The Elisa guidebook

P

Papain digestion, 124

Passive adsorption, 10, 45, 48, 49, 50

Protein A, 401

Protein G, 402

Pepsin digestion, 125

Periodate coupling, 397

Pipets, 148

calibration multichannel, 148, pipetting exercise, 149 single channel, 149 tips, 148

use, 148

Plates, 3, 45, 57¨C59

Practical aspects of ELISA, 9¨C44, 153¨C231 exercises, 153¨C231

direct ELISA, 155¨C165

indirect ELISA optimization, 165¨C170 indirect ELISA titration antibodies, 170¨C181

indirect ELISA single dilutions of test samples, 182¨C191 sandwich (capture) ELISA to titrate antigen, 192¨C202 sandwich (capture) ELISA to titrate antibodies, 202¨C205 competitive and inhibition ELISA 205¨C231

Prevalence (see Validation) Principles of ELISA, 9¨C44 Production of antisera, 403¨C405 Problems, 74¨C79

conjugates, 78 glassware, 75 incubation, 77 pipets, 76, plates, 76, reading, 78, 79,

sample addition, 78 stopping, techniques, 74 timing, 77

tips, 76 troughs, 76

troubleshooting guide, 80, 81 water, 75

Protein A, 402

Protein G, 402

Q

Quiz on ELISA, 407¨C413

Quality assurance (QA), 329

external quality assurance (EQA), 329

Quality control (QC), 329

Page 420

internal quality control (IQC), 329, 347¨C394 charting methods, 347¨C394

R

Random testing, 310 Reading, 3, 10, 72¨C74 by eye, 72, 73

spectrophotometer, 45, 73, 74 wavelengths for, 66

ROC analysis (see statistics)

S

Salt fractionation, 400

of immunoglobulins, 400

Sandwich ELISA, 11, 17¨C22, 192¨C205

competition assays for sandwich ELISA, 22¨C44, 206, 207 direct sandwich ELISA, 17¨C19, 192¨C201

indirect sandwich ELISA, 19¨C22, 202¨C205 Shelf life reagents, 331, 332

Standardization

(see Validation)

calibration standards, 324 in-house reference materials, 330

international standards, 330 working standards, 330

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

Substrates, 77, 154

reagents, (see Chromogens) Statistics, 6, 335¨C345, 356¨C358 basic terms, 6, 336, 356

charting methods for IQC (see IQC) frequency distributions, 185¨C189 confidence intervals, 342

normal distribution, 6, 187, 338¨C340 mean, median and mode, 6, 184, 338, 339

populations, 6, 185¨C189, 310, 336 standard deviation (SD), 6, 55, 184, 185, 356 ROC analysis, 317¨C320

variation, 6, 340

T

Test questions on ELISA, 407¨C413

Test answers, 408¨C413

Temperature, 3, 46, 48, 49, 57, 77

Theoretical considerations, 115¨C152

Timing of steps, 3, 48

Tips for pipets, 3, 56, 148

Titration of reagents, (see Chessboard titrations)

Troughs (reservoirs), 76

U

Units, 144 volumes, 144 weights, 144

V

Validation of tests using ELISA, 301¨C345 accuracy, 322, 324, 325

analytical sensitivity, 305, 322, 323 analytical specificity, 305, 322 diagnostic sensitivity, 307, 313, 316 diagnostic specificity, 307, 313, 316 gold standards, 308¨C310

Page 421

feasibility studies, 302 precision, 322, 323, ROC analysis, 317¨C320 repeatability, 305, 311 ruggedness testing, 326

selection of negative/positive thresholds, 311 Volume, units of, 144, 145

W

Washing, 3, 10

dipping methods, 51 gravity-feed methods, 52 special handwashing devices, 52 specialist machines, 52

wash bottles plus multiple delivery nozzles, 51, 52 Water, 75

quality, effect on ELISA, 75 Weights, units of, 144, 145

What is known already when setting up ELISA, 39, 115