Antisera are used to determine which antigens are on the red cells. The test using antisera and the patient's red cells is called the front cell group. The polyclonal antisera used in the front group are obtained from plasma apheresis of donors stimulated with soluble antigens (A substance from pig mucosa and B substance from horse mucosa). Alternatively, monoclonal antisera can be prepared from cultured cell lines. The development of monoclonal antibodies obtained from cultures of cells secreting antibodies called hybridomas has made available a new source of ABO typing reagents.

1. VISHNU MG
ASST.PROFESSOR
IPMS,MES MEDICAL COLLEGE
KERALA

2. Antisera are used to determine which antigens are on the red cells.
The test using antisera and the patient's red cells is called the
front cell group.
The polyclonal antisera used in the front group are obtained from
plasma apheresis of donors stimulated with soluble antigens (A
substance from pig mucosa and B substance from horse mucosa).
Alternatively, monoclonal antisera can be prepared from cultured
cell lines.
The development of monoclonal antibodies obtained from cultures
of cells secreting antibodies called hybridomas has made available
a new source of ABO typing reagents.

3. MONOCLONAL REAGENTS:
Murine (mouse) monoclonal anti-A and anti-B
reagents prepared using Hybridoma technology
These monoclonal antisera are very specific and very
potent (sensitive).
They are useful when investigating both weak and
extra antigens, e.g., weak subgroups of A or acquired
B phenomenon.

4. HYBRIDOMA
TECHNOLOGY FOR
MONOCLONALANTIBODY
PRODUCTION

5. The first step in making a Hybridoma is to generate
antibody producing B cells.
This is done by immunizing a mouse against the antigen
of interest.
Multiple immunizations are
performed over a period of weeks
until an appropriate titer is achieved.
Intra peritoneal injections are the most common method for
delivering the antigens into the mice.

6. Next, it must be determined if the mouse is
producing the antibodies of interest.
If the mouse is producing the desired antibody, its
spleen is removed and dissociated in culture
medium to release the resident B cells.
The culture medium also includes cells from
special mouse myeloma cell line. These tumor
cells can divide indefinitely but cannot produce
antibody.
When polyethylene glycol (PEG) is added to the
mixture, some of the two types of cells fuse to
form a Hybridoma cell.

7. The next step is to separate the fused Hybridoma cells from the
unfused B cells and myeloma cells.
The myeloma cells die when placed in a Special culture
medium called Hypoxanthine, Aminopterin and Thymidine
(HAT) medium.
In this medium, only fused cells survive because Aminopterin
in the medium blocks the de novo purine synthesis pathway.
Hence, unfused myeloma cells die as they cannot produce
nucleotides by de novo or salvage pathway.
Unfused B cells will die because they lack the ability to survive
in culture; they have a short life span.

8. In this way only the B cell-myeloma hybrids survive. These cells
produce antibodies (Property of B cells) and are immortal (a property of
myeloma cells).
The incubated medium is then diluted into multi-well plates to such an
extent that each well contains only 1 cell. Then the supernatant in each
well can be checked for desired antibody.
Since the antibodies in a well are produced by the same B lymphocyte,
they will be directed towards the same epitope, and are known as
monoclonal antibodies.
Hybridoma colony is established; it will continually grow in culture
medium like RPMI-l640 (with antibiotics and foetal bovine serum) and
produce the antibody.

9. NEWER APPROACHES
TO MONOCLONAL
ANTIBODY PRODUCTION

10. 1. Heterohybridoma:
This is a way of generation of human antibodies by
taking peripheral blood lymphocytes from individuals
sensitized to the Rh D antigen and immortalizing them
by infecting with Epstein-Barr virus (EBV).
Such EBV transformed cells can also be fused with
mouse myeloma cells (Heterohybridomas) to give
better results

11. 2. “Humanized” Mice
Mice with severe combined immunodeficiency (SCID)
can be humanized by infusing human peripheral blood
lymphocytes (PBL).
Responses have been obtained to tetanus toxoid and
group A and Rh D antigens. This strategy is currently
experimental but with modification may produce useful
antibodies in the future.

12. 3. Genetic Engineering:
Monoclonal antibodies maybe genetically engineered so
that the antigen specific binding site (hyper variable region)
is of rat and mouse origin but the remainder of the antibody
is human.
This procedure has the potential advantage of minimizing
the development of anti-mouse/rat antibodies an important
consideration when these are used therapeutically.

13. ANTISERAS USED FOR GROUPING IN
BLOOD BANK
Anti-A
Anti-B
Anti-AB
Anti D
Anti H Lectin
Anti A1lectin.

14. CONTENTS OF GROUPING RAGENT
Blood grouping reagents contain monoclonal murine
IgM antibodies in a buffer solution.
The solution is a phosphate buffer containing sodium
chloride, EDTA and bovine material.
This reagent contains < 0.1% sodium azide and the
government regulations specify that anti-A be colored
blue, anti-B be colored, yellow Anti-AB is colorless
and Anti D is colorless as a quality control (QC)
measure.

15. Anti A is blue because it uses a dyes called
Methylene blue or trypan blue, Patent Blue
that is mixed with Anti A
Anti B is colored yellow because of the dye
acriflavin or Tartrazine.
Anti-AB is colored pink because of using
eosin, now commercially available Anti AB is
colorless.

16. Anti-H Lectin
Anti-H Lectin blood grouping reagent is
prepared from an extract of Ulex
europaeus seeds, diluted with a sodium
chloride solution containing bovine
albumin.

17. Anti-A1 Lectin
Anti-A1 Lectin blood grouping reagent
is prepared from an extract of Dolichos
biflorus seeds, diluted with a sodium
chloride solution containing bovine
albumin

18. STORAGE AND STABILITY
Store the opened /unopened products at 2-8°C
until the expiry date detailed on the product
label.
Failure to store the products at the correct
temperature, for example, storage at higher
temperature or repeated freezing and thawing
may result in accelerated loss of reagent activity.