Production of polyclonal and

monoclonal antibodies in the lab

Dr. Pablo Baldi

FFYB-UBA
B CELL LIFE CYCLE
 A “NATURAL HISTORY” OF ANTIBODIES

Many BL having
B LYMPHOCYTE MATURATION IN
different specificities
are released from bone
1 BONE MARROW
marrow each day

ACTIVATION OF ANTIGEN-SPECIFIC B
ANTIGEN 2 CELLS (CLONAL EXPANSION)

ANTIBODY SECRETION AND

3 EFFECTOR FUNCTIONS
Genes encoding variable regions (VH and VL) of human Igs
GENE RECOMBINATION FOR VH
Different LB
epitopes in a single
protein molecule
 Polyclonal vs. Monoclonal
When an individual is immunized with an Ag…

Different clones of B cells

Ag recognize different epitopes from
the Ag, or the same epitopes but
with different affinity

Activation, clonal expansion

(proliferation) and…

production of specific Abs

Serum from the immunized host is always polyclonal → contains a mixture of

Ab produced by all the B cell clones that recognized the Ag
Polyclonal vs. Monoclonal

Ag

To obtain a monoclonal
Ab (MAb) you will need
to isolate a single B cell
clone and keep it viable
in vitro for a long time.
 Polyclonal vs. Monoclonal

Polyclonal Monoclonal
Ig isotypes and Mixture (IgM, IgG1, Only one (e.g. IgG1)
subisotypes IgG2a, IgG2b,IgG3,
IgA)

Light chains Mixture k and l k or l

Epitopes recognized Several Only one

Ab affinity Mixture Only one

Protein composition Mixture One single protein

Immunization plan for polyclonal Ab production
Adjuvants: substances that
activate APCs (enhanced
antigen processing and
presentation to T cells)

Any appropriate method

according to the characteristics
of the Ag
 Although polyclonal Ab have many applications in
biotechnology and research, for some uses it is
preferable to have an Ab (reagent) of constant and
defined chemical characteristics, specificity, affinity
and biological activity.

MAb
Ag Q: Is it possible to separate specific B
cell clones and grow them in vitro for
producing homogeneous Ab?
A: Yes, they can be isolated, but will
die in a few days

B lymphocytes

Antibodies
 César Milstein Georg Köhler
(1927-2002) (1946-1995)
Argentina Germany

“Continuous Cultures of Fused Cells Producing Antibody of

Predefined Specificity”
Nature 256: 495-97, 1975

Nobel Prize in Medicine and Physiology 1984

together with Niels K. Jerne (Denmark)
1 2
Antiserum (polyclonal)
Antigen
anti-1
3 anti-2
bleeding
anti-3
Hybridomas
(Mabs)

anti-1
PEG
Spleen
cells anti-2

anti-3
Fusion
Myeloma cells (HGPRT-) Clones
 Myeloma cells
Mouse tumor cells (lymphoid origin). NS0, NS-1 (from BALB/c) are the most
widely used.

✓Can grow indefinitely in vitro

✓Can induce tumors in vivo

✓Do not produce Ab per se (before fusion)

✓Deficient in the gen coding for HYPOXANTHINE-GUANINE-PHOSPHORIBOSYL-

TRANSFERASE (HGPRT) → enzyme required for the nucleotides synthesis
through the rescue pathway

Selection marker → these cells can only grow in media containing the compounds
required for nucleotides synthesis through the de novo pathway.
What do we obtain after this aleatory fusion?

Spleen cells Myeloma cells (NSO)

Limited life Unlimited life
span span (tumoral)
HGPRT+ HGPRT –

Fusion

Limited life Unlimited

Limited life span life span Unlimited
span HGPRT + Unlimited HGPRT- life span
HGPRT + life span HGPRT-
HGPRT+
 HGPRT: synthesis of
purines from guanine or
Purines synthesis
hypoxantin (H). Reuse
pathway

HGRPT H
DNA
X
de novo synthesis A synthesis

TK T

TK: synthesizes TMP

from thymidine (T)
TMP synthesis

AMINOPTERIN: antagonist of folic acid that inhibits the

de novo synthesis of purines and thymidine
 PRODUCTION AND SELECTION OF HYBRIDOMAS
HGPRT+
TK+

Secretes MAb

Spleen cell
(no proliferation in vitro) Synthesize purines
and pirimidines
PROLIFERATES
FUSION
Hybridoma
H
HGPRT- HGPRT+
TK+
A
TK+
T
No purines synthesis
Myeloma cell DEATH
(proliferates in vitro)
 Expansion and first screening

➢ Allow hybridoma growth for at least 1 week in HAT medium.

➢ FIRST SCREENING: which wells contain hybridomas that
produce Ab of interest (specific for our antigen)?
Specific Ab screening
ELISA
HR HR HR Anti-mouse Ig,
P P P
conjugated to
enzime (HRP)
Ab in culture
supernatant

Ag

-
+
 Cloning
Isolate the hybridomas of interest (Ag-specific) to get one clone per well
(monoclonality)

Limiting dilution

4.6 ml 230 cells/ml

Dispense 100 µl/well 5 cells/well

+ 4 ml medium
Dispense 100 µl/well 1 cell/well

+1 ml medium
Dispense 100 µl/well 0.5 cells/well

Incubate for 7-10 days

Check for wells with only one clone (optical microscope)
ELISA to check for specific Abs
ELISA (+) wells containing only one clone are the best candidates to be followed
Production of MAbs

MAb concentration is
up to 100 times
higher in ascitic fluid
Commercially available
antibodies
MAbs and PAbs are available against a vast range of
molecules and for many applications
 Detection of cell surface
or intracellular molecules
through
Flow Cytometry and
Fluorescence Microscopy
 Use of MAbs in research

Mamary carcinoma cells (MCF7) undergoing mytosis, displaying positive staining

for cytokeratin (green), tubulin (red) and DNA (blue)
Blocking/neutralizing Abs

Against Against
soluble membrane
mediators receptors
Cytokine-blocking antibodies
TLR-blocking
antibodies
Abs in quantitative capture ELISAs
 etc. etc.
etc.
etc. etc. etc.
etc.
etc.
Use of a home-made Mab in our lab

Preparation of CP antigen
Brucella
cytoplasmic
fraction

Anti-LPS mAb coupled

to gel matrix

LPS-free cytoplasmic
proteins
 Clinically used MAbs

❑Neutralization of cytokines (generally

proinflammatory ones) and other soluble factors

❑Blocking of cytokine receptors

❑Binding to celular surface molecules (opsonization

for cell depletion)
Limitations for the clinical use of murine MAbs

As murine Abs differ from human Abs in their AA sequence and glycosilation
patterns, human therapy with mice-derived Mabs results in the recognition of Mabs
by the human immune system leading to the production of …

Human anti-mouse antibodies (HAMA)

Subsequent doses of the immunotherapy can lead to

Hypersensitivity Reactions and/or Diminished Efficacy of
the treatment.
Development of recombinant MAbs

Murine V domain Murine CDR Immortalized

bound to human C bound to human B cells
domain human Ab
frame