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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).

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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

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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 Procedure
Day 0 Pre-bleed
1st immunization antigen + adjuvant IP 25µ
Day 7 1st bleed
Day 14 2nd bleed
Day 21 Boost antigen only IP 25µ or antigen + adjuvant
3rd bleed
Day 21 4th bleed
Day 28 5th bleed
Day 35 Boost antigen only IP 25µ or antigen + adjuvant
6th bleed
Day 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

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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.

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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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Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.

Baron, Samuel. (1996). Medical Microbiology. 4th edition, University of Texas Medical
Branch, Texas.

Brogden, K.A and J.M. Guthmiller (2002). Polymicrobial diseases, ASM Press.

Calabozo B, Duffort O, Carpizo JA, Barber D, Polo F (2001). Monoclonal antibodies against
the major allergen of Plantago lanceolata pollen, Pla 1 1: affinity chromatography
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APPENDICES

Proposed Time Table

Week Wee Week Week Week Week Week Week Week Week Week Week
1 k2 3 4 5 6 7 8 9 10 11 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