Understanding Antibodies: Structure, Production, and Labeling Techniques

Antibodies and

immunofluorescence labeling

http://www.cellsignal.com/common/content/content.jsp?id=apps-immunofluorescence

Green: primary antibodies against LAMP1 Protein (lysosomal

associated membrane protein 1)

What is an antibody?

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

http://www.biology.arizona.edu/immunology/tutorials/immunology/05t.html

Antibodies, or immunoglobulins, are glycoproteins

composed of 

2 heavy 

and 

2 light chains.

Both light and heavy chains have different 

variable

regions 

depending how the DNA is arranged and can

form 

more than 15,000,000 combinations

“Somatic

Recombination” is

the mechanism by

which some DNA

is removed from

the antibody

coding region

Variable regions

How do you create a specific

antibody?

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

http://en.wikipedia.org/wiki/Hybridoma_technology

1.

Immunize mouse to desired

antigen

2.

Isolate 

B cells 

(mortal) from

spleen

3.

Cultivate 

myeloma cells

(immortal)

4.

Fuse both cell types

5.

Separate individual cells and

cultivate

6.

Screen for antibody production

7.

Multiply and harvest successful

hybridoma cells

Different  immunolabeling

techniques

•

Immunofluorescence straining: antibodies are

conjugated with a fluorescent dye that can be

excited to emit detectable light

•

Immunoenzymological staining: antibodies are

conjugated with an enzyme that catalyzes the

transitions of an added substrate into an insoluble

particle that can be localized

•

Immunocolloidal gold staining: antibody is

conjugated with gold that is used as a marker

http://www.immunohistochemistry.us/immu

nohistochemistry-staining.html

QUIZ 1!

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

Why can different antibodies bind to so many

different biomolecules?

2.

Why do you think the B cells are fused with

myeloma cells?

3.

What is the main difference between

immunofluorescence staining, immunoenzymological

staining, and immunocolloidal gold staining?

Function of immunofluorescence

Antibodies target

specific biomolecules

(antigens) and reveal

their distribution and

density throughout the

sample via conjugated

fluorophore

Green = AlexaFluor 488nm dye conjugated to actin; 

Blue = DAPI

; 

Red = auto-fluorescence

http://alizeepathology.com/samples/1

Primary/Direct

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

http://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence

•

Uses a single type of ‘antibody

•

The ‘primary’ antibody binds directly to target

biomolecule (antigen) and fluoresces via the

attached fluorophore

Secondary/Indirect

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

http://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence

•

Uses 2 antibody types

•

The ‘primary’ antibody binds directly to the target

biomelocule (antigen) but has no attached

fluorophore

•

The ‘secondary’ antibody binds to the ‘primary’

antibody and fluoresces via the attached fluorophore

Example of primary/direct

immunofluorescence

http://www.scbt.com/datasheet-362617.html

SLBP (stem-loop

binding protein)

antibody binding to

hairpin loops on

mRNA in Hep G2

cells (liver tissue

with hepatocellular

carcinoma)

Example of secondary/indirect

immunofluorescence

http://www.birmingham.ac.uk/facilities/clinical-immunology-

services/autoimmunity/liver-associated-antibodies/Liver-antibodies.aspx

Smooth muscle

antibody binding to

actin, vimentin,

desmin, and tubulin

(all microfilaments)

in Hep G2 cells

(liver tissue with

hepatocellular

carcinoma)

Primary versus Secondary

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

TIME

Primary:

Less steps require less

time

Secondary:

Additional steps require

additional time.

Primary versus Secondary

immunofluorescence

Interatction with immunoglobulins

COMPLEXITY

Primary:

Less steps in the

protocol makes the

experiment less

complex

Secondary:

Additional steps,

selection of secondary

antibodies, and

potential background

interactions add

additional complexity

Primary versus Secondary

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

COST

Primary:

Primary antibodies with

an attached fluorophore

(conjugated) are

typically more expensive

than unconjugated

primary antibodies

Secondary:

Secondary antibodies

are less expensive and

can used against

different primary

antibodies

Primary versus Secondary

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

SENSITIVITY

Primary:

Less conjugated

antibodies bind overall,

so the signal is less

intense

Secondary:

Several secondary

antibodies bind to the

primary antibody and

produce an amplified

signal

Primary versus Secondary

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

FLEXIBILITY

Primary:

Pre-conjugated primary

antibodies have limited

flexibility

Secondary:

Ability to use different

secondary antibodies

increase flexibility of

experiment

Primary versus Secondary

immunofluorescence

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

SUMMARY!

Primary:

•

Less time required

•

Less complexity

•

Higher cost

•

Less sensitivity

•

Less flexibility

Secondary:

•

Higher time required

•

Higher complexity

•

Less cost

•

Higher sensitivity

•

Higher flexibility

QUIZ 2!

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

Is primary or secondary immunofluorescence

more complex?

2.

If you were attempting to detect a biomolecule

that likely has very low concentration in the

sample, would you choose to use secondary or

primary immunofluorescence?

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

C

o

a

t

c

o

v

e

r

s

l

i

p

s

i

n

p

o

l

y

e

t

h

y

l

e

n

e

i

m

i

n

e

(

f

o

r

i

n

c

r

e

a

s

e

d

a

d

h

e

s

i

o

n

)

2.

Rinse coverslips with water

3.

Dry coverslips

4.

Grow cells on glass coverslips

5.

Rinse with Phosphate-based saline (PBS)

General Procedure

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

E

i

t

h

e

r

I

n

c

u

b

a

t

e

c

e

l

l

s

w

i

t

h

1

0

0

%

m

e

t

h

a

n

o

l

o

r

4

%

(

p

a

r

a

)

f

o

r

m

a

l

d

e

h

y

d

e

i

n

P

B

S

2.

W

a

s

h

c

e

l

l

s

t

h

r

e

e

t

i

m

e

s

w

i

t

h

c

o

l

d

P

B

S

Fixation

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

I

n

c

u

b

a

t

e

s

a

m

p

l

e

s

w

i

t

h

P

B

S

a

n

d

0

.

1

-

0

.

2

5

%

T

r

i

t

o

n

X

-

1

0

0

(

a

d

e

t

e

r

g

e

n

t

t

h

a

t

i

m

p

r

o

v

e

a

n

t

i

b

o

d

y

p

e

n

e

t

r

a

t

i

o

n

)

2.

W

a

s

h

c

e

l

l

s

w

i

t

h

P

B

S

t

h

r

e

e

t

i

m

e

s

(

O

p

t

i

o

n

a

l

)

P

e

r

m

e

a

b

i

l

i

z

a

t

i

o

n

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

Incubate cells with

-

1

%

B

S

A

(

b

o

v

i

n

e

s

e

r

u

m

a

l

b

u

m

i

n

)

-

2

2

.

5

2

m

g

/

m

l

g

l

y

c

i

n

e

(

r

e

a

c

t

s

w

i

t

h

a

n

y

e

x

c

e

s

s

f

o

r

m

a

d

e

h

y

d

e

)

i

n

P

B

S

+

t

w

e

e

n

2

0

(

a

s

u

r

f

a

c

t

a

n

t

)

f

o

r

3

0

m

i

n

u

t

e

s

2

.

I

n

c

u

b

a

t

e

c

e

l

l

s

w

i

t

h

p

r

i

m

a

r

y

a

n

t

i

b

o

d

i

e

s

i

n

1

%

B

S

A

i

n

P

B

S

+

t

w

e

e

n

2

0

f

o

r

1

h

o

u

r

a

t

r

o

o

m

t

e

m

p

e

r

a

t

u

r

e

Blocking and incubation

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

3

.

W

a

s

h

c

e

l

l

s

t

h

r

e

e

t

i

m

e

s

i

n

P

B

S

4

.

I

n

c

u

b

a

t

e

c

e

l

l

s

w

i

t

h

s

e

c

o

n

d

a

r

y

a

n

t

i

b

o

d

y

i

n

1

%

B

S

A

f

o

r

1

h

o

u

r

a

t

r

o

o

m

t

e

m

p

e

r

a

t

u

r

e

i

n

t

h

e

d

a

r

k

5

.

W

a

s

h

a

n

t

i

b

o

d

y

s

o

l

u

t

i

o

n

t

h

r

e

e

t

i

m

e

s

w

i

t

h

P

B

S

i

n

t

h

e

d

a

r

k

Blocking and incubation

Immunofluorescence protocol for

staining cell cultures

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

Mount coverslip with a drop of mounting medium

2.

Seal coverslip

3.

Store in darkness

Mounting

LAST QUIZ!

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

If you were examining a human cell culture with

indirect immunofluorescence using bovine primary

antibodies and rabbit secondary antibodies, what

type of albumin serum would you use when

blocking and incubating the cell culture?

General references

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1.

http://www.bio.davidson.edu/genomics/method/IMF.html

2.

http://www.abcam.com/secondary-antibodies/direct-vs-

indirect-immunofluorescence

3.

http://www.microscopemaster.com/immunofluorescence-

microscopy.html

4.

http://en.wikipedia.org/wiki/Antibody

5.

http://atlasgeneticsoncology.org/Genes/CEACAM1ID40044

ch19q13.html

6.

http://www.biology.arizona.edu/immunology/tutorials/immun

ology/05t.html

7.

http://rockland-inc.com/BasePage.aspx?id=41002

8.

http://www.piercenet.com/method/overview-antibody-

production-purification

9.

http://www.scbt.com/datasheet-362617.html

Academic References

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

1. Agrawal A, Godar DE. Simultaneous Detection and

Semiquantification of DNA Damage in Normal and Apoptotic Cells:

Triple-Immunofluorescent Labeling Using DAPI, Antibodies, and TUNEL.

Applied Immunohistochemistry & Molecular Morphology

. 2012;20:402-

409.

2. Bordeaux J, Welsh AW, Agarwal S, et al. Antibody validation.

BioTechniques

. 2010;48:197-209.

3. Galluzzi L, Aaronson SA, Abrams J, et al. Guidelines for the use and

interpretation of assays for monitoring cell death in higher eukaryotes.

Cell Death Differ

. 2009;16:1093-1107.

4. Zinchuk V, Zinchuk O, Okada T. Quantitative colocalization analysis of

multicolor confocal immunofluorescence microscopy images: Pushing

pixels to explore biological phenomena. 

Acta Histochem Cytochem

.

2007;40:101-111.

Indirect fluorescence picture

example

A fluorophore is a fluorescent chemical

compound that can re-emit light upon light

excitation

DAPI = blue (nuclei)

TUNEL = green (apoptosis)

CPD (cyclobutane pyrimidine dimers)

antibody = red (DNA damage)

Agrawal A, Godar DE. Simultaneous Detection and Semiquantification of DNA Damage in Normal and Apoptotic Cells: Triple-Immunofluorescent Labeling

Using DAPI, Antibodies, and TUNEL. 

Applied Immunohistochemistry & Molecular Morphology

. 2012;20:402-409

S

k

i

n

a

n

d

v

a

g

i

n

a

l

t

i

s

s

u

e

s

e

x

p

o

s

e

d

t

o

U

V

d

a

m

a

g

e

a

t

t

i

m

e

o

f

d

a

m

a

g

e

(

a

,

b

)

,

1

h

o

u

r

p

o

s

t

-

d

a

m

a

g

e

(

c

,

d

)

,

a

n

d

2

4

h

o

u

r

s

p

o

s

t

d

a

m

a

g

e

(

c

,

f

)