Download Detection of Bacterial Flora in Biological Secretions

Department of Physics, Chemistry and Biology
Final Thesis
Detection of Bacterial Flora in Biological Secretions
Using Antibodies Developed In Vitro and Immobilized in a
Surface Plasmon Resonance System
Nakka Sravya Sowdamini
LiTH-IFM- Ex--2443--11
Supervisor: Fariba Nayeri, IKE.
Examiner: Mathiaas Laska, Linkoping University.
Department of Physics, Chemistry and Biology
Linköpings universitet
SE-581 83 Linköping, Sweden
Datum
Avdelning, Institution
Division, Department
Avdelningen för biologi
Instutitionen för fysik och mätteknik
Språk
Rapporttyp
Language
Report category
Date
3rd June 2011
ISBN
LITH-IFM-A-EX--—11/2443—SE
Svenska/Swedish
x Engelska/English
________________
Licentiatavhandling
x Examensarbete
C-uppsats
x D-uppsats
Övrig rapport
_______________
_________________________________________________
ISRN
__________________________________________________
Serietitel och serienummer
ISSN
Title of series, numbering
Handledare
Supervisor: Dr. Fariba Nayeri
URL för elektronisk version
Ort
Location: Linköping
Tite
Title:
Detection of bacterial flora in biological secretions using antibodies developed in vitro and
immobilized in a surface Plasmon resonance system
Författare
Author:
Nakka Sravya Sowdamini
Sammanfattning
Abstract:
Identification of pathogens living in biofilms of chronic infections has been difficult with PCR,
serological, biochemical and culture techniques. The study aims at the detection of bacterial
pathogens in biofilms of biological secretions using SPR analysis Biacore. The antibodies were
developed by isolating mononuclear lymphocytes from the blood of the patients who sustained
systemic infection. The isolated lymphocytes had antibody secreting B cells (plasma cells) which
were identified using flow cytometry analysis. The antibodies produced (n=4) were used to
immobilize CM5 chip of Biacore to detect the bacteria in ulcer secretions with wound secretions
of healthy volunteers as controls. The results from Surface Plasmon Resonance (SPR) analysis
and culture technique were compared and statistically there was no significant difference
obtained. The results from present study suggest that SPR analysis could be used as an
alternative system for detection of bacteria in poly-microbial samples and detect the organisms
that might not be discovered by culture or PCR method.
Nyckelord
Keywords:
Biofilms, antibodies, lymphocytes, B cells, flow cytometry, immobilize, SPR analysis, Biacore,
CM5 chip
Table of Contents
1. Abstract
1
2. Introduction
1
3. List of Abbrevations
1
4. Materials and Methods
3
4.1 Patients
4.2 Production of antibodies against bacteria
4.3 SDS-PAGE
4.4 Flow cytometry
4.5 Culturing bacteria
4.6 Biacore immobilization
4.6.1 Testing specificity of chip
4.6.2 Running Samples and detection of bacteria
4.6.3 Statistical Analysis
5. Results
5.1 Culturing Cells
5.2 Flow Cytometry Analysis
5.3 SDS PAGE
5.4 Biacore
5.4.1 Immobilization of chip
5.4.2 Specificity test of the chip
5.4.3 Sample run
3
4
4
4
5
5
5
6
6
6
6
7
8
9
9
10
10
6. Discussion
12
7. Conclusion
13
8. Future perspectives
13
9. Acknowledgements
14
10. References
14
1. Abstract
Identification of pathogens living in biofilms of chronic infections has been difficult with PCR,
serological, biochemical and culture techniques. The study aims at the detection of bacterial
pathogens in biofilms of biological secretions using SPR analysis Biacore. The antibodies were
developed by isolating mononuclear lymphocytes from the blood of the patients who sustained
systemic infection. The isolated lymphocytes had antibody secreting B cells (plasma cells) which
were identified using flow cytometry analysis. The antibodies produced (n=4) were used to
immobilize CM5 chip of Biacore to detect the bacteria in ulcer secretions with wound secretions of
healthy volunteers as controls. The results from Surface Plasmon Resonance (SPR) analysis and
culture technique were compared and statistically there was no significant difference obtained. The
results from present study suggest that SPR analysis could be used as an alternative system for
detection of bacteria in poly-microbial samples and detect the organisms that might not be
discovered by culture or PCR method.
Key words: Biofilms, antibodies, lymphocytes, B cells, flow cytometry, immobilize, SPR analysis,
Biacore, CM5 chip
2. List of Abbrevations
CM5- Carboxymethylated sensor chip
SPR- Surface Plasmon Resonance
PCR- Polymerase Chain Reaction
EDTA- Ethylenediamine tetra acetic acid
SDS-PAGE- Sodium dodecyl sulfate polyacrylamide gel electrophoresis
TBE- Tris Borate EDTA buffer
NHS- N-hydroxysuccinimide
EDC- N-ethyl-N‘-(dimethylaminopropyl) carbodiimide hydrochloride
HBS- HEPES buffered Saline
PBS- Phosphated buffered saline
L-15- Leibovitz-15 medium
CD45/CD3/CD19- Cluster of differentiation protein
IgG- Immunoglobulin G
3. Introduction
Inflammation is the response of a tissue to stimuli like irritation, damage or infection caused by
pathogens (Roitt et al., Immunology edition 6). Inflammation can be classified into acute and
chronic depending upon the severity of infection. Acute inflammation is an immediate response to
external stimuli and is characterized by redness, swelling, pain, increased blood flow, migration of
leukocytes and inflammatory mediators like cytokines to the site of injury or infection (Feghali and
Wright, 1997). The host immune system responds immediately to the external stimuli and a cascade
of events are triggered to initiate healing process by removing the cause of infection or damage to
1
the tissue. Chronic inflammation takes its form when the healing process is non- effective due to the
inability of host immune system to completely eliminate the cause of the inflammation such as
infection (Sibbald et al., 2003). Many studies have shown that the presence of bacteria and microbial
toxins in wound beds might be the reason for intensity of a non-healing wound (Bowler et al., 1999,
Mooseley et al., 2004). The healing process could be restored by proper administration of antibiotics
to eliminate the cause for pathogenesis at the site of infection during acute cases (Trengove et al.,
1999). If the infection persists long due to repeated invasion of pathogens then an acute phase could
take a chronic form.
Chronic form of ulcers is due to poly-microbial infections where the pathogens thrive in a symbiotic
relationship enabling suitable growth conditions to survive. When infectious agents build up
accommodations using body's own components to slip the host immune defense mechanisms, then
using antibiotics with a high dosage might not eliminate the infection. In other words a biofilm is
developed which is extremely difficult to investigate or to treat (Fux et al., 2003; Bjarnsholt et al.,
2005; Alhede et al., 2009; Van Gennip et al., 2009). It has been suggested that co-existence of
different bacteria might cause resistance to antibiotics that otherwise would be effective on bacteria
in their planktonic form. (Hill et al., 2010).
It has been years of research on setting platforms for diagnosis of pathogens invading host immune
system and causing dreadful diseases. Most of the disease diagnosis is based on isolation of colonies
on culture plates, biochemical tests and immunological assays. PCR-based methods have been
standardized for disease analysis and have been providing reliable results until recently but PCR is
time consuming and costly. When the bacteria have been associated strongly with each other, it
becomes very difficult to identify the bacteria and moreover, bacterial bio-films produce culturenegative results (Burmoll et al., 2010). At present, there are many serological tests that could
distinguish symptomatic and asymptomatic infections but are yet to be standardized as a diagnostic
tool (Reithinger et al., 2003).
The sensor chip used in Biacore is coated with a gold film adhered to a surface matrix of
carboxymethyl Dextran. The ligand (antibody) is immobilized and held firmly to the dextran layer
on the gold surface. When there is an interaction between ligand with it’s specific antigen then an
optical signal is measured as a result of change in mass and thus there is a change in refractive index.
This results in change in angular position from I to II (Figure 1) due to altered resonance state
(Leonard et al., 2003). The interaction could be measured in response units (RU) enabled by Biaevaluation software. Biacore is used in many studies for detecting pathogens. Some of the research
includes detection of S. enteritidis and E. coli (Waswa et al., 2006), and detection of serum
antibodies using Salmonella (Gortemaker et al., 2002).
2
Figure 1: Diagramatic representation of the Kretschmann prism arrangement used in Biacore
instrumentation (Kretschmann E 1971)
We intended primarily to investigate bacterial flora in biological excretions by immobilization of
specific bacterial antibodies on Biacore chip. These antibodies are not available commercially and
the traditional method of producing bacterial antibodies by injecting bacteria to the rabbits (coligan
et al., 1992) and collecting the serum did not yield proper results in our study. In fact the animals
produced antibodies against their own gastro-intestinal flora. Thus the serum contained antibodies
against several bacteria. In order to obtain pure antibodies we established a method using human Bcells separated from blood of patients that recently survived a bacterial infection and incubated the
same bacterial strain found in cultures taken from patient to the washed B-cells. The medium was
centrifuged in filtered tubes and the supernatant immobilized on chip in Biacore.
In the present study the results from analysis of ulcer secretion from healthy and patients with
chronic ulcer in Biacore is presented. The study aims to develop this method to establish an
alternative system for detection of bacteria in poly microbial samples and detect the organisms that
might not be discovered by culture or PCR method.
4. Materials and Methods
4.1 Patients
Ulcer secretions from eleven patients (44-89 years of age, median 76 years old) were included in the
study. The patients suffered from chronic leg ulcers (stable for at least six months). The aetiologies
of the ulcers were venous insufficiency or a combined venous and mild arterial insufficiency
determined by clinical judgement and physiological measurements (toe and ankle pressure
measurements). Patients with type 1 diabetes mellitus, serious arterial insufficiency, HIV, cancer or
who were on warfarin, heparin or low molecular heparin were excluded. Culture from the ulcers
revealed growth of bacteria (Staphylococcus aureus, Escherichia coli, Enterabacter
3
cloacae, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus
morganii). None of the patients suffered from liver or kidney disease.
mirabilis and Morganella
Ulcer secretions were also collected from twelve patients who had been operated within 2 weeks for
breast cancer without any signs of metastasis (all women, 31-85 years of age, median 64 years).
Cultures revealed growth of Staphylococcus aureus in two cases.
For controls, ulcer secretions were collected within 24 hours after the skin biopsies from the left arm
of healthy volunteers (10 women 40-60 years of age, median 54 years old). Ulcer secretions were
also collected by absorption using 1 cm2 of absorbent material (Mepilex, Mölnlycke Health Care
AB, Göteborg, Sweden) placed under the dressing and then transferred into a flask (scintillation vial
20 ml, Sarstedt AB, Landskrona, Sweden) containing 5 ml physiological sodium chloride. This was
then mixed in a Vortex (Vortex-Genie, Scientific Industries Inc., Bohemia, NY 11716, USA). The
suspension was centrifuged ( 3000 G for ten minutes) and the supernatant was transferred into tubes
(Nunc Cryo Tube, Nunc Brand Products, Denmark) and stored at -70 °C before the analysis. The
cultures from the ulcer secretions were negative. The study was approved by the local ethical
committee at Linköping University Hospital and all participants gave written consent.
4.2 Production of antibodies against bacteria
Blood from patients with systemic bacterial infection was obtained in EDTA vials from the
Department of infectious diseases, Linkoping University, Sweden. 5.0 ml of anti-coagulated blood (
EDTA anticoagulant) was carefully layered over the Polymorphprep solution in a 12 ml centrifuge
tube. Care was taken not to mix the layers and was centrifuged at 400g for 40 minutes (Whiteside
and Rowlands, 1977). Two leukocyte bands were obtained between the separating solution and
plasma. The top band containing mononuclear lymphocytes were carefully pipetted out without
mixing the layers (Ferante and Thong, 1980). Lymphocytes were cultured in Leibovitz L-15 medium
containing 10% FBS (Marsden et al., 1995). After 24 hours of incubation (37 ̊C), the same bacteria
as found in blood cultures previously was heat killed at 80 ̊C for 20 minutes and was added to the
medium. After 1-2 weeks of incubation, culture medium was centrifuged at 400g for 10 minutes to
settle down the cells and then was filtered using 0.45μm sterile filters. The filtered medium was
centrifuged again at 6000 rpm for 60 minutes in 100 KDa Amicon Microcon centrifugal filter
devices. Antibodies were left behind on the filter leaving the cell debris in the filtrate. Antibodies
collected from the filter were preserved by adding 5µl physiological Sodium Chloride solution.
4.3 SDS-PAGE
SDS-PAGE was performed with MINI-PROTEAN precast gels to determine the molecular weight
of antibodies as suggested by Ehle H and Horn A (1990). The antibody samples were diluted with
PBS (pH 7.4, Apotektet AB) and equal volume of loading dye was added. The samples were diluted
in a ratio of 1:1 with laemmli sample buffer and heated in the water bath at 98 ̊ C for 5 minutes to
denature proteins before loading into the wells. A 200 KDa Precision Plus Protein Standard Marker
(BIO-RAD, Sweden) was used as reference. 20μl of samples and marker were loaded in each well
and the gel was set to run for about an hour at 200V.
4.4 Flow cytometry
Blood cell components were identified using flow cytometry analysis. Blood from patients who
sustained systemic infection was obtained in EDTA vials and Ficoll density gradient centrifugation
4
was performed to isolate the Peripheral blood mononuclear cells (Ferante and thong, 1980). Cell
counting was done using Cell counter (Alfa SWELAB) to obtain the quantity and quality of
lymphocytes present in samples. 20 μl of a cocktail of cell surface markers (CD3, CD16+56, CD45,
CD19) tagged with flurochromes (FITC, PE, PerCP, APC) was added to 100μl of sample containing
lymphocytes and was incubated for 15 minutes in the dark. 450 μl of FACs lysing solution was
added in order to lyse the red blood cells and then was incubated in room temperature for 15 minutes
in dark. Cells in the mixture were sorted and identified in BD FACSCanto™ II. The fluorescence
was plotted on a log scale.
4.5 Culturing bacteria
Culture results for all the samples used in the study were available from the Department of
Infectious Diseases, Health University, Linkoping. Further, the bacteria used in the study were
cultured in Mueller-Hinton agar, Blood agar and Haeme agar plates before and after heat killing
(Barbera et al., 2000).
4.6 Biacore immobilization
Biacore 1000 SPR biosensor instrument (GE Healthcare Ltd, Peas institute, Sweden) was used to
detect antigen-antibody interaction. The antibodies prepared were immobilized onto the flow cells of
CM5 sensor chip using the amine coupling method (O’ Shannessy et al., 1992). The sensor surface
of the chip coated with carboxymethylated dextran layer was activated using N-ethyl-N‘(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)
mixture. The NHS esters produced upon activation of chip react with the ligands that contain
primary amino groups. The unreacted NHS esters were deactivated with Ethanolamine. Each
channel of the chip was immobilized with Antibodies (20µl) diluted with Acetate 4.5 (Waswa et al.,
2006).
Table1: Dilution speacification for each injection during immobilization of CM5 chip.
S.no
Content
Dilutions
Injection volume
Time
injection
1.
NHS+EDC
100 μl+100 μl
35 μl
7 minutes
2.
Antibodies
20μl in 80μl PBS 35 μl
Acetate 4.5
7 minutes
3.
Ethanolamine
70 μl
7 minutes
35 μl
of
4.6.1 Testing specificity of Antibodies
The chip was tested for the specificity of antibodies immobilized on it before running the samples.
Antibodies produced in vitro against Staphylococcus aureus, Staphylococcus epidermidis,
Pseudomonas aeruginosa and Enterococcus fecalis, were immobilized individually to one channel
at a time in Biacore 1000 instrument. Anti-human IgG and the bacteria (against which the antibody
was immobilized on that particular channel) were used as positive controls. Negative controls were
Anti-guinea pig IgG and other bacteria.
5
4.6.2 Running Samples and detection of bacteria
Ulcer secretions were diluted in the ratio of 1:1 with PBS ( pH 7.4, Apotektet AB) and were injected
at a flow rate of 5µl/min for 3 minutes. Surface of the sensor chip was washed with regeneration
buffer (equal volumes of 1M Sodium Hydroxide and Glycine (pH 2.0) at a flow rate of 5µl/min for 1
minute. The unbound molecules were removed during the run by using HEPES buffered Saline
(HBS) buffer (10mM HEPES, pH 7.4. 150mM NaCl, 3.4mM EDTA, 0.005% Biacore surfactant
P20). Angular deflection of the SPR signal is used for determining the interaction of bacteria
(antigen) with the antibody coated on the channel and the response was measured in response units
(RU).
Table 2: Specifications of dilutions during a single run in Biacore.
4.6.3 Statistical Analysis
Chi square test was performed to compare the results obtained with Biacore SPR analysis and
culture analysis. The degree of freedom was 1 in each case.
5. Results
5.1 Culturing Cells
Difference in size of the leukocytes cultured in L-15 medium was observed when stimulated with
heat killed bacteria (Figure 2B). Cells were also seen to clump together in the medium with bacteria
(Figure 2C) while cells in the medium without bacteria were seen as individual cells with distinct
nuclei (Figure 2A). This observation was true in all of the cultures.
A
B
C
Figure 2: Leukocytes separated by gradient centrifugation from blood (patients) gathered in EDTA
tubes and incubated at 37ºC in L-15 cell medium containing 10% FBS, before and after stimulation
with the same bacteria as grown in cultures (Resolution 40X).
6
5.2 Flow Cytometry Analysis
Lymphocytes separated from blood of patients and cultured in L-15 medium with and without
bacteria were used. This study was performed at the Department of Immunology, University hospital
of Linköping by staff using the same established method to identify the leukocytes in patients. Few
samples (n=3) were analysed and therefore the analysis for significance was not performed. The
cells were identified by their cell surface proteins. B cells were CD3- and CD19+ while T cells were
CD3+ and CD19-. Plasma cells were in different stage of maturation and some cell surface proteins
were not specified. But the CD3- cells that were larger in size and were not identified to be B or T
cells were identified as mature B cells (ie. plasma cells).
Lymphocytes were gated for identification of individual cell components by using cell surface
markers specific for all lymphocytes (CD45), B cells (CD19) and T cells (CD3) tagged with distinct
flurochromes during flow cytometry analysis (Figure 3). Medium containing lymphocytes in
medium stimulated with bacteria and incubated for 3 weeks when subjected to flow cytometry
analysis showed that B cells that were long living were antibody secreting plasma cells (Figure 4).
Figure 3: Flow Cytometry results of lymphocytes obtained from the blood of same patient cultured
in medium with and without bacteria. Lymphocytes were gated in the scatter plot against CD45
(surface marker for lymphocytes) tagged with flurochromes.
7
Figure 4: Flow Cytometry results of lymphocytes obtained from same patient incubated at 37 ̊C for
about 3 weeks in medium with and without bacteria. CD3- cells that survived even after 3 weeks in
medium stimulated with bacteria were identified as matured plasma cells while the number of
individual cell components in lymphocytes were found to have been decreased in the medium
without bacterial inoculation.
5.3 SDS PAGE
SDS PAGE was performed to analyze the molecular weight of the antibodies produced in each case.
Bands were obtained at 50 KDa and 25 KDa for various dilutions of antibodies (1:25, 1:50, 1:75,
1:100) with PBS (pH 7.4).
8
Figure 5: SDS PAGE results showing the light chain separation at 25 KDa and Heavy chain
separation at 50 KDa. The leukocyte culture medium was centrifuged in millipore tubes 100KDa
filter and the filterate was diluted in sodium chloride. Samples were prepared in the ratio1:25, 1:50,
1:75 and 1:100 with PBS and heated before adding loading buffer. BIO-RAD Mini-Protean Precast
gels were used.
5.4 Biacore
5.4.1 Immobilization of chip
The CM5 biosensor chip was immobilized with B-cell product after addition of Staphylococcus
aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Enterococcus fecalis per channel
respectively (Table 3).
Table 3: Response obtained for Immobilization of chip in
each channel showing the product of B cells after incubation
with each bacteria is shown in line 2.
9
5.4.2 Specificity test of the chip
After immobilization step, the chip is tested for the specificity of antibodies. Anti-guinea pig IgG
was used as negative control and Anti-human IgG was used as positive control. The chip was also
tested for different bacteria other than the bacteria against which the antibody was prepared and
immobilized in their respective channel.
Table 4 showing response obtained in Biacore against various
Controls for each of the channels immobilized with specific
antibodies. Nil = not done.
5.4.3 Sample Run
Healthy, acute and chronic ulcer secretions were run in Biacore 1000 instrument and the results
obtained were shown in Table 5.
Table 5 shows the results from the sample run using ulcer secretions from patients with chronic leg
ulcers (k1-k11), ulcer secretions from patients with acute ulcer post- breast cancer operation (1a12a), and ulcer secretion from skin biopsy of healthy volunteers (h1-h10). The culture results were
obtained from Department of Microbiology, US Linköping.* = the culture results correlate to
Biacore results.
Contents
S.aureus
S.epidermidis
P.aeruginosa
E.fecalis
Culture results
k1
positive
positive
negative
negative
k2
positive
negative
negative
negative
Staphyllococcus/Enterobacter
cloceae
S.aureus+E.coli
k3
k4
k5
k6
k7
positive*
negative
negative
negative
negative
negative*
negative
positive*
positive*
negative
negative*
negative
negative
negative
positive
negative
negative
positive*
negative
positive
Staphyllococcus + Pseudomonas
S.aureus
Proteus, KNS
negative
Pseudomonas, Enterococcus
10
positive
positive*
positive
negative
negative
negative
negative
negative
negative
negative
negative
positive*
positive*
positive*
positive*
positive*
negative
negative
negative
positive*
positive*
positive*
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
positive*
negative
negative
negative
negative
negative
negative
negative
negative
negative
S.aureus
S.aureus
Proteus, Morganella, KNS
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
positive*
positive*
negative
negative
negative
negative
negative
negative
negative
negative
negative
positive*
negative
negative
negative
negative
negative
negative
4a
5a
6a
7a
8a
9a
10a
11a
negative
negative
positive
positive
negative
negative
positive*
negative
negative
positive*
positive*
positive*
negative
negative
positive*
positive*
positive*
positive*
positive*
positive*
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
suspected
S.aureus
S.aureus
negative
negative
negative
negative
negative
negative
12a
positive*
positive*
negative
negative
negative
k8
k9
k10
k11
h1
h2
h3
h4
h5
h6
h7
h8
h9
h10
1a
2a
3a
Table 6: Results obtained during sample run using each of the channel of chip and that obtained in
culture were shown in numbers.
Bacteria
Positive hits (biacore)
No. of negatives (biacore)
Correlation with culture results
No correlation (culture)
Positive hits (culture)
negative hits (culture)
Total samples
S.aureus S.epidermidis P.aeruginosa E.fecalis
11
23
1
3
22
10
32
30
28
9
32
31
5
24
1
2
8
2
2
0
25
31
31
33
33
33
33
33
11
Positive and negative hits in Biacore SPR analysis were compared with culture results for the
bacteria S.aureus, S.epidermidis, P.aeruginosa and E.fecalis. 5, 1 and 2 out of 33 samples did not
correlate with culture results in channels immobilized with antibodies produced against S.aureus,
P.aeruginosa and E.fecalis respectively. While 24 non- correlating results were obtained with
channel immobilized against S,epidermidis bacteria.
From Figure 6 it could be seen that the culture results correlate with biacore results for all bacteria
with S.epidermidis as an exception. It could also be seen that the non-correlating results were much
less with all the bacteria except S.epidermidis.
Figure 6: A graph showing correlating and non correlating biacore results with the culture results.
Performing chi square test we have shown that the results (Biacore and culture from ulcer secretions)
were not statistically different in the case of Staphylococcus aureus (p < 0.05); Pseudomonas
aeruginosa (p > 0.5); Enterococcus fecalis (p < 0.10). However in case of Staphylococcus
epidermidis (p > 0.001) the difference reached significance.
6. Discussion
In the current study a new application for real time interaction and binding affinity in SPR analysis
using specific antibodies against bacteria to detect bacterial flora in body secretions was presented.
Results obtained in the study showed that the Bacterial diversity in biological materials might be
detected in poly-microbial solutions even when the other established methods like PCR and culture
technique could fail to identify.
Increase in size of cells and the presence of clumped cells in medium when stimulated with bacteria
shows that the cells were in the developing phase. Flow cytometry analysis further confirmed the
presence of antibody secreting plasma cells (matured B cells) along with B and T cell population. As
suggested by Matthias and Rolink (2005), survival of cells in medium even after 3 days also
12
confirms the presence of long lived plasma cells and this was showed in the study using flow
cytometry analysis for medium incubated with bacteria for three weeks.
Bands obtained at 50 KDa (heavy chain) and 25 KDa (light chain) in SDS-PAGE analysis (Figure 6)
showed the presence of IgG in the antibodies produced in vitro. This is because subsequent invasion
of pathogens triggers the host immune cells to produce IgG antibodies (Goldsby et al., 2003) and
during the study, the medium was stimulated with bacteria previously found in cultures of the patient
blood. Presence of IgG was also confirmed in the specificity test of chip where immoblized
antibodies on channel showed high positive interactions to Anti-human IgG (Table 4). Specificity
test of chip indicates that antibodies that were produced during the study were highly specific as
there was specific binding with positive controls used in each of the channels although there were
some cross reactions to anti-mouse IgG.
Previous studies have shown the use of SPR method for molecular detections by monitoring the
change in angle of refraction due to the mass bound to the analyte (kobayashi et al., 2011, Rich and
Myszka, 2008). Bacteria in sample were detected in the samples in response units when there is a
specific interaction with antibodies immobilized on channel. Further the results in SPR analysis were
compared with culture results of samples.
The biacore results were correlating with culture results in most cases other than S.epidermidis.
Most of the healthy controls showed positive response for S.epidermidis and the results for
S.epidermidis showed a markable difference when compared with culture results. This could
probably due to the fact that Staphylococcus epidermidis being a normal skin flora (Cogen et al.,
2010) could be present even in wound secretions of healthy controls. The ulcer secretions could have
been contaminated with S.epidermidis during sample collection from the patients.
7. Conclusion
Detection of bacterial flora in biological secretions using antibodies produced in vitro in SPR based
system was successfully presented in the study. SPR method could be used to identify multi bacterial
flora in biofilms simultaneously real time unlike PCR based methods and culture technique thereby
enabling proper administration of antibiotics as a treatment for chronic ulcers and other non healing
wounds.
8. Future Perspectives
SPR based method could be established for detection of anaerobic bacteria and further the method
could be standardized by comparing with previously established PCR method. An antibody
purification method such as affinity chromatography could be used for the purification of antibodies
produced and flow cytometry analysis could be performed with specific surface markers that could
distinguish plasma cells.
13
9. Acknowledgements
I would like to thank my supervisor Dr. Fariba Nayeri, Johanna Lonn and Faisal shahzad for their
valuable suggestions and moral support throughout the study. I am also grateful to Peas Institut for
financially supporting my study.
10. References
Alhede M., Bjarnsholt T., and Jensen P.O. Pseudomonas aeruginosa recognizes and responds
aggressively to the presence of polymorphonuclear leukocytes (2009). Microbiology 155: 3500–
3508.
Ankeny D.P., Guan Z., and Popovich P.G. B cells produce pathogenic antibodies and impair
recovery after spinal chord injury in mice. (2009).The journal of clinical investigation 119: 28812999
Bernasconi N.l., Traggiai E., and Lanzavecchia A, Maintenance of Serological Memory by
Polyclonal Activation of Human Memory B Cells (2002), Science 13 298: 2199-2202
Bjarnsholt T., Kirketerp-Moller K., Jensen P.O., Madsen K.G., Phipps R., Krogfelt K., Høiby
N., and Givskov M. Why chronic wounds will not heal: a novel hypothesis (2008). Wound Repair
Regeneration 16: 2–10.
Bjorck.L and Kronval.G. Purification and some properties of streptococcal protein G, a novel IgGbinding reagent.(1984). The Journal of Immunology 113: 969-974.
Bowler PG, Davies BJ. The microbiology of infected and noninfected leg ulcers (1999).
International Journal Dermatology 38:573–8.
Burmølle.M., Thomsen.T.R., Fazli.M., Dige.I., Christensen.L., Homoe.P., Tvede.M., Nyvad.B.,
Nielsen.T.T., Givskov.M., Moser.C., Møller.K.K., Johansen.H.K., Høiby.N., Jensen.P.O.,
Sørensen.S.J., and Bjarnsholt.T. Biofilms in chronic infections ‘ a matter of opportunity
‘monospecies biofilms in multispecies infections.(2003). Immunology & Medical Microbiology.
59(3): 324–336,
Cogen A.L., Yamasaki K., Sanchez K.M., Dorschner R.A., Lai Y., MacLeod D.T., Torpey
J.W., and Gallo R.L. Selective antimicrobial action is provided by phenol-soluble modulins derived
from staphylococcus epidermidis, a normal resident of the skin.(2010). The journal of investigative
dermatology 130 :192 -200.
Coligan, J.E., Kruisbeek, A.M., Margulies D.H., Shevach E.M. and Strober W. Current
Protocols in Immunology. (1992). Greene Publishing Associates, New York, NY.
Eriksson.G., Eklund.A.E., and Kallings.L.O. The Clinical Significance of bacterial growth in
venous leg ulcers (1984). Scandinavian Journal of infectious disease 16; 175-180.
Fux C.A., Stoodley P., Hall-Stoodley L., and Costerton J.W.Bacterial. Biofilms: a diagnostic and
therapeutic challenge (2003).Expert Review of Anti- Infective Therapy 1: 667–683.
14
Gendek-Kubiak.H and Gendek .E.G. Usefulness of some primary anti-human antibodies
in immunohistochemistry in guinea pig.(2004). Annales Academiae Medicae Bialostocensis 49(1):
192.
Goldsby RA, Kindt TK, Osborne BA and Kuby J.Immunology. (2003). 5th Edition, W.H.
Freeman and Company, New York.
Hannele R., Somer J., and Finegold S.M. Problems Encountered in Clinical Anaerobic
Bacteriology. (1984). Clinical Infectious Diseases 1: S45-S50.
Kobayashi H., Endo T., Ogawa N., Nagase H., Iwata M., Ueda H. Evaluation of the interaction
between β-cyclodextrin and psychotropic drugs by surface plasmon resonance assay with a
Biacore® system.(2011).Journal of Pharmaceutical and Biomedical Analysis 54: 258-263
Liu H., Gaza-Bulseco G., Chumsae C., Newby-Ke A. Characterization of lower molecular weight
artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE.(2007).
Biotechnology Letters 29:1611–162
Matthias P. and Rolink A.G. Transcriptional networks in developing and mature B cells (2005).
Nature Reviews Immunology 5, 497-508
Moseley R., Stewart J.E., Stephens P., Waddington R.J., Thomas D.W. Extracellular matrix
metabolites as potential biomarkers of disease activity in wound fluid:lessons learned from other
inflammatory diseases. (2004). British Journal of Dermatology 150: 401–13.
Ozinsky A., Underhill D.M., Fontenot J.D., Hajjar A.M., Smith K.D., Wilson C.B., Schroeder
L., and Aderem A. The repertoire for pattern recognition of pathogens by the innate immune
system is defined by cooperation between Toll-like receptors. (2000) The National Academy of
Sciences, 97(25): 13766–13771.
Pukstad B.S., Ryan.L., Flo T.H., Stenvik.J., Moseley.R., Harding.K., Thomas D.W., Espevik.T.
Non-healing is associated with persistent stimulation of the innate immune response in chronic
venous leg ulcers (2010). Journal of Dermatological Science 59: 115–122.
Reithinger R., Espinoza J.C., Courtenay O and Davies. Evaluation of PCR as a Diagnostic MassScreening Tool To Detect Leishmania (Viannia) spp. in Domestic Dogs (Canis familiaris). (2003).
Journal of clinical microbiology.1486–1493.
Reithinger R., Espinoza J.C., Courtenay O., and Davie C.R . Evaluation of PCR as a Diagnostic
Mass-Screening Tool To Detect Leishmania (Viannia) spp. in Domestic Dogs (Canis familiaris)
(2003). Journal of clinical microbiology. 1486–1493
Rich R.L and Myszka D.G. Survey of the year 2007 commercial optical biosensor literature
(2008). Journal of molecular recognition 21 :355–400
Sibbald R.G., Orsted H., Schultz G.S., Coutts P., and Keast D. Preparing the wound bed : focus
on infection and inflammation. (2003) Ostomy Wound Manage 49: 23–51.
15
Smock., Kristi J., Perkins., Sherrie L., Bahler., and David W. Quantitation of Plasma Cells in Bone
Marrow Aspirates by Flow Cytometric Analysis Compared With Morphologic Assessment.(2007) Archives
of Pathology and amp Laboratory Medicine 131:951-955
Van Gennip M., Christensen L.D., and Alhede M. Inactivation of the rhlA gene in Pseudomonas
aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear
leukocytes(2009). APMIS 117: 537–54
Wilson M. J., Weightman A. J. and Wade W. G. Applications of molecular ecology in the
characterisation of uncultured microorganisms associated with human disease (1997). Review of
Medical Microbiology 8: 91–101.
16