Principles, Advantages,
Disadvantages, Preparation of
Antisera, Titration, Avidity
Potency and Specificity
MLS 416 2024.
Introduction/Definition of Terms
● Antisera reagents contain antibodies against a specific antigen
● Antibody is a protein produced by B-lymphocytes that binds to a specific
antigen.
Types of Antiserum
1. Polyclonal antiserum: Contains antibodies to most of all the available epitopes
on the antigen
2. Monoclonal antiserum: Contains antibodies to one epitope. It is made using
identical immune cells that are all clones of a specific parent cell
Polyclonal Antibodies Preparation
● Monoclonal antibodies (mAbs) are generated by identical B cells which are
clones from a single parent cell. This means that the monoclonal antibodies
have monovalent affinity and only recognize the same epitope of an antigen.
● The process begins with an injection of the desired antigen into an animal,
often a mouse, multiple times
● Once the animal develops an immune response, the B-lymphocytes are
isolated from the animal's spleen and fused with a myeloma cell line,
creating immortalized B cell-myeloma hybridomas.
● The hybridomas, which are able to grow continuously in culture while
producing antibodies, are then screened for desired mAb.
Polyclonal Antibodies
Advantages:
● Short production time and low cost.
● Highly stable and tolerant of pH or buffer changes.
● High affinity. Since the antibodies bind to more than one epitope, they can
help amplify the signal from target protein even with low expression level.
This makes these antibodies ideal for immunoprecipitation and chromatin
immunoprecipitation.
● Tolerant of minor changes of antigen. Polyclonal antibodies are less
sensitive to antigen changes (slight denaturation, polymorphism,
heterogeneity of glycosylation) than monoclonal antibodies.
Disadvantages:
● Prone to batch to batch variability.
● Multiple epitopes make it important to check immunogen sequence for
any cross-reactivity.
Preparation of Monoclonal Antibodies
● Monoclonal antibodies (mAbs) are generated by identical B cells which are
clones from a single parent cell.
● Monoclonal antibodies are produced ex vivo using tissue-culture techniques.
The process begins with an injection of the desired antigen into an animal,
often a mouse, multiple times.
● Once the animal develops an immune response, the B-lymphocytes are
isolated from the animal's spleen and fused with a myeloma cell line, creating
immortalized B cell-myeloma hybridomas.
● The hybridomas, which are able to grow continuously in culture while
producing antibodies, are then screened for desired mAb.
Monoclonal Antibodies
Advantages:
● Highly specific recognition of only one epitope of an antigen
● Immortal hybridoma cell lines have the ability to produce unlimited
quantities of antibodies
● High consistency among experiments
● Minimal background noise and cross-reactivity
● Excellent for affinity purification
Disadvantages:
● Developing a monoclonal takes time and requires high technical skills.
● They can produce large amounts of specific antibodies but may be too
specific to detect in across a range of species.
● Vulnerable to the change of epitope. Even a slight change in
conformation may lead to dramatically reduced binding capacity.
Potency and Specificity
● Antisera must be specific for the antigen to be detected under the test
conditions
● The specificity of an antiserum can be established by testing equal volumes of
serum against 5% red cells suspension
● An agglutination reaction would only occur when reacted with coresponding
cells
● The serum to be emploed for grouping must have sufficient titre against the
corresponding cells in use as this determines the potency of the antisera
Titration and Avidity
● Avidity of an antiserum refers to the ability of the antiserum to react very
quickly and strongly with its corresponding antigen
● Avidity is based on affinity, specificity and strength of the serum
● It refers to the strength of binding of single epitope to single antigen binding
site
● A suitable antisera should readily produce complete agglutination in 30
seconds when mixed with corresponding cell suspension
● The avidity of anti A of clinical significance is its ability to detect weak
subgroups of A
Test for Avidity
● If anti A reacts against A cells, agglutination occurs within 10secs
● If anti B reacts against B cells, agglutination occurs within 10 secs
● If antiA + B reacts against B cells, agglutination occurs within 20 secs
** A cells reacts quicker and stronger than the B cells with the AB serum, this is
because the A component in the anti A + B is stronger than the B component
Antisera Titration
● A potent anti A serum for use as a good diagnostic reagent should have a titre
of 512 when titrated against A cells
● Anti B serum 256 titre when titrated against B cells
● Anti A + B titre of 512 and 256 respectively with A cells and B cells
