Cell Fusion for Production of Hybridomas

Question 1

Hybridoma cell

Answer 1

10^8 spleen cells BALB/c female

1-4 x 10^7 non-secreting myelomaas

Question 2

Myeloma cells

Answer 2

P3X63-Ag8.653
Derived from IgGl secreting BALB/c myeloma P3
Lacks HGRT enzyme for salvage pathway of nucleotide biosynthesis and cannot grow in HAT
2-3X size of B cells
1. Permanently growing. (Transformed cell lines)
2. Non antibody secretor.
3. Selection marker. Some way to select the cells from a successful fusion.
Myeloma parent cells should be in log phase growth for optimal fusion. Overgrowth leads to
problems. Keep cell density less than 1x106/ml.

Question 3

Aminopterin

Answer 3

Blocks main biosynthetic pathway for purines and pyrimidines

Kills unfused myelomas

Question 4

Ammonium chloride lysing buffer

Answer 4

Lyse rbc which could otherwise fuse w myelomas

Question 5

Incubation in CO2

Answer 5

B cells v sensitive to alkalinity

Question 6

PEG

Answer 6

Exposure time should be limited bc can cause cell death

Enhances membrane fusion

Question 7

Spleen cells

Answer 7

60-70% B cells
BALB/c female mice
Ig+

Question 8

Sigma adjuvant system

Answer 8

Consists of metabolizable oil (squalene) and modified bacterial products designed to provide immunopotentiation
First 3 injections with antigen+adjuvant
The final boost was antigen alone without adjuvant

Question 9

PMS

Answer 9

Mice are injected 3 times with antigen they are bled to obtain serum
Checked for antigen specific reactivity by ELISA before the spleen cells are used in fusion
Also be used in your screening ELISA assay as a positive control and has been pooled from all the immunized mice

Question 10

Uses for MAbs

Answer 10

1. Purification of proteins
2. Identification of cell populations
3. Diagnostic: E.g. detection of pathogens
4. Immunotherapy= tumor detection and killing.