MONOCLONAL ANTIBODY, HYBRIDOMA

Monoclonal antibodies are antibodies derived from a single clone of plasma cell. All
have the same antigenic specificity i.e. they are produced against a single epitope of
the antigen

Monoclonal antibodies are produced by Hybridoma technique developed by Georges

Kohler and Cesar Milstein – awarded Noble in 1984

Hybridoma technique is a process where a clone of B cell against a single epitope of

antigen is fused with an immortal cell capable of multiplying indefinitely (eg. Myeloma
cell) to produce a hybridoma cell

Steps of hybridoma technique:

1 – HARVEST MOUSE SPLENIC B CELLS:

• Inject a mouse with antigen of desired epitope – mouse splenic B cells are
activated against this antigen – wait for an interval and harvest these splenic
B cells

2 – MYELOMA CELLS:

• Source of immortal cells – cancerous plasma cells

• Closely resemble mouse B cell

• They produce their own antibody – genetically modified by double mutation –

lose ability to produce antibody, retain ability to multiply indefinitely

3 – FUSION:

• Mouse splenic B cells and mutated myeloma cells fused in polyethylene glycol
broth

• Three types of cells generated:

• Unfused myeloma cells

• Unfused mouse splenic B cells

• Fused hybridoma cells

 4 – PURIFICATION:

• Unfused cells are removed by subculturing on HAT medium which propogates

only the fused clone

FATE OF CELLS ON HAT MEDIUM:

• H – HYPOXANTHINE; A – AMINOPTERIN; T - THYMIDINE

• In mammalian cells –eg. Mouse splenic cells – purine synthesis is by de novo

pathway or salvage pathway

• Aminopterin blocks de novo pathway

• Mouse cells have to perform salvage pathway to survive

• Salvage pathway requires HGPRT and thymidine kinase

• Cells which lack HGPRT can’t grow on HAT – eg – myeloma cells

1. Unfused splenic cells: they can grow – but they are not immortal – do not
survive long

2. Unfused myeloma cells: they cannot grow – they lack HGPRT – cannot
perform salvage pathway

3. Hybridoma cells: GROW and SURVIVE

5 – SELECTION OF INDIVIDUAL HYBRIDOMA CELLS:

• Medium containing the hybridoma cells is diluted and dispersed into multi well
plates

• Diluted to such an extent that each well contains only one cell

• Hybridoma cells producing desired monoclonal antibody is selected by ELISA

using specific antigenic fragment

• Such cells are selectively proliferated

6 – MAINTENANCE OF MONOCLONAL ANTIBODY:

 Selected hybridoma cell is cultured to generate a clone of identical cells

 Concentration: 10 – 60 µg/ml
 Selected hybridoma cell is injected into peritoneal cavity of mouse – multiplies
and produces antibody in ascitic fluid
 Concentration: 1 – 10 mg/ml
Types of monoclonal antibodies:

 Mouse mAb:
Contains 100 % mouse derived proteins
 Chimeric mAb:
34% mouse protein (variable region) and 66% human protein (constant
region)
 Humanised mAb:
Only antigen binding site i.e. CDR is mouse derived (10%)
 Human mAb: 100% human derived proteins

Applications of monoclonal antibodies:

THERAPEUTIC USES:

• To treat various inflammatory and allergic disorders and cancer. Eg:

Infliximab, Rituximab

• As immunotoxin – mAb conjugated with bacterial toxin like diphtheria toxin –

mAb targets surface receptors of target cell – toxin kills the target cell

• Passive immunity – post exposure prophylaxis – Ig against Hepatitis B,

Rabies, Tetanus

• As enzyme – Abzyme – mAb with catalytic activity

DIAGNOSTIC USES:

• Specific mAb is tagged with dye or enzyme to detect specific antigen

• Detection of infection – hepatitis B , Serogrouping of Streptococci

• Tumour detection, Blood grouping, HLA typing, Pregnancy test

• Identification and separation of CD 4 and CD 8 T cells