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)

• Wound infections

• Sinus and Respiratory

infections

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 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

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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.

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 Significance of the Study
It can give more It could develop
ideas on the new diagnostic
interaction of the
approach and
antibody and
antigen regarding therapeutics
Proteus vulgaris regarding Proteus
antigen. 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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11/25/09 17
ACCLIMATIZATION ANTIGEN PREPARATION INJECTION OF MICE
OF MICE

Nutrient Agar
ANTIGEN ANTIBODY TESTING
CHARACTERIZATION
BLEEDING OF
MICE

IFAT

Western
SDS-PAGE 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

Streak the plate with

Dispense into the Proteus vulgaris.
Autoclave for 15 minutes petri dishes.
at 121°C.
8g 1 L yeast
Nutrient broth extract
powder

The yeast will be mixed Incubate and shake Incubate at

with Freund’s complete at room temp. for 37°C for 48 hrs.
adjuvant at 1:1 mixture &
Inoculate P.
48 hrs. vulgaris in nutrient
will be emulsified by
sonication. broth.

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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, air-
dried, 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

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• Freunds complete adjuvant from (5X10ml) P2500
• Freunds incomplete adjuvant (3x30 ml) P2250
• Mice (18 pcs) P3600
• Poly-L-Lysinecoated slides (72slides) P2750
• Trypsin (5x1g) P8800
• Triton X-100 (2x500ml) P1480
• peroxidase conj. AffiniPure Goat Anti-Human IgG (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

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• 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
• 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) P1850
• Mask (20pcs) P80
TOTAL P78971

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