12
A. Nowotny
Basic Exercises in Immunochemistry A Laboratory Manual
Second Revised and Enlarged Edition
With 62 Figures
Springer-Verlag Berlin Heidelberg New York 1979
ALOIS NOWOTNY, Professor of Immunology University of Pennsylvania 400l Spruce Street, Philadelphia, PA 19104/USA
ISBN-13: 978-3-540-09453-1 e-ISBN-13: 978-3-642-67356-6 DOl: 10.1007/978-3-642-67356-6
Library of Congress Cataloging in Publication Data. Nowotny, Alois. Basic exercises in immunochemistry. Bibliography: p. Includes index. I. Immunochemistry--Laboratory manuals. I. Title. QRI83.6.N68. 1979. 574.2'9'028. 79-14029.
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Under § 54 of the German Copyright Law. where copies are made for other than private use, a fee is payable to the publisher. the amount of the fee to be determined by agreement with the publisher.
© by Springer-Verlag Berlin Heidelberg 1969 and 1979
The use of registered names. trademarks. etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.
213113130-543210
Preface of the Second Edition
First we wish to say a few words about the appearance of this book, as compared to the first edition. The choice of printing and paper was simply for financial reasons. This book is written primarily for students who can barely afford to pay today's fees for higher education and are even less able to buy books, some of which are getting beyond the reach of the individual as well as less-well-endowed libraries. We have tried very hard to keep the price of this book low so that students can buy it.
As far as the content is concerned, we revised all the exer-cises which appeared in the first edition and omitted several of these which could be replaced with better ones, or which were found to be too complicated for student exercises. New refer-ences were added wherever they were needed, although in staying with the philosophy of the first edition, only a very small per-centage of the enormously increased literature has been cited. As we said in the introduction to the first edition, this book is not an exhaustive survey of the field. For such purposes numerous other publications should be studied first. On the oth-er hand, in this manual great emphasis has been placed on clar-ity of description, completeness of methodological details and reproducibility of the experiments.
Several old procedures were left in the second edition. We do not think that methodologies (or other publications, for that matter) should be ignored only because they were printed a few decades ago. We have still included those which are useful both for experimentation and for teaching purposes.
The 32 new exercises we have included were either developed after the first edition left the press, or have become appli-cable since then for inclusion in a collection of basic exer-cises. Radioimmunoassays, enzyme-labelled antibody methods, preparative thin-layer and affinity chromatographies, several new variations of electrophoretic procedures, membrane studies, immunocyte isolations and in vitro correlates of cell-mediated immune reactions are only a few examples of new exercises which we have included.
At the same time we realize fully that a great number of meth-odologies have been developed and published in various immu-nological and immunochemical laboratories which should have been included in this book. The obvious limiting factors were time and'space. It would defeat the purpose of this basic manual to try to achieve completeness, and it would be foolhardy for any-one to attempt to cover the entire field alone.
This brings me to the point of thanking all who helped me in this work. The deepest gratitude goes again to my wife, Anne Nowotny, who untiringly tested and corrected so many of the des-criptions and remained steadfast during this endeavor. My stu-dents, postdoctoral fellows and other research associates did
VI Preface of the Second Edition
their share in elaborating several of the methodological de-tails, without which the exercises simply would not function. I express here once more my heartfelt thanks for their devoted work. In addition to this, several descriptions were sent in draft form to the scientists who pioneered the developments of some procedures included in the new edition. Their comments, corrections, and additions were extremely helpful and we great-ly appreciate them. These scientists were:
Dr. C. Boone, National Cancer Institute, Bethesda, MDi Dr. G. Chen, University of Alabama, Birminghami Dr. T.P. King, Rockefeller University, New York, NYi Dr. F. Karush, University of Pennsylvania, Philadelphia, PAi Dr. J. Lightbody, Wayne State University, Medical School, Detroit, MIi Dr. J.J. Marchalonis, Frederick Cancer Research Center, Frederick, MDi Dr. R.H. Purcell, National Institute of Health, Bethesda, MDi Dr. L.A. Sternberger, University of Rochester, Rochester, NYi and Dr. R.M. Zacharius, U. S. Dept. of Agriculture, Philadelphia, PA.
Philadelphia, July 1979 A. NOWOTNY
Preface of the First Edition
This book intends to be neither a complete survey of the field nor an exhaustive source of references. For these purposes, the use of the extensive compilation Experimental Immunochemistry by E.A. Kabat and M.M. Mayer (1962), or the excellent methodological textbook, Methods in Immunology by D. H. Campbell, J. S. Garvey, E. E. Cremer and D.H. Sussdorf (1963), or the qUite comprehensive series Methods in Immunology and Immunochemistry by C.A. Williams and M.W. Chase (1967) are more suitable. The main purpose of this manual is to provide students with a simple book which will introduce them to some frequently occurring problems in the three major sections of the immunochemistry of natural products. These are the isola-tion of the materials, the chemical analysis of the constituents and their structure, and, finally, the assays of the most important biologic and immunologic activities.
In this manual the exercises are simplified and several short-cuts are taken in order to fit them into the framework of a teach-ing course. The introduction to each exercise gives a brief and elementary explanation of the reaction on which it is based. "Materials and Equipment" lists all tissues or cells, chemicals, glassware, and special equipment which must be available to carry out the exercise, although the very common laboratory tools are usually omitted from the list. The nProcedure" attempts to give a description so simple that students with minimal experience in this and related fields should be able to learn and carry out the procedure. The section is divided into several steps, and these have been set up so that the exercise may be interrupted and continued on the following day, starting with the next step. In the few cases where this could not be done, it is up to the instructor to make the students aware of this circumstance. The "Evaluation" either gives simple ways of obtaining quantitative data from the experiment or refers to other procedure given in this manual by which such information can be obtained. The para-graph headed "Use and Limitations" gives a short survey of the applicability of the method. It is perhaps most important in methodological handbooks to point out clearly what may be expected from the procedure, the purposes for which it may be used, and the realistic values of the data obtained. The "References" list only a few which were considered to be either the most important or the simplest. Several valuable contributions were not mentioned because it would not serve the purposes of this manual to include them.
The figures in the text illustrate only a few pieces of equip-ment, tools, or manipulations. The drawings of some pieces of equipment are taken from catalogs of supply companies, with their permission. Needless to say, a number of equally reliable and usefull similar tools are available from other sources. Wherever applicable, efforts have been made to introduce homemade eqUipment.
VIII Preface of the First Edition
Most of the procedures are described as they are routinely carried out in our laboratories. Some, which are not used fre-quently, were thoroughly investigated and tried out by us. Some of the exercises in this manual were used in graduate laboratory courses given at our school. In a few cases, handy modifications or recently developed improvements were learned from authentic experts in the corresponding field, and these have been incorpo-rated as "personal communications".
Although the manual does not cover all possible fields of immunochemical research, we have tried to bring in a few examples from various fields, and to give a useful selection of exercises from different areas of immunochemistry which will enable the teacher to choose those which he considers to be the most relevant to his teaching program. In the last chapter suggestions are given regarding a logical sequence of exercises which should be followed by the students. All these start with the isolation of a certain immunologically active natural product, and include a few steps of purification followed by both chemical analysis of the material obtained and the measurement of its biologic or immunologic activity in various assays.
I must express my deep gratitude to my wife, Anna M. Nowotny, and to those of my co-workers who tested the exercises in the laboratory and who contributed to this manual with suggestions and sound criticism, and last but not least, to Mrs. Alice Stone who edited, proofread and typed the manuscript.
Philadelphia, July 1969 A. NOWOTNY
Contents
ISOLATIONS AND PREPARATIONS
Exercise No. 1 Fractionation of Serum Proteins by Ammonium Sulfate .................................. .
Exercise No. 2 Isolation of Immunoglobulins by Gel Filtra-tion on Sephadex G 200 Column ............. 3
Exercise No. 3 Isolation of IgG by DEAE Cellulose ........ 8 Part A Column Chromatography .........•..•.. 9 Part B Batch-Type Operation .•...•.•..•..•.. 11
Exercise No. 4 Dissociation of Macroglobulins With 2-Mercaptoethanol ...............•..•...... 12 Part A ••••••••••••••••••••••••••••••••••••• 1 3 Part B •••••••••••.••••••••••••••••.•••.•••• 1 3
Exercise No. 5 Enzymatic Cleavage of IgG ....•.....•.....• 14 Exercise No. 6 Chromatographic Separation of the IgG
Fragments •.......................•........ 16 Exercise No. 7 Separation and Isolation of Hand L Chains
of IgG ......•..•..•..•..•........•..•..... 17 Exercise No. B Affinity Chromatography and Its Application
to Antibody Isolation ......•.....•..•..•.. 19 Exercise No. 9 Cellulose Acetate Paper Electrophoresis of
Human Serum .......•........•...•....•..... 23 Exercise No. 10 Disc Electrophoresis in Polyacrylamide Gel 26
Part A Separation of Serum Proteins .....••. 27 Part B SDS Gel Electrophoresis of Erythro-
cyte Membrane Proteins .............• 31 Part C Molecular Weight Determination by SDS
Gel Electrophoresis ................. 32 Exercise No. 11 Separation of Human Hemoglobins by Electro-
focusing ......•........................... 35 Exercise No. 12 Conjugation of Antibodies With Fluorescein
and Rhodamine .............•............... 41 Part A Fluorescein ..............•..•..•..•. 42 Part B Rodamine •..•..•.....•............•.. 42
Exercise No. 13 Enzymatic Labeling of Proteins ~Vith 125I .. 44 Part A ••••••••••••••••••••••••••••••••••••• 44 Part B ••••••••••••••••••••••••••••••••••••• 46
Exercise No. 14 Preparation of Peroxidase-Labeled Antibody 48 Exercise No. 15 Preparation of Soluble Dini trophenyl
Proteins .........................•........ 51 Exercise No. 16 Preparation of Dini trophenyl- and Trini-
trophenyl-Labeled Erythrocytes ........•..• 54 Part A ••••••••••••••••••••••••••••••••••••• 54 Part B ••••••••••••••••••••••••••••••••••••• 56
Exercise No. 17 Isolation of Cell r-1embranes From Ascites Tumor s .................................•.. 57
x Contents
Exercise No. 18 Preparation and Use of Synthetic Liposomes 61 Exercise No. 19 Isolation of Murine Transplantation Antigen
Preparation •...•..•.•.•..•••••••...•..•••• 63 Exercise No. 20 Isolation of Bacterial H-Antigens .••.••.•• 66 Exercise No. 21 Extraction of Bacterial 0 Antigens (Endotoxins
or Lipopolysaccharides) .•.....•••.•.....•• 69 Part A Trichloroacetic Acid Method .•..•..•• 69 Part B Phenol-Water Procedure •.••.•...•.... 71 Part C Chloroform-Methanol Extraction of
Endotoxic Glycolipid (EGL) From Rough r.1utants .•..•.••..••••.•...•.•...•..• 72
Exercise No. 22 Preparation of 1 4C-Labeled Endotoxin and Endotoxic Glycolipid (EGL) ..•..••.••.•••.• 73
Exercise No. 23 Isolation of Bacterial Capsular Polysac-charides, K Antigens ••••••.••••••••••••••. 76
Exercise No. 24 Extraction of Teichoic Acid ...•.•••.•.•.•• 78 Exercise No. 25 Extraction of Pneumococcus Type XIV Polysac-
charide ....•.••....•.......•..•••........• 80 Exercise No. 26 Isolation of Phytohemagglutinin .••.••.•..• 82 Exercise No. 27 A and B Blood Group Antigen Preparations
From Erythrocytes .•••..••..•.•••.••.•..•.. 84 Part A Solubilization With Dilute NaOH ••••. 84 Part B The Use of Detergent ..••.••...•.•••• 85
Exercise No. 28 Isolation of NN Blood Group Antigen From Human Erythrocytes .•..•..•....•••••...•••. 86
Exercise No. 29 Isolation of Forssman Hapten From Erythro-cytes ..................................... 88
Exercise No. 30 Isolation of Ragweed Pollen Allergens ••••• 90 Exercise No. 31 Ultrasonic Disruption of Bacteria and Den-
sity-Gradient Centrifugation of the Products 93 Exercise No. 32 Vacuum Distillation and Freeze Drying •••.• 96
2 STRUCTURAL STUDIES
2.1 QUALITATIVE METHODS 99
2.1.1 Hydrolytic Procedures •.•.••..••..•.•..•..•.....•.• 99
Exercise No. 33
Exercise No. 34 Exercise No. 35
Determination of Optimal Hydrolytic Condi-tions .•..••....••.••...•••••••...••..•.•.• 99 Part A Carbohydrate Liberation ••.......•••• 100 Part B Liberation of Amino Acids and Amino
Sugars ••.•..•.•.....•..••..••...••.. 101 Hydrolysis of Lipopolysaccharide With Acid 102 Partial Hydrolysis of Lipopolysaccharide With Ion Exchanger .••.••.••...•.••....••.. 104 Part A Discontinuous Method •..••....•.••..• 105 Part B Continuous System •••••.•••••••••••.• 107
2.1.2 Separation and Isolation of the Split Products ••.• 110
Exercise No. 36 The Methods of Analytic and Preparative Paper Chromatography ••.••••••••••..••••••• 110 Part A Preliminary Runs •.••.••••..••.•...•• 111 Part B Preparative Procedure ..•............ 113
Contents
Exercise No. 37
Exercise No. 38
Exercise No. 39
Exercise No. 40
Exercise No. 41
Exercise No. 42
XI
Separation and Isolation of Peptides or Oligosaccharides by High-Voltage Paper Electrophoresis ....•...................... 117 Demonstration of Ion Exchange Column Chro-matography of Amino Acids ................• 120 Gas-Liquid Chromatography for Fatty Acid Separation ...............................• 124 Carbohydrate Analysis by Gas-Liquid Chroma-tography ......................... , ....•... 127 Gas-Liquid Chromatographic Analysis of Amino Acids ..............•................ 131 Demonstration of Thin-Layer Chromatography 135
2.1.3 Some Other Qualitative Methods for Structural Studies ............................•.............. 140
Exercise No. 43
Exercise No. 44
Exercise No. 45 Exercise No. 46
Qualitative Analyses of the Constituents of an Unknown Natural Product ...............• Identification of N-Terminal and C-Terminal Amino Acids ......................•........ Part AN-Terminus ......................... . Part B C-Terminus ......................... . Part C Paper Chromatography of the Amino
Acids and Their Derivatives ........ . Staining of Gels for Carbohydrates ....... . Methylation of Carbohydrates ..•..•........
140
147 149 150
151 153 156
2.2 QUANTITATIVE ANALYTICAL DETERMINATIONS 159
Exercise No. 47
Exercise No. 48 Exercise No. 49 Exercise No. 50 Exercise No. 51
Exercise No. 52
Exercise No. 53
Exercise No. 54
Exercise No. 55 Exercise No. 56
Dry Weight and Inorganic Ash Determinations Part A Total Dry ~'ifeight .........•..•.....•. Part B Inorganic Ash ..............•.....•.. Microdetermination of Nitrogen ........•... Microdetermination of Phosphorus ........•. Protein Determination by the Biuret Method Determination of Primary Amino Compounds With Ninhydrin ..............•...........•. Carbohydrate Determination by Phenol-Sul-furic Acid ...........•.....•.....•..•..... Ultramicrodetermination of Reducing Car-bohydrates ................•............... Enzymatic Determination of Carbohydrates .. Part A Glucose ....•..............•..•..•... Part B Galactose .......................... . Determination of Amino Sugars •........•... Determination of Heptoses and Pentoses .... Part A Heptose Part B Pentose
159 159 160 162 166 168
169
171
173 175 176 177 178 180 180 181
Exercise No. 57 Determination of Sialic Acid and 2-Keto-3-Deoxyoctonic Acid Derivatives ..•..•..•.. 182 Part A Sialic Acid •..............•..•..•..• 183 Part B 2-Keto-3-Deoxyoctonic Acid .......... 184
Exercise No. 58 Chemical and Biologic Analysis of Compounds Separated by Preparative Thin-Layer Chroma-tography .........•..•..•.................. 185 Part A Chemical Analyses ..............•..•. 186 Part B Measurements of Biologic Activities. 189
XII Contents
Exercise No. 59 Oxydation of Carbohydrates With Periodate. Quantitative Spectrophotometric Determina-tion •.......•..••..••...•••..•••.....••..• 1 91
Exercise No. 60 Measurements of the Products of Periodate Oxydation ...••.••..•.....•.....•.......••. 194 Part A Formaldehyde Determination ......•••. 194 Part B Formic Acid Determination .•........• 195
Exercise No. 61 Reduction of Carbohydrates \Vi th NaBH 4 ••••• 196 Part A Determination of the Degree of Poly-
merization ..•..•.......•.••....•...• 197 Part B Identification of the Reducing Termi-
nal Carbohydrate ......•.•..•..•.•••. 198 Part C Investigation of the Glycosidic Link-
ages by Periodate and Borohydrate Treatment (Smith Degradation) ...•••. 200
Exercise No. 62 Quantitative Determination of Free Hydroxyl Groups .•..••....•..••...•...•••..•......•• 203
Exercise No. 63 Lipid Determination With the Hydroxylamine Method ...............•..•....••.•..•....•. 205
Exercise No. 64 Qualitative and Quantitative Analysis of Carboxylic Acids •....••....•.••....•...••• 207
3 IMMUNOLOGIC AND OTHER BIOLOGIC ASSAYS
3. 1 ANTIBODY PRODUCTION •...•......••••••.•••..•.•••••.•• 211
Exercise No. 65 Immunization and Adjuvant Effect •....••... 211 Part A Soluble Immunogen ...•...•.••.......• 212 Part B Particulate Immunogen •...•.••.•....• 213
Exercise No. 66 Demonstration of Antibody Production at Cellular Level (Immunoplaque Method) •.•.... 215
3.2 ANTIGEN-ANTIBODY INTERACTIONS •......••..•...•••..... 217
3.2.1 Methods of Agglutination ......•.............•....• 217
Exercise No. 67 Bacterial Agglutination................... 217 Exercise No. 68 Hemagglutination and Its Inhibition .•.•.•. 219 Exercise No. 69 Passive Hemagglutination and Its Inhibition 224
Part A Neter's Method •.••....••..•..•.••... 224 Part B Boyden's Procedure ..•.•..•.•..••.... 226 Part C Carbodiimide Coupling Method ..••..•. 226 Part D Inhibition of Passive Hemagglutination 227
Exercise No. 70 Charcoal Agglutination .............•.•.••. 230
3.2.2 Precipitation Methods .•...•..............•...•••.• 232
Exercise No. 71 Double Gel Diffusion/Ouchterlony Method .•. 232 Exercise No. 72 Immunoelectrophoresis. . . . . . . . • • . • . . . . . . • . . 235 Exercise No. 73 Quantitative Immunoelectrophoresis/Rocket
Method .....•......................•.....•. 237 Exercise No. 74 Crossed Immunoelectrophoresis ..••..••••••• 240 Exercise No. 75 Counter Immunoelectrophoresis .••......••.. 243 Exercise No. 76 Semiquantitative Micro-Precipitin Assays •. 245
Part A Microtitration in Gel .....•...•..... 245
Contents
Exercise No. 77 Exercise No. 78 Exercise No. 79
Exercise No. 80
Part B Determination of Optimal Antigen/ Antibody Ratio by Gel Diffusion .... .
Quantitative Precipitation ............... . Quantitative Radial Immunodiffusion ...... . Radioimmunoassays for Cyclic AMP and Cyclic GMP Determinations ....................... . Part A Cyclic AHP ......................... . Part B Cyclic GMP ......................... . Solid-Phase Radioimmunoassay ............ .
XIII
248 249 251
254 255 258 261
3.2.3 Other Reactions of Antibodies and Immunocytes ...... 263
Exercise No. 81
Exercise No. 82 Exercise No. 83
Exercise No. 84
Exercise No. 85
Exercise No. 86
Exercise No. 87 Exercise No. 88 Exercise No. 89 Exercise No. 90
Complement Fixation ...................... . Part A Preparation of Reagents ............ . Part B Hemolysin Titration ................ . Part C Titration of the Complement ........ . Part D Qualitative Complement Fixation
(Determination of the Proper Antigen Dose for Quantitative Complement Fixa-tion Test) ......................... .
Part E Anticomplementary Controls ......... . Part F Quantitative Complement Fixation ... . Total and Viable Cell Counts ............. . Direct and Indirect Staining of Bacteria With Fluorescein-Labeled Antibodies ...... . Unlabeled Antibody-Enzyme Method to Localize Antigens .............................•.... Isolation of Human Lymphocytes by Sedimenta-t ion ..................................... . Isolation of Adhering Leukocytes ......... . Part A •••••••••.•.....•••••...•••••.•.••••• Part B ..•...••••.•.•••....•..•..•..•.•••.•. Rosette-Forming Cell Assay ............... . Passive Cutaneous Anaphylaxis ............ . Lymphoblas t As say •........................ T Cell-Mediated Cytotoxicity of Influenza Virus-Infected Cells ..................... .
263 265 266 266
268 269 269 272
275
277
280 282 283 284 285 288 290
293
3.2.4 Miscellaneous Biologic Reactions ................•. 297
Exercise No. 91 Exercise No. 92
Exercise No. 93
Exercise No. 94 Exercise No. 95
The Local Shwartzman Phenomenon .......... . Measurement of the Activity of the Reti-culoendothelial System ................... . Enhancement of Nonspecific Resistance to Infection ................................ . Determination of TOxicity ................ . Measurement of Pyrogenicity .............. .
297
299
301 303 305
SUBJECT INDEX .....................•...................... 309
