Immunobiology Assignment:

TOPIC: humoral immunity, B cell activation, specific antibodies, Monoclonal

and polyclonal antibodies.

SUBMITTED TO: MA’AM TANZILA

SUBMITTED BY:
1. ISHA YASIN (L1F19BSBC0032)
2. IRAM ANWAR (L1F19BSBC0033)
3. IMAN ZAHIR (L1F19BSBC00
4. MANAL GUL (L1F19BSBC00
5. MAHRUKH (L1F19BSBC00
Humoral immunity
DEFINITION:
Humoral immunity is the type of adaptive immunity. In this immunity, B-lymphocytes and
plasma cells generate the antibodies against any antigen which then stimulate the T-lymphocytes
to kill that foreign substance or antigen that had entered in our body. These antibodies are also
responsible for generating the stimulants or chemicals like interferons and complement proteins
etc, that helps in the removing the antigen from body.

INTRODUCTION:
Immunity and immune system are the most important terms of science and our body. Immunity
is basically termed as the capability of our body against any microorganism or pathogen which
let us stay stable under these condition and protect our body. Many of the cells, tissues, fluids
and secretions take part in developing this capability of our body which all together considered
as the immune system. Immunity could be of two major types; Innate immunty and Adaptive
immunity.

Humoral immunity which is the type of adaptive immunity, is also termed as antibody-mediated
immunity. Humoral immunity leads to the production of specific molecules in case of entry of
any pathogen or antigen. That production of antibody by this immunity circulate and move
around the blood and enter tissues in response to that antigen. They are present in the
extracellular spaces and are considered as most effective microbes or toxins. It is considered as
the part of both innate and adaptive immunity. Immunoglobulins and antibodies are those
specific glycoproteins that are generated by our body in response to any foreign substance or any
antigen that has entered are body and these are produced by B-lymphocytes and plasma cells.
Antibodies are considered as the prime factors in stimulating the humoral immunity.
HUMORAL RESPONSE:

Humoral or humoral mediated immunity is named as this because of the difference in its
mechanism of action which shows how it works when any of the foreign substance enters our
body. It works by the mechanism in which it requires the specific substance that is present in our
body fluids termed as humor.

It is also known as antibody-mediated immunity because of how it works by production of the

specific antibody for specific antigen which means that it is very specific in its mechanism of
action. Specific humoral response is generated in response to each of the antigen or any foreign
substance.

The most important purpose of humoral immunity is to protect the extracellular spaces because
when the intracellular substances enters the body, they move from one host to another host to
spread the infection throughout the body and they navigate from one cell to another kne through
the extracellular path so the humoral immunity's main function is to protect that extracellular
pathway which plays the important route in spreading it throughout the body. So thats the main
reason the antibody production in this immunity is in B cells which is then secreted for the
further development of immunity against pathogens.
Figure 1- humoral immune response

HUMORAL V/S CELL MEDIATED IMMUNITY:

Humoral and cell-mediated immunity, both have many differences but both of them are adaptive
in nature because both of them are types of adaptive immunity which means they get develop
after the birth other than the innate immunity.

CHARACTERISTICS HUMORAL CELL MEDIATED

IMMUNITY IMMUNITY

Antigen specific antibodies Yes No

Antigen recognizing receptors BCR receptors TCR receptors

Triggered by B-lymphocytes T-lymphocytes

Mediation via B-cells, T-cells and Cytotoxic T cells, helper T-

macrophages cells
Time of action Rapid Slow
Secretory cells B-cells T-cells

Major secretion Antibodies Cytokines

ROLE OF ANTIBODY IN HUMORAL IMMUNITY:

Antibodies play an important role in the recognization of antigen and specific receptors. and play
an instrumental role in generating the immune response. It identifys the antigen that has entered
the body or pathogens and neutralize them. They are very specific to different type of antigens.
Moreover, folowing are the processes that are done by antibodies:

 Blocking viral receptors

 Agglutination
 Precipitation
 Stimulate immune response

B cell activation: 
Introduction:

B cell activation is the process that precedes the transformation of B cells to plasma cells and
memory B cells. The plasma cells in turn produce antibodies. The antibodies serve a critical role
in an immune response against foreign attackers in your body.

Importance of Antibodies as products of B Cell Activation

Antibodies are proteins produced by the B cells in response to antigen challenges. The antibodies
mediate humoral immunity which is one arm of the adaptive immune system. Once, produced
antibodies coordinate several effector mechanisms in your body to eliminate the attacking
antigen.

Mechanism:

Neutralization of the antigens

The antibodies in this case specifically bind to all the epitopes (active sites) of the antigens
making the continued multiplication of the antigen/microorganisms come to a halt. This
effectively helps the other cells of the immune system that have receptors for those antibodies to

clear the infection.

Activation of Complement
Through the classical pathway of the complement system, antibodies combine with antigens to
make complexes and initiate the complement activation process on the cell walls of
microorganisms. The ultimate outcome of this process is the formation of the membrane attack
complex (MAC) which helps in making pores on the walls of bacteria which kills them.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

Some cells like the cytotoxic T lymphocytes and the natural killer cells are guided by antibodies
which specifically tag the attacking antigens. These cells have receptors for the Fc regions of the
antibodies and bind them. This binding causes these cells to release their Cytotoxicity granules
(Granzymes and perforins) that help kill the microorganism.

B cell Activation Process:

The process of B cell activation occurs in the secondary lymphoid organs. The secondary
lymphoid organs involved here include the spleen and various lymph nodes around your body.
Firstly, the B cells develop in the bone marrow through seven B cell development stages to
become mature B cells, the last stage of their development.  

The mature B cells are now available in your blood circulation ready to encounter antigens. The
B cells recognize antigens through their B cell receptors (BCRs). For this to happen there must
be specificity between the epitopes (Ab-binding active parts) of the antigens and the BCR
(membrane-bound immunoglobulin).

This activation eventually gives rise to plasma cells as well as memory B cells. The activation of
B cells is important in your body.  During an attack, it is only through this process that your
immune system can make antibodies.  Antibodies are the cornerstones of the humoral immune
response that eliminates the attacker through several effector mechanisms. 

Step-By-Step B cell Activation

The mature B cells are always available in your blood circulation. They can access different
secondary lymphoid organs through the blood vessels. On the other hand, the attacking antigens
are also taken to the secondary lymphoid organs (Spleen, lymph nodes) through the lymphatic
system.

In the spleen and/or lymph nodes, the B cells through BCRs interact with the antigens. The B
cells will scan antigens and the B cells with BCR specific to the antigens will bind. This binding
will act as a switch or key to a complex signaling process that will lead to the multiplication of
that clone of B cells to several copies (clonal expansion).
Germinal Centers’ Activities:

Germinal centers (GCs) are structures that are formed during an immune response in the B cell
zones or follicles. This occurs within the spleen or lymph nodes. We have two paths through
which the B cells can be activated here. This can be through the T-Independent path or through
the T-dependent path of the immune response.

B cell activation sometimes occurs through T-help cells

In the T-independent path immune response, the BCR interacts directly with the specific epitopes
of the attacking antigen. On the other hand, in the T-dependent path, the T helper cells must be
available to help the B cells. Therefore, where the T helper cells’ assistance is needed the B cells
move closer to the T cell zones border.

The T cell through CD40L on its surface interacts with CD40 coreceptor on the surface of the B
cell. There is also going to be upregulation of the cytokine receptors on the B cell surface. The
cytokines whose receptors will be expressed here include IL-21, IL-4 and IL-10.

The T cells (TH2) produce important cytokines (IL-21, IL-4 and IL-10) that help fasten the
process of B cell activation and proliferation in the germinal centers. It is important to note that
in the active state, the mature B cells have IgM/IgD serving as BCR.
However, after activation, the B cells can change (Hyper mutate) to IgG, IgA, or IgE The B cells
may also retain IgM on their surface while some IgM may be available as free serum
immunoglobulin. This act of changing one immunoglobulin isotype to the other is known as
class switching.  

End-Product of B cell Activation – Plasma Cells and Memory B cells

Whichever path they use the B cells once activated will end up being either plasma cells or
memory B cells. Plasma cells are short-lived cells that produces antibodies specific to the
original antigen that was recognized by the mature cells’ BCRs.

The plasma cells will exit the germinal centers and migrate to the bone marrow where they help
clear antigens therein. The memory B cells on the other hand will exit to go into the blood
circulation where they can live for many years. They confer long-lived protection.

Whenever the same antigen they are specific for comes around, it will quickly be dealt with
before sickness sets in your body. Therefore, the long-term protection induced by vaccines in the
process of immunization is partly provided by the memory B cells. The other part is about
memory T lymphocytes. 

In a nutshell, the B cells are activated through the following stages as indicated in the diagram in
this section. The activation process begins with mature B cells interacting with antigens, then
formation of lymphoblast, then GC B cells, and finally, differentiates to either plasma B cells or
memory B cells.
 The flow diagram of the B cell activation process (istock)

Both plasma B cells and memory B cells possess high-affinity BCR for their specific antigen.
Review the diagram above for conceptualization of how this process happens.  Note that plasma
cells are the only cells in the diagram that produce antibodies. They are also the only cells in
your immune system that produce antibodies.
What are specific antibodies?

Each individual IgG molecule is uniquely designed to protect against a specific pathogen. These
molecules are called specific antibodies, and they are usually formed in response to natural
exposure to bacteria and viruses, or through exposure to vaccines.

Antibody production

Antibody production involves preparation of antigen samples and their safe injection into
laboratory or farm animals so as to evoke high expression levels of antigen-specific antibodies in
the serum, which can then be recovered from the animal.

Successful antibody production depends upon careful planning and implementation with respect
to several important steps and considerations:

 Synthesize or purify the target antigen (e.g., peptide or hapten)

 Choose an appropriate immunogenic carrier protein
 Conjugate the antigen and carrier protein to create the immunogen
 Immunize animals using appropriate schedule and adjuvant formula
 Screen serum (or hybridoma) for antibody titer and isotype (also called antibody
characterization)

Antibody purification

Antibody purification involves isolation of antibody from serum (polyclonal antibody), ascites
fluid or culture supernatant of a hybridoma cell line (monoclonal antibody). Purification methods
range from very crude to highly specific:

 Crude—precipitation of a subset of total serum proteins that includes immunoglobulins

 General—affinity purification of certain antibody classes (e.g., IgG) without regard to
antigen specificity
  Specific—affinity purification of only those antibodies in a sample that bind to a
particular antigen molecule

Which level of purification is necessary to obtain usable antibody depends upon the intended
application(s) for the antibody.

Antibody characterization

Antibody characterization involves three kinds of activities that are usually performed at various
stages throughout an entire antibody production and purification project:

 Screening—identifying antibody samples having antigen-binding specificity

 Titering—measuring antibody concentration and functional assay titer
 Isotyping—determining a monoclonal antibody class and subclass identity

Screening is first required during production to identify which animals and hybridoma clones
are producing a high level of antigen-specific antibody. This is usually accomplished using
ELISA techniques. Antibody concentration can be estimated using either a general protein assay
or a species- and immunoglobulin-specific method, such as with specialized micro agglutination
assay kits.

Antibody titer is related to concentration but refers more specifically to the effective potency of
a given antibody sample. Measuring titer usually means determining the functional dilution of an
antibody sample necessary for detection in a given assay, such as ELISA.

Isotyping involves determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a)
of a monoclonal antibody. This is a critical step in antibody production, as it is necessary for
choosing an appropriate purification and modification method for the molecule. Isotyping is
most easily accomplished with commercial, ready-to-use antibody isotyping kits.
Antibody fragmentation

Purified antibodies can be modified for particular uses by several methods including
fragmentation into smaller antigen-binding units, conjugation with enzyme or other detectable
markers, and immobilization to solid supports. Most often antibodies are used in whole-molecule
form. However, the performance of some techniques and experiments can be improved by using
antibodies whose nonessential portions have been removed.
Antibody fragmentation refers to procedures for cleaving apart whole antibody molecules and
removing portions that are not necessary for binding antigen. Fab and F(ab)'2 are antibody
fragments of IgG that are most frequently created and utilized by researchers.

Antibody labeling and immobilization

Antibodies are produced and purified for use as antigen-specific probes. However, their utility in
any given technique (ELISA, western blotting, cellular imaging, and immunohistochemistry)
depends upon having a mechanism to secondarily detect the antibody.

Techniques that utilize antibodies for immunoprecipitation or other form of affinity purification
depend upon mechanisms for attaching or immobilizing them to chromatography media (e.g.,
beaded agarose resin). Strategies for accomplishing this involve the same considerations and
chemical methods as antibody labeling.

Monoclonal Antibodies

Monoclonal antibodies are a type of medicine that has changed the way we prevent and treat
diseases ranging from cancer and immune system disorders to childhood viral infections.They
are based on natural antibodies, which are proteins produced by the body to defend itself against
disease, but they are created in a laboratory and mass-produced in factories.

More than 30 years ago, the first monoclonal antibody product was licenced. Millions of people
have benefited from more than 100 such treatments since then. In the last six years,
approximately 50 of these have been introduced to the market.

This is one of the fastest-growing areas of biomedical research and a growing segment of the
pharmaceutical market.

Monoclonal antibodies work :-

They bind to their specific target while causing no harm to anything else in their path. This target
does not always represent a 'foreign intruder,' such as a virus. Antibodies can be programmed to
attach to specific molecules in the body, for example, to suppress the immune response when it
overreacts; this phenomenon, which occurs in some Covid-19 patients, is known as a 'cytokine
storm.'Monoclonal antibodies have been used safely and effectively to treat a growing number of
diseases, some of which were previously difficult to treat, due to their numerous applications.

Production:-

Production of monoclonal antibodies is a time-consuming and costly process.

First, scientists isolate the appropriate antibodies from human blood. They then replicate and
mass produce them in large quantities.

The majority of monoclonal antibodies are produced in Chinese hamster ovary cells, which are
grown in large bioreactors for 10 to 15 days. The resulting antibodies are then purified and
packaged for easy administration. This entire process takes a long time and requires expensive
materials.

Manufacturers are investigating ways to reduce production costs, such as through novel
technologies and the use of alternatives to hamster cells (such as algae, yeast, and plants), which
would change the way these drugs are made.

Application:-

 The majority of monoclonal antibodies on the market are used to treat noncommunicable
diseases such as rheumatoid arthritis and cancer.
  Cancer immunotherapies have saved the lives of millions of people worldwide over the
last few decades. Monoclonal antibodies have revolutionised the treatment of a variety of
cancers, including breast cancer, for which the drug Herceptin has been a game changer.
 Only seven of the more than 100 licenced monoclonal antibodies are for treating and
preventing infectious diseases, despite the fact that many more are in the works,
including candidates for SARS-CoV-2, the virus that causes Covid-19.
 Monoclonal antibodies have the potential to revolutionise the way we treat and prevent
infectious diseases. There are already encouraging signs.
 Two experimental Ebola antibody therapies are being used successfully as part of an
emergency access programme in the Democratic Republic of the Congo.
 Several antibodies that can fight different strains of HIV are also being developed.

Polyclonal Antibodies
Polyclonal antibodies are antibodies produced by various B cell lineages in the body (whereas
monoclonal antibodies come from a single cell lineage). They are a group of immunoglobulin
molecules that react against a specific antigen, each of which recognises a different epitope.

Dean and Abelseth (1973) obtained concentrated specific antibodies from hyperimmune serum
by using inactivated rabies virus (RABV)-infected mouse brain suspensions as a source of
antigens for immunisation of animals. Larger mammals are frequently preferred because they
can collect more serum. An antigen is injected into the mammal, which causes B lymphocytes to
produce immunoglobulin G (IgG) specific for that antigen.
The primary goal of animal antibody production is to obtain high-titered, high-affinity antisera
for use in research or diagnostic tests. Chickens, goats, guinea pigs, hamsters, horses, mice, rats,
and sheep are commonly used in PAb production.

Production:-
To initiate an amplified immune response, an antigen/adjuvant conjugate is injected into a
suitable animal. The animal is expected to have developed antibodies against the conjugate after
a series of injections over a set period of time. The animal's blood is then extracted and purified
to obtain the desired antibody.

Inoculation is carried out on an appropriate mammal. Adjuvants are substances that are used to
improve or enhance an immune response to antigens. Most adjuvants include an injection site,
antigen depot, and slow release of antigen into draining lymph nodes.

Many adjuvants contain or function directly as:

 surfactants that promote protein antigen molecule concentration over a large surface area,
and
 Immunostimulatory molecules or characteristics Adjuvants are commonly used in
conjunction with soluble protein antigens to boost antibody titers and induce a prolonged
response with memory.

Such antigens are generally ineffective immunogens. Most complex protein antigens induce
multiple B-cell clones during the immune response, resulting in a polyclonal response. Adjuvants
generally improve or worsen immune responses to non-protein antigens, and there is no system
memory.

Chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep are commonly used for
polyclonal antibody production. For this purpose, the rabbit is the most commonly used
laboratory animal. It based on;

 The amount of antibody required,

  The relationship between the donor of the antigen and the recipient antibody producer
(generally, the more distant the phylogenetic relationship, the greater the potential for
high titer antibody response)
 The characteristics [e.g., class, subclass (isotype), complement fixing nature] of the
antibodies to be made should all be considered when selecting animals. Immunization
and phlebotomies are stressful procedures, and specific pathogen free (SPF) animals are
preferred when using rabbits and rodents. The use of such animals has been shown to
significantly reduce morbidity and mortality caused by pathogenic organisms,
particularly Pasteurellamultocida in rabbits.

The size, extent of aggregation, and nativity of protein antigens can all have a significant impact
on the quality and quantity of antibody produced. To increase immunogenicity and provide T
cell epitopes, small polypeptides (10 ku) and non-protein antigens must be conjugated or
crosslinked to larger, immunogenic carrier proteins. The larger the immunogenic protein, the
better. Larger proteins, even in small amounts, usually result in better engagement of antigen
presenting antigen processing cells, resulting in a more satisfying immune response. Injection of
soluble, non-aggregated proteins is more likely to induce tolerance than a satisfactory antibody
response.
Advantages:-
Polyclonal antibodies have advantages over monoclonal antibodies. The technical skills required
to create polyclonal antibodies are not as difficult. They are relatively inexpensive to produce
and can be completed in a matter of months. Because PAbs are heterogeneous, they can bind to a
wide variety of antigen epitopes. PAbs are more likely to bind to a specific antigen because they
are made up of a large number of B cell clones. PAbs remain stable in a variety of environments,
such as changes in pH or salt concentration, making them more useful in certain procedures.
Furthermore, depending on the quantity required, PAbs can be manufactured in large quantities
in relation to the size of the animal used.

Pharmaceutical uses:-
 Digoxin Immune Fab is an antigen-binding fragment of polyclonal antibodies raised to a
Digitalis derivative as a hapten bound to a protein that is used to reverse life-threatening
digoxin or digitoxin toxicity.
 Rho(D) immune globulin is created by pooling human plasma from Rh-negative donors
who have antibodies to the D antigen. It is used to provide passive immune binding of
antigen, preventing a maternal active immune response that could result in newborn
hemolytic disease.
 Rozrolimupab is an anti-RhD recombinant human polyclonal antibody composed of 25
distinct IgG1 antibodies that is used to treat immune thrombocytopenia purpura and to
prevent isoimmunization in Rh-negative pregnant women.
 REGN-COV2 (Regeneron Pharmaceuticals) - a potential treatment for COVID-19 as well
as a means of preventing SARS-CoV-2 coronavirus infection.
References

1. https://immunostudies.com/blog/b-cell-activation/
2. https://www.ncbi.nlm.nih.gov/books/NBK27142/
3. https://www.ncbi.nlm.nih.gov/books/NBK27142/
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New York, NY.
5. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
6. Sites, D.P., et al. (1976). Basic & Clinical Immunology. Lange Medical Publication,
Los Altos, CA.