Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1987 The production and characterization of anti- idiotypic antibodies to syngeneic monoclonal and xenogeneic polyclonal antibodies to soybean mosaic virus Raymond LeRoy Mernaugh Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons Recommended Citation Mernaugh, Raymond LeRoy, "The production and characterization of anti-idiotypic antibodies to syngeneic monoclonal and xenogeneic polyclonal antibodies to soybean mosaic virus " (1987). Retrospective Theses and Dissertations. 9280. https://lib.dr.iastate.edu/rtd/9280 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. 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Other The production and characterization of anti-idiotypic antibodies to syngeneic monoclonal and xenogeneic polyclonal antibodies to soybean mosaic virus by Raymond LeRoy Mernaugh A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major: Microbiology Approve Signature was redacted for privacy. ^fn CRarqe of Maj^r Work Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1987 ii TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 MATERIALS AND METHODS 22 Origin of Virus Antigens and Idiotype Antibodies 22 Production of Antibody-Secreting Hybridomas 22 Dulbecco's modified Eagle's medium (DMEM) 22 lOOX L-glutamine 23 Regular growth medium (DMEM-R) 24 8-azaguanine medium (DMEM-8-aza) 24 Conditioned medium (CM) 24 Phosphate buffered saline 25 Cell viability stain 25 Polyethylene glycol 1000 (PEG-1000) 25 Cloning-thawing medium (DMEM-CT) 25 Hypoxanthine-aminopteri n-thymidi ne medium (HAT) 26 Cell freezing medium 26 Growth and maintenance of SP2/0 cells 26 General scheme for ascites production 26 Protein A purification of SI IgG from mouse ascites 27 G1utara1dehyde fixation of 31 hybridoma cells 28 Immunization of mice with SI 28 Immunization of mice with rabbit Abs 29 Immunization of rats with anti-idiotype cells and/or ascites 29 Cell fusion protocol 30 Cell freezing protocol 32 Cell thawing protocol 33 Cell cloning protocol 33 Enzyme Linked Immunosorbent Assays 34 Basic ELISA protocol 34 Mouse serum screen for anti-Sl idiotope Ab production 37 Mouse serum check for anti-idiotope Ab production to SMV Ab 37 ii1 Page Optimization of idiotype Ab concentration for ELISA 38 Identification of hybridoma anti-idiotope Abs by indirect ELISA 39 Optimization of anti-idiotope ascites dilutions for ELISAs 40 Competition ELISAs 42 Competition assay between SMV and syngeneic anti-idiotope Ab in ascites for bound SI 42 Competition between biotinylated SI and SMV for bound syngeneic anti-idiotope Ab 43 Competition between SMV and xenogeneic anti-idiotope Abs for bound SMV Abs 44 Reactivity of anti-idiotope Abs with mono­ clonal and polyclonal Abs and SMV 45 Reactivity of anti-idiotope Abs with SMV and other viruses 46 Reactivity of anti-Sl idiotope Abs with SMV 47 Competition between SMV and SMV Ab for bound la anti-idiotope Abs 48 Competition between SI and SMV Abs for bound la anti-idiotope Abs 49 Optimization of anti-idiotope la and A4D ascites concentration for detection of SMV Abs 49 Reactivity of IgG Abs from rats injected with either anti-idiotope Ab la or 3 50 Reactivity of biotinylated SI with IgG2a kappa McAbs 51 Competition between biotinylated SI and SMV for bound SI 52 Immunoglobulin class and subclass determina­ tion 53 Competition Assay Between SMV and Anti-idiotype Abs on Detached Leaves 54 RESULTS 56 Immunization of Mice with the SI Idiotype Ab 56 Immunization of Mice with Rabbit Abs 56 Success Rates for Cell Fusion, Freezing and Thawing 57 Optimization of the Basic ELISA Protocol 57 iv Page Identification of Anti-Si Idiotope Hybridomas by Indirect ELISA 59 Identification of Anti-SMV Ab Idiotope Hybri­ domas by Indirect ELISA 61 Syngeneic Anti-idiotope Ab Recognition of SI 63 Optimization of Anti-idiotope Ascites Dilution for Competition ELISAs 66 Competition Between SMV and Anti-Si Idiotope Abs for Bound SI 66 Competition Between SMV and Biotinylated SI for Bound Anti-idiotope Ab 73 Reactivity of Anti-Si Idiotope Abs with Varying Concentrations of SMV 74 Reactivity of Anti-Si Idiotope Abs with SMV and Other Viruses 74 Reactivity of Rabbit Anti-SMV Abs with Anti- idiotope Ab la Coated Plates 80 Competition Between SMV and SMV Ab for Bound Anti-idiotope Ab la 80 Competition Between SI and SMV Ab for Bound Anti-idiotope Ab la 85 Interaction Between Biotinylated SI and Bound SI 85 Competition Between Biotinylated SI and SMV for Bound SI 90 Competition Between SMV and Anti-idiotope A4D for Bound SMV Ab 90 Reactivity of Rabbit Anti-SMV Ab with A4D Coated Plates 95 Competition Between Anti-idiotope Ab A3A and SMV for Bound SMV Ab 95 Anti-idiotope Ab Isotype Characterization 100 V Page Anti-idiotope Ab Inhibition of SMV Local Lesion Formation 101 Production of IgG Abs by Rats Injected with Anti-idiotope Abs la or 3 106 DISCUSSION 109 LITERATURE CITED 128 ACKNOWLEDGEMENTS 141 vi LIST OF TABLES Page Table 1. Anti-Si idiotope Ab interactions with SI or other Abs or with SI plus SMV 60 Table 2. Hybridoma Ab interactions with SMV Ab, TRSV Ab, SMV or SMV Ab plus SMV 62 Table 3. Anti-Si idiotope Ab interactions with plant and animal viruses 79 Table 4. Hybridoma Ab isotype and inhibition of SMV lesion formation on Top crop bean leaves 104 Table 5. Anti-Si idiotope Ab inhibition of SMV local lesion formation 106 Table 6. Production of IgG Abs in rats injected with either anti-Si idiotope Ab la or 3 108 vi i LIST OF FIGURES Page Figure 1. ELISA results for anti-Sl idiotope Ab interactions with bound SI 65 Figure 2. Optimization of anti-Si idiotope Ab concentration for use in ELISA 68 Figure 3. ELISA results for competition assays between SMV and anti-Sl idiotope Abs for bound SI 70 Figure 4. ELISA results for competition assays between SMV and anti-Sl idiotope Abs for bound SI 72 Figure 5. ELISA results for competition assays between SMV and biotinylated SI for bound anti-Sl idiotope Abs 76 Figure 6. ELISA results for interactions between anti-Sl idiotope Abs and bound SMV 78 Figure 7. ELISA results for the interactions between SMV Ab or TRSV Ab and bound anti-Sl idiotope Ab la 82 Figure 8. ELISA results for competition assays between SMV and SMV Ab for bound la 84 Figure 9. ELISA results for competition assays between SI and SMV Ab for bound la 87 Figure 10. ELISA results for the interaction between biotinylated SI and bound SI 89 Figure 11. ELISA results for competition assays between biotinylated SI and SMV for bound SI 92 Figure 12. ELISA results for competition assays between SMV and anti-idiotope Ab A4D for bound SMV Ab 94 V i i i Page Figure 13. ELISA results for the interaction between SMV Ab or TRSV Ab and bound A4D 97 Figure 14.