Index

Antibodies, 5, 10, 121animals, production in, 137, 154,

403,404combining site, 120diagnostic potential, 140digestion, 124immunization, 130

live vaccines, 131passive vaccines, 131,

infectious agents, responses to,132-134

bacteria, 133, 135fungi, 133helminths, 133S 136protozoa, 133, 135viruses, 133, 134

labeling with enzymes, 69,395-398

maternal, 141structure, 121, 123, 124

Anamnestic response, 126, 128Antigen, 7, 10, 117, 118

affinity and avidity, 5, 7, 127,128,129

affinity maturation, 126, 129antigens and antibodies, forces

between, 5,127, 129antigenic commonness, 130,

143antigenic determinant {see

Epitope)

antigenic site, 5, 11 8antigenicity, 116

improving of, 404immunogenicity, 116,403,404plastics, adsorption to, 45, 46,

50desorption, 49

purity, 46assays, effects on, 46-48size considerations, 1! 7

Assay choice, 39-44, 115Avidin/biotin systems, 399

ABC system, 399BRAB system, 399LAB system, 399

BBasic principles of ELISA,

9-44Binding ratios, 100-102, 106,

108Bound and free reactants, 10Buffers blocking, 3, 14, 60

BSA containing, 62inert proteins containing,

62reasons needed, 61Tween-20 containing, 62

Buffers coating, 3, 49carbonate/bicarbonate, 49phosphate-buffered saline (PBS),

49Buffers stopping, 3, 10, 72

415

416 Index

Capture ELISA, (see sandwichELISA)

antigen titration, 192-202antibody titration, 202-205

JgG as capture reagent, 199serum as capture reagent, 200problems ins 205

Charting methods, (see IQC,Standards and Validation)

Chessboard or checkerboardtitrations (CBT), 83-113

for direct ELISA reagents, 84-92for indirect ELISA reagents,

92-103for direct sandwich ELISA, 103for indirect sandwich ELISA, 112

Chromogens, 4, 10, 154ABTS (2,2"-azino di-

ethylbenzothiazolinesulfonic acid), 65

color change, 4, 665-AS (5-aminosalicy!ic acid), 4,

65color change 66

o-nitrophenyl-betagalactopyranoside, 4, 65

color change, 66OPD (o^o-phenylaraine-

diamine), 4, 65color change, 66

pnpp (para-nitrophenylphosphate),4,65

color change, 66TMB (tetra methyl benzidine), 4,

65color change, 66

Urea, 4, 65color change, 66

Coating, 3,45-51adsorption, passive, 3, 46-50antigen attachment, covalent, 50desorption, 49plastics, binding capacity, 3, 46time and temperature, 3,46,48

Color development, 3, 4, 66, 69, 72stopping color development, 4, 72stopping agents, 3, 4, 72

Competition ELISA, 11,27-38,definition (with respect to

inhibition ELISA), 22practical exercises, 205—231

Conformational epitope, 119Conjugates, 4, 10

availability, 68gluteraldehyde coupling, 396labels, (see Enzymes and

Antibodies labeling),alkaline phosphatase, (see

Enzymes)beta-galactosidase, (see

Enzymes)horseradish peroxidase, (see

Enzymes)urease, 71 (see Enzymes)

periodate coupling, 397Continuous epitope, 119Curve shapes, 176-180

D

Definitions, 10affinity and avidity, 120antibody combining site, 120antigen, 10antigenicity, 116antispecies antibodies, 10

Index 417

conformational epitope, 119continuous epitope, 119,discontinuous epitope, 119epitope, 118epitype, 118immunogenicity, 116linear epitope, 119monoclonal antibody (mAb), 121,

122paratope, 120polyclonal antibodies, 121

Descriptions of assays, 12—44,153-231

ELISA competition, 22, 23, 24,205-231

ELISA direct, 12, 13, 14,155-165

ELISA indirect, 14, 15, 16,165-191

ELISA sandwich 17-22, 192-205Dilutions, 4, 144

calculations of, 144-148in chessboard titrations (CBT),

83-113series, 150, 151

Direct ELISAchessboard titrations of antigen

and labeled antibody,84-92

exercises, 155-165principles, 12—14,

labeled antibody, 12-14

E

ELISA, 1exercises using nonpathogenic

materials, 153-231troubleshooting, 79-81stages, 45-82

systems, 9—44definition of components of

ELISA, 1-8, 10Enzymes, 10, 64,-72

alkaline phospliatase, 4, 70beta-galactosidase, 4, 64, 68horseradish peroxidase, 4,

70urease, 71

Enzyme stability, 72Enzyme systems, 64—68

alkaline phospliatase plus para-nitro phenylphosphate, 4,65, 66, 67,

beta-galactosidase, pluso-nitrophenyl-betagalactopyranoside, 4, 65, 66,68

horseradish peroxidase plushydrogen peroxide/ABTS, 4,65, 66,

horseradish peroxidase plushydrogen peroxide/5AS, 4,65, 66, 67

horseradish peroxidase plushydrogen peroxide/OPD, 4,64, 65, 66,

horseradish peroxidase plushydrogen peroxide/TMB, 4,65, 66,

urease, pH change andbromocresol purpteindicator, 4, 65, 66,68

Epitope, 118antigenic site, 118continuous epitope, 119discontinuous epitope, 119epitype, 119

418 Index

Establishment of control negativesera, 311

External quality assurance (EQA),329

Fab, 124Fab2, 125Fc, 125Frequency distribution (see

Statistics)

G

Gluteraldehyde, 396

H

Heavy chain (see Antibodystructure),! 21, 122

Herd immunity, 142

I

IgA,5, 124, 126,129,134,135, 136

IgD, 5, 126IgE, 5, 126, 136IgG, 5, 123, 126,128, 129, 134,

135, 136, 154IgM, 5, 124, 126, 128, 129, 134,

135,136Immunosorbents, 402, 403Immobilization of antigen (see

Practical aspects of ELISA,coating)

Incubation, 57plate rotation,

57-59advantages, 59

stationary incubation, 60timing of steps, 48

Indirect ELISA exercises,170-192

Internal Quality Control (IQC),347-394

Ion exchange chromatography, 401

K

Kits, 327-329,definition of, 327ruggedrtess reagents, 326shelf life reagents, 331, 332

Labeling antibodies with enzymes,69, 395-398,,

Light chains (see Antibodystructure)

Linear epitope, 119

M

Maternal antibody, 121, 122Microtiter plates,Molarities, 151-152Monoclonal antibodies (mAbs), 19,

129,233—299availability, 234comparison to polyclonal

antibodies, 129examples of use of mAbs in

ELISA, 256, 257,268-298

isotypes, 236isotyping, 260-262purification, 257, 259—261screening for activity, 235,

249-251competition ELISA, 237-249indirect ELISA, 252-255

stability, 258

Index 419

systems for using mAbs,264-268

direct ELISA, 265indirect ELISA, 265sandwich ELISA direct. 266sandwich ELISA indirect, 267

N

Negative sera and control sera,Nonspecific binding, 10, 14, 46, 50

O

Overview of ELISA, 1-8scientific disciplines needed, 1--8

P

Papain digestion, 124Passive adsorption, 10, 45, 48, 49,

50Protein A, 401Protein G, 402Pepsin digestion, 125Periodate coupling, 397Pipets, 148

calibrationmultichannel, 148,pipetting exercise, 149single channel, 149tips, 148use, 148

Plates, 3, 45, 57-59Practical aspects of ELISA, 9-44,

153-231exercises, 153-231

direct ELISA, 155-165indirect ELISA optimization.,

165-170indirect ELISA titration

antibodies, 170-181

indirect ELISA single dilutionsof test samples,182-191

sandwich (capture) ELISA totitrate antigen, 192-202

sandwich (capture) ELISA totitrate antibodies,202-205

competitive and inhibitionELISA 205-231

Prevalence (see Validation)Principles of ELISA, 9-44Production of antisera,

403^05Problems, 74-79

conjugates, 78glassware, 75incubation, 77pipets, 76,plates, 76,reading, 78, 79,sample addition, 78stopping,techniques, 74timing, 77tips, 76troughs, 76troubleshooting guide, 80, 81water, 75

Protein A, 402Protein G, 402

QQuiz on ELISA, 407-^13Quality assurance (QA),

329external quality assurance (EQA),

329Quality control <QC), 329

420 Index

internal quality control (IQC),329, 347-394

charting methods, 347-394

R

Random testing, 310Reading, 3, 10,72-74

by eye, 72, 73spectrophotometer, 45, 73,74wavelengths for, 66

ROC analysis (see statistics)

S

Salt fractionation, 400of immunoglobulins, 400

Sandwich ELISA, 11, 17-22,192-205

competition assays for sandwichELISA, 22-44, 206, 207

direct sandwich ELISA,17-19,192-201

indirect sandwich ELISA, 19-22,202-205

Shelf life reagents, 331, 332Standardization (see Validation)

calibration standards, 324in-house reference materials, 330international standards, 330working standards, 330

Solid phase, 10,45microtiter plates, 45, 46

Substrates, 77, 154reagents, (see Chromogens)

Statistics, 6, 335-345, 356-358basic terms, 6, 336, 356charting methods for IQC (see

IQC)frequency distributions,

185-189

confidence intervals, 342normal distribution, 6,187,

338-340mean, median and mode, 6.

184,338,339populations, 6, 185-189,310,336standard deviation (SD), 6, 55,

184, 185,356ROC analysis, 317-320variation, 6, 340

TTest questions on ELISA, 407-413

Test answers, 408^413Temperature, 3, 46, 48,49, 57, 77Theoretical considerations, 115-152Timing of steps, 3, 48Tips for pipets, 3, 56, 148Titration of reagents, (see

Chessboard titrations)Troughs (reservoirs), 76

U

Units, 144volumes, 144weights, 144

V

Validation of tests using ELISA,301-345

accuracy, 322, 324, 325analytical sensitivity, 305, 322,

323analytical specificity, 305, 322diagnostic sensitivity, 307, 313,

316diagnostic specificity, 307, 313,

316gold standards, 308-310

Index 421

feasibility studies, 302 gravity-feed methods, 52precision, 322, 323, special handwashing devices,ROC analysis, 317-320 52repeatability, 305, 31 ] specialist machines, 52ruggedness testing, 326 wash bottles plus multiple

selection of negative/positive delivery nozzles,thresholds, 311 51,52

Volume, units of, 144, 145 Water, 75w quality, effect on ELISA, 75

Weights, units of, 144,145Washing, 3, 10 what is known already when setting

dipping methods, 51 u p ELISA, 39, 115