Monoclonal Antibodies: What are they ?

Why are they important in science?

Monoclonal antibody discovery

Monoclonal antibodies were first

produced in a laboratory by Kohler and
Milstein in 1975.
What is an antibody?

Los anticuerpos
Antibodies are son glicoproteínas
glycoproteins
producidas y secretadas en gran
produced and secreted in large
cantidad por las células plasmáticas
amounts by plasma
resultantes de la activación y
diferenciación de los linfocitos B.
The essential function of antibodies
La función esencial de los
is toanticuerpos
activate es
effector mechanisms
activar mecanismos
that allow the removal
efectores que of the antigen
permitan la
fromeliminación
the body. del antígeno del
organismo.
Ab structure?
Antibody structure
Variable region of heavy
chain
Antigen binding site

Light chain Variable region of

light chain
constant region of
light chain

Heavy chain

Constant region of heavy

chain
Polyclonal & monoclonal Abs

Polyclonal Abs Monoclonal Abs

Protein

Cell lines
Peptides
cDNA
 Polyclonal & monoclonal Abs
Animal blood
containing Abs

Antigen injected
into the animal

Memory B cell

Animal
selection Secreting plasma
depending cells
on their size
Antigen activated B
cells

Time of production: 2 months

Time of production: 4-6 months

Polyclonal & monoclonal Abs

Polyclonal & monoclonal Abs 20% of the Abs

are specific
limited amount
of Ab

100% of the Abs

are specific

unlimited amount
of Ab
 Ab production process

Antigen
2 Fusion

PEG

+
1 Immunization
Hybridomas
3 selection

Ab characterization and IHC

4 ELISA
validation
1. Antigens

Different types of antigens (any substance which provokes an

adaptive immune response)

Peptides proteins cDNA Cell lines

2. Immunization

1st immunization

Different types of antigen 14 days

2nd immunization

14 days
3rd immunization

Peptides 10 days

Neg Serum testing Pos

Proteins
4th immunization

cDNA
3 days

Cell lines

FUSION
3. Fusion
HPRT- HPRT-

HPRT-
HPRT-

PEG HPRT-

HAT Selection
 3. Hybridomas selection (I)

10 days in the
incubator

More than 24.000

hybridomas

S
C
R
E
E
N
I
N
G 10 days 9 days 7 days 5 days 3 days 1 day
 3. Hybridomas selection (II)

WB/IP

Immunohistochemistry ELISA
Flow Cytometry
Double Screening

C+
Cloning technique

96 cells in
19,2 ml
Count the cell Medium

Monoclonal Ab

ELISA

Pos

IHC
Ab production

Freezing and supernatant production

Supernatant
Monoclonal Ab

Monoclonal Ab

Freezing down the hybridomas

Ab characterization
Ab 1 Ab 2 Ab 3
4. Abs characterization and validation

IF
CF

WB ChIP
IHC
 Abs in basic research

Location of DNA
Chromatin
binding sites Chromatin
Immunoprecipitation
Immunoprecipitation Isolate cell
ELISA populations
Dot Blot ELISA
Dot Blot
Presence of Flow Cytometry
Flow Cytometry
a substance Western Blotting
in a sample Western Blotting
Immunofluorescence
Immunoprecipitation Immunofluorescence
Immunoprecipitation Analyze cells or
Immunohistochemistry subcellular
Immunohistochemistry localization

Radioimmunoassay
Radioimmunoassay
Measure the amount of Localize antigens
a substance by in cells or in a tissue
a radioactive label