RIA 2014.Key

http://www.chemistryexplained.com/images/chfa_04_img0954.jpg http://www.bio-world.com/images/reagents.jpg http://www.nlm.nih.gov/visibleproofs/media/detailed/iii_d_142b.jpg Radioimmunoassay 2310302 RAD TRACER

Lerson Tanasugarn, Ph.D. Department of Biochemistry, Faculty of Science, Chulalongkorn University Klum 515, Phone 662-218-5424, Departmental FAX 662-218-5418 Email: [email protected] Handout: [email protected]/public/biochemistry/2010 2310302 RIA.pdf

• History of radioimmunoassay • Principles of radioimmunoassay • Non-isotope immunoassay • Methods • Applications

http://2.bp.blogspot.com/ http://www.aps.org/publications/ Rall, J. E. 1990. Solomon A. http://www.invention-iﬁa.ch/ _DZH2cmCoois/SLFpb8J_GPI/ apsnews/199810/images/ Berson 1918-1972. National images/Rosalyn_Yalow2.jpg AAAAAAAAFvc/4fpGNpLZ6YE/s400/ rosalyn_sussman_yalow.jpg Academy of Sciences. Nobel_Laureate_1977_Rosalyn_Yalow .bmp Rosalyn Sussman Yalow 1921- Solomon Berson 1918 - 1972 • City College of NY - BS and MSc • Born in NYC (NY+German) • Anatomy instructorship NYU • Hunter College BS • MD (NYU 1945) • PhD in physics (U. Illinois 1945) • Intern (Boston City Hospital 1945-46) • Joined the Bronx V.A. Hospital to set up • US Army (1946-48) its radioisotope service • Recruited by Yalow to work in the Radioisotope • Recruit Berson to form a research team Service of the Bronx V.A. Hospital (to support her work) • Chief of Radioisotope Service (forerunner of the Nuclear Medicine Service) 1954

• Rall, J. E. 1990. Solomon A. Berson 1918-1972 A Biographical Memoir. National Academy of Sciences Washington, D.C. http://books.nap.edu/html/biomems/sberson.pdf • Yalow, R. 1977. Autobiography, http://nobelprize.org/nobel_prizes/medicine/laureates/1977/yalow-autobio.html • Yalow, Rosalyn S. 1999. Development and proliferation of radioimmunoassay technology. J. Chem. Edu. 76(6):767-768. Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 3 1951 - isotope & blood volume

from Biography of Sol Berson

His initial publicationsSOLOMO (1951-1960)N A. BERSON are listed below63 SELECTED BIBLIOGRAPHY 1951 With R. S. Yalow. The use of K42-tagged erythrocytes in blood volume determinations. Science, 114:14-15. • isotope dilution technique1952 used for blood volume determinationWith R. S. Yalow. The effect of cortisone on the iodine accumulat- ing function of the thyroid gland in euthyroid subjects. J. Clin. Endocrinol. Metab., 12:407-22. With R. S. Yalow, J. Sorrentino, and B. Roswit. The determination 1S1 of thyroidal and renal plasma I clearancCopyright © e2010 rate by Lersons a Tanasugarn.s a routin All rights ereserved. 4 diagnostic test of thyroid dysfunction. J. Clin. Invest., 31:141— 58. With R. S. Yalow. The use of K42 or P32 labeled erythrocytes and I131 tagged human serum albumin in simultaneous blood volume determinations. J. Clin. Invest., 31:572-80. With R. S. Yalow, A. Azulay, S. Schreiber, and B. Roswit. The bio- logical decay curve of P32-tagged erythrocytes. Application to the study of acute changes in blood volume. J. Clin. Invest., 31:581-91.

1953 With R. S. Yalow, J. Post, L. H. Wisham, K. N. Newerly, M. J. Vil- lazon, and O. N. Vazquez. Distribution and fate of intravenously administered modified human globin and its effect on blood volume. Studies utilizing I131-tagged globin. J. Clin. Invest., 32:22-32. With R. S. Yalow, S. S. Schreiber, and J. Post. Tracer experiments with I131-labeled human serum albumin: Distribution and degradation studies. J. Clin. Invest., 32:746-68.

1954 With R. S. Yalow. The distribution of I131-labeled human serum albumin introduced into ascitic fluid: Analysis of the kinetics of a three compartment catenary transfer system in man and spec- ulations on possible sites of degradation. J. Clin. Invest., 33:377-87. With S. S. Schreiber, A. Bauman, and R. S. Yalow. Blood volume SOLOMON A. BERSON 63 SELECTED BIBLIOGRAPHY 1951 With R. S. Yalow. The use of K42-tagged erythrocytes in blood vol- 1952ume determinations - radioisotope. Science, 114:14-15 iodine. exchange

1952 With R. S. Yalow. The effect of cortisone on the iodine accumulat- ing function of the thyroid gland in euthyroid subjects. J. Clin. Endocrinol. Metab., 12:407-22. With R. S. Yalow, J. Sorrentino, and B. Roswit. The determination of thyroidal and renal plasma I1S1 clearance rates as a routine diagnostic test of thyroid dysfunction. J. Clin. Invest., 31:141— 58. With R. S. Yalow. The use of K42 or P32 labeled erythrocytes and I131 tagged human serum albumin in simultaneous blood volume determinations. J. Clin. Invest., 31:572-80. With R. S. Yalow, A. Azulay, S. Schreiber, and B. Roswit. The bio- logical decay curve of P32-tagged erythrocytes. Application to the study of acute changes in blood volume. J. Clin. Invest., 31:581-91. • uptake of radioactive 195iodine3 by thyroid With R. S. Yalow, J. Post, L. H. Wisham, K. N. Newerly, M. J. Vil- lazon, and O. N. Vazquez. Distribution and fate of intravenously administered modified human globin and its effect on blood volume. Studies utilizing I131-tagged globin. J. Clin. Invest., 32:22-32. Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 5 With R. S. Yalow, S. S. Schreiber, and J. Post. Tracer experiments with I131-labeled human serum albumin: Distribution and degradation studies. J. Clin. Invest., 32:746-68.

1954 With R. S. Yalow. The distribution of I131-labeled human serum albumin introduced into ascitic fluid: Analysis of the kinetics of a three compartment catenary transfer system in man and spec- ulations on possible sites of degradation. J. Clin. Invest., 33:377-87. With S. S. Schreiber, A. Bauman, and R. S. Yalow. Blood volume • plasma protein labeled with radioactive iodine for studying the metabolism of these proteins

Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 6 64 BIOGRAPHICAL MEMOIRS alterations in congestive heart failure. J. Clin. Invest., 33:578- 86. With R. S. Yalow. Quantitative aspects of iodine metabolism. The exchangeable organic iodine pool, and the rates of thyroidal secretion, peripheral degradation and fecal excretion of endog- enously synthesized organically bound iodine. J. Clin. Invest., 33:1533-52. 1955 With R. S. Yalow. Critique of extracellular space measurements with small ions: Na24 and Br82 spaces. Science, 121:34-36. With R. S. Yalow. The iodide trapping and binding functions of the thyroid. J. Clin. Invest., 34:186-204. With M. A. Rothschild, A. Bauman, and R. S. Yalow. Tissue distribution of I131-labeled human serum albumin following intra- venous administration. J. Clin. Invest., 34:1354-58. With A. Bauman, M. A. Rothschild, and R. S. Yalow. Distribution and metabolism of I131-labeled human serum albumin in congestive heart failure with and without proteinuria. J. Clin. Invest., 34:1359-68. 1956 With R. S. Yalow, A. Bauman, M. A. Rothschild, and K. Newerly. Insulin-I131 metabolism in human subjects: demonstration of 1953-59 insulin binding globulin in the circulation of insulin-treated subjects. J. Clin. Invest., 35:170-90. •retarded production of insulin? 1957 excessively rapid degradation of normally secreted insulin? • With R. S. Yalow. Chemical and biologicalmaturity-onset alterations induced by •too few or defective insulin receptors?irradiatio n of I131-labeled human serum albumin. J. Clin. In- •abnormality in the insulin responsevest. pathway, 36:44-50 inside. the cells? diabetes With M. A. Rothschild, A. Bauman, and R. S. Yalow. The effect of used synthesized labeledlarge doses o finsulin desiccated thyroito followd on the distributio degradationn and metabolism of albumin-I1M in euthyroid subjects. J. Clin. Invest., 131 36:422-28Insulin-I. Insulin-I131 slowly disappears due to Normal SubjectWith R. S. Yalow. Serum protein turnovenormalr in multipl degradatione myeloma. J. Lab. Clin. Med., 49:386-94. Subject who has 131 With R. SInsulin-I. Yalow. Ethanol fractionatioInsulin-In of plasm131 disappearsa and electropho even- been treated with retic identification of insulin-binding antibody. J. Clin. Invest., slower than before insulin (diabetic, shock therapy)36:642-47.

serum analyzed by paper electrophoresis found radioactivity associated with the beta and gamma regions of serum proteins Their study of reversible equilibrium --> formation of soluble Ag-Ab complex that between a ligand and a binding protein were metabolized more slowly than free insulin led to an idea that a method can be developed to measure a very small

amount of material Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 7 General Theory of Radioimmunoassay • R = receptor or antibody or any substance (almost always a protein) that binds L L = ligand or antigen or any substance (almost always a small molecule) that binds R • IF R + L RL R + L* RL* and if there is a way to separate bound ligand (PL + PL*) from free ligand (L + L*) • Then a standard curve can be constructed to map the ratio of free and bound to the concentration of free unlabeled ligand.

Insulin Labeled Receptor or Antibody Competition Insulin against insulin

Standard Curves

• Construct the standard curve where unlabeled and labeled ligands compete for the same binding site on the same receptor • Plot bound vs free or bound vs total --> the more unlabeled ligand the less radioactivity in the bound form • Redo the experiment with sample insulin & read from Standard Curve

Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 9 SOLOMON A. BERSON 65 With R. S. Yalow. Apparent inhibition of liver insulinase activity by serum and serum fractions containing insulin-binding antibody. J. Clin. Invest., 36:648-55. With R. S. Yalow, S. Weisenfeld, M. G. Goldner, and B. W. Volk. The effect of sulfonylureas on the rates of metabolic degradation of insulin-I131 and glucagon-I131 in vivo and in vitro. Dia- betes, 6:54-60. With A. Bauman, M. A. Rothschild, and R. S. Yalow. Pulmonary circulation and transcapillary exchange of electrolytes. J. Appl. Physiol., 11:353-61. With R. S. Yalow. Studies with insulin-binding antibody. Diabetes, 6:402-7. 1958 With R. S. Yalow. Insulin antagonists, insulin antibodies and insulin resistance. Am. J. Med., 25:155-59. With A. B. Gutman, T. F. Yu, H. Black, and R. S. Yalow. Incorpo- ration of glycine-1-C1959-196014, glycine-1-C14 and glycine-N15 into uric acid in normal and gouty subjects. Am. J. Med., 25:917-32. 1959 With S. Weisenfeld and M. Pascullo. Utilization of glucose in nor- • Showed that insulin mal and diabetic rabbits. Effects of insulin, glucagon and glucose. Diabetes, 8:116-27. is immunogenic With R. S. Yalow. Quantitative aspects of reaction between insulin and insulin-binding antibody.}. Clin. Invest., 38:1996-2016. With R. S. Yalow. Species-specificity of human anti-beef, pork in- • Showed no sulin serum. J. Clin. Invest., 38:2017-25. With R. S. Yalow. Assay of plasma insulin in human subjects by immunological methods. Nature, 184:1648-49. degradation product With R. S. Yalow. Recent studies on insulin-binding antibodies. Ann. N.Y. Acad. Sci., 82:338-44. from insulin labeling 1960 With R. S. Yalow. Immunoassay of endogenous plasma insulin in • Separated clean man. J. Clin. Invest., 39:1157-75. With R. S. Yalow. Plasma insulin in man (Editorial). Am. J. Med., labeled insulin after 29:1-8. With R. S. Yalow. Plasma insulin concentrations in nondiabetic and early diabetic subjects. Diabetes, 9:254-60. the labeling step • Demonstrated that juvenile-onset diabetics are insulin deﬁcient • Many older diabetics have normal or even elevated blood insulin • Patients treated with (animal) insulins developed antibodies to it Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 10 Awards Sources have been identiﬁed in earlier slides. • Nobel Prize in Chemistry or Medicine 1977 awarded to Rosalyn Yalow. (Sol Berson died in 1972) • Many other awards. • Postage stamps set of Sierra Leone in Africa.

http://stamp-search.com/images/sie9534co2nobel9women.jpg

Maria Goeppert-Mayer Irène Joliot Curie Mother theresa Discovery of nuclear shell French chemist, radium nucleus, Catholic nun with Indian structure created artiﬁcial radioactivities, citizenship - looks after the poor 1963 Nobel Prize in Physics 1935 Nobel Prize in Chemistry - 1979 Nobel Peace Prize

Rosalyn Yalow Selma Lagerlöf Co-developer of RIA Dorothy Crowfoot Hodgkin, Swedish author 1977 Nobel Prize in Physiology Protein crystallographer 1909 Nobel Prize in Literature or Medicine 1964 Nobel Prize in Chemistry

Rita Levi-Montalcini Italian neurologist Mairead Corrigan Betty Williams http://stamp-search.com/images/sie9534co2nobel9women.jpg co-discoverer NGF (with Cohen) Founder of the Northern Ireland Founder of Northern Ireland 1986 Nobel Prize in Physiology Peace Movement - 1976 Nobel Peace Movement - 1976 Nobel or Medicine Peace Prize Laureate Peace Prize Laureate

• Assume Ag and Ag* have identical binding constants and the number of binding sites (Ab) is limited

Ag + Ag* + Ab --> Ag-Ab + Ag*-Ab

initial concentration [Ag]i [Ag*]i [Ab]i 0 0

ﬁnal concentration [Ag]i-X [Ag*]i-Y X Y

• If the Ab binding sites are saturated, • X+Y = [Ab]i • X/Y = [Ag]i/[Ag*]i

• Ab is present only in one chemical form. Other antibodies may be present but Ab is the only protein that binds Ag or Ag* • Ab has a high specificity for Ag and Ag* and does not bind other antigens. • The binding characteristics of Ag and Ag* are not significantly different. • Sufficient time is allowed for each chemical organization. • The bound and free forms of the labeled antigen can be separated without disturbing the equilibrium • There are no co-operative or allosteric effects in the binding reaction. • Radioactive measurements are proportional to the concentration of Ag* present regardless of its chemical form.

• Do a series of experiment, varying the concentration of unlabeled Ag and keeping constant

• the concentration of labeled antigen [Ag*]i

• total antibody [Ab]i • When unlabeled Ag is absent, about 50% of the Ag* is bound. Nonzero Ag will cause the bound Ag* to decrease. This is how the standard curve is constructed.

• Let B0 = radioactivity of Ag*-Ab when [Ag]i=0 B = radioactivity of Ag*-Ab when [Ag]i>0 F = radioactivity of Ag* when [Ag]i>0 T = total radioactivity of all antigens, Ag* and Ag*-Ab = B+F

• Measurable quantities: B0, T and (B or F) • Unknown quantities: actual [Ag*], [Ag*-Ab], [Ab]

• Hold [Ag*]i and [Ab]i constant for a given assay series only ratios such as B/B0, B/F and B/T are required

• Plot B/B0 or B/F or B/T against [Ag]

Non-Isotope Immunoassays http://www.genprice.com/elisa_1.jpg • Convenience of not having to use radioactive facilities • Several techniques exist, for example: • ELISA • EMIT Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 18 Enzyme-Linked Immunosorbent Assay (ELISA)

• AKA Enzyme-Linked Immunoadsorbent Assay • Combine the sensitivity of a spectrophotometric assay of an enzyme (ampliﬁcation factor) with the speciﬁcity of antibodies • Several techniques • Competitive ELISA • Direct ELISA • Double Antibody ELISA • Sandwich ELISA • Spot ELISA (ELISPOT)

Wikipedia Encyclopedia polystyrene

Wikipedia Encyclopedia

• The process • Specific Ab’s are adsorbed onto a solid phase • Incubate with a mixture of a fixed amount of Ag- Enz and a varying amount of pure, unlabeled Ag • Wash off unbound Ag & Ag-Enz • Add substrate and allow color to develop • Disadvantages • Difficulties in preparing enzyme-linked Ag • Having to prepare enzyme-linked Ag for each Ag

http://www.neogen.com/LifeSciences/images/ELISA_Generic.jpg

• The process • Adsorb Ag on surface • Block nonspeciﬁc adsorption e.g. with BSA • Add Ab-Enz Direct Detection Antibody • Wash excess Ab-Enz • Add substrate • Disadvantages

http://media.wiley.com/CurrentProtocols/MB/mb1102/mb1102-ﬁg-0002-1-full.gif

Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 22 Indirect ELISA (no capture Ab, for detecting Ab) • The steps • Ag adsorbed, e.g. 30-60 min. at high pH • cover nonspecific binding sites with powdered milk or BSA • Add Ab or serum sample to be tested for specific Ab • Wash off unbound Ab • Add anti-Ig that binds the Fc region of the Ab This second Ab is linked to a chromogenic enzyme • Add the substrate and observe the color • Advantages http://www.microvet.arizona.edu/courses/mic419/ToolBox/elisa3.jpg • No need to prepare a second Ab for each

• The steps • Capture Ab adsorbed on the surface of a microtiter well • Non speciﬁc binding blocked with BSA or powdered milk • Incubate with pure Ag or serum sample and wash • Incubate with enzyme-labeled second Ab and wash • Add substrate and measure the enzymatic activity • Advantages • Not having to prepare Ag-Enz conjugate

Indirect detection Ab

Capture Ab

Comparing with competitive ELISA

http://www.chemicon.com/resource/ANT101/a2C.asp

Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 25 Spot ELISA (ELISPOT) Visualization of the secretory product of individual activated or responding cells • The steps • Monoclonal Ab captured on polyvinylidene ﬂuoride (PVDF) substrate and blocked with BSA • Cells grown and washed away • Add biotinylated polyclonal Ab against the antigen and wash • Add Avidin-HRP • Add substrate

250,000 each human PBMC cells on PVDF membrane Incubate 40 h with human interleukin-4 Left: unstimulated, Right: stimulated with nickel http://www.mabtech.com/elispot_images.asp?view=02 http://www.sigmaaldrich.com/etc/medialib/life-science/custom-dna- Animation => http://www.elispot-analyzers.de/ oligos/elispot-assay-procedure.Par.0001.Image.573.gif english/elispot-animation.html Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 26 Practical Aspects of ELISA

• Enzyme • stable, easy to assay, high turnover number, products have high molar absorptivity • Examples: dehydrogenases (G6PD, ADH, etc.) alkaline phosphatase + p-nitrophenylphosphate --> p-nitrophenol (aborb at 495 nm) • Solid surface • Cross-linked dextrans, polyacrylamide, ﬁlter paper, nitrocellulose paper, polypropylene, polystyrene microtiter plates

• Binding of Ab to Ag-Enz reversibly inhibits enzymatic activity • Free Ag competes with bound Ag, resulting in some Ag-Enz not bound by Ab and can exhibit enzymatic activity Biotol (1993) Technological Applications of Biocatalysts • Use for low mw Ag’s

• Preparation of Antigens • Preparation of Antibodies • Labeling • Separation of bound and free forms

• MW 150 KD • IgG valency = 2 10 • Kass ~ 10 • No cooperativity

Sarma et al (1971) J Biol Chem 246:3753.

• Immunogen vs antigen • IgG from antiserum • Immunogenicity • Polyclonal vs monoclonal antibody • Adjuvant • Hapten Eisen (1974) Immunology

Deﬁnition

Discovery

Complete and incomplete

Relationship to biosignaling pathways

• Immunization of laboratory animals (usually with adjuvants, e.g. Complete Freund’s Adjuvant) • Poorly immunogenic proteins may need to be partially denatured • Low mw substances may need to be covalently linked (as haptens) to high mw immunogenic carrier proteins • Booster shot (with incomplete Freund’s Adjuvant)

[AbL] Total binding • Speciﬁc binding

Specific binding • Nonspecific binding • Rule of thumb Nonspecific binding for telling what

Total ligand added = [L] + [AbL] kind of binding

Ab + Ag ⇌ AbAg r If then = K(n − r) R + L ⇌ RL c c = concentration of free ligand concentration of bound ligand AbL r = = [ ] total antibody concentration [Ab]initially added K = association constant n = number of identical, independent binding sites

Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 35 Scatchard Plot Plot of r/c vs r [bound/free vs bound] r • vertical axis = r/c = K(n − r) horizontal axis = r c • slope = -K • If ligand is added in excess, the concentration of free ligand becomes large, and r/c approaches 0 -K(n-r)=0; therefore n=r http://www.graphpad.com/prism/tutorials/scatchard/SNAG-0024.gif • The number of ligand bound per Ab molecule approaches the number of ligand binding sites, or the valency of Ab Copyright © 2010 by Lerson Tanasugarn. All rights reserved. 36 Equilibrium Dialysis and Scatchard Plot

• Separation of free and bound species • Equilibrium dialysis • Attachment to a surface • Use your own immagination • Experimental data: free and bound concentrations • Output: association constant (~105 to 1010 litre/mole) and valency • Note the structures of the immunogen and the antigen Eisen & Karush (1949) J Am Chem Soc 71:363.

r log = alog K + alogc n − r

• Based on Sips Distribution a = index of dispersion of K

about K0 http://www.discourses.org.uk/statistics/MODELING_ﬁles/image080.gif • Plot log[r/(n-r)] vs log c • Slope = a (a positive value) • Here, valency n = 2 if r=1, log[r/(2-r)]=0, so K0 is the reciprocal of the value of c

where log[r/(n-r)]=0 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/Sips1.gif

• Intrinsic labeling • Preferentially iodination with 125I • Substitution of H on a phenyl ring of tyrosine (at positions 2 and 6) • Extrinsic labeling • Used when the antigen cannot be labeled directly with iodine • Radioactively label a molecule that can be covalently attached to the target antigen

• 125I, a γ emitter, has a half life of 60 days • The speciﬁc activity of a 125I-protein having 1 atom of iodine per molecule is about 100 μCi/μg at mw = 20 Kd (or 1,000 μCi/μg at mw = 2 Kd) • Techniques • Iodine monochloride method - too harsh • Chloramine-T method - harsh but useful for some • Electrolytic method (large scale manufacturing) • Lactoperoxidase method - gentle method for enzymes

• Chloramine-T

[H3C-Φ-SO2-N-Cl]-[Na]+ • Reaction initiated by chloramine-T, stopped by sodium metabisulﬁte and diluted by cold KI • Gel ﬁltration by Sephadex G-50 in a pasteur pipette column

• First peak is labeled protein Hunter (1973) Radioimmunoassay. In Weir (ed) Handbook of Experimental Immunology

• Alteration due to substitution of iodine for hydrogen in the structure of protein • Radiation damage • Chemical damage due to reagent used • Chemical damage due to noxious substance in the radioactive reagent

Hunter (1973) Radioimmunoassay. In Weir (ed) Handbook of Experimental Immunology

• Double antibody method • Conventional methods • Precipitation by solvents, salts, etc. • Centrifugation • Chromatography • Adsorption • Ion exchange • Electrophoresis • Gel ﬁltration

• Sensitivity - because of radioactivity • Speciﬁcity - because of antibody

• Endocrine physiology • Clinical endocrinology • Drug abuse • Forensic medicine • Whenever you want to quantify minute amounts of a (biochemical) substance in a mixture of other chemicals (like blood serum)