1. Review of: Continuous cultures of
fused cells secreting antibody of
predefined specificity Köhler G,
Milstein C.-
Name: Eman Abd El-raouf Ahmed

2. introduction
THE manufacture of predefined specific antibodies by means of permanent tissue culture cell
lines is of general interest.
There are at present a considerable number of permanent cultures of myeloma cells1,2 and
screening procedures have been used to reveal antibody activity in some of them.
We describe here the derivation of a number of tissue culture cell lines which secrete anti-
sheep red blood cell (SRBC) antibodies.
The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an
immunised donor. To understand the expression and interactions of the Ig chains from the
parental lines, fusion experiments between two known mouse myeloma lines were carried
out.

3. Important terms
1-An autoradiograph is an image on an x-ray film or nuclear emulsion produced
by the pattern of decay emissions (e.g., beta particles or gamma rays) from a
distribution of a radioactive substance.
2-The direct Plaque assay or Jerne technique, allows the enumeration in agar of
cells producing antibodies to sheep RBC's or other erythrocytes. A
suspension of spleen cells from a previously injected animal is mixed with
the test erythrocytes and molten agar. After the agar has solidified the
mixture is incubated at 37C for one hour, during which time the antibody
producing cells will release antibody that will bind unto the antigen (the
sheep RBC's). Subsequent addition of complement will lyse the antibody
coated erythrocytes, causing the appearance of a clear plaque with the
antibody producing cell in the middle.

4. Indirect Plaque Assay
Erythrocytes will lyse in the presence of complement if
the antibody is of
the lgM class Those cells coated with antibody of the lgG
class will not
lyse because the antibodies are too far apart to bind
complement. However,
the system may be modified to detect lgG producing
cells. The DPA direct plaque
assay technique is DPA Method performed and the
plaques are counted and marked. Anti-lgG is
added and the plate is re-incubated with more
complement. Any new plaques
are the result of the IgG producing cells.

5. HAT Medium
(hypoxanthine-amino
pterion-thymidine
medium) is a selection
medium for mammalian
cell culture

6. practical

7. Continue-practical

8. Analysis of data –figure 1

9. Analysis of the practical figure 2

10. Analysis:

11. analysis

12. Steps for reviewing the article:
Describe the derivation of anumber of tissue culture cell lines which secrete anti-sheep red
blood cell antibodies.
Each cell expresses only one of 2 alleles
2 antibody-producing cells are fused
Products: both parental lines are expressed
Experiment firmed by fusing 2 myeloma cells of the same mouth strain.
*aim of the study:
Provide the background for the derivation and understanding of antibody –secreting hybrid lines
in which one of the parental cells is an antibody –producing spleen cell.
Preparation:
2 myeloma cell lines ofBalB/c origin were used
The first: pi Bul is resistant to 5-bromo-2-deoxyuridine
The second: p3-x63Ag8 prepared from p3 cells
Note:
Equal numbers of cells from each parental fine fused using inactivated sedai virus.
Result from the experiment:
Shows the iso electric focusing pattern of the secreted products of different lines

13. Comment for the first result:
Figure 1 A shows clones derived from different hybrid-sation
experiments and from sub clones of one line are
indistinguishable.
Figure 1h:in which the heavy chain of P3 is no longer observed.
*note:
The cell line p-3 dies when exposed to HAT medium
Spleen cells from an immunized mouse also die in growth
medium.
When both cells are fused by sendai virus and the resulting
mixture is grown in HAT medium, surviving clones can be
observed to grow and become established after a few weeks.

14. Second-figure:
Figure 2a:used SRBC as immunogen which enabled us after
culturing the fused lines, to determine the presence of specific
antibody –producing cells by a plaque assay technique.
Figure 2b:
The hybrid cells were cloned in soft agar and clones producing
antibody were easily detected by an overlay of SRBC and
complement
Figure 2-c:
Individual clones were isolated and shown to retain their
phenotype as almost all the clones of the derived purified line are
capable of lysing SRBC.
Figure 2d:
The clones were visible to the naked eye

15. Figure 3
Figure 3 a:shows the IEF pattern of the material secreted
by 2 such sp hybrid clones
The IEF bands derived from the parental P3 line are
visible in the pattern of the hybrid cells ,although
obscured by the presence of a number of a new bands.

16. Notes:
Cell fusion technique are a powerful to produce specific antibody directed
against a predetermined antigen.
Three different fusion experiments were successful in producing a large
number of antibody producing cells.
3 weeks after the initial fusion 3%clones were positive by the direct plaque
assay.
The cloning efficiency in the experiment was 50%
In another experiment: the proportion of positive clones (0.2%)
In the third experiment: the hybrid population was studied by limiting dilution
analysis.

17. Figure.4
Shows the inhibition of SRBC lysis by a specific anti –IgM antibody.

18. References:
http://www.nature.com/nature/journal/v256/n55
17/abs/256495a0.html
http://web2.uwindsor.ca/courses/biology/fackrell
/Immunology/Ref_Shelf/Jerne/frmain.htm

19. Thanks