Ultrasensitive and Rapid Enzyme Immunoassay (eBook)

Front Cover 1
Ultrasensitive and Rapid Enzyme Immunoassay 4
Copyright Page 5
Contents 8
Preface 6
Chapter 1. Introduction: classification of immunoassays 18
Chapter 2. History of ultrasensitive enzyme immunoassays 22
Chapter 3. Factors limiting the sensitivity of noncompetitive heterogeneous solid phase enzyme immunoassays 24
3.1. Factors limiting the sensitivity to antigens and antibodies 24
3.2. Factors limiting the sensitivity to antibodies 26
3.3. Factors limiting the sensitivity to antigens 31
Chapter 4. Methods to minimize effects of factors limiting the sensitivity 34
4.1. Methods to minimize the nonspecific bindings of antigen- and antibody-enzyme conjugates 34
4.2. Method for efficient trapping of antigens and antibodies 51
4.3. Methods using as full reactivities of antigens and antibodies as possible 57
4.4. Methods to reduce serum interference 81
Chapter 5. Principle of the immune complex transfer enzyme immunoassay 96
5.1. Immune complex transfer enzyme immunoassay of antibodies 96
5.2. Immune complex transfer enzyme inmunoassays of antigens 108
5.3 Immune complex transfer enzyme immunoassay for simultaneous detections of antigens and antibodies 110
Chapter 6. Potential of the immune complex transfer enzyme immunoassay to improve the sensitivity and its limitations 114
6.1. Improvement of sensitivity to antibodies 114
6.2. Improvement of sensitivity to antigens 132
6.3. Simultaneous detection of antigens and antibodies by the immune complex transfer enzyme immunoassay 137
6.4. Further improvement of sensitivity 141
6.5. Assay variation 141
6.6. Factors limiting improvement in sensitivity of the immune complex transfer enzyme immunoassay 142
Chapter 7. Ultrasensitive and rapid enzyme immunoassay 158
7.1 . Shaking for immunoreactions on solid phases 158
7.2. Use of larger sample volumes 164
7.3. Use of solid phases with larger surfaces 165
7.4. Use of circulating thin aqueous layers covering solid phase surfaces 166
7.5. Advantages of circulating thin aqueous layer immunoassay 172
7.6. Circulating thin aqueous layer immunoassay for antibodies 173
7.7. Circulating thin aqueous layer irnmunoassay for antigens 187
Chapter 8. Protocol of immune complex transfer enzyme immunoassay 194
8.1. Choice of enzymes as labels 194
8.2. Choice of enzyme-labeling methods 194
8.3. Use of inactive forms of enzyme labels 195
8.4. Preparation of 2,4-dinitrophenyl-bovine serum albumin for immunization 195
8.5. Preparation of biotinyl- and 2,4-dinitrophenyl-bovine serum albumin for irnmunoassays 196
8.6. Affinity-purification of (anti-2,4-dinitrophenyl group) IgG 198
8.7. Coating of solid phases with proteins 199
8.8. Preparations of 2,4-dinitrophenyl.(biotinyl )-antigens and antibodies and antigen- and antibody-enzyme conjugates 199
8.9. Typical protocol of immune complex transfer enzyme immunoassay V for antibody IgG 200
8.10. Typical protocols of immune complex transfer enzyme immunoassays IX and X for antigens 204
Chapter 9. Ultrasensitive immunoassay for haptens 210
9.1. Introduction 210
9.2. Principle of noncompetitive (hetero-two-site) immunoassay 211
9.3. Feasibility of the principle 213
9.4. Measurement of haptens in plasma and urine 222
9.5. Other methods 227
Chapter 10. Properties of the reagents for enzyme-labeling 228
10.1. Solubility 228
10.2. Stability and reactivity 229
10.3. Purity 238
Chapter 11. Enzyme-labeling of antibodies 240
11.1. Introduction 240
11.2. Hinge method 242
11.3. Nonhinge method 248
11.4. Monomeric antibody-enzyme conjugates 250
11.5. Affinity-purified antibody-enzyme conjugates 251
11.6. Microscale preparation of antibody-enzyme conjugates 252
11.7. Characterization and evaluation of antibody-enzyme 253
Chapter 12. Preparation and affinity-purification of IgG fragments 266
12.1. Preparations of F(ab..)2 and Fab' 266
12.1. Preparations of F(ab') 2 and Fab' 266
12.2. Preparations of affinity-purified IgG, F(ab')2 and Fab' 270
Chapter 13. Protocol for enzyme-labeling of antibodies 278
13.1. Introduction 278
13.2. Labeling of Fab' with peroxidase by the hinge method using maleimide derivatives 278
13.3. Labeling of Fab' with ß-D-galactosidase by the hinge method using maleimide derivatives 281
13.4. Labeling of Fab' with alkaline phosphatase by the hinge method using maleimide derivatives . 282
13.5. Labeling of Fab' with glucose-6-phosphate dehydrogenase by the hinge method using maleimide derivatives 284
13.6. Labeling of IgG with ß-D-galatosidase by the hinge method using maleimide derivatives 285
13.7. Labeling of Fab' with peroxidase by the hinge method using pyridyl disulfide derivatives 287
13.8. Nonhinge method 288
Chapter 14. Protocol for labeling avidin with 2,4-dinitrophenyl groups and ß-D-galactosidase 294
14.1. Labeling of avidin with 2,4-dinitrophenyl groups 294
14.2. Labeling of avidin with 2,4-dinitrophenyl-bovine serum albumin 296
14.3. Labeling of avidin with ß-D-galactosidase 298
Chapter 15. Enzyme-labeling of antigens 300
Chapter 16. Protocol for enzyme-labeling of antigens 306
16.1. Labeling of recombinant HIV- I reverse transcriptase (rRT) with 2,4-dinitrophenyl groups 306
16.2. Labeling of recombinant HIV-I p17 antigen (rp17) with 2,4-dinitrophenyl groups 310
16.3. Labeling of recombinant HIV-I p24 antigen (rp24) with 2,4-dinitrophenyl groups 312
16.4. Labeling of recombinant HIV-I reverse transcriptase (rRT) with ß-D-galactosidase: Method I 314
16.5. Labeling of recombinant HIV-I reverse transcriptase (rRT) with ß-D-galactosidase: Method I1 315
16.6. Labeling of recombinant HIV-I reverse transcriptase (rRT) with ß-D-galactosidase: Method III 316
16.7. Labeling of recombinant HIV-I p17 (rp17) with ß-D-galactosidase 318
16.8. Labeling of recombinant HIV-I p24 (rp24) with ß-D-galactosidase 318
Chapter 17. Assays of enzymes 320
17.1. Introduction 320
17.2. Assay of peroxidase 321
17.3. Assay of ß-D-galactosidase 322
17.4. Assay of alkaline phosphatase 323
17.5. Assay of glucose-6-phosphate dehydrogenase 325
References 328
Subject Index 344