The Elisa guidebook
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Page 415
Index
A
Antibodies, 5, 10, 121
animals, production in, 137, 154, 403, 404 combining site, 120
diagnostic potential, 140 digestion, 124 immunization, 130
live vaccines, 131 passive vaccines, 131,
infectious agents, responses to, 132¨C134 bacteria, 133, 135
fungi, 133 helminths, 133, 136 protozoa, 133, 135 viruses, 133, 134
labeling with enzymes, 69, 395¨C398 maternal, 141
structure, 121, 123, 124 Anamnestic response, 126, 128 Antigen, 7, 10, 117, 118
affinity and avidity, 5, 7, 127, 128, 129
affinity maturation, 126, 129
antigens and antibodies, forces between, 5, 127, 129 antigenic commonness, 130, 143
antigenic determinant (see Epitope) antigenic site, 5, 118
antigenicity, 116 improving of, 404
immunogenicity, 116, 403, 404 plastics, adsorption to, 45, 46, 50
desorption, 49 purity, 46
assays, effects on, 46¨C48 size considerations, 117
Assay choice, 39¨C44, 115
Avidin/biotin systems, 399
ABC system, 399
BRAB system, 399
LAB system, 399
B
Basic principles of ELISA, 9¨C44
Binding ratios, 100¨C102, 106, 108
Bound and free reactants, 10
Buffers blocking, 3, 14, 60
BSA containing, 62
inert proteins containing, 62 reasons needed, 61 Tween-20 containing, 62
Buffers coating, 3, 49 carbonate/bicarbonate, 49 phosphate-buffered saline (PBS), 49
Buffers stopping, 3, 10, 72
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C
Capture ELISA,
(see sandwich ELISA) antigen titration, 192¨C202 antibody titration, 202¨C205
IgG as capture reagent, 199 serum as capture reagent, 200 problems in, 205
Charting methods, (see IQC, Standards and Validation)
Chessboard or checkerboard titrations (CBT), 83¨C113
for direct ELISA reagents, 84¨C92 for indirect ELISA reagents, 92¨C103 for direct sandwich ELISA, 103
for indirect sandwich ELISA, 112 Chromogens, 4, 10, 154
ABTS (2, 2''-azino diethylbenzothiazoline sulfonic acid), 65 color change, 4, 66
5-AS (5-aminosalicylic acid), 4, 65 color change 66
o-nitrophenyl-beta galactopyranoside, 4, 65 color change, 66
OPD (ortho-phenylamine-diamine), 4, 65
color change, 66
pnpp (para-nitro phenylphosphate), 4, 65 color change, 66
TMB (tetra methyl benzidine), 4, 65 color change, 66
Urea, 4, 65
color change, 66 Coating, 3, 45¨C51
adsorption, passive, 3, 46¨C50 antigen attachment, covalent, 50 desorption, 49
plastics, binding capacity, 3, 46 time and temperature, 3, 46, 48 Color development, 3, 4, 66, 69, 72
stopping color development, 4, 72 stopping agents, 3, 4, 72
Competition ELISA, 11, 27¨C38,
definition (with respect to inhibition ELISA), 22 practical exercises, 205¨C231
Conformational epitope, 119
Conjugates, 4, 10
availability, 68 gluteraldehyde coupling, 396
labels, (see Enzymes and Antibodies labeling), alkaline phosphatase, (see Enzymes) beta-galactosidase, (see Enzymes) horseradish peroxidase, (see Enzymes) urease, 71 (see Enzymes)
periodate coupling, 397 Continuous epitope, 119 Curve shapes, 176¨C180
D
Definitions, 10
affinity and avidity, 120 antibody combining site, 120 antigen, 10
antigenicity, 116 antispecies antibodies, 10
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conformational epitope, 119 continuous epitope, 119, discontinuous epitope, 119 epitope, 118
epitype, 118 immunogenicity, 116 linear epitope, 119
monoclonal antibody (mAb), 121, 122 paratope, 120
polyclonal antibodies, 121 Descriptions of assays, 12¨C44, 153¨C231
ELISA competition, 22, 23, 24, 205¨C231
ELISA direct, 12, 13, 14, 155¨C165
ELISA indirect, 14, 15, 16, 165¨C191
ELISA sandwich 17¨C22, 192¨C205
Dilutions, 4, 144
calculations of, 144¨C148
in chessboard titrations (CBT), 83¨C113 series, 150, 151
Direct ELISA
chessboard titrations of antigen and labeled antibody, 84¨C92 exercises, 155¨C165
principles, 12¨C14,
labeled antibody, 12¨C14
E
ELISA, 1
exercises using nonpathogenic materials, 153¨C231 troubleshooting, 79¨C81
stages, 45¨C82 systems, 9¨C44
definition of components of ELISA, 1¨C8, 10 Enzymes, 10, 64,¨C72
alkaline phosphatase, 4, 70 beta-galactosidase, 4, 64, 68 horseradish peroxidase, 4, 70 urease, 71
Enzyme stability, 72
Enzyme systems, 64¨C68
alkaline phosphatase plus para-nitro phenylphosphate, 4, 65, 66, 67, beta-galactosidase, plus o-nitrophenyl-beta galactopyranoside, 4, 65, 66, 68 horseradish peroxidase plus hydrogen peroxide/ABTS, 4, 65, 66, horseradish peroxidase plus hydrogen peroxide/5AS, 4, 65, 66, 67 horseradish peroxidase plus hydrogen peroxide/OPD, 4, 64, 65, 66, horseradish peroxidase plus hydrogen peroxide/TMB, 4, 65, 66,
urease, pH change and bromocresol purple indicator, 4, 65, 66, 68 Epitope, 118
antigenic site, 118
continuous epitope, 119 discontinuous epitope, 119 epitype, 119
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Establishment of control negative sera, 311
External quality assurance (EQA), 329
F
Fab, 124
Fab2, 125
Fc, 125
Frequency distribution (see Statistics)
G
Gluteraldehyde, 396
H
Heavy chain (see Antibody structure), 121, 122
Herd immunity, 142
I
IgA, 5, 124, 126, 129, 134, 135, 136
IgD, 5, 126
IgE, 5, 126, 136
IgG, 5, 123, 126, 128, 129, 134, 135, 136, 154
IgM, 5, 124, 126, 128, 129, 134, 135, 136
Immunosorbents, 402, 403
Immobilization of antigen (see Practical aspects of ELISA, coating) Incubation, 57
plate rotation, 57¨C59 advantages, 59
stationary incubation, 60 timing of steps, 48
Indirect ELISA exercises, 170¨C192
Internal Quality Control (IQC), 347¨C394
Ion exchange chromatography, 401
K
Kits, 327¨C329,
definition of, 327
ruggedness reagents, 326 shelf life reagents, 331, 332
L
Labeling antibodies with enzymes, 69, 395¨C398,,
Light chains (see Antibody structure)
Linear epitope, 119
M
Maternal antibody, 121, 122
Microtiter plates,
Molarities, 151-152
Monoclonal antibodies (mAbs), 19, 129, 233¨C299
availability, 234
comparison to polyclonal antibodies, 129
examples of use of mAbs in ELISA, 256, 257, 268¨C298 isotypes, 236
isotyping, 260¨C262 purification, 257, 259¨C261
screening for activity, 235, 249¨C251 competition ELISA, 237¨C249 indirect ELISA, 252¨C255
stability, 258
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systems for using mAbs, 264¨C268 direct ELISA, 265
indirect ELISA, 265 sandwich ELISA direct, 266 sandwich ELISA indirect, 267
N
Negative sera and control sera,
Nonspecific binding, 10, 14, 46, 50
O
Overview of ELISA, 1¨C8
scientific disciplines needed, 1¨C8