Thesis Presentation

What are Antibodies? • Soluble proteins by B cells which interact with antigens

•Most important tools for the diagnosis & components for vaccines

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Polyclonal Antibodies • Derived from different B-cell lines. • Mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.

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Proteus vulgaris Gram stain: Gram-negative Growth Temp.: 37°C Shape: Rod-shaped bacillus Motility: Motile with many flagella Respiration: Facultative anaerobe

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Causes •

Urinary Tract Infection (UTI)

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Wound infections •

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Sinus and Respiratory infections 5

Causes • Sepsis neonatorum and bacteremia with fever and neutropenia.

• Involved in synergistic nonclostridial anaerobic myonecrosis

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Pathophysiology • Possess an extracytoplasmic outer membrane, lipid bilayer, lipoproteins, polysaccharides, lipopolysaccharides • Initial inoculum size is important

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Pathophysiology • Produce urease and can alkalinize the urine

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Motility • Has peritrichous flagella

•Ability to swarm in the agar plates.

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Treatment • Ciprofloxacin

• Ceftazidime

• Netilmicin

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Treatment • Sulbactam

• Meropenem

• Tazobactam 11/25/09

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Objectives To produce murine polyclonal antibodies against Proteus vulgaris. To quantify the amount of antibody produced by ELISA. To characterize the antigen through molecular weight determination by Western blotting. To indicate the occurrence of a specific antigen-antibody reaction by IFAT. 11/25/09

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Significance of the Study It can give more ideas on the interaction of the antibody and antigen regarding Proteus vulgaris antigen.

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It could develop new diagnostic approach and therapeutics regarding Proteus infection.

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Significance of the Study Further understanding in the pathogenicity of Proteus vulgaris.

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Scope and Limitations This study will determine only: Production, characterization of murine polyclonal antibodies Antigen detection through IFAT and ELISA Characterization of highly immunogenic protein in Proteus vulgaris through Western blotting

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Scope and Limitations This study will not include: Study of properties of antibodies Application on therapeutics Study of mechanism of antibodies Further purifications of polyclonal antibodies Cross-reactivity of polyclonal serum generated

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ACCLIMATIZATION ANTIGEN PREPARATION OF MICE

INJECTION OF MICE

Nutrient Agar

ANTIGEN CHARACTERIZATION

ANTIBODY TESTING BLEEDING OF MICE IFAT

SDS-PAGE

Western blot ELISA

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Mouse Acclimatization 5 -8 weeks old female BALB/c mice will be used in the experiment. Acclimatize for 2-3 weeks.

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Antigen Preparation Nutrient agar

23 g nutrient 1 L distilled agar water

Autoclave for 15 minutes at 121°C.

The yeast will be mixed with Freund’s complete adjuvant at 1:1 mixture & will be emulsified by sonication. 11/25/09

Incubate and shake at room temp. for 48 hrs.

Streak the plate with Proteus vulgaris.

Dispense into the petri dishes. 8g Nutrient broth powder

1 L yeast extract

Inoculate P. vulgaris in nutrient broth.

Incubate at 37°C for 48 hrs.

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Injection of Mice Intraperitoneal injections will be given in the first injection (antigen + CFA). Subsequent IP boost (antigen + ICFA) will be given after 21 days and 35 days.

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Immunization Schedule

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Bleeding of mice A sharp end glass capillary tube will be used to penetrate the orbital conjunctiva and rupture the orbital sinus.

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IFAT Drops (approximate 1μl) containing bacteria will be applied to the wells of Teflon-coated glass slides, spread, airdried, fix in absolute methanol for 10 mins.

Undiluted polyconal antibody for 30 min at 370C 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.

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ELISA PBS will be used for washing. BSA will be used for blocking of plates. Horseradish peroxidase conjugated anti-mouse immunoglobulin will be used as secondary antibody. TMB will be the substrate. Reaction will be stopped by H2SO4. The plate will be read at 450 nm.

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SDS-PAGE

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Western blot Gel: SDS Polyacrylamide gel with 12% resolving and 4% stacking Blocking: BSA Membrane: Nitrocellulose Developer: Diaminobenzidine tetrahydrochloride 11/25/09

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• • • • • • • • • • • • • • •

Freunds complete adjuvant from (5X10ml) Freunds incomplete adjuvant (3x30 ml) Mice (18 pcs) Poly-L-Lysinecoated slides (72slides) Trypsin (5x1g) Triton X-100 (2x500ml) peroxidase conj. AffiniPure Goat Anti-Human IgG (3x0.5ml) TMB/H2O2 substrate (2x100ml) Diaminobenzidine tetrahydrochloride (2x5g) nitrocellulose 10.5 x 12.8 cm, (20 sheets) Coomasie brilliant blue (500ml) Glycine (500g) Tris-base (500g) SDS (2x100g) Acrylamide/bis 29:1 30% (W/V) (2x100ml)

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P2500 P2250 P3600 P2750 P8800 P1480 P18600 P4740 P2840 P3250 P1800 P1220 P1605 P2160 P1820

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• • • • • • • • • • • • • • • •

Tris-HCl (1L) Ammonium Persulfate (2x100g) TEMED (2x50ml) Bromophenol blue Glycerol (1L) 2-Mercaptoethanol (2x100ml) Tween-20 (500ml) Molecular weight standards Waterer (3x32oz) Modified cages (3 pcs) Capillary Tubes (50pcs) Syringes (50pcs.x1 mL) Gloves (1box) Microfuge tubes (1pack) Food of mice (10 kg) Mask (20pcs)

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TOTAL

P1230 P2970 P2420 P1210 P1625 P1470 P621 P2040 P900 P1500 P250 P500 P300 P590 P1850 P80 P78971

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