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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
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
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!
General Method of the Preparation of Bacterial Vaccines, Toxoids, Viral Vaccine, Antitoxins, Serum-immune Blood Derivatives and Other Products Relative to Immunity.
General Method of the Preparation of Bacterial Vaccines, Toxoids, Viral Vaccine, Antitoxins, Serum-immune Blood Derivatives and Other Products Relative to Immunity.
