Proteus Vulgaris

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, 1

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 antigenantibody 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 5

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). 6

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, 7

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 Abcontaining 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 cellmediated 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 antigenspecific 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 14

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 Day 0 Day 7 Day 14 Day 21 Day 21 Day 28 Day 35 Day 42

Procedure Pre-bleed 1st immunization antigen + adjuvant IP 25µ 1st bleed 2nd bleed Boost antigen only IP 25µ or antigen + adjuvant 3rd bleed 4th bleed 5th bleed Boost antigen only IP 25µ or antigen + adjuvant 6th bleed 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.

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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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References: Andreoli, T.E., J.C. Bennett, C.C.J. Carpenter and F. Plum M.D. (1997). Cecil Essentials of Medicine, 4th edition, W.B. Saunders Company, Philadelphia, Pennsylvania 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 purification of the allergen and development of an ELISA method measurement. Allergy. Volume 56, 429-35. Carpenter-Rose, Emily A. (2007). Candidiasis. American Academy of Emergency Medicine, American College of Emergency Physicians, American Medical Association, American Medical Women's Association, and Christian Medical & Dental Society. E-medicine from the WebMD. Cartledge, C., C. McLean, and J. Landon (1992). Production of polyclonal antibodies in ascitic fluid of mice: Time and dose relationships. J. Immunoassay, Volume 13, 339-353. Cheung HY, Chan KM, Ng TB, Cheng CH (2002). Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a noncompetitive antigen-capture ELISA. Comp. Biochem. Physiol. Biochem. Mol. Bio., Volume 131, 37-46. Fridkin, S. K., and W. R. Jarvis. (1996). Epidemiology of nosocomial infections. Clin. Microbiol. Rev. Volume 9, pp. 499-511. Gallagher S, Winston SE, Fuller SA, Hurrell JGR. 1998. Immunoblotting and immunodetection. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, Current Protocols in Immunology. New York: John Wiley and Sons, Inc. p 8.10.1-8.10.21. Goldsby, R.A., T. J. Kindt and B. A. Osborne (2000) , Kuby Immunology, 4th edition, W. H. Freeman and Company, New York. Gonzalez, Gus M.D. (2006). Proteus infection. American College of Physicians-American Society of Internal Medicine. E-medicine from the WebMD. Harlow E, Lane D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press.

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Harlow E, Lane D. (1999). Using Antibodies: A Laboratory Manual. Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press. Hornbeck P. I(1991). Enzyme-linked immunosorbent assay. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, eds. Current Protocols in Immunology. New York: John Wiley and Sons, Inc. p 2.1.22.1.22. Hau, J, and C. F. M. Hendriksen (2005). Refinement of Polyclonal Antibody by Combining Oral Immunization of Chickens with Harvest of Antibodies from the Egg Yolk. ILAR Journal, Volume 46, 294-298. Huwei, T., L. Deng, L. Nie, S. Yao (1997). Detection and analysis of The Growth Characteristics of Proteus vulgaris With a Bulk Acoustic Wave Ammonia Sensor. Analyst. Volume 122, 179-184. Janeway, C.A. Jr., P. Travers, M. Walport and M.J. Shlomchik (2001).Immunobiology: the immune system in health and disease, 5th edition, Garland Science: Taylor Francis Group, New York. Jennings, V.M. (1995). Review of Selected Adjuvants used in Antibody Production. ILAR Journal. Volume 37. Johnston, B. A., H. Eisen, and D. Fry. (1991). An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab. Anim. Sci. Volume 41, 1521. Karu, A.E. (1993). Monoclonal antibodies and their use in measurement of environmental contaminants. Hazard Assessment of Chemicals, Volume 8, 205-322. Kenney, J. S., B. W. Hughes, M. P. Masada, and A. C. Allison. 1989. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods Volume 121, 157-166. Kurpisz, M., S.K. Gupta, D.L. Fulgham, and N.J. Alexander (1988). Production of large amounts of mouse polyclonal antisera. J. Immunol. Methods, Volume 115, 195-198. Lacy, M.J., and E.W. Voss, Jr. (1986). A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells, J. Immunol. Methods, Volume 87,169-177. Lipman, N.S., L.R. Jackson, L.J. Trudel and F.W. Garcia (2005). Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR Journal, Volume 46, 259-268. Lipman, N. S., L. J. Trudel, J. C. Murphy, and Y. Sahali. (1992). Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and Ribi adjuvants in mice. Lab. Anim. Sci. Volume 42, 193-197. 25

Madigan, M.T., J.M. Martinko and J. Parker (2003). Brock Biology of Microorganisms, 10th edition, Pearson Education, Inc., New Jersey. Mahana, W., and A. Paraf. (1993). Mice ascites as a source of polyclonal and monoclonal antibodies. J. Immunol. Methods, Volume 161, 187-192. Maurer, P.H., and H.J. Callahan (1980). Protein and polypeptides as antigens. Methods Enzymol. Volume 70, 49-70. Morrison, C. J., and M. D. Lindsley (2001). Serological approaches to the diagnosis of invasive fungal infections, p. 667-716. In R. Cihlar and R. Calderone (ed.), Fungal pathogenesis: principles and clinical applications. Marcel Dekker, New York, N.Y. Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology, 4th ed., McGraw Hill. Smith, D. E., M. E. O'Brien, V. J. Palmer, and J. A. Sadowski. (1992). The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits. Lab. Anim. Sci. Volume 42, 599-601. Standard Operating Procedure for Antibody Production in Mice Using TiterMax Adjuvant, April 1997. Takahashi, H., T. Takeshita, B. Morein, S. Putney, R. N. German, and J. A. Berzofsky. (1990). Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMS. Nature Volume 344, 873-875. Tan, K.S.W., M. Ibrahim, G.C. Ng, A.M.A. Nasirudeen, L.C. Ho, E.H. Yap and M. Singh (2001). Exposure of Blastocystis Species to a Cytotoxic Monoclonal Antibody, Parasit. Res. Volume 87, 534-538. Tung, A.S. (1983). Production of large amounts of antibodies, nonspecific immunoglobulins, and other serum proteins in ascitic fluids of individual mice and guinea pigs. Methods Enzymol, Volume 93, 12-23. Verdoliva A., Basile G., Fassina G. (2000). Affinity purification of immunoglobulinss from chicken egg yolk using a new synthetic ligand. J. Chromatogr. B. Volume 749, 233-42. Vincent, W. H. (2005). Infections Cause by the Genus Proteus. Quest Diagnostics. Volume 12, 23-27. Wagner, John A. (1997). Making and Using Antibodies. Cornell University Medical College. Wezel R.P. (1995) Nosocomial candidemia: risk factors and attributable mortality. Clin. Infect. Dis., Volume 20, 1531–1534.

26

APPENDICES Proposed Time Table Week 1

Wee k2

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

27

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 28

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

29

P78971