ANTICORPI MONOCLONALI. Adapted from M ilstein (1980) Scientific American , Oct. p.58. 1. 1. 2. 2. 3. 3. 4. 4. 1. m. 3. m. 2. m. 4. m. Antigen. Spleen cells. Immunization. + Myeloma. B cell. 1. 3. Cell fusion. 2. 1. 2. 3. 4. 4. x. 1. 2. 3. 4. Monoclonal
Pure single Ab
A mixture of all Ab
If a single B cell was picked up and cultured, then it will produce only one kind of antibody.
But B cell can not survive well in the culture.
Each B cell produces only one kind of antibody, which binds to its specific antigen.
Conventional antiserum is the mixture of all antibodies produced by B cells from spleen.
If B cell is fused with myeloma, the fused cell might be cultured and produce antibody.
Each hybridoma line can produce pure single antibody, called monoclonal antibody.
Myeloma cell can be cultured in the test tube, but can not produce useful antibody.
After immunization, the mouse spleen contains B cells producing specific antibodies.
Individual B lymphocyte hybridoma
is cloned and cultured.
Secreted antibodies are collected
from culture media
Antibodies that are collected
from sera of exposed animal
multiple antigenic sites
of injected biochemical.
ONE antigenic site
of injected biochemical
1975 Kohler and Milstein
Multiple myeloma (human disease)
by Sendai virus
Bence Jones proteins in urine
1. Hyperimmunize mouse with Ag
2. Fuse B cells with tumor fusion partner (+ PEG)
3. Limiting dilution (96 wells)
to fractionate fused cells
in HAT media selection
HAT media (hypoxanthine, aminopterin, thymidine).
B cells die (mortal, HGPRT-positive)
Tumor cells die (HGPRT deficient and cannot utilise salvage pathway)
Fusions live (HAT Resistant and immortal)
The de novo pathway can be inhibited using aminopterin,
which inhibits the transfer of methyl groups
from activated dihydrofolic acid.
cells needHypoxanthine and Thymine as sources of purines and pyrimidines for the salvage pathway.
Myeloma cells are HGPRT- and cannot create nucleotides in the salvage pathway.
Plasma cells are HGPRT+ and can utilize hypoxanthine in the salvage pathway.
in vitro material is less concentrated
contain high [mAb]
and minor contamination
with mouse Ig
Or use Ag or epitope
Technique can be used to purify specific protein from a mixture
Co-immunoprecipitation – interacting proteins also found in the immunoprecipitateImmunoprecipitation
Modelli di precipitazione
con la doppia immuno-
diffusione eseguita su un
antisiero e 2 diversi
A: epitopi identici
B:nessun epitopo in
Separazione elettroforetica degli Ag
Archi di precipitazione
Ponendo in grafico l’altezza degli archi contro la concentrazione
Si otterrà una retta di taratura che può servire per determinare
la concentrazione di Ag in un campione incognito
(Presenza o assenza di gonadotropina)
IFA of Ehrlichia chaffeensis
in DH82 cells, 400X
Bullous pemphgoid with linear deposits
of IgG at dermal-epidermal junction
Very good rapid diagnostics
The dark blue spot represents a positive clone.
The light blue spots are positive controls,
the next two wells to the right are negative controls
% conc. Ag
Marcato legato a Ab
Conc. Ag non marcato
Could be cleaved out by papain digest;
pepsin digestion even better, as it produces F(ab’)2
Often it is enough
for diagnostic purposes –
and it is easier to produce
Anti Botulinum toxin
Equine FAB fragment product
made by DoD at time of Gulf War,
The size and specificity
of the ScFv fragment
may allow for attachment
to cryptic sites
Determination of HER2-protein overexpression
(semiquantitative DAKO Hercep Test™)
Fluorescence in situ hybridisation (FISH)
of HER2 amplification
Clone those genes
and express them
as a recombinant protein
in the protein expression system
Sales of Rituxan/
MabThera by Year
Common side effect –
Vascular leak syndrome.
Could be relieved
by usage of mutated toxin
Bunch of other toxins
could be used
single chain ribosome inactivating proteinSaponin (from soapwort)
For T-cell leukemia could be
targeted via CD7
Monoclonal antibodies, which are specific for a certain antigen, i.e. prostate carcinoma cells, can be tagged with a radioactive tracer,which when injected into the body will localize in areas of these recurrent carcinoma cells
OctreoScan in a patient
with metastatic small cell carcinoma of the prostate