monoclonal antibody production hybridization and charecterization hybridoma screening, priming, methods of antibody charecterization

1. Monoclonal antibody production
by hybridoma technology &
characterization
Submitted by
Ranjitha H.B
P-2082
BTY

2. • Antibody- Y-shaped protein produced mainly
by plasma cells which neutralize pathogens such
as bacteria and viruses.
• Polyclonal antibodies- secreted by different B cell ,
each identify a different epitope
• Monoclonal antibodies- made by identical immune
cells, which bind to the same epitope

3. Monoclonal Antibodies
• Class of highly specific antibodies produced by the clones of a
single hybrid cell
• Produced by fusing a B cell secreting the antibody with a
myeloma cell capable of growing indefinitely - Hybridoma
technology
• Fused cell called hybridoma
• Uses - Proteomics research, diagnosis of diseases & treatment
of diseases (infections & cancer).

4. •The term hybridoma was coined by Leonard Herzenberg
1964- Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using
HAT selection media
•Hybridoma technology- Kohler & Milstein in 1975
Discovery of hybridoma technology

6. HAT medium hypoxanthine, aminopterin & thymidine
Nucleotide synthesis is essential for cell survival
De nova pathway
Salvage pathway
De nova pathway is blocked in all cells
Simple sugars nucleotides
Aminopterin
B cell
HGPRT+
Myeloma cell
HGPRT-
Hybrid cells
HGPRT+
Hypoxanthine Guanine
HGPRT
Myeloma cell
HGPRT-

7. How hybrid cells are selected in HAT medium
HAT medium
Unfused B cells
undergo normal
cell death
Large scale culture of
hybrid cells. HGPRT+
from B cell
Myeloma cells
cannot grow in
HAT medium
Myeloma cells
are HGPRT- &
de nova
pathway is
blocked by
aminopterin.
So cannot
grow in HAT
medium

8. PRODUCTION OF MONOCLONAL
ANTIBODY

9. Materials
Mice (Balb/c or A/J), 6 to 10 weeks old
P3.653 myeloma
Antigen (125 μg per mouse is optimal): Small antigens can be conjugated to
keyhole limpet hemocyanin
Adjuvants (alum or Freund’s adjuvant; complete and incomplete)
TCD (T-Cell depletion) buffer: Hanks’s balanced salt solution + 10mM HEPES + 0.3% BSA
NH4Cl, 0.16M
Antimouse Thy 1.2 antibody 1:500
Rabbit complement- Reconstitute in 1mL of cold UPW, dilute 1:12 in TCD buffer, and
filter sterilize
serum-free medium: MEM

10. PEG (polyethylene glycol): Melt 10.5mL of PEG 1450 (Sigma) in a 56◦C water bath; add
19.5mL of warm sterile MEM(pH 8.3 to pH8.7)
HAT medium:
SCM (spleen-conditioned medium
8-Azaguanine stock, 10mM
MEM, 10% fetal calf serum, 0.1mM 8-azaguanine (for maintenance of P3.653 myeloma)

11. Step 1: - Immunization Of Mice
Antigen
Mice (Balb/c or A/J),
6 to 10 weeks old
Antigen: Intact cells, whole
membranes, &
microorganisms
+ Adjuvants
Immunized every 2-3 weeks

12. Step 2: Screening of Mice for Antibody
Production
After several weeks of
immunization
Spleen cells
Antigen
Measurement of serum antibodies
ELISA & Flow cytometry
Antibody titer is high
Cell fusion can be performed
titer is too low
Boost

14. • Step 3: Preparation of Myeloma Cells
To ensure their sensitivity
to hypoxanthine-
aminopterin-thymidine
(HAT) selection
Maintain the P3.653
myeloma cell line in MEM
+ 10% fetal calf serum + 8-
azaguanine- week before
cell fusion

15. • Step 4: Fusion of Myeloma Cells with
Immune Spleen Cells
ratio of 4:1

16. Hybridoma Development
• Single immunized spleen cells with myeloma cells in
presence of PEG
• Cells are plated on 96 well plates containing feeder cells
derived from saline peritoneal wash of mice(murine BM
derived MΦ)

17. • Step 5: Cloning of Hybridoma Cell Lines by “Limiting
Dilution” or Expansion and Stabilization of Clones by
Ascites Production

18. Screening
• Selection in HAT medium
• Limiting dilution
• Clonal culture
• Sub cloning

19. Production in cell culture ( In-vitro)
Batch tissue culture method:
• Grow hybridoma cells in batches
• Purify Mabs from the culture media
• Fetal bovine serum commonly used
• low concentration (below 20 mg/ml)
• denaturation during concentration
Semi permeable membrane based system :
• A barrier – hollow fibre or a membrane
• Larger compartment containing culture media
• Smaller chamber to isolate cells and Mabs
• High concentrations (10-160 mg)
• Method of choice for large scale production

20. Production in animals (in vivo)
Mouse ascites method:
• Hybridoma cells injected in mouse
• Produce ascites
• Fluid contains high concentration of
Ab’s
• No further concentration required
• Purification required
• Easy and inexpensive

21. Priming
• Generation of ascites fluid
• Most commonly used- Pristane (tetramethyl penta
decane), others- Freund’s Incomplete Adjuvant (FIA)
• Prime adult female mice of at least 6 weeks of age or
retired breeders

22. Cont……
• If administering Pristane as the priming agent, do not
exceed a volume of 0.2 ml
• Administer FIA IP once (not to exceed 0.3 ml)
• Standard interval between priming & inoculation of
hybridoma cells is 10-14 days

23. Hybridoma Inoculation(In vitro)
• Test hybridoma cells for presence of pathogens using
PCR-based or species-specific antibody production assay
• Inject primed mice 10^5- 10^7 hybridoma cells IP 0.1-0.5
ml
• Monitor mice at least once daily for the first 7 days

24. Ascites/Tumor Growth
• Abdominal distension will usually be noted 7 to 10 days
after inoculation
• 3 survival taps
• weight gain should not exceed 20% of the original body
weight
• No signs of pain or distress (18-22G needle to remove
excess fluid)

25. Ascites fluid collection
• Each mouse will yield from 2.0 ml to over 5.0 ml
of ascites fluid
• Euthanize animals by cervical dislocation
• Clean abdomen with 70% ethanol
• Make an incision in the skin over the abdomen
while holding the mouse over the collection
container
• Drain all the fluid into container and transfer into
centrifuge tube

26. Cont…
• Centrifuge ascites fluid at 1500 rpm for 5 minutes
• Separate supernatant from blood cells and store at
-80°C if necessary
• Animals will be kept for a maximum of 30 days
• If ascites has not developed within this timeframe,
animals will be euthanized as per Rodent
Euthanasia SOP

27. Ethical issues :
• Freund’s complete adjuvant (FCA) (to enhance the immune response): painful
lesions at the injection site.
• Pristane as a "priming" agent - granulomatous reactions
• Respiratory distress: due to ascites.
• Shock - rapid fluid loss
• FCA should not be used more than once in individual mice.
• The volume of FCA and pristane used should not exceed 0.1ml and 0.2ml
respectively.
• Individual mice should not be inoculated with adjuvant more than 3 times.
• Ascites fluid should only be harvested once at the time of euthanasia

29. Characterization of MABs
Identification of class and sub class, antigen
specificity

30. Antibody purification
• Affinity chromatography: reversible interaction between a
binding protein (e.g. antibody) and a ligand (e.g. antigen or
Protein A)
i) Fc binding protein purification
ii) Antigen-specific purification

31. Methods for antibody characterization

32. Enzyme-linked immunosorbent assay,
or ELISA
• For the detection and quantification of MABs in
supernatants

33. Western blotting
• Identification of proteins in complex mixtures
• validating that antibodies bind to a target protein of
the expected size
• posttranslational modifications

34. Immunohistochemistry
• Information on protein expression in tissues, at
both cellular and subcellular levels, can be
provided by immunohistochemical (IHC)
analysis

35. Immunofluorescence microscopy
• Discrimination between nuclear or cytoplasmic
staining is achievable and in some cases membranous
staining can be recognized
Fixation of cells
permeabilization
primary antibody
secondary antibody conjugated with
fluorescent tag
immunofluoroscent microscopy

36. Epitope mapping
• Method used to determine binding sites, or
'epitopes', of antibodies on their target antigens
• X-ray crystallography
• mutagenesis of the antigen
• site-directed masking of epitopes

37. Isotyping of murine monoclonal
antibodies
• Qualitative determination of isotype of MABs IgG1, IgG2a,
IgG2b, IgG3, IgM or IgA
• By capture enzyme-linked immunosorbent assay (ELISA)

38. IsoStrip Mouse Monoclonal Antibody Isotyping Kit ( Sigma Aldrich)
Pierce™ Rapid Antibody Isotyping Kit – Mouse
( Thermo Fisher Scientific)
Mouse Monoclonal Antibody Isotyping Test Kit from AbD Serotec
( Bio-Rad )
Antibody Isotyping (eBioscience)
Isotyping Kit for Mouse Monoclonal Antibody (Sino Biological Inc.)
RapidYield Mouse Monoclonal Antibody Isotyping Kit (Crystal Chem's )
 Mouse Antibody Isotyping Kit (Cell Biolabs, Inc.)

39. • THANK YOU