Presented To:

Dr. Snober Mir

Associate Professor,
Department of Bioengineering,
Integral University
Presented By:
Aiman Fatima
1800100346
Ayushi Rastogi
1800100263
Zoinks! Scooby Doo, where are you?
Save me from the werewolf !

Oh! I’m dying of my sickness. I

can’t come.

Oh! Come on, boy. You are way too

much drama. Your antibody army
will take care of you.
 Good old days

Year Scientists Contribution

1890 Emil von Behring and Described antibody activity against

Shibasaburo Kitasato diphtheria toxins

1897 Paul Ehlrich Side-chain theory, coined the term

‘antibody’

1945 Linus Pauling Suggested lock and key interaction of

antigen and antibody

Late Herman Nathaniel Divalent nature of antibody

1940s Eisen and Fred Karush
Angel of the West (antibody sculptor made by Julian Voss-Andreae)
1950 Porter Cleavage of antibody into Fab and Fc

1952 Douglas H. Campbell Specific combining site of antibody < 700Å

and Bullman

Svedberg Mol wt. of antibody 1,60,000 Da

1960 Henry Bence Jones Bence Jones protein

1961 Edelman and Poulik Discovered light and heavy chains

1963 Porter Suggested Y shaped structure of antibody

1970 Kabat and Wu Hypervariable region

1972 Rodney R. Porter and Chemical structure of antibodies

Gerald M. Edelman

1976 Susumu Tonegawa Genetic rearrangement as means of variation

in antibodies [1]
What are antibodies?
Classification of antibodies
Parameter Ig A IgD IgE IgG IgM

Mol wt. 150,000 180,000 190,000 150,000 900,000

(Da)

Sed. 9S 7S 9S 7S 19S
coefficient

Heavy chain α δ ε γ µ

% in serum 10-15 0.2 0.002 80 5-10

Valency 2 2 2 2 10
Subclasses IgA1, IgA2 - - IgG1, IgG2 -
IgG3, IgG4

Presence Tears, saliva, Present on Present on Internal Newborn

mucous, mature B cell mature B cell bodily fluids
breast milk, surface surface
bronchial ,
digestive tract
and
genitourinary
tract
secretions,
Function Protects external Lymphocyte Type 1 Combats Primary
body surface by activation and hypersensitivi microbes and agglutinin
preventing suppression ty, protects their toxins, [1][2][3]
attachment of external activates
bacteria and virus mucosa complement
system
 Antigen-antibody interaction
The antigen-antibody interaction is analogous to the enzyme-substrate
reaction. This interaction is non-covalent wherein antigenic determinant or
epitope interact with VL/VH domain of antibody, especially hypervariable
regions. The region of antibody that binds to epitope is called paratope. The
noncovalent interaction are reinforced through hydrogen bonds, ionic
bonds, van der Waal and hydrophobic interactions. Antibodies incorporate
either of three mechanisms:
a) Complement activated system by IgG and IgM
b) Cell mediated cytotoxicity by immunoglobulins
c) Opsoninization [4]
 Polyclonal Antibody
 A polyclonal antibody is a collection of many immunoglobulins, each
generated from a different B-cell clone. These antibodies target different
epitopes, or binding sites, on a single antigen.
They are secreted via different B cell lineages.
 Production of Polyclonal Antibody
1. ANIMAL SELECTION:
a) Amount of PAb needed
b) Ease of obtaining blood samples
c) Phylogenetic relationship between the antigen and the animal species
d) Intended use of the PAb
e) Age of animal
f) Health
2. ANTIGEN PREPARATION:
a) Size
b) Extent of aggregation
c) Relative nativity of protein antigens
d) Antigen quantity
e) Asepticity
f) Toxicity
g) Reactivity
h) Purity
i) Quality control of injection process
j) Cross-reactivity
3. ADJUVANT SELECTION: Adjuvants are a group of structurally
heterogenous compounds which evoke or increase immune response to
an antigen.
• Freund’s complete adjuvant
• Freund’s incomplete adjuvant
•Aluminium compounds like phosphates (AlPO4) and hydroxides
(Al(OH)3)
• Quil-A® adjuvant
• Iscoms
• Montanides
• TiterMax™
•RIBI™
• Specol
4. INJECTION PROTOCOL:
a) Routes of administration: The choice of injection route is shaped to
some extent by the choice of the animal species and adjuvant, as well
as by the character, quantity, and volume of the antigen.
The most frequently used routes of injection for PAb production are
subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.), intraperitoneal
(i.p.), and intravenous (i.v.). The choice of animal species may also
eliminate some injection routes. The i.d. route is not recommended in small
rodents (mouse, rat, and hamster), and the i.p. route is not recommended in
larger animals (rabbit and larger).
b) Volume: The maximum injection volumes depend on animal species,
injection route, and injection mixture.
c) Number of Injection Sites: The antigen/adjuvant volume can be
administered as a single injection or as multiple injections of low volumes.
In several European countries, a maximum of four injection sites is
suggested when emulsions are used but it is preferable to administer
antigen/adjuvant mixtures at one site only.
d) Booster Injections: In general, a booster can be considered after the
antibody titer has reached a plateau or begins to decline. When the first
immunization is performed without a depot-forming adjuvant, the antibody
titer usually peaks 2 to 3 wk after immunization. When a depot-forming
adjuvant is used, a booster injection likely follows at least 4 wk after the
first immunization. Usually, a maximum of two or three booster injections
are recommended.
 Primary injection (Day 0) Booster injection(s)
(Day 28 and/or
later)

With adjuvant Without adjuvant With adjuvant Without adjuvant

Subcutaneous Intravenous Subcutaneous Subcutaneous

Intramuscular Subcutaneous Intramuscular Intramuscular

Intradermal Intramuscular Intradermal Intravenous

Intraperitoneal Intraperitoneal

Intradermal Intradermal
5. POST-INJECTION OBSERVATION: After immunization, animals
should be monitored daily and examined for specific side effects at least
three times per week. Examination and palpation of the injection site are
essential to evaluate side effects of the injected mixture. After booster
injection via the i.v. or i.p. route, it is imperative to monitor the animals
during subsequent hours for any anaphylactic reactions. In mammals,
antibody responses during the experiment can be monitored by obtaining
and evaluating a blood sample for antibodies in the serum. In chickens,
because antibodies are excreted in the eggs, titers can be studied in the egg
without an invasive action. Upto 10% of the circulating blood volume can
be taken on a single occasion from healthy animals. After this maximum
blood volume has been collected, animals need to rest for 3 to 4 wk.
6. COLLECTION AND PURIFICATION OF THE ANTIBODIES:
Blood is then extracted from the animal and then purified to obtain the
antiserum. Exsanguination must be performed under general anaesthesia
and is best carried out by heart puncture. It should result in the death of the
animal. Euthanasia should be in accordance with the 2000 Report of the
American Veterinary Medicine Association (AVMA 1 ) panel on euthanasia.
Antiserum obtained from animals will not only contain antibodies against
the antigen artificially introduced in the laboratory, but it will also contain
antibodies to any other antigens to which the animal has been exposed
during its lifetime. For this reason, antisera must first be “purified” to
remove other antibodies before using the antibodies for research or
diagnostic assays. [4] [5] [6]
 Polyclonal antibody in therapy
• Digoxin Immune Fab is the antigen binding fragment of polyclonal
antibodies raised to Digitalis derivative as a hapten bound to a protein and
is used for the reversal of life-threatening digoxin or digitoxin toxicity.
• Rho(D) immune globulin is made from pooled human plasma provided by
Rh-negative donors with antibodies to the D antigen. It is used to provide
passive immune binding of antigen, preventing a maternal active immune
response which could potentially result in hemolytic disease of the
newborn.
• Rozrolimupab is the anti-RhD recombinant human polyclonal antibody
composed of 25 unique IgG1 antibodies and is used for the treatment of
immune thrombocytopenia purpura and prevention of isoimmunization
in Rh-negative pregnant women.
 Advantages of Polyclonal antibody
•The technical skills needed to produce polyclonal antibodies is not as
demanding.
•They're inexpensive to make and can be generated fairly quickly, taking up to
several months to produce.
•PAbs are heterogeneous, which allows them to bind to a wide range of antigen
epitopes.
•Because PAbs are produced from a large number of B cell clones, they're more
likely to successfully bind to a specific antigen.
•PAbs remain stable in different environments, such as a change in pH or salt
concentration, which allows them to be more applicable in certain procedures.
•Additionally, depending on the amount needed, PAbs can be made in large
quantities in relation to the size of the animal used. [7]
 References
1. Khan, FH. (2009). “Antibodies.” The Elements of Immunology. Pearson
2. Kuby J et al. (2007). “Antibodies: Structure and function”. Immunology. (Ed. 5th)
3. Khan, FH. (2009). “Antibodies.” The Elements of Immunology. Pearson
4. Rao, CV. (2005). “Antigen and antibody interaction.” Immunology. Narosa
Publishing House
5. Janeway, CA. (2012). “The Humoral Immune Response.” Immunobiology. Taylor
and Francis.
6. Leenaars, M and Hendriksen, CFM. (2005). “Critical Steps in the Production of
Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations.” ILAR
Journal, Volume 46, Issue 3, Pages 269–279, Oxford Academics.
7. Lipman NS et al. (2005). “Monoclonal Versus Polyclonal Antibodies:
Distinguishing Characteristics, Applications, and Information Resources”. ILAR
Journal.
 Images
1. https://scoobydoo.fandom.com/wiki/Werewolf_(Who%27s_Afraid_of_the_Big_Ba
d_Werewolf)
2. https://tenor.com/view/sick-scooby-doo-ihate-the-flu-gif-11219220
3. https://comicbook.com/gaming/2019/03/02/mortal-kombat-11-shaggy-roster-leak/
4. https://en.wikipedia.org/wiki/Angel_of_the_West#/media/File:AngeloftheWest.jpg
5. Screenshot from Kuby’s Immunology
6. Screenshot from Kuby’s Immunology
7. https://www.labome.com/method/Mouse-Antibody.html
8. https://www.amoebasisters.com/parameciumparlorcomics/antibody-hats
9. https://nanocomposix.com/pages/antibody-selection-and-purification-for-lateral-
flow-rapid-tests#target
10. https://courses.lumenlearning.com/microbiology/chapter/polyclonal-and-
monoclonal-antibody-production/
Thank You!