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In all cases we have filmed the best available copy. University Microfilms International 300 N. ZEEB RD.. ANN ARBOR, Ml 48106 8207261 Sealfon, Michael Stephen REACTIONS OF MONOCLONAL AND POLYCLONAL ANTI-GAMMA GLOBULINS WITH NATIVE AND CONFORMATIONALLY ALTERED IGG The Ohio State University Ph.D. 1981 University Microfilms International 300 N. Zeeb Road, Ann Arbor, M I 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages 2. Colored illustrations, paper or print ✓ 3. Photographs with dark background ______ 4. Illustrations are poor copy ______ 5. Pages with black marks, not original copy ______ 6. Print shows through as there is text on both sides of page ______ 7. Indistinct, broken or small print on several pages ______ 8. Print exceeds margin requirements ______ 9. Tightly bound copy with print lost in spine ______ 10. Computer printout pages with indistinct print ______ 11. Page(s) ____________ lacking when material received, and not available from school or author. 12. Page(s) ____________ seem to be missing in numbering only as text follows. 13. Two pages numbered ____________ . Text follows. 14. Curling and wrinkled pages ______ 15. Other _________________________________________________________________________ University Microfilms International REACTIONS OF MONOCLONAL AND POLYCLONAL ANTI-GAMMA GLOBULINS WITH NATIVE AND CONFORMATIONALLY ALTERED IgG DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Michael S. Sealfon, M.S. The Ohio State University 1981 Reading Committee: Approved By John Lott, Ph.D. Howard Newman, Ph.D. George Ecker, Ph.D. Q-%tr Russell Skavaril, Ph. D. Adviser irtment of Pathology ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. Penn, my advisor, for his support, guidance, and insight, and Dr. Lott, my co-advisor, for his encouragement and patience during my graduate studies at Ohio State University. I would also like to express my appreciation to Dr. Furman, the laboratory staff, and the administration at W.C.A. Hospital, Jamestown, New York for their cooperation. Special thanks is due to M r s . Lorrie Bush for her perserverance and patience. Special acknowledgement is due my wife, Lee and my two children, David and Aimee for their love and devotion during this endeavor. This dissertation is dedicated to them. This work was supported by N.I.H. Training Grant #5-T01-GM01805-09. VITA March 2, 1944 Born - New York, New York 1961 Graduated from Pascack Valley Regional High School, Hillsdale, New Jersey 1966' B.S., Pennsylvania State University, University Park, Pennsylvania 1974 M.S., University of Tennessee, Center for the Health Sciences, Memphis, Tennessee 1974-1981 N.I.H. Trainee, Division of Clinical Chemistry, Department of Pathology Ohio State University, Columbus, Ohio PUBLICATIONS Sealfon, M.S., Reactions of Monoclonal and Polyclonal Anti-Gamma Globu­ lins with Native and Conformationally Altered IgG, Clin. Chem. 25, 1106 (1979). FIELD OF STUDY Major Field: Clinical Chemistry TABLE OF CONTENTS ACKNOWLEDGEMENTS................................................. ii VITA.............................................................. iii LIST OF TABLES . vi LIST OF FIGURES ................................................. vii INTRODUCTION..................................................... 1 Chapter I. Antigen-Antibody Reactions ............................ 4 II. Rheumatoid Factors ..................................... 23 III. Immune Complexes ....................................... 40 MATERIALS AND METHODS ........................................... 76 Patients 76 Selection of Patients...................................... 77 Protein Isolation Techniques .............................. 78 Ammonium Sulfate Precipitation........................ 78 Gel Filtration Chromatography ........................ 78 Ion Exchange Chromatography ........................ 79 Reduction and Alkylation of Immunoglobulins ........ 79 Papain Digestion .................................... 80 Protein Characterization Techniques........................ 80 Ouchterlony Double Diffusion ........................ 80 Quantification of Immunoglobulins ................... 80 Immunoelectrophoresis ................................ 81 Protein Quantification Procedures ................... 81 Preparative Density Gradient Ultracentrifugation . 81 Demonstration of Rheumatoid Factor Activity............... 82 Rheumatoid Factor Tests .............................. 82 Radioimmunoassay Procedures .............................. 82 (125-1) Precipitation Radioimmunoassay — Rheumatoid Factor Binding Assay .............................. 82 (125-1) Inhibition Radioimmunoassay — Solid Phase Rheumatoid Factor Assay .............................. 83 IV TABLE OF CONTENTS (CONT.) Additional Techniques .................................. 85 Trace Labelling of Immunoglobulins ............... 85 Heat Denaturation of Gamma Globulins ............. 85 Fluorescent Anti-Nuclear Antibody Test ........... 86 Urea Denaturation of I g G .......................... 86 Anti-Streptolysin 0 Activity ..................... 86 Heterophil Antibody Activity ..................... 87 RESULTS ................................................ 88 DISCUSSION ................................................. 164 BIBLIOGRAPHY ................................................. 186 V LIST OF TABLES Table Page 1 PHYSIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF SELECTED ANTI-GAMMA GLOBULINS .......... 89 2 ESTIMATION OF INTERMEDIATE COMPLEXES IN SERUM BY EVALUA­ TION OF THE DIFFERENCE BETWEEN GAMMA-GLOBULIN AND IgG CON­ CENTRATIONS............................................. 103 3 INTERACTION OF THREE RHEUMATOID FACTOR BINDING ASSAYS (RFBA) WITH MATERIAL CONTAINING SIMULATED 22S COMPLEXES. 129 12 5 4 INTERACTIONS BETWEEN ( I)-LABELED IgM RHEUMATOID FACTORS AND IMMUNOGLOBULIN MATERIALS AS DEMONSTRATED BY SOLID PHASE RHEUMATOID FACTOR ASSAYS (SPRFA) ................... 149 5 COMPOSITE RESULTS OF REACTIONS WITH SOLID PHASE RHEUMATOID FACTOR ASSAY SYSTEMS (SPRFA) USING SELECTED HUMAN SERA . 156 6 SUMMARY OF MEDICAL CHARACTERISTICS AND IMMUNOLOGICAL RE­ ACTIONS OF SELECTED PATHOLOGIC S E R A ....................... 158 vi LIST OF FIGURES Figure Page 1 COMPLEMENT ACTIVATION SEQUENCE... .......................... 19 2 IMMUNOELECTROPHORESIS PATTERNS OF SERUM To ................... 90 3 IMMUNOELECTROPHORESIS PATTERNS OF SERUM Wa ................... 91 4 IMMUNOELECTROPHORESIS PATTERNS OF SERUM Kr .............. 92 5 SEPARATION OF T o ., W a ., and K r . AMMONIUM SULFATE PRE­ CIPITATES ON SEPHADEX G-200 94 6 CHROMATOGRAPHY OF A PURPURA HYPERGAMMAGLOBULINEMIA SERUM (Mu.) ON SEPHAROSE 6B AT pH 7 . 4 ............................ 98 7 CHROMATOGRAPHY OF AN IgA MYELOMA SERUM (Cl.) DEMON­ STRATING HIGH LEVELS OF RHEUMATOID FACTOR ACTIVITY ON SEPHAROSE 6B AT pH 7 . 4 ............ 99 8 CHROMATOGRAPHY OF A RHEUMATOID ARTHRITIS SERUM (Kr.) ON SEPHAROSE 6B AT pH 7 . 4 ................................. 100 9 SEPARATION OF HEAT AGGREGATED IgG (COHN FRACTION II) ON PREVIOUSLY CALIBRATED SEPHAROSE 6B ................... 105 10 10-40% SUCROSE DENSITY GRADIENT FRACTIONATION OF HEAT AGGREGATED IgG (COHN FRACTION II) 106 11 DEMONSTRATION OF RHEUMATOID FACTOR ACTIVITY USING HEAT AGGREGATED IgG IN AGAR G E L .............................. 107 12 COMPARISON OF PRECIPITIN REACTIONS BETWEEN SELECTED RHEUMATOID FACTOR ISOLATES (IgM)