proteus vulgaris
TRANSCRIPT
Chapter 1
INTRODUCTION
Antibody production is important in researches, medical industry and can be
indispensable tools to analyze protein functions in the broad area of life science. Antibodies
are one of the most important tools for the diagnosis and components for vaccines. Animals
like rabbit, mouse, guinea pig, chicken, goat, sheep, hamster and rat have been used
frequently for the production of antibodies.
Antibodies are soluble proteins by B cells, which interact with antigens. Antigens are
molecules capable of interacting with specific components of the immune system (Madigan,
M. T. et al., 2003). Polyclonal antibodies are antibodies that are derived from many different
cells or cell lines, similar to the mixture of antibodies found in sera. Polyclonal antibodies are
therefore a mixture of much different specificity. This is in contrast to monoclonal antibodies
which are derived from one clone (Wagner, 2007). There were researches conducted for the
production and characterization of polyclonal antibodies. Polyclonal antibody production in
mammals is generally associated with multiple injections of antigens, adjuvants and repeated
blood sampling procedures (Hau, J. et al., 2005). Certain antibodies can be produced to
protect the body from different pathogens like bacteria, fungi or viruses.
Proteus is a genus of Enterobacteriaceae, which occurs widely in humans and
animals and in the environment, and can be easily recovered from sewage, soil, garden
vegetables and many other materials. Proteus strains are often found as concomitants of
Shigella and make their appearance in the stools of patients recovering from bacillary
dysentery. They can cause many illnesses, such as kidney stones, bladder stones, peritonitis,
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septicaemia, pyelitis and infection. Also, the diagnosis of pyelonephritis and urinary tract
infection can be made when the Proteus concentration in urine is greater than 105 cells/ml.
Therefore, the detection and analysis of the growth characteristics of Proteus is of
importance in clinical microbiology, environmental science, food technology, toxicology and
biotechnology (Tan, H. et al., 1997)
Proteus vulgaris is an opportunistic pathogen and has been associated with cases of
bacteremia, pneumonia and focal lesions. It can cause many different types of infection
including urinary tract infections and wound infections and is a common cause of sinus and
respiratory infections, especially in South East Asia, which can be extremely hard to
eradicate in sinus and respiratory tissues (Baron, S. et al., 1996).
Proteus vulgaris is gram-negative facultative anaerobe that has a growth temperature
of 37°C. Proteus vulgaris occurs naturally in the intestines of humans and a wide variety of
animal, manure, soil and polluted waters. Proteus vulgaris is highly motile and often swarm
across the surface of agar plates, giving a distinct appearance different than distinct colonies
normally seen.
Mice are used frequently for production of monoclonal antibodies. Their small size
has generally been considered an impediment to collection of enough serum for harvesting
adequate quantities of polyclonal antibodies. However, the use of mice for polyclonal
antibody production may be particularly advantageous when the quantity of antigen is limited
to a few or when only a limited amount of the antibody is needed. On the other hand, mouse
size may no longer be a major drawback in terms of quantity of antibodies that can be
harvested, as polyclonal antibodies can be harvested from ascitic fluid. A number of
methods have been developed for producing ascites in mice (Cartledge et al., 1992; Kurpisz
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et al., 1988; Lacy and Voss, 1986; Mahana and Paraf, 1993; Maurer and Callahan, 1980;
Tung, 1983) with variable success.
The objectives of this study are to obtain high-titer, high-affinity antisera in a manner
consistent with the welfare of the mice being immunized in order to produce polyclonal
antibodies against a specific antigen which is Proteus vulgaris, to quantify the amount of
antibody produced by ELISA, to characterize the antigen through molecular weight
determination by Western blotting and to indicate the occurrence of a specific antigen-
antibody reaction by IFAT.
Polyclonal antibodies that can be produced can be used to fight the pathogen Proteus
vulgaris. This research can give more ideas on the interaction of the antibody and antigen
regarding Proteus vulgaris antigen. It could develop new diagnostic approach and
therapeutics regarding Proteus infection. The scope of the study is for the production,
characterization and quantification of murine polyclonal antibodies only. It also includes
characterization of highly immunogenic protein in Proteus vulgaris through Western
blotting. It does not include the study of properties, application on therapeutics, study of
mechanism of antibodies, further purifications and cross-reactivity of polyclonal serum
generated.
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Chapter 2
REVIEW OF RELATED LITERATURE
Bacterial infection is one of the common infections encountered in the Philippines. It
causes diseases in man which can be harmful in their health. Proteus vulgaris is one of the
bacteria that cause bacterial infection. It is usually found in soil, fecal matter, and sewage.
2.1 Proteus species
Proteus is a genus of Gram-negative Proteobacteria, which includes pathogens
responsible for many human urinary tract infections (Baron, S. et al., 1996). Because of the
rapid motility of Proteus cells, colonies growing on agar plates often exhibit a characteristic
swarming phenomenon. Cells at the edge of the growing colony are more rapidly motility
than those in the center of the colony. The former move a short distance away from the
colony in a mass and then undergo a reduction in motility, settle down and divide forming a
new crop of motile cells that again swarm. As a result, the mature colonies appear as a series
of concentric rings, with higher concentrations of cells altering with lower concentrations
(Madigan, M. T. et al., 2003).
Proteus species do not usually ferment lactose, but have shown to be capable lactose
fermenters depending on the species in a TSI test, Triple Sugar Iron. Since it belongs to the
family of Enterobacteriaceae, general characters are applied on this genus. Its oxidase
negative, but catalase and nitrase positive. Specific tests include positive urease (which is the
fundamental test to differentiate Proteus from Salmonella) and phenylalanine deaminase
tests. On the species level, indol is considered reliable as it's positive for Proteus vulgaris but
negative for Proteus mirabilis (Ryan and Ray, 2004).
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2.1.1 Proteus vulgaris
Proteus vulgaris is a rod-shaped bacillus, Gram negative bacterium that inhabits the
intestinal tracts of animals, but it can also be found in soil, stagnant water, standing water,
fecal matter, raw meats and dust. It is in the Proteobacteria and considered to be pathogenic.
In humans, it can cause many different types of infection including urinary tract infections
and wound infections and is a common cause of sinus and respiratory infections, especially
in South East Asia, which can be extremely hard to eradicate in sinus and respiratory tissues
(Baron, S. et al., 1996).
A typical sinus and or respiratory infection caused by P. vulgaris can take weeks or
even months to eradicate in humans, even using the few antibiotics that the P. vulgaris
pathogen is sensitive to. P. vulgaris can be deadly when in the sinus or respiratory tissues, if
left untreated or is treated with antibiotics that have only an intermediate effect on P. vulgaris
(Andreoli et al., 1997). The isolation of P. vulgaris is associated with post-antibiotic
superinfection, particularly in immunosuppressed patients receiving extended courses of
antibiotic therapy.
2.1.2 Pathophysiology of Proteus vulgaris
Proteus vulgaris possess an extracytoplasmic outer membrane, a feature shared with
other gram-negative bacteria. In addition, the outer membrane contains a lipid bilayer,
lipoproteins, polysaccharides, and lipopolysaccharides (Gonzalez G., 2006).
Infection depends on the interaction between the infecting organism and the host
defense mechanisms. Various components of the membrane interplay with the host to
determine virulence. Inoculum size is important and has a positive correlation with the risk of
infection. Certain virulence factors have been identified in bacteria. The first step in the
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infectious process is adherence of the microbe to host tissue. Proteus vulgaris induce
apoptosis and epithelial cell desquamation. Bacterial production of urease has also been
shown to increase the risk of pyelonephritis in experimental animals. Urease production,
together with the presence of bacterial motility and fimbriae, may favor the production of
upper urinary tract infections (UTIs) by organisms such as Proteus vulgaris (Gonzalez G.,
2006).
The ability of Proteus vulgaris to produce urease and to alkalinize the urine by
hydrolyzing urea to ammonia makes it effective in producing an environment in which it can
survive. This leads to precipitation of organic and inorganic compounds, which leads to
struvite stone formation. Struvite stones are composed of a combination of magnesium
ammonium phosphate (struvite) and calcium carbonate-apatite. Struvite stone formation can
be sustained only when ammonia production is increased and the urine pH is elevated to
decrease the solubility of phosphate. Both of these requirements can occur only when urine is
infected with a urease-producing organism such as Proteus vulgaris. Urease metabolizes urea
into ammonia and carbon dioxide: Urea → 2NH3 + CO2. The ammonia/ammonium buffer
pair has a pK of 9.0, resulting in the combination of highly alkaline urine rich in ammonia
(Gonzalez G., 2006).
2.1.3 Motility of Proteus vulgaris
Due to the presence of many flagella completely around the organism (these are
called “peritrichous flagella”), these organisms can propel themselves across an agar plate at
an astounding speed. In terms of scale miles, the microorganisms would be traveling at
speeds in excess of 100 mph if it were the size of a human being. The extraordinary ability to
swarm across an agar plate very much facilities its identification (Vincent, W.F, 2005).
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2.1.4 Causes of Proteus vulgaris
Hospital-acquired infections are usually caused by interruption of the closed sterile
system by hospital personnel. Proteus vulgaris also cause sepsis neonatorum and bacteremia
with fever and neutropenia. Proteus vulgaris are also involved in synergistic nonclostridial
anaerobic myonecrosis, which may involve subcutaneous tissue, fascia, and muscle
(Gonzalez G., 2006).
2.1.5 Treatment of Proteus vulgaris
Known antibiotics that P. vulgaris is sensitive to are Ciprofloxacin, Ceftazidime,
Netilmicin, Sulbactam or Cefoperazo, Meropenem, Piperacil or Tazobactam, Unasyn.
Antibiotics should be introduced in much higher doses than normal when P. vulgaris has
infected the sinus or respiratory tissues (Vincent, W.F, 2005).
2.2 Antibodies
Antibodies or immunoglobulins are found as cell-surface antigen receptors on B cells,
or in soluble form in high concentrations in serum and other body fluids, where Ig functions
to neutralize and opsonize foreign antigens (Madigan, M. T. et al., 2003). Antibodies were
the first specific product of the adaptive immune response to be identified found in the fluid
component of blood, or plasma, and in extracellular fluids. Because body fluids were once
known as humors, immunity mediated by antibodies is known as humoral immunity
(Janeway, C.A. Jr. et al., 2001).
Antibodies are important tools used by many investigators in their research and have
led to many medical advances. Mammalian sera represent a remarkable and economical
source of immunoglobulins widely used in diagnostic and therapeutic applications
(Gallacher, 1993; Gathumbi et al., 2001). In biochemical and biological researches,
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polyclonal antibodies are routinely used as ligands for the preparation of immunoaffinity
columns (Shin et al., 2001) and as coating or labeling reagents for the qualitative and
quantitative determination of molecules in a variety of assays such as enzyme linked
immunosorbent assay (ELISA), double diffusion, radial immuno-diffusion, Western blot and
radioimmunoassay (Calabozo et al., 2001; Cheung et al., 2002; Verdoliva et al., 2000).
2.2.1 Polyclonal and Monoclonal Antibodies
The decision regarding whether to use a PAb or MAb depends on a number of
factors, the most important of which are its intended use and whether the antibody is readily
available from commercial suppliers or researchers. PAbs can be generated much more
rapidly, at less expense and with less technical skill than is required to produce MAbs
(Lipman, N.S. et al., 2005).
One can reasonably expect to obtain PAbs within several months of initiating
immunizations, whereas the generation of hybridomas and subsequent production of MAbs
can take up to a year or longer in some cases, therefore requiring considerably more expense
and time. The availability of an “off the shelf” reagent eliminates the issues of time and
frequently, cost (Lipman, N.S. et al., 2005).
The principal advantages of MAbs are their homogeneity and consistency. The
monospecificity provided by MAbs is useful in evaluating changes in molecular
conformation, protein-protein interactions, and phosphorylation states, and in identifying
single members of protein families. It also allows for the potential of structural analysis
(e.g., X-ray crystallography or gene sequencing) to be determined for the antibody on a
molecular level. However, the monospecificity of MAbs may also limit their usefulness.
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Small changes in the structure of an epitope (e.g., as a consequence of genetic polymorphism,
glycosylation and denaturation) can markedly affect the function of a MAb. For that reason,
MAbs should be generated to the state of the antigen to which it will eventually need to bind.
In contrast, because PAbs are heterogeneous and recognize a host of antigenic epitopes, the
effect of change on a single or small number of epitopes is less likely to be significant. PAbs
are also more stable over a broad pH and salt concentration, whereas MAbs can be highly
susceptible to small changes in both. Another key advantage of MAbs is that once the
desired hybridoma has been generated, MAbs can be generated as a constant and renewable
resource. In contrast, PAbs generated to the same antigen using multiple animals will differ
among immunized animals, and their avidity may change as they are harvested over time.
The quantity of PAbs obtained is limited by the size of the animal and its lifespan (Lipman,
N.S. et al., 2005).
PAbs frequently have better specificity than MAbs because they are produced by a
large number of B cell clones each generating antibodies to a specific epitope, and polyclonal
sera are a composite of antibodies with unique specificities. However, the concentration and
purity levels of specific antibody are higher in MAbs. The concentration of specific antibody
in polyclonal sera is typically 50 to 200 g/mL and the range of total Ig concentration in sera
is between 5 and 20 mg/mL. In comparison, MAbs generated as ascites or in specialized cell
culture vessels are frequently 10-fold higher in concentration and of much higher purity
(Lipman, N.S. et al., 2005).
MAbs are not generally useful for assays that depend on antigen cross-linking (e.g.,
hemagglutination) unless dimeric or multimeric antigens or antigens bound to a solid phase
are used. Additionally, they may not activate complement readily because activation requires
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the close proximity of Fc receptors. Modification of antibodies by covalently linking a
fluorochrome or radionuclide may also alter antibody binding. This potential is less of a
concern when using PAbs, which recognize a host of epitopes, but it can be significant for
MAbs if the change affects its monospecific binding site. Many of the disadvantages of
MAbs can be overcome by pooling and using multiple MAbs of desired specificities. The
pooled product is consistent over time and available in limitless quantity. However, it is
frequently difficult, too expensive, and too time consuming to identify multiple MAbs of
desired specificity (Lipman, N.S. et al., 2005).
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Figure 1. Polyclonal and Monoclonal Antibodies (Goldsby, R.A. et al., 2000).
11
2.2.2 Mouse for Polyclonal Antibody Production
Inbred strains of mice are available, which allows the investigator to select the highest
responders from among a number of strains tested with the Ag-of-interest. Although mice
are used frequently for production of monoclonal Abs, their small size has generally been
considered an impediment to collection of enough serum for harvesting adequate quantities
of polyclonal Abs. However, the use of mice for polyclonal Ab production may be
particularly advantageous when the quantity of Ag is limited. On the other hand, mouse size
may no longer be a major drawback in terms of quantity of Ab that can be harvested, as
polyclonal Abs can be harvested from ascitic fluid. A number of methods have been
developed for producing ascites in mice (Cartledge et al., 1992; Kurpisz et al., 1988; Lacy
and Voss, 1986; Mahana and Paraf, 1993; Maurer and Callahan, 1980; Tung, 1983) with
variable success. Karu has recently developed a method using T-180 sarcoma cells to induce
ascites in polyclonal Ab-producing Swiss Webster mice, such that 10-40 mL of Ab-
containing ascitic fluid can be collected from a single mouse (Karu, 1993; Ou et al., 1993;
Sartorelli et al.). This method appears to be superior to other reported methods for ascites
production being relatively reliable giving high yields and not requiring repeated injections
of Freund's adjuvant or pristane.
2.2.3 Adjuvants
Immunologic adjuvants are agents that nonspecifically increase immune responses to
specific antigens that are weakly immunogenic. The three adjuvants that are commonly used
are Freund's-type mineral oil adjuvant emulsions, Ribi Adjuvant System, and TiterMax®
(Lipman et al., 1992; Smith et al., 1992; Johnston et al., 1991).
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Whether used for research or production of antisera or for developing vaccines, the
objective of using adjuvants differs greatly. Adjuvants intended for production of antisera
need to induce high titers of high avidity antibody within a short time. Adjuvants intended for
vaccine use need only induce protective titer, although the duration of the response and
induction of immunologic memory are critical for maintaining protection. Induction of cell-
mediated immunity is a requirement for protection against many etiologic agents but is also
partly responsible for side effects of adjuvants (Jennings, V.M., 1995).
Adjuvants or immunopotentiators were initially thought of as agents capable of
promoting an augmented and more sustained antibody response. However, new evidence has
shown that adjuvants influence the titer, duration, isotype, and avidity of antibody, as well as
affecting properties of cell-mediated immunity (Hunter et al., l995). For example, adjuvants
have been shown to induce class I-restricted CD8-positive cytotoxic T lymphocytes and
modulate the specificity of antibody among available epitopes on protein antigens (Takahashi
et al., 1990; Kenney et al., 1989).
Adjuvants can be been categorized according to their origins (whether they are
derived from mineral; bacterial; plant; synthetic; or host product, such as Interleukin 1 and 2
and according to their proposed mechanism of action. Certain adjuvants such as aluminum
compounds, oil emulsions, liposomes, and synthetic polymers act through the effect of
antigen localization ("depot" effect), which leads to slow delivery of the antigen. Most
adjuvants also induce complex cell interactions between macrophages and lymphocytes
(Jennings, V.M., 1995).
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No one adjuvant works best with all antigens, animal species, or experimental
conditions. Each adjuvant has advantages and disadvantages, and the antigen's properties
must also be taken into account to select the adjuvant most likely to give the best results
(Jennings, V.M., 1995).
2.2.4 Analysis
Immunoblots is one of the basic methods by which antibodies are used to establish
whether an antigen or related molecule is in a prepared solution (i.e., cell or tissue lysate)
(Bonifacino et al. 2001; Gallagher et al. 1998; Harlow and Lane, 1999). Immunoblots
involve transferring soluble antigen onto a suitable membrane (nitrocellulose or positively
charged nylon/polyvinylidene fluoride [PVDF]), blocking the membrane to prevent
subsequent nonspecific binding, and then probing it with an antigen-specific antibody
(primary antibody). The primary antibody-antigen complex is then identified by incubating
the blot with a secondary antibody against the isotype of the primary antibody which is
conjugated to an enzyme (i.e., horseradish peroxidase [HRP]) or radionuclide-labeled
antibody to facilitate detection. Western blots are immunoblots preceded by protein
separation, usually based on size, utilizing a polyacrylamide gel (Lipman, N.S. et al., 2005).
The enzyme-linked immunosorbent assay (ELISA) is another basic application used
to analyze soluble antigens (Hornbeck 1991). This approach allows the simultaneous
processing of many small samples. It requires two antigen specific antibodies. One antigen-
specific antibody is coated onto a solid substrate (typically a 96-well plate) to capture the
antigen from the applied solution while the other is used to detect the immobilized antigen.
As with immunoblots, a HRP-conjugated secondary antibody is normally used for detection
purposes. The capture and primary antibodies must be different isotypes, if not from different
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species, so that the secondary antibody will only detect the presence of the primary antibody
and correctly indicate that the antigen has been captured in the well (Lipman, N.S. et al.,
2005).
The spatial expression of an antigen relative to an individual cell or in the context of
whole tissue can be analyzed with antibodies using immunofluorescence and
immunohistochemistry, respectively (Harlow and Lane 1999). Both applications involve
preparing samples (cells or tissue sections) in a manner that retains their three-dimensional
structure, immobilizing them on glass slides, probing them with antibodies and visualizing
the antigen antibody microscopically. The antibodies used in these applications are either
conjugated to fluorophores that emit light when excited by light of the appropriate
wavelength or are conjugated to an enzyme, such as HRP, which produces a detectable color
when a chromagen is present. PAbs are used more frequently in these applications for two
main reasons: (1) They recognize multiple independent epitopes and therefore have a better
chance of binding epitopes that are still available in fixed samples and (2) it is generally
impractical to screen hundreds to thousands of cultures for MAbs that work in
immunohistochemistry. The use of PAbs can result in nonspecific background staining
however, affinity purification, using the desired antigen immobilized on a solid support, can
be used to minimize or eliminate the problem. Background staining may also result from
binding of the antibody’s Fc region to Fc receptors in the sample (Lipman, N.S. et al., 2005).
2.2.5 Applications
The ability of antibodies to selectively bind a specific epitope present on a chemical,
carbohydrate, protein or nucleic acid has been thoroughly exploited through the years, as
evidenced by the broad spectrum of research and clinical applications in which they are
15
utilized. Applications include simple qualitative and/or quantitative analyses to ascertain the
following: (1) whether an epitope is present within a solution, cell, tissue or organism, and if
so, where: (2) methods to facilitate purification of an antigen, antigen-associated molecules,
or cells expressing an antigen; and (3) techniques that use antibodies to mediate and/or
modulate physiological effects for research, diagnostic, or therapeutic purposes (Lipman,
N.S. et al., 2005).
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CHAPTER 3
METHODOLOGY
In this study, the production of polyclonal antibodies for Proteus vulgaris in mice will
be done. Immunization of mice will be done by injecting the mixture of antigen, Proteus
vulgaris cells, and an adjuvant, Freund’s Complete adjuvant at the intraperitoneal side of the
mice. Serum from the mice will be collected and tested for the presence of polyclonal
antibody by IFAT (Indirect Fluorescence Antibody Test). The quantitation of polyclonal
antibody produced will be determined by ELISA (Enzyme Linked Immunosorbent Assay).
Finally, the antibody will be characterized by Western blotting.
3.1 Antigen preparation
The antigen will be the bacteria Proteus vulgaris obtained from UP Los Baños
Culture Collection. The bacteria will be cultured in nutrient agar at 37°C for 48 hours. The
bacteria will be incubated at 80°C for 30 minutes thrice with intermittent cooling at room
temperature for two hours each time. It will then be mixed with Freund’s Complete adjuvant
at a 1:1 mixture. The mixture will be emulsified by sonication.
3.1.1 Nutrient Agar
Suspend 23 g of the nutrient agar powder in 1 L distilled water and mix thoroughly.
Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Autoclave at 121°C for 15 minutes (Bacteriological Analytical Manual, 1998).
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3.1.2 Nutrient Broth
Suspend 8 g of nutrient broth powder in 1 L distilled water. Autoclave at 121°C for
15 minutes.
3.2 Immunization of mice
Five to eight weeks BALB/c old mice will be obtained from St. Luke’s Medical
Center, Research and Biotechnology Development.
3.2.1 Injection of mice
Intraperitoneal (IP) injections with a maximum volume of 0.25 mL (antigen +CFA)
will be given for the first injection. The subsequent IP boosts (antigen+ICFA) will be given
at 14 days intervals.
According to Standard Operating Procedure for Antibody Production in Mice Using
Freund’s Adjuvant (2007), injection schedules will vary from 10 days to 2-3 week intervals.
Briefly, the mouse is grasped and tilted, head-downward at a 45°C angle. The injection is
given in the abdomen (alternating sides for booster immunizations) and the needle inserted
perpendicular to the spine in the area bordered by the midline, groin and the top of the hip
(These land marks delineate a triangular injection area). Before injection, the syringe should
be aspirated to assure that the needle is not within a blood vessel or a loop of bowel. The
injection should be made smoothly and slowly. The maximum volume for an IP injection is
0.25cc.
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3.2.2 Bleeding of mice
Bleeding will be done via the orbital sinus (Procedure for Submandibular Blood
Sampling and Blood Withdrawal Using the Orbital Sinus). For retroorbital bleeds, mice are
anesthetized and a capillary tube is inserted at the medial canthus of the eye and directed
caudally behind the globe to the medial-posterior aspect of the orbit. The tube is lightly
twisted to disrupt the vascular plexus at this site and blood collected as it flows out with the
hematocrit tube. For facial vein or submandibular bleeds, the mouse is manually restrained
and a special lancet is used to puncture the facial vein located on the lower cheek behind the
curve or the mandible. The blood is collected in a microhematocrit tubes as it flows out. The
volume collected at one time is two (2) microhematocrit tubes (75μl total volume/tube) (see
Table 1).
3.3 Antibody Testing
Indirect fluorescence antibody test (IFAT) will be used for testing. Drops (approximate
1μl) containing organism will be applied to the wells of Teflon-coated glass slides, spread,
air-dried, fix in absolute methanol for 10 mins. Undiluted polyconal antibody for 30 min at
37 0C will be reacted. The slides will be washed air-dried and will be reacted with a 1:10
dilution of fluorescin-conjugated antimouse immunoglobulin for 30 min at 370C. The slides
will be washed and examined under a fluorescence microscope (Tan, K.S.W. et al., 2001).
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Immunization Schedule ProcedureDay 0 Pre-bleed
1st immunization antigen + adjuvant IP 25µDay 7 1st bleedDay 14 2nd bleedDay 21 Boost antigen only IP 25µ or antigen + adjuvant
3rd bleedDay 21 4th bleedDay 28 5th bleedDay 35 Boost antigen only IP 25µ or antigen + adjuvant
6th bleedDay 42 Last bleed
Table 1. Immunization schedule and procedure
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3.4 Antibody Quantification
ELISA will be used to determine the interaction between the antigen and the antibody
to quantify the amount of polyclonal antibody produced.
Polyvinyl chloride microtiter plates will be coated with appropriate antigens in
varying concentrations (0.05 - 0.1 μg/well) in 50 mM PBS. The plates will be placed at room
temperature for 2 hours. Unbound antigens will be washed off with PBST (50 mM PBS
containing 0.2% Tween-20). Further, the wells will be treated with 1% fraction V-bovine
serum albumin (BSA) in 50mM PBS at 37°C for 2 hours. The excess BSA will be washed
off and 100 μl of various serum dilutions, PBS and control serum will be added to
appropriate wells. The antigen-antibody reaction will be allowed to take place at 37°C for 1
hour. After washing the wells thoroughly with PBST, secondary antibody at a dilution of
1:5000 (100 μl/well) will be added for 1 hour. The plates will be washed thrice with PBST
followed by washing thrice with PBS. The reaction will be stopped with H2SO4 and the plates
will be read at 450 nm using an ELISA plate reader. The reaction is developed by incubating
TMB/H2O2 substrate prepared in distilled water.
3.5 Characterization of antibody
3.5.1 SDS- Polyacrylamide gel electrophoresis
Proteins will be separated on polyacrylamide gel based on size.
SDS-polyacrylamide gel electrophoresis will be made in 12 % ( wt/vol) resolving and
4% (wt/vol) stacking gel. 1.75 ml of water will be mixed with 1.25 ml of 1.5 M Tris-Base
(pH 8.8), 0.4% SDS, with 2 ml of 30% acrylamide, 0.8% bisacrylamide, 10% Ammonium
21
persulfate, and 3 μL of TEMED for resolving gel. The stacking gel will be prepared by
mixing 3.1 ml of water, 1.25 ml of 0.5 M Tris-HCl (pH 6.8), 0.4% SDS, 0.65 ml 30%
acrylamide, 0.8% bisacrylamide, 50 μL 10% Ammonium persulfate, and 5 μL of TEMED.
Running buffer will be prepared by mixing 192 mM glycine, 25 mM Tris-base, 10 g
SDS in 1 L of distilled water.
3.5.2 Preparation of sample
The yeast will be suspended in 2x sample buffer, which is a solution of 50 mM Tris–
HCl (pH 6.8), 4% SDS, 0.004% bromophenol blue, 20% glycerol, 10% 2-mercaptoethanol.
3.5.3 Running the gel
Load 30μL of pre stained molecular weight standards (marker) and sample. Run the
gel at 100 V and observe.
After running the electrophoresis, transfer proteins at 4°C from the SDS-PAGE gel to
either a nitrocellulose by using a 5x transfer buffer containing 10 mM Tris-HCl, 15 mM Tris
Base, 192 mM glycine, and 0.05% (v/v) SDS, dissolved into 1 L of distilled water (Harlow
and Lane, 1988).
3.5.4 Western blot
In a Western blot, proteins can be separated on Polyacrylamide gels on the basis of
size, transferred to a membrane, detected with antibodies and visualized by the addition of an
enzyme substrate.
22
After the transfer is completed, membranes will either be air-dried or blocked
immediately with 3-5% of either BSA or non-fat dry milk in TBST for at least 1 h. The gel
will be stained with Coomassie Brilliant Blue R250 to check if transfer is complete.
Primary antibody will be applied at 1-3 µg/mL or serum at 1:100-1:1000 dilutions.
Dilute in blocking solution (BSA or milk concentration can be decreased 2-5 fold) in TBST
for 1-2 h at room temperature or at 4°C overnight. Membrane will be washed in TBST at
room temperature (4x) for 5 min/wash. A secondary antibody, horseradish peroxidase (HRP)
conjugated against the host species of the primary antibody will be applied. It will be
incubated at 0.1-1.0 µg/mL in blocking solution for 1-2 h at room temperature with shaking.
Membrane will be washed in TBST at room temperature (4x) for 5 min/wash. Blot will be
developed using diaminobenzidine tetrahydrochloride (ABTS).
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APPENDICES
Proposed Time Table
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Week 7
Week 8
Week 9
Week 10
Week 11
Week 12
Preparation of things: cages, distilled water, feed, beddings, waterer, order cultures, prepare media, autoclave
Mice (already in the lab for adaptation), cell culture available (for increasing population)
Test run for IFAT and Western blot Antigen preparation Mouse Injection Blood collection, Immunization, Boosting ELISA, IFAT, Western blot
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BUDGET PROPOSAL (good for 3 persons)
Freunds complete adjuvant from (5X10ml) P2500
Freunds incomplete adjuvant (3x30 ml) P2250
Mice P3600
Poly-L-Lysine coated slides (72slides) P2750
Trypsin (5x1g) P8800
Triton X-100 (2x500ml) P1480
peroxidase conjugated AffiniPure Goat Anti-Human IgG (H+L) (3x0.5ml) P18600
TMB/H2O2 substrate (2x100ml) P4740
Diaminobenzidine tetrahydrochloride (2x5g) P2840
nitrocellulose 10.5 x 12.8 cm, (20 sheets) P3250
Coomasie brilliant blue (500ml) P1800
Glycine (500g) P1220
Tris-base (500g) P1605
SDS (2x100g) P2160
Acrylamide/bis 29:1 30% (W/V) (2x100ml) P1820
Tris-HCl (1L) P1230
Ammonium Persulfate (2x100g) P2970
TEMED (2x50ml) P2420
Bromophenol blue P1210
Glycerol (1L) P1625
2-Mercaptoethanol (2x100ml) P1470
Tween-20 (500ml) P621
Molecular weight standards P2040
Waterer (3x32oz) P900
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Modified cages (3 pcs) P1500
Capillary Tubes (50pcs) P250
Syringes (50pcs.x1 mL) P500
Gloves (1box) P300
Microfuge tubes (1pack) P590
Food of mice (10 kg) P 1850
Mask (20pcs) P80
TOTAL P78971
29