Optimization, Validation and Application of Radioimmunoassays for Plant Growth Substances in Avocado (Persea americana Mill.) Fruits

CONTENTS

(note: page numbers correspond to original document)

CHAPTER	PAGE
DECLARATION	(i)
ACKNOWLEDGEMENTS	(ii)
LIST OF ABBREVIATIONS	(iv)
INTRODUCTION	1
1 REVIEW OF LITERATURE	4
1.1 Introduction	4
1.2 Radioimmunoassay	5
1.2.1 Terminology	5
1.2.2 Basic principle of RIA	6
1.2.2.1 Binder dose-response curves	8
1.2.2.2 Standard dose-response curves	9
1.2.3 The binder or antibody	11
1.2.3.1 Cellular events	11
1.2.3.2 Chemistry of antibodies	13
1.2.3.3 The immune response	15
1.2.3.4 Immunogens	16
1.2.3.5 Haptens	17
1.2.3.6 Adjuvants	19
1.2.3.7 Antibody production	20
1.2.4 The ligand	20
1.2.4.1 Purified ligand	21
1.2.4.2 Tracer ligand	21
1.2.5 Separation of bound and free ligand	23
1.2.5.1 The ideal separation technique	23
1.2.5.2 Differential migration of bound and free fractions	24
1.2.5.2.1 Paper chromato-electrophoresis	24
1.2.5.2.2 Gel filtration	25
1.2.5.2.3 Gel equilibrium	25
1.2.5.3 Adsorption methods	26
1.2.5.3.1 Charcoal	26
1.2.5.3.2 Silicates	27
1.2.5.4 Fractional precipitation	27
1.2.5.5 Double antibody method	28
1.2.5.6 Solid phase methods	29
1.2.6 Statistical analysis of RIA data	30
1.3 Immunological Assays for PGSs	32
1.4 Avocado Fruit Development and Senescence	34
1.4.1 PCS regulation of fruit growth	35
1.4.2 PCS regulation of senescence	38
2 RADIOIMMUNOASSAY FOR ISOPENTENYL-TYPE CYTOKININS	40
2.1 Materials and Methods	40
2.1.1 Conjugation of IPA to BSA	40
2.1.2 Production of antisera	41
2.1.2.1 Immunization	41
2.1.2.2 Purification and storage	42
2.1.2.3 Titer determination	42
2.1.3 Radioimmunoassay	43
2.1.3.1 Optimization	43
2.1.3.1.1 Effect of separation technique	43
2.1.3.1.2 Effect of charcoal concentration	45
2.1.3.1.3 Determination of incubation time	45
2.1.3.1.4 Effect of pH	46
2.1.3.1.5 Tracer preparation	46
2.1.3.2 Standard procedure	47
2.1.3.3 Statistical analysis and calculation of results	50
2.1.4 Validation	51
2.1.4.1 Extract dilution curves	51
2.1.4.2 Antibody selectivity (cross-reaction studies)	53
2.1.5 Biological sample preparation	53
2.1.5.1 Extraction	54
2.1.5.2 Purification	54
2.1.5.3 Isolation	55
2.2 Results	56
2.2.1 IPA-BSA conjugate	56
2.2.2 Response to Immunization	58
2.2.3 Radioimmunoassay	61
2.2.3.1 Effect of separation technique	61
2.2.3.2 Effect of charcoal concentration	66
2.2.3.3 Effect of incubation time	66
2.2.3.4 Effect of pH	69
2.2.3.5 Tracer properties	69
2.2.3.6 Standard dose-response curve	69
2.2.4 Validation	73
2.2.4.1 Extract dilution, curves	73
2.2.4.2 Cross-reactivities to Anti-IPA	77
2.3 Discussion	78
3 RADIOIMMUNOASSAY FOR ABSCISIC ACID	81
3.1 Materials and Methods	81
3.1.1 Conjugation of (+)ABA to BSA	81
3.1.2 Production of antisera	82
3.1.2.1 Immunization	82
3.1.2.2 Purification and storage	82
3.1.2.3 Titer determination	83
3.1.3 Radioimmunoassay	83
3.1.3.1 Optimization	83
3.1.3.1.1 Effect of separation technique	83
3.1.3.1.2 Determination of incubation time	83
3.1.3.1.3 Effect of charcoal concentration	84
3.1.3.1.4 Effect of pH	84
3.1.3.2 Standard procedure	84
3.1.4 Validation	85
3.1.4.1 Extract dilution curves	85
3.1.4.2 Antibody selectivity (cross-reaction studies)	86
3.1.5 Biological sample preparation	86
3.1.5.1 Extraction	87
3.1.5.2 Purification	87
3.1.5.3 Isolation	87
3.2 Results	88
3.2.1 ABA-BSA conjugate	88
3.2.2 Response to Immunization	90
3.2.3 Radioimmunoassay	90
3.2.3.1 Effect of separation technique	90
3.2.3.2 Effect of charcoal concentration	96
3.2.3.3 Effect of pH	96
3.2.3.4 Effect of incubation time	96
3.2.3.5 Standard dose-response curve	96
3.2.4 Validation	100
3.2.4.1 Extract dilution, curves	100
3.2.4.2 Cross-reactivities to Anti-ABA	100
3.3 Discussion	100
4 RADIOIMMUNOASSAY FOR INDOLE-3-ACETIC ACID	106
4.1 Materials and Methods	106
4.1.1 Conjugation of IAA to BSA	106
4.1.2 Production of antiserum	108
4.1.2.1 Immunization	108
4.1.2.2 Purification and storage	108
4.1.2.3 Titer determination	109
4.1.3 Radioimmunoassay	109
4.1.3.1 Optimization	109
4.1.3.1.1 Effect of separation technique	109
4.1.3.1.2 Effect of charcoal concentration	110
4.1.3.1.3 Determination of incubation time	110
4.1.3.1.4 Effect of pH	111
4.1.3.1.5 Effect of methanol	111
4.1.3.2 Standard procedure	111
4.1.4 Validation	112
4.1.4.1 Extract dilution curves	112
4.1.4.2 Antibody selectivity	113
4.1.5 Biological sample preparation	114
4.1.5.1 Extraction	114
4.1.5.2 Purification	114
4.2 Results	115
4.2.1 IAA-BSA conjugates	115
4.2.2 Response to Immunization	116
4.2.3 Radioimmunoassay	119
4.2.3.1 Effect of separation technique	119
4.2.3.2 Effect of charcoal concentration	119
4.2.3.3 Effect of pH	121
4.2.3.4 Effect of incubation	121
4.2.3.5 Effect of methanol	121
4.2.3.6 Standard dose-response curve	121
4.2.4 Validation	124
4.2.4.1 Extract dilution, curves	124
4.2.4.2 Cross-reactivities to Anti-IAA-75	124
4.3 Discussion	126
5 PLANT GROWTH SUBSTANCE TRENDS IN AVOCADO FRUIT	129
5.1 Fruit Growth and Development	129
5.1.1 Materials and methods	129
5.1.1.1 Fruit material	129
5.1.1.2 Preparation of material and RIA	130
5.1.2 Results	131
5.1.2.1 Fruit growth morphology	133
5.1.2.2 Auxin (Indole-3-acetic acid)	136
5.1.2.2 Isopentenyl adenine and isopentenyl adenosine	141
5.1.2.2 Free abscisic acid	141
5.1.3 Discussion	141
5.2 Post-harvest PCS Trends in Avocado Fruit	148
5.2.1 Materials and methods	148
5.2.1.1 Avocado material	148
5.2.1.2 Preparation of material and RIA	148
5.2.2 Results	150
5.2.2.1 General	150
5.2.2.2 Abscisic acid	151
5.2.2.2 Isopentenyl-type cytokinins	154
5.2.2.2 Ethylene	157
5.2.3 Discussion	157
OVERALL DISCUSSION AND CONCLUSIONS	162
SUMMARY	169
LITERATURE CITED	172