History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem

History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem
Core Module Immunology Doctoral Training Group GK1660 Erlangen  2011 History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg

TIME LINE - History of Immunology
Discovery of cells and germs ( ) Prevention of Infection (1840 – today) Start of Immunology ( ) The antibody problem: Immunochemistry ( ) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

TOPCIS: Start of Immunology
Preventive Immunization Jenner (1789)-1. designed immunization (1798) Pasteur (1880) – chicken cholera generalized Jenner‘s small pox approach Cellular Immunity Methnikoff (1884) - discovers phagocytic activity Humoral Immunity & Serotheraphy Bering (1890/91) – Tetanus/Diphtheria Ehrlich‘s Sidechain Theory (1897) Cytotoxic humoral immunity and complement Bordet (1899): substance sensibilisatrice + Buchner‘s Alexin Ehrlich (1899): Amboreceptor + Komplement Serodiagnostic (Start of Serology) Widal (1896) – Widal agglutination test for typhoid fever Bordet (1901) - Complement fixation test Wassermann (1905) – Syphilis test Landsteiner (1901) – Blood goups in human Anaphylaxis and Related Disorders (harmless antigens make us sick) Portier & Richet (1902) - Anaphylaxis Arthus reaction (1903) Von Pirquet (1906) - Serum sickness – Allergie Wolff_Eisner (1906) - Heufieber Meltzer (1910) - Asthma C. Bogdan (July) Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1930 Nobel 1913 S. Finotto (July)

TIMELINE: Serum Therapy of Diphtheria
Safe Active Vacci-nation Ramon Klebs discovers bacteria on material from diceased diphtheria patient Behring& Ehrlich (Berlin) 1st serum therapy in humans Roux and Yersin idenify soluble diphtheria toxin Behring 1st Serum therapy (diphtheria) in guinea pigs Roux develops antisera in horses Behring-werke in Marburg 1883 1884 1888 1890 1891 1892 1893 1894 1904 1924 Löffler identifies C. diphtheriae as the cause of diphtheria Behring & Kitasato 1st serum therapy (tetanus) in mice Hoechst (Behring) Industrial production of antisera in sheep Roux & Chaillon (Paris) Serum therapy in humans Park & Williams (NYC) Production of antisera in

Summary: Humoral immunity (1905)
SOLUBLE, INDUCIBLE & SPECIFIC IMMUNITY (Antitoxins, Immunkörper, Amboceptor, Zwischenkörper, Immunisin, substance sensibilisatrice, copula, Desmon, philocytase, fixateur) Antitoxins Behring (1890) Bacteriolysins Pfeiffer (1895) Agglutinins von Gruber (1896) Precipitins Kraus (1897) Hemolysins Belfanti & Crabone (1898) Opsonins Wright (1903) The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[67] In 1905 it was not clear that all these humoral activities can be traced back to the same class of inducible compounds (i.e., the antibody molecule) Today, Antikörper (Antibody) is a neutral term for the common component in all the different biological activities of immune sera Eichmann, Klaus (2000). The network collective: rise and fall of a scientific paradigm JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen‘ Scand. J. Immunol., 19,

The first paradigm in immunology
Discovery of a inducible, soluble and specific activity in the blood (later termed „antibodies“) in 1890 The first paradigm in immunology „Specific immunity induced by antigens is associated with the formation of antibodies“

Another Paradigm in Immunology
„Infections are cleared by cellular and humoral immunity“ 1908 Paul Ehrlich: Ehrlich (1908). Über Antigene und Antikörper. Einleitung in „Handbuch der Immunitätsforschung“. P.1 -10 Very nice overview about the knowledge of antibody and antigen in 1908.

IMMUNOLOGY: Own Discipline
PAUL-EHRLICH-INSTITUT (devoted to serum therapy Paul-Ehrlich Institute für Serum-forschung und Serumprüfung (1896) bis Paul-Ehrlich Institute Bundesamt für Sera und Impfstoffe (1990) Königliches Institut für experimentelle Therapie, Frankfurt 1899 Königliches Institut für experimentelle Therapie + Georg-Speyer-Haus 1922. Frankfurt Ab 1947 Paul-Ehrlich-Institut für Exp. Therapie Paul-Ehrlich-Institut im Jahre 1990 als Bundesamt für Sera und Impfstoffe in Langen bei Frankfurt/Main Das Paul-Ehrlich-Institut für Serumprüfung und Serumforschung Jahre1896 in Steglitz bei Berlin Georg-Speyer-Haus, 1906, Frankfurt

IMMUNOLOGY: Own Discipline
JOURNALS Zeitschrift für Immunitätsforschung (1908) J. of Immunology (1913) Eur. J. Immunology (1970) PROFESSIONAL SOCIETIES American Associaten of Immunologist (1913) Deutsche Gesellschaft für Immunologie = DGfI (1953)

TIME LINE - History of Immunology
Discovery of cells and germs ( ) Prevention of Infection (1840 – today) Start of Immunology ( ) The antibody problem - Immunochemistry ( ) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

THE ANTIBODY PROBLEM ( )

Immunochemistry of Antibodies
Antigens Antibody-Antigen Interaction Purification Detection Identification Quantification Structure

TOPICS: THE ANTIBODY PROBLEM
Immunochemistry Antigens - Features and Origin of Term Antibodies are proteins Antibody quantitation Antibody structure Variable and Hypervariable regions (paratop) Crystal structure Monoclonal antibodies

THE ANTIBODY PROBLEM Antigens

Side Visit - Antigens

Antigen – Orgin of the Term
László Detre a.k.a. Ladislas Deutsch, Ladislaus Deutsch ( , Hungary) Since he believed in Buchner‘s theory, he called in his first publication (1899, in French) the hypothetical substance that induces immunity „Substances immunogenes ou antigenes“ i.e., a substance between a toxin and an antitoxin (just like zymogen is a precursor of an enzyme) In the German version of his article published in 1903, Deutsch accepted Ehrlich‘s theory and used the noun ‚Antigen‘ and states that this is a contraction of ‚Antiisomatogen = Immunkörperbildner‘ Oxford EnglishDictionary indicates that the logical construction should be anti(body)-gen for antibody generating In 1899, Ladislas Deutsch (Laszlo Detre) ( ) named the hypothetical substances halfway between bacterial constituents and antibodies "substances immunogenes ou antigenes". He originally believed those substances to be precursors of antibodies, just like zymogen is a precursor of an enzyme. But, by 1903, he understood that an antigen induces the production of immune bodies (antibodies) and wrote that the word antigen is a contraction of "Antisomatogen = Immunkörperbildner". The Oxford English Dictionary indicates that the logical construction should be "anti(body)-gen" [6] . Immunkörper.[67] László Detre (October 29, 1874, Nagysurány - May 7, 1939, Washington, DC (a.k.a. Ladislas Deutsch, Ladislaus Deutsch [1] ) was a Hungarian physician and microbiologist, [2] the founder and first director of the Hungarian Serum Institute in Budapest. [3] Detre suggested the term "antigen". [1] [4] He is also a codiscoverer of the Wasserman reaction, publishing this finding on humans just two weeks after Wasserman published his findings on apes. [5] JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen‘. Scand. J. Immunol., 19, Eichmann, K. (2008). The network collective: rise and fall of a scientific paradigm. Birkhäuser Verlag,

Antigens - Definitions
Any Substance that combines directly after the key-lock principle (→ Emil Fischer, 1894) with B cell receptor or antibodies (→ Paul Ehrlich) or T cell receptors or MHC Immunogen Substance that induces a humoral or T cell-mediated immune response Hapten Antigen that binds to immune receptors but does not induce an immune response Allergen A substance that provokes an allergic reaction Tolerogen A substance that invokes a specific immune non-responsiveness Superantigen A class of antigens that cause non-specific activation of T-cells, resulting in polyclonal T cell activation and massive cytokine release. IMMUNOGEN. A molecule which can elicit the production of specific antibody upon injection into a suitable host. ANTIGEN. A molecule which can be specifically recognized and bound by an antibody. 2-1. DEFINITIONS a. An antigen is traditionally defined as any substance that will cause production of antibodies and which reacts specifically with those antibodies. This term, however, is incomplete because it emphasizes the production of immunoglobulins. Therefore, the term immunogen was introduced to include biological processes involving proliferation of lymphocytes and synthesis of specific substances or recognition molecules which can specifically combine with the inducing antigen. In physiochemical terms, antigens are macromolecules that possess a high degree of internal chemical complexity. They are soluble or easily solubilized by phagocytic cells of the animal and are foreign to the animal. b. A hapten is defined as a small molecule which by itself cannot stimulate antibody synthesis but will combine with the antibody once formed. When the hapten is conjugated to a protein molecule called a carrier, it can elicit an immune response. 2-2. IMMUNOGENICITY Immunogenicity may be defined as that property of a substance (immunogen) that endows it with the capacity to provoke a specific immune response. Antigenicity, on the other hand, is the property of a substance (antigen) that allows it to react with the products of the specific immune response. Substances that are immunogenic are always antigenic, but antigens are not necessarily immunogenic. a. Antigen Factors. (1) Molecular weight. As a general rule, for a molecule to be immunogenic it should have a molecular weight of 10,000 or more. The greater the molecular weight of a substance, the more likely it is to function as an antigen. (2) Molecular complexity. Large molecular size alone is not enough to confer antigenicity on a substance. A molecule must possess a certain degree of chemical complexity; generally, immunogenicity increases with structural complexity. (3) Solubility. Molecules that are insoluble in body fluids and cannot be converted to a soluble form by tissue enzymes are poor antigens. (4) Foreignness. A substance that the body does not recognize as belonging to or being a part of itself. b. Host-Related Factors. (1) Nonspecific factors. The response to a given immunogen is not only a function of the physiochemical properties of the substance, but also is influenced by several host-related factors, including genetic makeup, age, and host environmental and nutritional status. Existing disease in the host may alter immune response capability. (2) Antigen dose and administration route. As a rule, low antigen doses induce the formation of small amounts of antibody with high affinity and specificity. Low doses injected frequently over long periods of time will induce greater response than large doses over a short period of time. The route of antigen administration greatly affects the nature of the immune response.

Superantigen

Antigens Nach Herkunft Nach chemischen Gesichtspunkten
Natürlich (Proteine, Kohlenhydrate, Nukleinsäuren, bakt. Toxine, Zellen) Synthetisch (Haptene, Polypeptide) Nach chemischen Gesichtspunkten Proteine Kohlenhydrate Nukleinsäuren Konjugierte Proteine (Hapten-Protein) Polypeptide Lipide Nach genetischer Beziehung zwischen Spender und Empfänger Autoantigen - aus dem zu immunisierenden Individuum Isoantigen - aus einem genetisch identischen (syngenen) Spender (Inzucht) Alloantigen - aus einem nichtverwandeten Spender derselben Spezies Xenoantigen - aus einem Spender einer anderen Spezies

Botenstoffe (z.B, Zytokine, Chemokine, Lymphotoxine
Antigens – Recognition by B- and T cells B-Zell- Rezeptor (BZR) Ag Naive Lympho-zyten B T (CD4) T (CD8) MHC II MHC I Ag-Prozessierung & Präsentation Dendritische Zelle THelfer TKiller Effektor- zellen Plasma TRegs Botenstoffe (z.B, Zytokine, Chemokine, Lymphotoxine Effektor- Moleküle T-Zell- Rezeptor (TZR) AK Humorale Immunität Zelluläre Immunität

Epitope und Paratope (Schlüssel-Schloß)
Ag Ag Epitop (Determinante) → Bereich auf dem Antigen, der an den Ag-Rezeptor bindet Paratop → Bereich auf dem Antikörper, der mit dem Epitop des Antigens interagiert VH VL Loops („fingers“) that form the paratop are also called hyper-varibale regions (HV) or complementary determining regions (CDR3)

T Cell Epitopes TH TK B Almost allways processed peptides
Rarely lipids and phospopeptides Must be presented by MHC molecules to TCR Co-receptors are required to stablize binding TH TK Erklärt wieso CD8+ T-Killerzellen nur Zellen mit MHC-Klasse I CD4+ T-Helferzellen nur Zellen mit MHC-Klasse II erkennen TZR CD4 CD8 (α) MHC II (I) + Peptid B Ziel B

FR = framework (Gerüstregion); HV = hypervariabel
Hypervariable Regions or CDRs Die loops that form the paratop are also called hypervaribale regions (HV) or complementary determining regions (CDR3) Wu-Kabat-Plot: AA comparison of VH regions Ag VH VL L-CDR1 L-CDR2 L-CDR3 H-CDR1 H-CDR2 H-CDR3 CDR1 CDR CDR3 Variability AA position FR = framework (Gerüstregion); HV = hypervariabel Kuby, 4th edition

Induction of Antibodies (1924)
Cells Erythrocytes (Belfanti & Carbone) Bacteria (Pfeiffer) Spermien (Landsteiner, Metschnikow, Moxter) Flimmerepithel (von Dungern) Nierenzellen (Metschnikow) Proteins e.g., Toxins (Ehrlich, Behring, …. Albumin Organic compounds coupled to carrier protein (Landsteiner, 1920) Carbohydrates (Heidelberger & Avery, 1924)

B Cell Epitopes - Composition
Ig-Rezeptoren erkennen Proteine Lipide Nukleinsäuren Kohlenhydrate Organische Moleküle oder Haptene (Halb-Ag) Metalle Plastik Aber nur Proteine sind gute T-Zell-abhängige Antigene Die Welt der Antigene (Antikörper generierend) IgM Kurzlebige Plasmazelle IgM +/-TH IgG, IgA, IgE Ag IgD Langlebige Plasmazelle Naive B-Zelle +TH Gedächtnis- B-Zelle

B Cell Epitopes - Conformations

B Cell Epitopes - Conformations
Conformational epitope Linear epitope Neo- Epitope polypeptide chain Denaturation Epitope is lost Epitope remains intact Epitope is new

Antitoxin - Mode of Action

Antitoxins: Mechanism of action (Ehrlich, 1897)
Mechanisms of antitoxic effect of Behring‘s serum therapy? Hypothesis 1 : Antitoxins destroys toxin. Disproved since toxins could be detected on toxin/anti-toxin mixtures Hypothesis 2 (e.g., Roux und Buchner): „Antitoxin soll keine aktive Wirkung auf das Toxin ausüben, sondern in erster Reihe auf die Zellen einwirken und dieselben gewissermassen gegen die Giftwirkung immunisieren“. Hypothesis 3 (Ehrlich): „Gift und Gegengift paaren in den Gewebsflüssigkeiten zu einer Art Doppelverbindung, welche nicht mehr in bestimmten Geweben fixiert wird und welche daher keine Krankheitserscheinungen mehr auslöst.“ Seit B ehr in gs Entdeckung der antitoxisclien Functionen hat die Frage nach dem Wesen dieser Erscheinung andauernd das Interesse und die Arbeit fast alles Vertreter der modernen Richtung gefesselt, wie die zahlreichen diesbezüglichen Publilrationen beweisen. Nachdem die ursprüngliche Annahme von einer Zerstörung des Giftes ' durch den Antikörper als unhaltbar sich erwiesen hatte, nachdem sie11 ergeben hatte, dass in den xphysiologi,cjch neutralem Toxin- Antitoxingemischen noch beide Componenten als solche enthalten sind, stehen sich zur Zeit zwei Meinungen schroff gegenüber, Es kann nach der einen Anschauung sich Gift und Gegengift in den Gewebsiiüssi~eiten zii einer Art Doppelverbindung paaren, welche niclit mehr in bestimmten Geweben fiairt wird und welche daher keine Kranldieitser~cheinun~emn ehr auslöst. Im Gegensatz zu dieser chemisclien Auffassung nehmen einige Autoren, insbesondere R o ux und Buchn er, eine meh indirecte Wirkung des Antitoxins an ; dasselbe soll keine active Wirkung auf das Toxin ausüben, sondern in erster Reihe auf die Zellen einwirken und dieselben gewissermassen gegen die Giftwislcung immunisiren. Die S~hwieri~lredietr Entscheidung Probem: In Tierversuchen keine Untertsuchung möglich Abhilfe: Reagenzglasversuche. P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

- Buchner …. -

1st Theory to Explain Antitoxin (Buchner 1893)
1893 Hans Buchner, Emil Roux, Emmerich & Loew: Toxins are transformed in the body into their corresponding antitoxin Antitoxin, instead of acting directly on the toxin, act direct ly on the living elements (cells) of the organism, preserving them from intoxication. BUCHNER (893). Münchener med. Wochenschr. Ueber Bacteriengifte und Gegengifte. p. 480. EMMERICH, R., LOEW, O. (1901). Über biochemischen Antagonismus. Zentralbl. Bakteriol. Mikrobiol. Hyg. (A) 30:552 EHRLICH (1901). Üeber Toxine und Antitoxine. Die Therapie der Gegenwart. Mai, p.193

- Ehrlich -

Serum and ricin before addition to RBC
Antitoxins: Mechanism of action (Ehrlich, 1897) Mix of anti-ricin Serum and ricin before addition to RBC Experiment: Tubes with blood from un-immunized rabbits Ricin Ricin mediates clumbing of RBC - + + Ricin Oberservation → Anti-ricin toxin prevents ricin (lectin)-mediated clumping of red blood cells in a concentration dependent manner Conclusion → Cellular explanation of Roux and Buchner (hypothesis 2) disproved → First evidence for direct (from mix in vitro) and chemical interaction (from titration) of antitoxin with toxin P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

Ehrlich The antotoxic substance inhibits the morbigenic action of the toxin by neutralizing the toxin, combining with the latter to form a compound of a chemical nature which is devoid of toxicity and without action on the organism. According to this theory, the influence of the antitoxin on the toxin is direct, and does not require the intervention of the living cellular protoplasm. Such was also the belief of Prof. Ehrlich.

Antitoxin – Toxin Interaction
- Chemical reactions -

Antitoxin/Toxin: Chemical Interaction
1. Direct chemical interaction of antitoxin and toxin in solution P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

Strength of interaction of antitoxin and toxin is affected by concentration and temperature Interaction is a chemcal reaction Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299

Start of Immunochemistry
Servate Arrhenius (1907) “I have given to these lectures the title "Immunochemistry" and wish with this word to indicate that the chemical reactions of the substances that are produced by the injection of foreign substances into the blood of animals, i.e. by immunisation [sic], are under discussion in these pages. From this it follows also that the substances with which these products react, as proteins and ferments, are to be here considered with respect to their chemical composition.” Arrhenius, S. Immunochemistry: The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies; The Macmillan Company: New York, 1907, vii. P. 31 I have given to these lectures the title "Immuno-chemistry" and wish with this word to indicate that the chemical reactions of the substances that are produced by the injection of foreign substances into the blood of animals, i.e. by immunisation [sic], are under discussion in these pages. From this it follows also that the substances with which these products react, as proteins and ferments, are to be here considered with respect to their chemical composition. [19] Arrhenius, S. Immunochemistry: The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies; The Macmillan Company: New York, 1907, vii. Considerably more important was the work of Karl Landsteiner, the most recognized pioneer in the field of immunochemistry. In the 1930s, he investigated many of the fundamental principles of this field using an elegant molecular level approach to understanding antibody recognition. He made extensive use of immunoprecipitation, [20] which he called a "serological reaction", as a method to detect the binding of antibodies to antigens. In his authoritative text, The Specificity of Serological Reactions, he wrote: For a considerable period of time after the discovery of serological phenomena and despite an abundance of observations, a method was yet wanting for the systematic investigation, along chemical lines, of specificity in serum reactions. It was indeed clear that serological reactions must somehow be dependent upon the chemical properties of the substances involved ... but insufficient chemical knowledge concerning the available antigens (proteins) ... made a closer analysis impossible. [21]

Savante Arrhenius Svante Arrhenius Sweden Nobel Prize Chemistry 1903 Developed theoretical basis for electro- lytic dissociation and chemical reaction → Nobel Prize Chemisty in 1903 The Arrhenius equation: Formuates emperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction.

Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen
Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Winau F, Westphal O, Winau R (2004). "Paul Ehrlich--in search of the magic bullet". Microbes Infect. 6 (8): 786–9.

Savante Arrhenius ↔ Paul Ehrlich
Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction (irreversible) ARRHENIUS Reversible process with equilibration (A + B AB) In a mixture of antitoxin and toxin, there is a certain quantity of free toxin and antitoxin. Although both believed that the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction, they disagreed on the degree of binding Controversy was negative for Ehrlich since Arrhenius was member of the Noble Prize committee in Stockholm

Antitoxin – Toxin is a Chemical Reaction
Ehrlich and Angelo Knorr demonstrated that neutralization is less rapid in dilute solutions than in concentrated ones. Svante Arrhenius demonstrated the occurrence of limited reactions between antitoxin and toxin analogous to the esterification of an alcohol by an acid, and in such a manner that there always exists, in a mixture of these two substances, a certain quantity of free toxin and antitoxin. the antitoxin inhibits the noxious action of the toxin, even outside the living organism, by uniting with it to form a compound in identically the same manner as when a strong base and a strong acid are brought together. Toxins and Venoms and Their Antibodies. By EM. Pozzi-Eso-T. Authorized Translation by Alfred I. Cohn, Phar.D. i2mo, vii pages.

Ehrlich The antotoxic substance inhibits the morbigenic action of the toxin by neutralizing the toxin, combining with the latter to form a compound of a chemical nature which is devoid of toxicity and without action on the organism. According to this theory, the influence of the antitoxin on the toxin is direct, and does not require the intervention of the living cellular protoplasm. Such was also the belief of Prof. Ehrlich.

1. Direct chemical interaction of antitoxin and toxin in solution P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

Strength of interaction of antitoxin and toxin is affected by concentration and temperature Interaction is a chemcal reaction Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299

Savante Arrhenius Svante Arrhenius Sweden Nobel Prize Chemistry 1903 Developed theoretical basis for electro- lytic dissociation and chemical reaction → Nobel Prize Chemisty in 1903 The Arrhenius equation: Formuates emperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction.

Antitoxin – Toxin Interaction
- Chemical reactions -

Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen
Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Winau F, Westphal O, Winau R (2004). "Paul Ehrlich--in search of the magic bullet". Microbes Infect. 6 (8): 786–9.

Savante Arrhenius ↔ Paul Ehrlich
Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction (irreversible) ARRHENIUS Reversible process with equilibration (A + B AB) In a mixture of antitoxin and toxin, there is a certain quantity of free toxin and antitoxin. Although both believed that the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction, they disagreed on the degree of binding Controversy was negative for Ehrlich since Arrhenius was member of the Noble Prize committee in Stockholm

Antitoxin – Toxin is a Chemical Reaction
Ehrlich and Angelo Knorr demonstrated that neutralization is less rapid in dilute solutions than in concentrated ones. Svante Arrhenius demonstrated the occurrence of limited reactions between antitoxin and toxin analogous to the esterification of an alcohol by an acid, and in such a manner that there always exists, in a mixture of these two substances, a certain quantity of free toxin and antitoxin. the antitoxin inhibits the noxious action of the toxin, even outside the living organism, by uniting with it to form a compound in identically the same manner as when a strong base and a strong acid are brought together. Toxins and Venoms and Their Antibodies. By EM. Pozzi-Eso-T. Authorized Translation by Alfred I. Cohn, Phar.D. i2mo, vii pages.

Immunochemistry regarding the chemical description of the immune response, and Many researchers perceived the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction Ehrlich: the toxin-antitoxin reaction is not bound to living matter, but can also take place in vitro. 9 Controversy between Arrhenius and Ehrlich, as well as the larger part of the debate within the Karolinska Institute over a Nobel prize to Ehrlich, Ehrlich: Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction, Arrhenius looked at it as a reversible process of equilibration, which followed general physical laws.

Ehrlich was extremely absent-minded, aloof, brusque and, in his later
years, eccentric [27]. As head of the Frankfurt Institute for Experimental Therapy he developed a highly demanding and autocratic style of leadership.

The crux of the dispute, then, was the nature of the immune response
The crux of the dispute, then, was the nature of the immune response. Focusing on the molecular structure of the immune reactants, Ehrlich claimed that the interaction of toxin and antitoxin implied a high degree of specificity and, thus, resulted in definite chemical bonds. In principle, he presumed the process to be irreversible, although he admitted that, under certain in vitro conditions, it could be reversible. Apart from some remarkable exceptions which will be discussed later, Arrhenius agreed with Ehflich about the pertinent facts; his criticism lay in their appropriate interpretation. Concentrating on the reactants' physical properties, Arrhenius studied the kinetics and stoichiometry of the process and concluded that the reactions in question were similar to those taking place in highly dissociated electrolytes, so that the result of the process were loose, reversible chemical bindings. According to him, the physical laws which underlay chemical reactions in general should also be applied in immunochemistry. As later developments in biochemistry have shown, neither of these standpoints was totally incorrect, and during the following decades the differences between the two positions gradually disappeared. At that time, however, they seemed unsurmountable.

Als seine bedeutendste Leistung ist die Ausarbeitung der Grundlagen der elektrolytischen Dissoziation anzusehen. Svante Arrhenius Sweden Nobel Prize Chemistry 1903 in Stockholm) war ein schwedischer Physiker und Chemiker und Nobelpreisträger für Chemie. The Arrhenius equation is a simple, but remarkably accurate, formula for the temperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction.

Antigen-Antibody Reactions
THE ANTIBODY PROBLEM Antigen-Antibody Reactions

Two most important advances in the attack on the problem of the nature of
immunological reactions were the discovery that the specific precipitate contains both antigen and antibody (7) and the discovery that antibodies, which give antisera their characteristic properties, are proteins. The verification of these facts was provided by the work of many investigators over a score of years. This work, which is summarized in Marrack’s monograph (6, chap. II), culminated in the preparation of purified antibody by Felton and Bailey (S), Heidelberger and collaborators (9), and others, and the determination of its properties, including amino-acid composition and molecular weight, which show that it is very closely related to normal serum globulin (6, chap. II). Pauling Review

LANDSTEINER The most strikung feature of the humoral immune response is the specific nature with antigen Even a single amino acid change can e distuingished by antibodies Can also distinge distinguish between D and L amino acids

By the mid-1930s, Pauling was beginning to understand that simply knowing the structures of individual proteins was not enough. The essence of life resulted not from individual molecules, but from the interactions between them. How did organisms make offspring that carried their specific characteristics? How did enzymes recognize and bind precisely to specific substrate molecules? How did the body produce antibodies that recognized and bound to specific foreign, invading antigens? How did proteins, these flexible, delicate, complex molecules, have the uncanny ability to recognize and interact with specific molecules? These questions all fell under the heading of biological specificity. To this topic Pauling directed much of his attention during the late 1930s and 1940s. To understand biological specificity, Pauling decided to work first with antibodies and antigens, the understanding of which immunologists such as Karl Landsteiner were beginning to perfect. Pauling met and spoke with Landsteiner on several occasions, and he began his own research program in the late 1930s combining Landsteiner's methods with his own most recent chemical techniques. During a decade of antibody experiments, carried out through the late 1940s, Pauling built a detailed picture of the binding of antibody and antigen at the molecular level. His findings were surprising. Pauling demonstrated that the precise binding of antigen to antibody was accomplished not by typical chemical means, but rather through the shapes of molecules. He discovered that an antibody fits an antigen as a glove fits a hand. Their shapes were complementary. When the fit was tight, the surfaces of antibody and antigen came into very close contact, making possible the formation of many weak bonds that operated at close quarters and were relatively unimportant in traditional chemistry--van der Waals' forces, hydrogen bonds, and so forth. To work, the fit had to be incredibly precise. Even a single atom out of place could significantly affect the binding. Having demonstrated the importance of complementary structure with antibodies, Pauling extended his idea to other biological systems, including the interaction of enzymes with substrates, odors with olfactory receptors, and even the possibility of genes composed of two complementary molecules. Pauling's idea that biological specificity was due in great part to complementary "fitting" of large molecules to one another was essential in the development of molecular biology. The path of his research now formed a broad arc, from early work on the chemical bond as a determinant of molecular structure; through finding out the structures of large molecules, first inorganic substances, then biomolecules; and, finally, to elucidating the interactions between large biomolecules. By the early 1950s, Pauling felt that he had discovered the essentials of life at the molecular level. He was ready for something new.

„Introduction of Haptens“ („Halb-Antigen)
Landsteiner realized that while proteins were immunogenic (capable of inducing an immune response) the lack of structural knowledge of proteins made using them to study the chemical specificity of "serological reactions" impossible. He turned to the use of small molecules which had a defined chemical composition to study antibody specificity. Landsteiner was the first to systematically develop and chart the use of small molecules as probes of antibody recognition. One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies.

Antibodies againstInduction of Antibodies (1924)
One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies. One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies.

Immunity againts Haptens (Landsteiner 1920-21)
Antisera againts small organic molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [(a) Erlanger, B. F. In Methods in Enzymology; Academic Press: San Diego, 1980; Vol. 70, pp (b) Brinkley, M. Bioconj. Chem. 1992, 3, 2-13. 22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies. Landsteiner demonstrated that antibodies exhibited regioselective binding behavior R designates the acid groups (COOH or SO3H or AsO3H2) Figure 3. Reproduction of Table 21 taken from reference 21 showing the cross-reactivity between serum derived via immunization with a 3-aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates. One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies. Landsteiner realized that while proteins were immunogenic (capable of inducing an immune response) the lack of structural knowledge of proteins made using them to study the chemical specificity of "serological reactions" impossible. He turned to the use of small molecules which had a defined chemical composition to study antibody specificity. Landsteiner was the first to systematically develop and chart the use of small molecules as probes of antibody recognition.

serum derived from immunization with 3-aminobenzenesulfonic acid exhibits very limited cross-reactivity with the corresponding ortho and para regioisomers. However, as would be expected, considerable precipitation is observed when the hapten itself is used. These results indicated that shape-selectivity plays a major role in antibody recognition. Figure 3. Reproduction of Table 21 taken from reference 21 (Landsteiner, K. The Specificity of Serological Reactions; Harvard University Press: Cambridge, Massachusetts, 1945, p 156. ) showing the cross-reactivity between serum derived via immunization with a 3-aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates. R designates the acid groups (COOH or SO3H or AsO3H2) Figure 3. Reproduction of Table 21 taken from reference 21 showing the cross-reactivity between serum derived via immunization with a 3-aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates.

serum derived from immunization with 3-aminobenzenesulfonic acid exhibits
p.169

p. 173

As early as 1898 with his assistant Tito Carbone he
commenced a careful biochemical study of the nature of antitoxins, and after numerous experiments on the fractionation of antitoxic sera reached the conclusion that antitoxins in horse serum are associated with globulins precipitated by half saturation with ammonium sulphate. Michael Heidelberger was the first to apply mathematics to the reaction of antibodies and their antigens (the "precipitin reaction"). Heidelberger's calculations launched decades of research that helped reveal the specificity, function, and origin of antibodies.

Antibodies are proteins
Heidelberger and Avery’s early experiments hinged on a simple element: nitrogen. Based on its presence or absence, the duo showed that the antibody- reactive substances on pneumococcal bacterial capsules were nitrogen-free carbohydrates, and that the reacting antibodies were nitrogen containing proteins (see “How Heidelberger and Avery sweetened immunology” JEM 202:1306). The discovery that antibodies are proteins was a notable achievement, particularly at a time when the nature of antibodies—substances known only to reside in proteinaceous serum— remained a mystery

Early StudiesANTIBODIES ARE PROTEINS
As early as 1898 with his assistant Tito Carbone he commenced a careful biochemical study of the nature of antitoxins, and after numerous experiments on the fractionation of antitoxic sera reached the conclusion that antitoxins in horse serum are associated with globulins precipitated by half saturation with ammonium sulphate.

Immunbodies againts carbohydrates (1924
serum derived from immunization with 3-aminobenzenesulfonic acid exhibits

Stärke-Elektrophoresese
Antibodies are g-Globulins (1939) Aus: Kuby, Immunology unbehandelt Serum Stärke-Elektrophoresese Absorption an Ovalbumin Ovalbumin g-Globuline sind Antikörper

QUANTITATION of Ag/Ab reactions
[Antibody] [Antigen = SSSIII] Heidelberger, M., and KendaIl, F. E. (1929). A QUANTITATIVE STUDY OF THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND PURIFIED HOMOLOGOUS ANTIBODY J. Exp. Med., 1929,60, 809.

Menge der Ak-Ag-Komplexe Antigen-Konzentration Antikörper-Konzentration Aus Janeway: Immunobiology

The study of antibodies began in 1890 when Emil von Behring and Kitasato Shibasaburō described antibody activity against diphtheria and tetanus toxins. Behring and Kitasato put forward the theory of humoral immunity, proposing that a mediator in serum could react with a foreign antigen.[71][72] Their idea prompted Paul Ehrlich to propose the side chain theory for antibody and antigen interaction in 1897, when he hypothesized that receptors (described as “side chains”) on the surface of cells could bind specifically to toxins – in a "lock-and-key" interaction – and that this binding reaction was the trigger for the production of antibodies.[73] Other researchers believed that antibodies existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies coated bacteria to label them for phagocytosis and killing; a process that he named opsoninization.[74]

The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[67]

In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies were made of protein.[75] The biochemical properties of antigen-antibody binding interactions were examined in more detail in the late 1930s by John Marrack.[76] The next major advance was in the 1940s, when Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions between antibodies and antigens depended more on their shape than their chemical composition.[77] In 1948, Astrid Fagreaus discovered that B cells, in the form of plasma cells, were responsible for generating antibodies.[78]

Further work concentrated on characterizing the structures of the antibody proteins. A major advance in these structural studies was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light chain,[79] and their realization that this protein was the same as the Bence-Jones protein described in 1845 by Henry Bence Jones.[80] Edelman went on to discover that antibodies are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody-binding (Fab) and antibody tail (Fc) regions of IgG were characterized by Rodney Porter.[81] Together, these scientists deduced the structure and complete amino acid sequence of IgG, a feat for which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine.[81] The Fv fragment was prepared and characterized by David Givol.[82] While most of these early studies focused on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi discovered secretory antibody (IgA)[83] and David S. Rowe and John L. Fahey identified IgD,[84] and IgE was identified by Kikishige Ishizaka and Teruki Ishizaka as a class of antibodies involved in allergic reactions.[85]

Genetic studies identifying the basis of the vast diversity of these antibody proteins when somatic recombination of immunoglobulin genes was by Susumu Tonegawa in 1976.[86]

The concept of the idiotype of immunoglobulins emerged in the early 1960s from two different approaches, one by Oudin with rabbit antibodies, the other by Kunkel and analysis of the immunochemical characteristics of human myeloma proteins. At the time, Oudin had described the allotypic specificities as defining antigenic characteristics of a group of individuals within a given animal species. This was observed for rabbit immunoglobulins and was the consequence of allelic variants of both the heavy and light chains. It was then found that when rabbit antibodies prepared against Salmonella typhi were injected into another rabbit expressing the same (known) allotypes, they induced the synthesis of antibodies that specifically recognized the anti-Salmonella antibodies raised in the first rabbit. It was also shown that they did not react with normal rabbit serum taken before the Salmonella injection, thereby excluding that they identified a new allotypic specificity. Idiotypic specificities were thus defined as antigenic specificities characteristic of one antibody (the idiotype) produced by one animal and specific for one given antigen. Antibodies produced by the second animal were termed anti-idiotypic antibodies. Idiotypes are also referred to as Ab1 while anti-idiotypes are referred to as Ab2, in a simplified nomenclature. At the same time, Kunkel was comparing the immunochemical characteristics of human myeloma proteins with those of normal immunoglobulins. He found that antibodies raised against one given myeloma protein, which cross-reacted against normal immunoglobulins, became specific for the immunizing protein once extensively adsorbed on normal immunoglobulins. This was taken as indicative of determinants specific for any given monoclonal product, although it was not entirely clear whether it was also linked to the pathological "abnormal" nature of the myeloma protein. Structural analysis confirmed, as anticipated, that idiotypic determinants, or idiotopes, were present on the Fab fragments and, more precisely, on the variable regions of H and/or L chains (see Immunoglobulin Structure), depending on the idiotype. Extensive analysis of idiotype structure performed by a combination of immunochemical and structural analysis of anti-hapten antibodies revealed several types of idiotopes. Some Ab1-Ab2 interactions could be inhibited by a hapten of Ab1, indicating that the corresponding idiotopes were part of the antibody combining site, whereas other were not. Genetic analysis also revealed that some idiotypic specificities were common to several idiotypes, whereas others were strictly specific for one given Ab1, leading to the distinction of public and private idiotypes, a notion that was directly related to the dual origin of antibody diversity, germline encoded and somatically generated. Idiotypy was studied extensively when it was discovered that the cascade Ag(X) ^ Ab1 ^ Ab2 could be continued and amplified in a large idiotypic network of interactions, providing a basis for autoregulation of the immune system. An especially interesting observation was that Ab2 could induce certain Ab3 molecules that resembled Ab1 in that they could bind the original Ag(X) antigen. This led to the definition of the "internal image" of the antigen, proposed by Jerne, containing the idea that the collection of normal immunoglobulins of an individual could represent a huge population of internal images of the outside antigen world. It also stimulated approaches of idiotypic vaccines that could have been used in place of classical antigens, an interesting idea whenever antigens were either difficult to identify or poorly immunogenic.

Side visit: Edman degradation
Isoltion of myeloma protein Reduction of disulfide bonds Cleavage of isolated H and L chains by CNBr and pepsin, trypsin etc Separation of fragments by ion exchnage and gel chromatography Determination of N- (by Edman clevage) and C-terminal AA Stepwise release of N-terminal aa by the danysl Edman degradation method Deetecion of cleaved off aa by paper chromatography

The Complete Sequence of hu IgG1 (1969)
Isoltion of myeloma protein Reduction of disulfide bonds Cleavage of isolated H and L chains by CNBr and pepsin, trypsin etc Separation of fragments by ion exchnage and gel chromatography Determination of N- (by Edman clevage) and C-terminal AA Stepwise release of N-terminal aa by the danysl Edman degradation method Deetecion of cleaved off aa by paper chromatography Edelman et al. (1969). THE COVALENT STRUCTURE OF AN ENTIRE TG IMMUNOGLOBULIN MOLECULE. PNAS VOL. 63, 79, 1969

Hypervariable regions are discovered (1970)
Number of different amino acids at a given position Variability = Frequency of the most common amino acid at that position Wu-Kaba Plot Thus at position 7 63 proteins were studied, serine occurred 41 times and 4 different amino acids, Pro, Thr, Ser, and Asp, have been reported. The frequency of the most common is 41/63 = 0.65 and the variability is then 4/0.65 = 6.15. Reproduced from The Journal of Experimental Medicine, 1970, 132: 211–250. Copyright 1970

Hypervariable regions form paratop

Tertärstruktur einer Ig-Faltdomäne
Antigen- Bindnungs stelle Merkmale einer Ig-Faltdomäne Anzahl der Aminosäuren Zylindrische, globuläre Form aus Aminosäuren Anzahl und Orientierung der b-Stränge 7 (C-Region) bzw. 8 (V-Region) anti-parallele Ketten in b- Struktur  β-Stränge (Sekundärstruktur) Anzahl der Faltblätter Zwei Lagen anti-paralleler b-Stränge bilden zwei b-Faltblätter, die durch eine Disulfidbrücke miteinander verbunden sind Ig-Superfamilie Ig-Faltdomänen kommen in vielen anderen Proteinen vor (CD4, CD8, MHC Klasse I und II, T-Zellrezeptor, ICAMs…. V C Disulfid- rücke b-Strang Janeway Immunobiology

Antigenbindungstelle
Die Antigenbindungsstelle (Paratop) Je drei Schlaufen (Finger) der VH- und VL-Domäne (Hände) bilden die Antigenbindungstasche (Schloss) oder Paratop des Antikörpers Paratop ist der Teil des Antikörpers, der mit dem Epitop (Schlüssel) des Antigens interagiert In 1960, Niels Jerne coined the term epitope when he proposed that an antigen particle carries several epitopes (Jerne, N., Ann. Rev. Microbiol., : p ) Antigenbindungstelle = Paratop VH VL H CL L

The hybrioma technique and monoclonal antibodies
Georges Köhler & Cesar Milstein Nobelprize 1984

Immunisierung von Labortieren mit Antigenen
Polyclonal - monoclonal Immunisierung von Labortieren mit Antigenen Polyklonale Antiseren Heterogenes Gemisch von Antikörper, die ver-schiedene Epitope auf dem Immunogen erkennen Immunisieren von Tieren mit Antigen in komplettem Freunds Adjuvans [besteht aus Mineralöl (→ Depot-wirkung) und abgetöteten Tuberkelbazil-len→(unspezifische Aktivierung von DZ u. MF)] Immunisierungen werden mehrmals wiederholt (Boosts) Monoklonale Antikörper homogener Antikörper, der nur ein Epitop auf dem Immunogen erkennt Immunisierung von Maus, Ratte, Hamster, Kaninchen oder (Mensch) Generierung von Hybridomen 91

Polyclonal - monoclonal
Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope. These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal’s serum.

The road to monoclonal antibodies
xxxxx Köhler and Milstein Hybridoma Littlefield introduces HAT selection to select for fused somatic cell hybrids First mamamilan cell hybrids Kunkel shows that myelomas produce Ig 2000 xxxx 1962 1964 1973 2001 2002 Michael Potter estabishes Ig-producing mouse myelomas Milstein and Cotton succesfully fused a mouse and rat myeloma cell xxx

Overview immortalising B cells via somatic cell hybridisation
they built on several advances Somatic cell hybdriisatin technique Selection for hybrid cells Usage of meyloma as a fusion partner

Selection for purine synthesis (HAT selection)
DNA sysnthesis in mammalian cells proceed through a main (de novo) pathway which requires glutamine and aspartate respectively (as well as activated phosphate) as initial substrates for a series of reactions for the synthesis of purine-type (DATP and dGTP) and pyrimidine-type (dCTP and dTTP) dNTPs. Several of the reactions involved can be blocked by aminopterin, an analogue of dihydrofolate (another one is methotrexate) that binds with very high affinity and blocks the enzyme dihydrofolate reductase. As a result, de novo synthesis of dATP, dGTP, dCTP and dTTP is blocked. Mammalian cells, however, survive culture in the presence of aminopterin because they can utilise two salvage pathways. The first converts hypoxanthine in IMP, a reaction catalysed by the enzyme hypoxanthine-guanine phosporybosyl transferase (HGPRT). The second converts thymidine in dTMP, a reaction catalysed by thymidine kinase (TK). Thus a mutation in either the HGPRT or the TK gene would lead to normal growth in standard culture medium but to death in Littlefield's HAT medium (aminopterin, hypoxanthine and thymidine). The hybrid produced between such myeloma line and a B cell, however, would survive because it would utilise the normal HGPRT or TK gene of the B cell. Littlefield, J. W Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145:

Hybridomatechnik: Gewinnung monoklonaler Antikörper
(Köhler und Milstein, 1976, Nobelpreis 1984) Aminopterin blockiert De Novo Purin- und Pyrimidinsynthese Milz-B Myelom HGPRT + Ig+ sterblich HGPRT - Ig- unsterblich PEG De Novo Nukleotide DNA, RNA Salvage Pathway Thymidin Thymidin- kinase (TK+) HAT-Medium: Hypoxanthin, Aminopterin, Thymidin Hypoxanthin Hypoxanthin- Guanin- Phospho- ribosyl- transferase (HGPRT+) B-Zell/Myelom-Hybrid Ig+ HGPRT + unsterblich Amin- opterin Aus Kuby 96

Mouse Plasmacytoma Lines (M. Potter xxx)
Generation of HGPRT- or TK- myeloma lines Both these genes reside on the X chromosome and thus a single mutation is sufficient for generating HGPRT- or TK- phenotypes. These are produced by culturing the cells in the presence of 6-thioguanine or 8-azaguanine for selection of HGPRT- mutants or in the presence of bromodeoxyuridine for selection of TK- mutants. 6-thioguanine or 8-azaguanine are hypoxanthine analogues and bromodeoxyuridine is a thymidine analogue. Thus in the presence of HGPRT and TK these analogues are incorporated into DNA. Thus HGPRT+ or TK+ cells will die but HGPRT- or TK- cells will survive and be selected (see Base analogues for selection of HGPRT- and TK- myeloma cell lines).

Somatic cell hybrids – Time line

Fusion of myeloma pairs (Milstein 1973)
Fusion of myeloma pairs. Before conducting the fusion experiment involving B lymphocytes and myeloma cells, Cesar Milstein and his colleague utilised a similar approach. This experiment established that the antibody H and L chains of each partner were expressed in a co-dominant manner in the hybrid (see Fig Fusion of two Ig producing hybridoma line). This result was very important because it suggested that, in a B lymphocyte-myeloma hybrid, the H and L chains of the B lymphocytes could be expressed. The actual fusion experiment that established monoclonal antibodies is reported in the Fig: Continuous cultures of fused cells secreting antibody of predefined specificity. This experiment confirmed the co-dominant expression of the antibody chains and led to the isolation of the first monoclonal antibody with predefined antigen specificity after somatic cell fusion (see additioal Fig). Fusion of myeloma pairs.

The final experiment (Koehler & Milstein 1974)
Continuous cultures of fused cells secreting antibody of predefined specificity. Kohler G and Milstein C. Nature 256: 495, 1975

Why hybridoma technology is so effective
Why hybridoma technology is so effective. The procedure of somatic cell hybridisation developed by Kohler and Milstein has revolutionised immunochemistry. Why is it so effective ? It is so effective for two reasons: The first is that the antibodies produced in this way employ B cells from hyper-immune animals. These cells therefore have undergone stringent antigen selection and affinity maturation and, when immortalised, will yield high-affinity, monoclonal antibodies. The second reason is that the fusion procedure itself selects for blast cells. Therefore, if a final boost is carried out 2-3 days before fusion, the frequency of antigen-specific clones among the population of hybrid myelomas is very high.

Impact of hybridoma technique
Tools to detect proteins in single cells Tools for practical application in biotechnology and medicine → Diagnosis and therapy of diseases Final confirmation of the clonal selection theory (one B cell – one antibody) also others already provided strong evidence (see lecture in repertoire) Provided material for elucidating the mechanisms governing the genetic control of antibody synthesis and diversity (e.g., class switch, somatic hyper mutation, mRNA stability, genetic control elemenst, antibody assembly … )

One example: Discovery of BiP

Monoclonals as Biologics

Monoclonals as Biologics
Humanisation of rodent antibodies in order to facilite their therapeutic applications was carried in two steps (see Fig Protein engineering of mouse monoclonal antibodies for therapeutic applications). In the first the VH and VL domains are cloned and sequence and inserted in plasmids carrying sequences corresponding to CH1, CH2 and CH3 domains for the Heavy chain and to the CL domain for the Light chain and the plasmids used in order to enable expression of a recombinant form of the antibody in suitable mammalian cell hosts. These chimeric antibodies proved to be much less imunognic than their mouse counterparts. In a second step the CDRs of the mouse antibody were grafted onto human V scaffold yielding, essentially a human antibody. These 'humanised' antibodies are the proteins of choice for applications in patients in vivo.

History of Immunology Who named it? The antibody resuurce page Bacterology Online Bacteriology & Immunology Textbook Chronology Klinisches Wörterbuch

One example: Discovery of BiP

Antikörper gegen Immunglobuline
Durch Immunisieren z.B. eines Kaninchens mit gereinigtem Maus-IgM aus einer weißen Maus = BALB/c wird die Produktion folgender Kaninchen-Antikörper induziert. Anti-idiotypische AK (gegen Epitope in V-Regionen der H- und L-Kette) Gegen eine bestimmte (private= idio) Form eines AK BALB/c C57Bl/6 Die konstante Region der mH-Kette aus einer BALB/c-Maus (weiß) und einer C57Bl/6-Maus (schwarz) unterscheidet sich in einer Aminosäure  verschiedene Allotypen codiert durch Varianten desselben Gens bzw. Allels) BALB/c Anti-allotypische AK (gegen ein bestimmtes Epitop In C-Region) Gegen verschiedene Formen eines bestimmten AK (allo) Anti-isotypische AK (gegen Epitope in C-Regionen der H- und L-Kette) Gegen verschiedene AK-Klassen (iso) 108

Richard Lerner, San Diego, 1986; Peter Schultz, Berkely 1986)
Catalytic Antibodies Richard Lerner, San Diego, 1986; Peter Schultz, Berkely 1986) Conventional Catalytic Antibody Antibody Binds antigen but Binds antigen and cleaves it does not cleave Is regenerated after reaction Is " used " up during reaction

Periodatoxidation von p-Nitro-Toluol-methyl-Sulfid zu Sulfoxid
Hapten (Stabiles Analog zum Übergangszustand) Instabiler Übergangszustand Amino- phosphonsäure L.C.Hsieh-Wilson, P.G.Schultz, and R.C.Stevens. Proc. Natl. Acad. Sci. USA, 93: (1996).

Enzymatische Spaltung und Inaktivierung von Kokain
(Landy and coworkers, New York 1993) O H C O O 3 N N H C OCH H C 3 N HO 3 3 O OCH 3 OCH 3 O O O O OH + C HO HO Cocaine Unstable Cleavage Products (active) Transition State (inactive) O H C N 3 OCH 3 O O P HO Stable Transition State Analog

In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p149

DNA is the substance of heridtiy

Oswald Avery (Rockefeller) showed
xxxxx Köhler and Milstein Hybridoma Oswald Avery (Rockefeller) showed Miescher isolates nucleic acids from puss (Tübingen) 1871 1928 1964 1952 2001 2002 Griffiths shows that a heat-resistance substance from pathologic S-pneumococcus transformed hamless R-fom into a pathologic strIN Hersey-Case confirmed that DNA is the material of heridity xxx

Falsifying famous dogmas

The Central Dogma 1958 Francis Crick
The Central Dogma: Sequential information transfe ris unidriectional from DNA to RNA to protein Reverse trasnfe ris forbidden Exception 1971 david Baltimore and Howard Temin independtyl showed that certains RNA viruses use an enzym the trasnforms RNA back to DNA 1982 Pruisiner (UCSF): Prions (proteins) can tranfer structural information Is Cricks hypothesis falsified. No ist not since most processes of infomation retrival follow his hypotheis. What was falsified was the term DOGMA since dogma menas „without exception“, so it became a theory

Contovery Formation of Antibody specificity
Started 1930 and lasted 20 years Instructional theory: All antibody have the same aa sequence and binding of antigen fixed binding pocketz and thus specificty . Once folded, the confoamtiaon was stable Clonal selection: one cell one antibody The latter became adogma, even so Milstein showed in 1994 that But this exeption did not falsify the clonal selction theory

History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem

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