 722; Lane A3, S. aureus E1978; Lane A4, S.
417
aureus NCIM 2792; Lane A5, ATCC 43300; Lane A6: S. aureus NCIM 2127; Lane B1, S.
418
aureus ATCC 700699; Lane B2, S. aureus NCIM 2901; Lane B3, S. aureus NCIM 2121; Lane
419
B4, S. aureus E2533; Lane B5, S. aureus E2279; Lane B6, S. aureus FI 4.
Ac ce p
te
d
M
413
19 Page 19 of 25
Table
Table 1: Bacterial strains used in the study: Strains
Sources
Escherichia coli
pLysS
Invitrogen, Bangalore, Karnataka, India
cr
Staphylococcus aureus
ip t
Escherichia coli BL21 (DE3)
ATCC: American Type Culture Collection, Manassas, Va
FRI 722
Food Research Institute, India
ATCC 43300
ATCC: American Type Culture Collection, Manassas, Va
ATCC 29213
ATCC: American Type Culture Collection, Manassas, Va
ATCC 6538
ATCC: American Type Culture Collection, Manassas, Va
NCIM 2079
National Collection of Industrial Microorganisms, Pune, India
NCIM 2901
National Collection of Industrial Microorganisms, Pune, India
NCIM 2654
National Collection of Industrial Microorganisms, Pune, India
NCIM 2672
National Collection of Industrial Microorganisms, Pune, India
NCIM 2792
National Collection of Industrial Microorganisms, Pune, India
NCIM 2127
National Collection of Industrial Microorganisms, Pune, India
Ac ce p
te
d
M
an
us
ATCC 700699
NCIM 2124
National Collection of Industrial Microorganisms, Pune, India
NCIM 2121
National Collection of Industrial Microorganisms, Pune, India
NCIM 2122
National Collection of Industrial Microorganisms, Pune, India
RAB (DRDE)
Defence Research and Development Establishment, India
IVRI
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India
E 2147
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E2533
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E2279
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1345
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1357
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Page 20 of 25
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1975
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1977
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1978
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
FI 1
Food isolate from local market Mysore, Karnataka; India
FI 2
Food isolate, this work
FI 3
Food isolate, this work
FI 4
Food isolate, this work
FI 5
Food isolate, this work
FI 6
Food isolate, this work
FI 7
Food isolate, this work
CI 1
Clinical isolate from pus, this work
CI 2
Clinical isolate from wound infection, this work
CI 3
Clinical isolate from abscess, this work
CI 4
Clinical isolate from pus, this work
CI 5
Clinical isolate from wound infection, this work
CI 6
Clinical isolate from pus sample, this work
Ac ce p
Other Bacterial Strains
te
d
M
an
us
cr
ip t
E1988
Vibrio vulnificus ATCC 27562
ATCC: American Type Culture Collection, Manassas, Va
Shigella flexneri
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Salmonella typhimurium
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Proteus vulgarisATCC 6380
ATCC: American Type Culture Collection, Manassas, Va
Aeromonas hydrophila ATCC
ATCC: American Type Culture Collection, Manassas, Va
7966 Bacillus cereusATCC 14579
ATCC: American Type Culture Collection, Manassas, Va
Listeria monocytogenes
ATCC: American Type Culture Collection, Manassas, Va
Page 21 of 25
ATCC 13932 Klebsiella pneumonia
ATCC: American Type Culture Collection, Manassas, Va
Table 2: Primers used for the cloning of luk-S gene: Sequence
Accession number
luk-S For
5’ CGCggatccATCACTCCTATTGCTACTTCG 3’
AB678714.1
luk-S Rev
5’ CCGggtaccGCCATAGTGTGTTGTTCTTCT 3’
Amplicon (bp)
cr
Primer
ip t
ATCC BAA2146
size
us
798 bp
an
The sequence corresponding to restriction enzymes were given in lowercase.
M
Table 3: Specificity of MAb1 compared with Western Blot, Plate ELISA and Dot ELISA: Strains
Ac ce p
te
d
Staphylococcus aureus ATCC 700699 Staphylococcus aureus Japan TSST Staphylococcus aureus FRI 722 Staphylococcus aureus ATCC 43300 Staphylococcus aureus ATCC 29213 Staphylococcus aureus ATCC 6538 Staphylococcus aureus NCIM 2079 Staphylococcus aureus NCIM 2901 Staphylococcus aureus NCIM 2654 Staphylococcus aureus NCIM 2672 Staphylococcus aureus NCIM 2792 Staphylococcus aureus NCIM 2127 Staphylococcus aureus NCIM 2124 Staphylococcus aureus NCIM 2121 Staphylococcus aureus NCIM 2122 Staphylococcus aureus RAB (DRDE) Staphylococcus aureus IVRI Staphylococcus aureus E 2147 Staphylococcus aureus E2533 Staphylococcus aureus E2279 Staphylococcus aureus E1345 Staphylococcus aureus E1357 Staphylococcus aureus E1988 Staphylococcus aureus E1975 Staphylococcus aureus E1977 Staphylococcus aureus E1978 Staphylococcus aureus FI 1
Western Blot
Plate ELISA
Dot ELISA
+ + + + + + + + + + + -
+ + + + + + + + + + + -
+ + + + + + + + + + + -
Page 22 of 25
ip t
+ + + + + + -
cr
us
+ + + + + + -
an
+ + + + + + -
Ac ce p
te
d
M
Staphylococcus aureus FI 2 Staphylococcus aureus FI 3 Staphylococcus aureus FI 4 Staphylococcus aureus FI 5 Staphylococcus aureus FI 6 Staphylococcus aureus FI 7 Staphylococcus aureus CI 1 Staphylococcus aureus CI 2 Staphylococcus aureus CI 3 Staphylococcus aureus CI 4 Staphylococcus aureus CI 5 Staphylococcus aureus CI 6 Escherichia coli Vibrio vulnificus Shigella spp Salmonella typhii Proteus vulgaris Aeromonas spp Bacillus cereus Listeria spp Klebsiella spp
Page 23 of 25
Figure
an
us
cr
ip t
Fig 1:
Ac ce p
te
d
M
Fig 2:
Page 24 of 25
Ac ce p
Fig 4:
te
d
M
an
us
cr
ip t
Fig 3:
Page 25 of 25
S0944-5013(14)00060-3 http://dx.doi.org/doi:10.1016/j.micres.2014.05.002 MICRES 25677
To appear in: Received date: Revised date: Accepted date:
26-11-2013 5-5-2014 9-5-2014
Please cite this article as: Poojary NS, Application of Monoclonal Antibodies Generated Against Panton Valentine Leucocidin (PVL-S) Toxin for specific identification of Community Acquired Methicillin Resistance Staphylococcus aureus, Microbiological Research (2014), http://dx.doi.org/10.1016/j.micres.2014.05.002 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.
Application of Monoclonal Antibodies Generated Against Panton Valentine Leucocidin
2
(PVL-S) Toxin for specific identification of Community Acquired Methicillin Resistance
3
Staphylococcus aureus
4
Niveditha Sundar Poojary1, Shylaja Ramlal1, Radhika Madan Urs1, Murali Harishchandra
5
Sripathy1, Harsh Vardhan Batra*1
Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore-
8
*Author for Correspondence
9
Dr. Harsh Vardhan Batra
us
570011. Karnataka, India.
7
cr
1
an
6
ip t
1
Director, outstanding scientist ‘H’
11
Defence Food Research Laboratory,
12
Siddartha Nagar, Mysore-570011. Karnataka, India.
13
E-mail: [email protected]
14
Phone: +91-821-2473671
15
Fax: +91-821-2473468
16
Co-authors:
17
Niveditha Sundar Poojary
18
E-mail: [email protected]
Ac ce p
te
d
M
10
19
1 Page 1 of 25
20
Dr. Shylaja Ramlal
21
e-mail: [email protected]
23
Radhika Madan Urs
24
e-mail: [email protected]
ip t
22
Dr. Murali Harishchandra Sripathy
27
e-mail: [email protected]
us
26
cr
25
Running title : Detection of CA-MRSA
M
29
an
28
30
Abstract:
32
Panton-Valentine
33
Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing
34
pneumonia comprised of two fractions namely PVL S and PVL F. In the present study, three
35
monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against
36
recombinant PVL-S (35 kDa) protein of Staphylococcus aureus. All the three MAbs specifically
37
reacted to confirm PVL- S positive strains of S. aureus recovered from clinical samples in
38
Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14
39
different pathogenic bacteria belonging to other genera and species in Western blot analysis.
40
Furthermore, a simple dot-ELISA method was standardized for the identification of PVL–S toxin
41
containing S. aureus strains. Initially in dot ELISA, Protein A (Spa) of S. aureus posed
42
background noise problems due to the non-specific binding of antibodies resulting in false
(PVL)
produced
by
Community
Acquired
Methicillin
Ac ce p
te
Leukocidin
d
31
2 Page 2 of 25
positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5%
44
milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL
45
monoclonal antibodies in dot- ELISA. Once standardized, this simple dot ELISA was evaluated
46
with nine PVL positive strains recovered from food, environmental and clinical samples and the
47
results were compared with PCR assay for the presence of PVL toxin genes and also with
48
Western blot analysis.
49
Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA
50
system can be of immense help in providing, rapid detection of the PVL toxin containing S.
51
aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of
52
CA-MRSA.
53
Key words: Staphylococcus aureus, Panton-Valentine leukocidin (PVL), Dot-ELISA,
54
Diethylpyrocarbonate
55
INTRODUCTION
ip t
43
te
d
M
an
us
cr
A 100% correlation was found between dot-ELISA, PCR assay and
Community associated Methicillin resistant Staphylococcus aureus (CA-MRSA) has
57
emerged as a major public health problem worldwide and epidemiologic data suggest that the
58
Panton–Valentine leukocidin (PVL) toxin is expressed by a large majority of CA-MRSA strain
59
(over 90%) and this could contribute to severe human infections, particularly in young and
60
immunocompetent hosts (Ching Wen Tseng et al. 2009). Rising antibiotic resistance is the
61
looming problem associated with clinical management of S. aureus. PVL is a pore forming toxin,
62
consists of two subunits, LukS-PV and LukF-PV, which in an octamer structure that is essential
63
for pore formation on host cells. PVL presumably breaches the body’s defense system by lysing
64
human polymorphonuclear cells (PMNs). CA-MRSA proliferates within elderly persons,
Ac ce p
56
3 Page 3 of 25
65
newborn infants and cancer patients and causes pneumonia, sepsis and proven to be a cause of
66
high mortality with the CA-MRSA strains bearing PVL (Jayasinghe and Francis JS 2005). Previously CA-MRSA was confined to hospitals and nursing homes and now it has
68
encroached upon immunocompetent populations and poses a growing threat to public health
69
worldwide (CDC). In India, probably due to overcrowding and poor hygiene, CA-MRSA has
70
spread and pronounced in bacteremias affecting neonates, especially from lower economic
71
sections, and breast abscesses in lactating mothers, becoming increasingly common in urban
72
areas (Namitha Desouza et al. 2010).
us
cr
ip t
67
To date, the diagnosis of PVL producing strains is mainly based on Polymerase Chain
74
Reactions (PCRs), Real-Time PCR assays and nucleic acid-hybridization kits (AdvanDx® PVL
75
Evigene™) for the detection of lukF-PV and lukS-PV genes (Yi-Wei Tang et al. 2007; Carrol and
76
Karen 2008). PCR systems require prior isolation of bacterial DNA, preparation of enzyme
77
reaction mix, and expensive instruments for nucleic acid amplification and sophisticated
78
laboratory facility. Moreover, quantitative measurement of toxin may be a better prognostic
79
marker than checking for the presence of PVL genes. The conventional method of identifying
80
Staphylococcus aureus is labour-intensive and time-consuming procedure (4 to 5 days) and the
81
detection of MRSA strains by antibiotic sensitivity test is also labour-intensive and time-
82
consuming procedure which is inadequate for making timely assessments on the microbiological
83
safety of foods and also in clinical laboratories (Samia et al. 2007).
Ac ce p
te
d
M
an
73
84
ELISA could be a better alternative in terms of simplicity, low cost and reliability but 99
85
% of S. aureus are capable of producing soluble and secretory Staphylococcal protein A (SpA)
86
which normally would interferes with the ELISA as Fc region of IgG antibodies would be
87
reacting to the SpA present in S. aureus resulting in false positive results (Goding et al. 1978).
4 Page 4 of 25
To prevent protein A interference specifically in ELISA systems several techniques have been
89
attempted (Hein et al. 2010). All these approaches are relatively expensive and most of them are
90
not fully adequate to remove SpA interference. DEPC has been suggested to inhibit non-specific
91
binding of Spa to IgG (Haake et al. 1982).
ip t
88
In the present study, we describe the generation of mouse monoclonal antibodies against
93
r-PVL-S fraction of S. aureus and also we report the development of a simple and novel dot
94
ELISA method employing these MAb for the specific detection of PVL-S toxin containing S.
95
aureus strains. The described dot-ELISA method does not require sophisticated instruments and
96
can be easily integrated into any diagnostic laboratories and might contribute towards timely
97
therapy of PVL associated infections.
d
100
MATERIALS AND METHODS
2.1 Bacterial cultures, chemicals and reagents:
te
99
M
98
an
us
cr
92
Bacterial strains used in this study are listed in Table 1. All the media used in this study
102
were purchased from Hi-Media labs (Mumbai, India). The S. aureus strains were grown in Brain
103
Heart Infusion broth (BHI) and plated on Baird Parker agar (BPA) supplemented with Egg yolk
104
tellurite emulsion. The plates were incubated at 37 OC for 24 hours. The isolates were identified
105
morphologically and biochemically by standard laboratory procedures (Murray et al. 2007) and
106
maintained in 15% glycerol stocks in -80 OC. Cloning host E. coli BL21 (DE3) pLysS was
107
grown and maintained in Luria Bertani broth/agar.
108
2.2 Construction of LukS-PV recombinant protein:
Ac ce p
101
109
The primers (Table 2) used in the construction of recombinant luk-S gene was designed
110
using the Generunner software. The gene sequences were retrieved from NCBI database. Each
5 Page 5 of 25
PCR reaction was performed with 1x pfu buffer with 2 mM MgSO4, 200 μM of dNTPs, 10
112
picomole each of forward and reverse primers, 50 ng of DNA template, one unit of pfu DNA
113
polymerase and the final volume was adjusted to 20 µl with nuclease free water. The PCR was
114
performed as follows: initial denaturation of 4 minutes at 94 OC followed by 30 cycles of
115
denaturation for 30 seconds at 94 OC, annealing at 57 OC for 30 seconds, extension of one minute
116
at 72 OC followed by a final extension of ten minutes at 72 OC. PCR products were analysed in
117
2% agarose with ethidium bromide. The PCR product was digested purified and digested with
118
BamHI and HindIII and inserted into pRSET A vector pre-digested with BamHI and HindIII.
119
The ligated product was transformed in to BL21(DE3)-pLysS E. coli host strains. The resultant
120
colonies were screened by colony PCR for the presence of insert by flanking T7 sequencing
121
primers. The plasmid was extracted from transformed strain and sequenced with T7 primers.
M
an
us
cr
ip t
111
The E. coli host cells harboring recombinant plasmids were grown overnight at 37 OC in
123
LB broth with 100 µg/ml ampicillin. One ml of this culture was further inoculated in to flasks
124
containing 25 ml LB broth containing 100 µg/ml ampicillin and grown at 37 OC with vigorous
125
shaking until the OD600 reached 0.7. One mM IPTG was added to the cells and allowed to grow
126
at 37 OC with vigorous shaking. One ml of cells were collected after 5 hrs of induction, lysed,
127
subjected to 12 % SDS-PAGE and stained with Coomassie brilliant blue to determine the
128
expression of the proteins. The proteins separated on SDS-PAGE was transferred on to a
129
nitrocellulose membrane by Western blot procedure, blocked with phosphate buffered saline
130
(PBS) containing 5% skim milk for 1 h followed by incubation with mouse monoclonal anti-
131
histidine antibodies. After washing with PBST (PBS + 0.05% Tween 20), the membrane was
132
incubated for 30 minutes with anti-mouse HRP labelled antibodies raised in goat. The membrane
133
was thoroughly washed with PBST and developed with 3,3`, 5, 5` -d Diaminobenzidine tetra
Ac ce p
te
d
122
6 Page 6 of 25
chloride and 0.004 % H2O2 in PBS. Later the reaction was stopped by washing the membrane
135
with distilled water. For bulk production, the culture was inoculated in 100 ml LB broth with
136
antibiotics. The recombinant protein was purified under denaturation conditions using urea as
137
denaturant by immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose
138
(Qiagen, Germany) as chelating resin and amino terminal hexa-histidine residues as affinity tag
139
according to manufacturer’s recommendations. The purity of the purified protein was analyzed
140
by 12% SDS-PAGE. The elutions were pooled and the protein concentration was quantified by
141
Lowry’s calorimetric assay using BSA as standard (Niveditha et al. 2013).
142
2.3
an
Primary and secondary antibody preparation:
us
cr
ip t
134
Four different groups of 8 female BALB/c mice aged between 6–8 weeks were
144
immunized intramuscularly in hind legs with 50 g of purified r-PVL-S proteins at an interval of
145
14 days (0, 14, 28 and 42 days). The first immunization was with Freund’s complete adjuvant
146
(FCA) and subsequent doses were provided with incomplete Freund’s adjuvant (IFA)
147
intramuscularly.
148
2.4
te
d
M
143
Ac ce p
Generation of monoclonal antibodies:
149
Approximately 108 SP 2/0 myeloma cells were fused with homogenized spleen cell
150
suspensions prepared from the spleens of immunized mice. Stable hybrid cell lines were selected
151
with Littlefield hypoxanthine-aminopterin thymidine (HAT) medium. The fused cells were
152
plated in 96 well tissue culture plates at several dilutions to ensure single clones per well. The
153
colonies were fed every fifth day with freshly prepared HAT medium. Approximately 2 weeks
154
after fusion, wells containing single and double macroscopic clones were tested for antibody
155
production and toxin specificity by indirect ELISA. Wells containing positive cells were cloned
156
by the limiting dilution method into 96-well tissue culture plates at least three times to ensure 7 Page 7 of 25
monoclonality. Of the 192 wells examined, only three clones gave strong positive signals in the
158
indirect ELISA with the highest affinity. Therefore, the supernatant of these three clones were
159
aspirated from the fusion well and subjected to limiting dilution for hybridoma selection. After
160
limiting dilution and ELISA screening, clones were used for the production of culture
161
supernatant for further large scale production of antibody.
162
2.5
cr
ip t
157
us
Isotyping of monoclonal antibodies:
Monoclonal antibodies obtained from immunized mice were used for detection of
164
specific antibody and antibody isotypes by enzyme-linked immunosorbent assay (ELISA) by
165
using a mouse MAb Isotyping kit Roche. Briefly, microtitre plates were coated with 100µl of
166
purified proteins (10 µg ml-1) in carbonate-bicarbonate buffer and the unbound sites were
167
blocked with skimmed milk powder (5 %) in PBS. Serially diluted test sera were added to the
168
wells and maintained at 37°C for 1 h, followed by similar incubation with Horse Radish
169
Peroxidase (HRPO)-conjugated goat anti-mouse IgG, IgM, IgG2a, IgG2b and IgG3 antibodies
170
(sigma aldrich India). Colour was developed with the substrate O-phenylenediamine
171
dihydrochloride (OPD) in presence of 0.4% H2O2 and absorbance was measured in ELISA plate
172
reader (Infinite M200 pro; Tecan, Grodig, Austria) at 470nm (Leary SE 1995; Uppada SB 2011).
173 174
2.6
Ac ce p
te
d
M
an
163
Dot immunobinding assay:
175
The crude protein preparations and purified recombinant proteins were coated on to
176
nitrocellulose membrane. The blots were blocked with phosphate buffered saline (PBS)
177
containing 5% skim milk along with different concentration of diethylpyrocarbonate (DEPC) for
178
1 h followed by incubation with 1:1000 dilution of anti-rPVL-S, mouse serum followed by 1 h
179
incubation with goat anti-mouse IgG-HRP conjugate (Sigma chemicals, USA). The membrane 8 Page 8 of 25
was thoroughly washed with PBST and chromogenic reaction was observed by incubating with
181
3,3`, 5, 5` -d Diaminobenzidine tetra chloride and 0.004 % H2O2 in PBS. Later the reaction was
182
stopped by washing the membrane with distilled water. For the assessment of sensitivity,
183
overnight culture of PVL positive S. aureus was serially diluted by using saline (0.9% of NaCl).
184
100 microlitre of sample drawn from each dilution and further processed for DNA extraction by
185
boiling lysis method. The DNA (1.0 μl) was used as template in PCR assay. 500 microlitre of
186
sample drawn from each dilution were pelleted in carbonate-bicarbonate buffer and Dot-ELISA,
187
was performed.
188
2.7
an
us
cr
ip t
180
Western blot for LukS-PV:
The crude protein preparations and purified recombinant proteins were resolved on 12%
190
polyacrylamide gel and were transferred on to nitrocellulose membrane (Towbin et al. 1979) and
191
blocked with phosphate buffered saline (PBS) containing 5% skim milk for 1 h. Incubation with
192
anti PVL-S MAbs followed by 1 h incubation with goat anti-mouse IgG-HRP conjugate (Sigma
193
chemicals, USA). Chromogenic reaction was observed by incubating with Diaminobenzidine
194
(DAB)-H2O2 substrate.
d
te
Ac ce p
195
M
189
196
RESULTS
197
3.1 Construction and Cloning of luk-S gene of PVL:
198
The individual gene luk-S was amplified from S. aureus genomic DNA (Fig.1a). After
199
cloning these genes into pRSETA vector, they were sequenced to determine if any mutations
200
have appeared. Positive clones were induced with 1mM IPTG for 5 hrs at 37 OC and the
201
expression of recombinant protein was detected in 12% SDS-PAGE gel stained with Coomassie
202
blue. The relative sizes of the proteins r-luk-S was in agreement with the predicted size i.e. 35 9 Page 9 of 25
203
kDa (Fig.1b). Subsequently, the expression or the proteins was confirmed by Western blot
204
analysis with anti-Histidine antibodies.
205
3.2 Purification of the recombinant protein: The recombinant proteins were purified from 100 ml induced culture by using
207
immobilized metal affinity chromatography on Ni-NTA agarose column (Qiagen). The cells
208
were induced for 5 h with 1 mM IPTG and they were collected and purified by urea denaturation
209
according to manufacturer’s protocol (Qiagen) (Fig.1b). The purified proteins were pooled and
210
dialysed into PBS + 10 mM Urea for 2 h at 4OC. After buffer exchange, the protein concentration
211
of luk-S protein was quantified to be 4.0 mg/ mL by Lowry’s calorimetric assay using bovine
212
serum albumin as a standard.
213
3.3 Monoclonal antibody to PVL-S toxin secreting clones:
M
an
us
cr
ip t
206
Stable hybrid cell lines were selected with Littlefield hypoxanthine-aminopterin thymidine
215
(HAT) medium. The fused cells were plated in 96 well tissue culture plates, from the 192 wells
216
examined, twenty hybridoma clones were obtained. From that only three clones, MAb1, MAb9
217
(B2, C1), MAb10 (C11, D12) gave strong positive signals in the indirect ELISA with the highest
218
affinity (Fig.2). Therefore, the supernatant of these three clones were aspirated from the fusion
219
well and subjected to limiting dilution for hybridoma selection. After limiting dilution and
220
ELISA screening, clones were used for the production of culture supernatant for further large
221
scale production of antibody.
222
Isotyping of monoclonal antibodies:
Ac ce p
te
d
214
223
To assess the specific antibody isotype in all three monoclonal antibodies, isotyping was
224
carried out by Indirect plate ELISA. MAb1 was found to be significantly higher in producing
225
IgG1 antibody isotype. MAb10 and MAb9 were biased towards the IgG2b and IgG3 isotype
10 Page 10 of 25
226
respectively, but they were statistically insignificant (p < 0.01) (Fig.3). Results were presented as
227
the means value ± standard error (SE) and the graph was created with the help of Graph Pad
228
Prism6 software. Statistical difference were analyzed and assumed with p value (p < 0.01).
230
ip t
229
3.4 Specificity:
The monoclonal antibodies raised against PVL-S protein were tested for their reactivity
232
by Indirect ELISA, Dot-ELISA and Western Blot analysis. MAb 1 reacted very strongly with
233
native toxin, which was shown by Indirect ELISA, Dot-ELISA and Western Blot analysis. The
234
MAb1 did not exhibit any cross-reactions with other organisms tested when compared with other
235
two monoclonal antibodies (Table 3).
236
3.6 Sensitivity:
M
an
us
cr
231
Sensitivity was determined by the comparative analysis between PCR and Dot-ELISA
238
assays through serial dilution method. PCR could detect upto 109 to 101 CFU/ml and Dot-ELISA
239
is also sensitive upto detect low cell concentration of 101 with all 3 monoclonal antibodies.
240
Hence the newly standardised Dot-ELISA based method is giving 100% correlation with PCR
241
(supplementary data). Reproducibility also checked with repeated testing of a newly developed
242
Dot-ELISA assay.
243
3.7 Simple Dot- ELISA assay with DEPC treatment:
Ac ce p
te
d
237
244
The MAb1 raised against recombinant luk-S protein was tested for their reactivity by
245
simple Dot ELISA assay by the addition of 10mM concentration of DEPC in blocking step has
246
reduced the interference of protein A in assay when compared with different concentrations like
247
1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM and15.0 mM. Assay showed that MAb1 reacted strongly
248
with native toxin of PVL positive Staphylococcus aureus strains which were compared with PVL
11 Page 11 of 25
249
negative Staphylococcus aureus strains (Fig.4). This assay correlates with the PCR method of
250
identification of 87 S. aureus isolates.
251
DISCUSSION A strong association of Panton-Valentine Leukocidin both in CA-MRSA related
253
outbreaks as well as CA-MRSA involved in chronic diseases has implications both for
254
epidemiological and clinical studies. Until recently, the CA-MRSA strains were susceptible to
255
many antibiotics other than the beta-lactams; however, resistance seems to be increasing and
256
multiple antibiotic resistant strains have started to emerge. Rapid identification of PVL in
257
methicillin-resistance S. aureus isolates causing severe infections is necessary in order to prevent
258
their potentially devastating consequences. PVL, a bi-component exotoxin consists of two
259
separately secreted and non-associated proteins (class S and class F components). Both the
260
components
261
permeabilization of target cells and leading to their lysis (König et al. 1995; Siqueira et al. 1997).
262
Employing a specific antibody and targeting the PVL molecule through a simple
263
immunoassay system would be a workable strategy for the quick identification of CA-MRSA
264
strains. Monoclonal antibodies overall have been utilized in large number of immunoassays for
265
the detection of toxins and pathogens.
266
generated against PVL-S fraction of S. aureus by using a recombinant PVL-S protein as the
267
immunogen. The spleen from a single immunized BALB/c mouse resulted in 19 antibody
268
producing hybrids which after cloning by limiting dilution, yielded 3 stable clones. MAb 1
269
belonged to IgG1 class and MAb 10 was IgG2b in nature. It implies that produced monoclonal
270
antibodies were directed towards Th2 and Th1 mediated antibody response against PVL-S toxin.
271
All the three MAbs generated were tested for specificity by dot ELISA and by Western blot
sequentially
and
synergistically,
inducing
the
activation
and
Ac ce p
te
d
interact
M
an
us
cr
ip t
252
In the present study, monoclonal antibodies were
12 Page 12 of 25
analysis. No reaction was seen against E. coli, S. flexneri, S. boydii, Klebsiella, S. typhi, S.
273
typhimurium, Proteus sp, Citrobacter, B. cereus, A. hydrophila, Y. enterocolitica and other
274
relevant pathogenic bacteria. All the three MAbs generated were also tested for sensitivity by
275
comparing with PCR and Dot-ELISA. However, non PVL producing S. aureus strains showed
276
false positive signal due to the presence of SpA. Different methods have been tried by several
277
workers to prevent SpA interference and there are addition of porcine IgG coupled to insoluble
278
matrix has been used to remove protein A from culture supernatants (Berdal et al. 1981; Fey and
279
Burkhard 1981). Revealing rabbit antibodies have been biotinylated so that the binding site of
280
Spa on the IgG molecule is masked (Hahn et al. 1986). Using IgG antibodies raised in rats and
281
sheep as a capturing antibody may contribute towards improved positive signal because of their
282
weak binding to SpA (Rogemond et al. 1991; Freed et al. 1982). In addition, chicken anti-protein
283
A Ig Y has been used to sequester protein A, since chicken IgY does not bind protein A in the Fc
284
region (Hoffman et al. 1996; Reddy et al. 2012). DEPC has also been suggested to inhibit non-
285
specific binding of Spa to IgG (Haake et al. 1982; Hein et al. 2010). Hence an attempt was made
286
to standardize a dot ELISA method by the incorporation of DEPC at concentrations ranging from
287
1 mM to 15 mM. Incorporation of 10 mM DEPC along with 5% milk in PBS in the blocking
288
step completely prevented non-specific binding of Spa to mouse anti-PVL monoclonal
289
antibodies in simple dot- ELISA. Once standardized, this simple dot ELISA was evaluated with
290
nine PVL positive and large numbers of PVL negative strains of S. aureus. False signals were
291
not observed in the DEPC treated dot ELISA. Similar results were also observed in Western blot
292
analysis (Table 3). The results are obtained within 3 hours. The isolates which were identified as
293
PVL producing strains by this newly described dot ELISA method were unequivocally detected
Ac ce p
te
d
M
an
us
cr
ip t
272
13 Page 13 of 25
294
positive by the in-house developed PCR format. Newly developed detection system is sensitive
295
upto to detect as low as 101cells. The importance of PVL detection from community-acquired MRSA has been gaining
297
attention. A method for amplifying and detecting the PVL gene by the PCR method, which is
298
currently used as a PVL detection method, requires expensive laboratory infrastructure and well-
299
experienced technicians and time-consuming. Moreover, at times the presence of genes might
300
not correlate with the expression of toxin. Specific and highly sensitive detection of the toxin
301
protein is difficult with the use of conventional techniques and also limited to specialized
302
laboratories with equipment and experience to perform such assays (Kalyan et al. 2008). To
303
facilitate the rapid detection of PVL toxin by routine clinical microbiology laboratories, the
304
newly described monoclonal antibody based dot-ELISA method reported here, can be of
305
immense help in providing, rapid and specific detection of the PVL toxin containing MRSA
306
strains at a relatively low cost which are added advantages over existing methods. This kit would
307
serve as a valuable tool for the reliable identification of PVL producing CA-MRSA strains from
308
clinical, food and environmental sources to arrest their dissemination and further expansion.
310
cr
us
an
M
d
te
Ac ce p
309
ip t
296
Acknowledgments:
311
Authors are thankful to the Director, DFRL for providing necessary facilities to conduct the
312
study. Niveditha. S is a Senior Research fellow funded by the Indian Council of Medical
313
Research (ICMR), India.
314 315
References:
14 Page 14 of 25
316
Berdal BP, Olsvik O, Omland T. (1981). A sandwich ELISA method for detection of
317
Staphylococcus aureus enterotoxins. Acta Pathol Microbiol Scand B89, 411–415.
318
Carrol and Karen C. (2008). Rapid Diagnostics for MRSA: current status, Molecular diagnosis
322
(1999). Four pediatric deaths from community-acquired methicillin resistant
cr
321
CDC.
Staphylococcus aureus-Minnesota and North Dakota. JAMA 282, 1123–1125. Ching Wen Tseng, Pierre Kyme,Jennifer Low,Miguel A. Rocha,Randa Alsabeh,Loren G. Miller,Michael
Arditi,Binh
An
Diep,
Victor
Nizet,Terence
M.
324
Doherty,David O. Beenhouwer, George Y. Liu. (2009). Staphylococcus aureus Panton
325
Valentine Lecocidin contributes to inflammation and Muscle Tissue injury, PLoS
326
one4(7), e6387.
M
an
323
Fey H, Burkhard G. (1981). Measurement of staphylococcal protein A and detection of protein
328
A-carrying
strains
329
ofImmnological Methods47, 99–107.
by
a
competitive
ELISA
method. Journal
te
staphylococcus
d
327
Otto,Moshe
us
320
ip t
and Therapy, Infectious diseases. 12, 1, 15-24.
319
Francis JS, Doherry MC, Johnston C, Sinha G, Ross T, Cai M S. (2005). Severe Community-
331
onset pneumonia in healthy adults caused by Methicillin resistant Staphylococcus aureus
332
carrying the Panton Valentine Leukocidin genes. Clin Infect Dis40, 100-7.
333 334
335 336
Ac ce p
330
Goding JW.(1978). Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 20, 241–53.
Haake DA, Franklin EC, Frangione B. (1982). The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate. J Immunol 129,190–2.
15 Page 15 of 25
337
Hahn IF, Pickenhahn P, Lenz W, Brandis H. (1986). An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. J Immunol Methods92, 25–9.
338
Hien M. Nguyen1, Migue A. Rocha, Koteswara R. Chintalacharuvu, and David O. Beenhouwer.
340
(2010). Detection and quantification of Panton-Valentine leukocidin in Staphylococcus
341
aureus cultures by ELISA and Western blotting: diethylpyrocarbonate inhibits binding of
342
protein A to IgG, J Immunol Methods 356(1-2), 1–5.
cr
ip t
339
Hoffman WL, Ruggles AO, Tabarya D. (1996). Chicken anti-protein A prevents Staphylococcus
344
aureus protein A from binding to human and rabbit IgG in immunoassays and eliminates
345
most false positive results. J Immunol Methods198, 67–77.
an
us
343
Jayasinghe, L and H. Bayley. (2005). The leukocidin pore: evidence for an octamer with four
347
LukF subunits and four LukS subunits alternating around a central axis. Protein Sci.14,
348
2550–2561.
d
M
346
Kalyan T.D,Balakrishna K, Murali H.S and Batra H.V. (2008). Construction of a recombinant
350
intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL
351
of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of
352
Staphylococcus aureus. 2008. Journal of Medical Microbiology 58, 577-583. Konig
Ac ce p
353
te
349
B,PrevostG,
PiemontY,
and
KonigW.(1995).
Effects
of
Staphylococcus
354
aureusleukocidins on inflammatory mediator release from human granulocytes. Journal
355
of Infectious Diseases171, 607–613.
356
Leary SE, Williamson ED, Griffin KF, Russell P, Eley SM, Titball RW. (1995). Active
357
immunization with recombinant V antigen from Yersinia pestis protects mice against
358
plague. Infect Immun63, 2854-2858.
16 Page 16 of 25
359
Namita D'Souza, Camilla Rodrigues and Ajita Mehta (2010). Molecular Characterization of
360
Methicillin-Resistant Staphylococcus aureus with Emergence of Epidemic Clones of
361
Sequence Type (ST) 22 and ST 772 in Mumbai, India. J. Clin. Microbiol 48(5), 1806-09. Niveditha Sundar Poojary*, Shylaja Ramlal, Murali Harishchandra Sripathy, Harsh Vardhan
363
Batra (2013). Application of Recombinant LUK-SF Protein of Panton-Valentine Toxin
364
of Community Acquired Methicillin Resistant Staphylococcus aureus for Toxin
365
Detection and Neutralization Properties. International Journal of Advanced Research 1,
366
1(10), 129-138.
us
cr
ip t
362
Reddy PK, Aravind S, Joseph Jeyabalaji Kingston, Murali HS, HV Batra (2013).Evaluation of
368
IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus
369
aureus without staphylococcal protein A interference. J Immunol Methods391,31–38.
M
an
367
Rogemond V, Da Costa Castro JM, Delaunay T, Cazaux C, Guinet RM. (1991). Protein A
371
insensitive ELISA detection of staphylococcal enterotoxin B. Lett Appl Microbiol13,
372
175–8.
te
d
370
Samia Perwaiz, Qamaruddin Barakzi, BadarJehan Farooqi, Nazia Khursheed, Nasim Sabir
374
(2007). Antimicrobial susceptibility pattern of clinical isolates of Methicillin Resistant
375
Staphylococcus aureus, J Pak Med Assoc57(1), 1-4.
Ac ce p
373
376
Siqueira J. A., Speeg-schatz c., Freitas f. I. S., Sahel j., Monteil'f H. and G. Prevost. (1997).
377
Channel-forming leucotoxins from Staphylococcus aureus cause severe inflammatory
378
reactions in a rabbit eye model. J. Med. Microbiol 46, 486-494.
379
Towbin H, Staehelin T, Gordon J. (1979). Electrophoretic transfer of proteins from
380
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl
381
Acad Sci U S A76(9), 4350-4.
17 Page 17 of 25
Uppada SB, Bhat AA, SahA, Donthamshetty RN. (2011). Enhanced humoral and mucosal
383
immune responses after intranasal immunization with chimeric multiple antigen peptide
384
of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice. Vaccine
385
29,9352-9360.
ip t
382
Yi-Wei Tang, Abdullah Kilic, Qunying Yang, Sigrid K. McAllister, Haijing Li, Rebecca S.
387
Miller, Melinda McCormac, Karen D. Tracy, Charles W. Stratton, Jian Han, and Brandi
388
Limbago. (2007). StaphPlex System for Rapid and Simultaneous Identification of
389
Antibiotic Resistance Determinants and Panton-Valentine Leukocidin Detection of
390
Staphylococci from Positive Blood Cultures, J Clin Microbiol 45(6), 1867–1873.
an
us
cr
386
Figure Legends:
392
Fig. 1
393
A. Agarose gel showing amplification of luk-S gene: Lane 1, PCR amplification of luk-S gene
394
(798 bp); Lane 2, 1 kb DNA ladder
395
B. Coomassie blue stained SDS-PAGE gel showing expression and purification of r-luk-S
396
protein by using immobilized metal affinity chromatography: Lane1, Prestained protein
397
ladder; lane 2, Induced E. coli BL21 (DE3) carrying pRSET A-luk-S vector (35kDa); lane 3,
398
Unstained protein ladder; last lanes 4, 5 & 6 are the elutions of Purified r-luk-S.
d
te
Ac ce p
399
M
391
400
Fig. 2 Plate ELISA showing reactivity of three Monoclonal antibody (MAb1, MAb9,
401
MAb10) against r-luk-S protein (10 µg ml-1) in duplicate wells: Lane A1, B1, C1, E1,
402
Negative control (PBS); Lane D1, positive control (PoAb as primary antibody); Lane A2 & A3,
18 Page 18 of 25
403
MAb 9; Lane B2 & B3, MAb 9; Lane C2 & C3, MAb 10; Lane D2 & D3, MAb 10; Lane E2 &
404
E3, MAb 1.
405
Fig. 3 Isotyping of Monoclonal antibodies:
407
Antibody isotypes specific to r-luk-S was evaluated by ELISA using isotype-specific monoclonal
408
antibodies for IgG1, IgG2a, IgG2b, IgG3 and IgM. The level of isotypes was expressed as
409
absorbance (A470 nm) of the colored complex developed in the Immunosorbent assay. Data are
410
represented as the mean value ± standard error (SE) and graph was created with the help of
411
Graph Pad Prism6 software. A statistical difference was assumed with p value (p < 0.01).
an
us
cr
ip t
406
412
Fig. 4 Dot ELISA showing reactivity of MAb1 against PVL positive and PVL negative S.
414
aureus strains
415
Lane A1-A6 are PVL positive and Lane B1-B6 are PVL negative S. aureus strains.
416
Lane A1, S. aureus E2147; Lane A2, S. aureus FRI 722; Lane A3, S. aureus E1978; Lane A4, S.
417
aureus NCIM 2792; Lane A5, ATCC 43300; Lane A6: S. aureus NCIM 2127; Lane B1, S.
418
aureus ATCC 700699; Lane B2, S. aureus NCIM 2901; Lane B3, S. aureus NCIM 2121; Lane
419
B4, S. aureus E2533; Lane B5, S. aureus E2279; Lane B6, S. aureus FI 4.
Ac ce p
te
d
M
413
19 Page 19 of 25
Table
Table 1: Bacterial strains used in the study: Strains
Sources
Escherichia coli
pLysS
Invitrogen, Bangalore, Karnataka, India
cr
Staphylococcus aureus
ip t
Escherichia coli BL21 (DE3)
ATCC: American Type Culture Collection, Manassas, Va
FRI 722
Food Research Institute, India
ATCC 43300
ATCC: American Type Culture Collection, Manassas, Va
ATCC 29213
ATCC: American Type Culture Collection, Manassas, Va
ATCC 6538
ATCC: American Type Culture Collection, Manassas, Va
NCIM 2079
National Collection of Industrial Microorganisms, Pune, India
NCIM 2901
National Collection of Industrial Microorganisms, Pune, India
NCIM 2654
National Collection of Industrial Microorganisms, Pune, India
NCIM 2672
National Collection of Industrial Microorganisms, Pune, India
NCIM 2792
National Collection of Industrial Microorganisms, Pune, India
NCIM 2127
National Collection of Industrial Microorganisms, Pune, India
Ac ce p
te
d
M
an
us
ATCC 700699
NCIM 2124
National Collection of Industrial Microorganisms, Pune, India
NCIM 2121
National Collection of Industrial Microorganisms, Pune, India
NCIM 2122
National Collection of Industrial Microorganisms, Pune, India
RAB (DRDE)
Defence Research and Development Establishment, India
IVRI
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India
E 2147
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E2533
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E2279
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1345
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1357
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Page 20 of 25
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1975
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1977
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
E1978
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
FI 1
Food isolate from local market Mysore, Karnataka; India
FI 2
Food isolate, this work
FI 3
Food isolate, this work
FI 4
Food isolate, this work
FI 5
Food isolate, this work
FI 6
Food isolate, this work
FI 7
Food isolate, this work
CI 1
Clinical isolate from pus, this work
CI 2
Clinical isolate from wound infection, this work
CI 3
Clinical isolate from abscess, this work
CI 4
Clinical isolate from pus, this work
CI 5
Clinical isolate from wound infection, this work
CI 6
Clinical isolate from pus sample, this work
Ac ce p
Other Bacterial Strains
te
d
M
an
us
cr
ip t
E1988
Vibrio vulnificus ATCC 27562
ATCC: American Type Culture Collection, Manassas, Va
Shigella flexneri
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Salmonella typhimurium
SDM College of Medical Sciences and Hospital isolates, Dharwad, Karnataka; India
Proteus vulgarisATCC 6380
ATCC: American Type Culture Collection, Manassas, Va
Aeromonas hydrophila ATCC
ATCC: American Type Culture Collection, Manassas, Va
7966 Bacillus cereusATCC 14579
ATCC: American Type Culture Collection, Manassas, Va
Listeria monocytogenes
ATCC: American Type Culture Collection, Manassas, Va
Page 21 of 25
ATCC 13932 Klebsiella pneumonia
ATCC: American Type Culture Collection, Manassas, Va
Table 2: Primers used for the cloning of luk-S gene: Sequence
Accession number
luk-S For
5’ CGCggatccATCACTCCTATTGCTACTTCG 3’
AB678714.1
luk-S Rev
5’ CCGggtaccGCCATAGTGTGTTGTTCTTCT 3’
Amplicon (bp)
cr
Primer
ip t
ATCC BAA2146
size
us
798 bp
an
The sequence corresponding to restriction enzymes were given in lowercase.
M
Table 3: Specificity of MAb1 compared with Western Blot, Plate ELISA and Dot ELISA: Strains
Ac ce p
te
d
Staphylococcus aureus ATCC 700699 Staphylococcus aureus Japan TSST Staphylococcus aureus FRI 722 Staphylococcus aureus ATCC 43300 Staphylococcus aureus ATCC 29213 Staphylococcus aureus ATCC 6538 Staphylococcus aureus NCIM 2079 Staphylococcus aureus NCIM 2901 Staphylococcus aureus NCIM 2654 Staphylococcus aureus NCIM 2672 Staphylococcus aureus NCIM 2792 Staphylococcus aureus NCIM 2127 Staphylococcus aureus NCIM 2124 Staphylococcus aureus NCIM 2121 Staphylococcus aureus NCIM 2122 Staphylococcus aureus RAB (DRDE) Staphylococcus aureus IVRI Staphylococcus aureus E 2147 Staphylococcus aureus E2533 Staphylococcus aureus E2279 Staphylococcus aureus E1345 Staphylococcus aureus E1357 Staphylococcus aureus E1988 Staphylococcus aureus E1975 Staphylococcus aureus E1977 Staphylococcus aureus E1978 Staphylococcus aureus FI 1
Western Blot
Plate ELISA
Dot ELISA
+ + + + + + + + + + + -
+ + + + + + + + + + + -
+ + + + + + + + + + + -
Page 22 of 25
ip t
+ + + + + + -
cr
us
+ + + + + + -
an
+ + + + + + -
Ac ce p
te
d
M
Staphylococcus aureus FI 2 Staphylococcus aureus FI 3 Staphylococcus aureus FI 4 Staphylococcus aureus FI 5 Staphylococcus aureus FI 6 Staphylococcus aureus FI 7 Staphylococcus aureus CI 1 Staphylococcus aureus CI 2 Staphylococcus aureus CI 3 Staphylococcus aureus CI 4 Staphylococcus aureus CI 5 Staphylococcus aureus CI 6 Escherichia coli Vibrio vulnificus Shigella spp Salmonella typhii Proteus vulgaris Aeromonas spp Bacillus cereus Listeria spp Klebsiella spp
Page 23 of 25
Figure
an
us
cr
ip t
Fig 1:
Ac ce p
te
d
M
Fig 2:
Page 24 of 25
Ac ce p
Fig 4:
te
d
M
an
us
cr
ip t
Fig 3:
Page 25 of 25