Southern Blot Analysis

Washing and Detection

Hybridization with Probe

Digoxigenin label
of probe
Bases of DNA
extending out from
membrane

UCGAUCGGU
AGCTGGCCA

Complementary base
pairing with probe DNA
Phosphodiester
backbone of DNA
bound to membrane

Synthesis of Probe
(Question 1)

Method: Random primed synthesis

Combine:
DNA template (complementary to desired
sequence of probe)
dNTPs (for DNA synthesis)
Labeled dNTP (to detect presence of probe)
Hexanucleotide mixture (random sequences;
primers for DNA synthesis)
DNA polymerase

Synthesis of Probe
(Questions 1, 2)
Template DNA

hexanucleotides

label

newly-synthesized DNA

Label:
Digoxigenin-dUTP

Principle of Probe Specificity

(Question 3)
Lack of
complementarity:
probe doesnt bind

1) Phosphodiester backbone of
ssDNA crosslinked to membrane;
bases available for pairing

2) Labeled ssDNA probe binds

to complementary ssDNA only;
this is sequence specific

3) Only area where probe binds

to DNA is visualized

Washing of Membrane
(Questions 4-6)
Hybridize with 5 X SSC (+ formamide)
Appropriate stringency for maximum binding of
probe

Wash with 2 X SSC (no formamide) @ RT

Decrease [salt] to increase stringency
Remove any unbound probe

Wash with 0.1 X SSC (no formamide) @ 68C

Increase stringency further
Probe bound specifically to complementary DNA

IgG Structure
(Question 7)

Site of
attachment
of enzyme in
conjugate

IgG molecule composed of 4 separate polypeptide chains

2 H chains + 2 L chains
Chains connected by disulfide bonds

IgG Structure
(Question 7)
Variable: varies with antigen

Constant: same in each host

(e.g., all rabbits have ~ the same
constant region)

Antigen binding site

composed of VH+VL
2 Ag binding sites per
molecule

Digoxigenin Label
(Question 9)
Non-radioactive:
Can be reused
indefinitely

Bulky chemical group: easy to raise

antibodies that bind to this group.

Steps in Protocol
(Questions 10-13)

Wash in Buffer 1 (Tris, NaCl): to

equilibrate membrane in solution used in
next step
Incubate in Buffer 2 (blocking reagent): to
prevent non-specific binding of antibody
Wash in Buffer 1: to wash off blocking
reagent
Incubate with antibody-enzyme conjugate:
to allow binding of antibody to digoxigenin

Steps in Protocol
(Questions 14-17)

Wash twice with Buffer 1: to remove any

unbound or non-specifically bound antibody
conjugate
Incubate in Buffer 3: to adjust pH to 9.5 (pH
optimum for alkaline phosphatase)
Add substrate and incubate: to develop blue
color
Wash with Buffer 4 (water): to stop reaction
Examine bands and compare to gel
photograph

Detection of Southern Blot

Substrate
BCIP/
NBT

Product
Blue color
Antibody:enzyme conjugate

UCGAUCGGU
AGCTGGCCA

Digoxigenin label of probe

DNA probe
DNA bound to membrane

If done properly, should only see color where

probe binds DNA

Color Reaction
(Questions 18, 19)
Enzymatic reaction:
Alkaline phosphatase

BCIP

Colorless
Phosphate + indolyl

BCIP = 5-bromo-4-chloro-3-indolyl phosphate

Chemical reaction:
Indolyl + NBT

Blue color

NBT = nitroblue tetrazolium

Serological Assays
Agglutination

Binding of Antibody to Antigen

(Questions 20, 21)
Epitopes

Antigen

Antibodies

ANTIGEN = ANTIbody GENerating

Each antigen has many potential epitopes
Each Antibody recognizes ONE specific epitope

Gram Negative Cell Wall

(Question 22)

immunodominant

MICROBIOLOGY: Diversity, Disease, and the Environment

by Salyers and Whitt, 2001 Fitzgerald Science Press, Inc.

Salmonella typhimurium LPS

(Question 23)
Core

Lipid A

N
OH

Abequose

O
O
COCH3
HO
O O
O

O
OH

OH O

OH
Mannose

OH

Rhamnose

OH O
OH O
Galactose

O antigens of S. typhimurium : O4, O5, and O12

Agglutination Materials
(Question 24)
S. typhimurium LT2 (O4, O5, O12)
LT2 strain

S. typhimurium LT2 oafA::TndKn (O4, O12)

O5 negative strain

S. typhimurium LT2 DgalE (no galactose)

(O-antigen negative control: O negative strain)

Difco anti-O12 antisera

Difco anti-O5 antisera

Agglutination Reactions
(Questions 25, 26)
Ag with multiple Ab
binding sites

Bivalent antibodies
Insoluble matrix of antibodies and antigen molecules forms
Has appearance of clumps

Agglutination Reaction
(Question 26)

Mix cells of
unknown bacteria
with antisera
(specific for cellsurface molecule)

Negative
Cloudy appearance
of cell suspension

Positive
Clumps indicate
agglutination

Serological Assays
ELISA

ELISA:
Enzyme-Linked ImmunoSorbent Assay
96-well
microtiter
plate
Run multiple
tests on one
plate

Small volumes
Use less of
reagents

Can use bound Ab to quantitate soluble antigen

Can use bound antigen to quantitate serum Ab

Steps in the ELISA

(Question 28)

Partially purified,
inactivated HIV
antigens pre-coated
onto an ELISA plate
ELISA plate wells already coated with HIV antigen

Steps in the ELISA

(Question 29)

Primary (1) antibody

(body fluid sample)

Patient serum added which contains antibodies to

HIV antigen

Steps in the ELISA

(Question 30)

Primary (1) antibody

Variable domain binds
antigen; constant domain
free
Unbound antibodies washed away

Steps in the ELISA

(Question 31)
e

e e

Secondary (2) antibody

(conjugated to horse
radish peroxidase)
Variable domain of 2
antibody binds constant
domain of
1 antibody

Antisera added which contains anti-human

secondary antibody-enzyme conjugate

Steps in the ELISA

(Question 32)
e
e

Secondary (2) antibody

Variable domain of 2
antibody binds constant
domain of 1 antibody
Unbound antibodies washed away

Steps in the ELISA

(Question 33)

Chromagen or substrate added

(tetramethylbenzidine or TMB)

Addition of chromagenic substrate results in color reaction

Color intensity increases with concentration (titer) of antibodies
Darker color = higher titer

Steps in the ELISA

Primary (1) antibody

NEGATIVE REACTION:
There are no antibodies present that bind HIV antigen

Steps in the ELISA

NEGATIVE REACTION:
All primary antibodies washed away

Steps in the ELISA

(Question 34)
e
e

e e

Secondary (2) antibody

NEGATIVE REACTION:
There are no primary antibodies for the
secondary antibodies to bind

Steps in the ELISA

NEGATIVE REACTION:
All secondary antibodies washed away

Steps in the ELISA

Substrate added

NEGATIVE REACTION:
There is no enzyme to carry out the color reaction
NO COLOR CHANGE

Scoring ELISA
(Question 35)

Compare color to positive and negative

controls
Indicate the range of dark blue
The larger the range of color, the more
concentrated the antibodies
Highest concentration of antibodies in
those samples that were originally
infected