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4 Characterization of Conjugation Methods
Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
5. Gosling, J. P., Immunoassays: A Practical Approach, Oxford University Press, New York, 2000.
6. Deshpande, S. S., Enzyme Immunoassays: From Concept to Product Development, Chapman & Hall,
New York, 1996.
7. Ishikawa, E., Ultrasensitive and rapid enzyme immunoassay, in Laboratory Techniques in Biochemistry
and Molecular Biology, Vol. 27, Pillai, S. and van der Vliet, P. C. (eds.), Elsevier, Amsterdam, the
8. Hornbeck, P., Enzyme-linked immunosorbent assays, Curr. Protoc. Immunol., Chapter 2, Unit 2.1, 2001.
9. Crowther, J. R., Stages in ELISA, Methods Mol. Biol., 516, 43, 2009.
10. Voller, A., Bartlett, A., and Bidwell, D. E., Enzyme immunoassay with special reference to ELISA techniques, J. Clin. Pathol., 31, 507, 1978.
11. Schuurs, A. H. W. and van Weemen, B. K., Enzyme immunoassay: A powerful analytical tool,
J. Immunoassay, 1, 229, 1980.
12. Freytag, J. W., Affinity column mediated immunoenzymometric assays, in Enzyme-Mediated
Immunoassay, Ngo, T. T. and Lenhoff, H. M. (eds.), Plenum Press, New York, 1985, p. 277.
13. Kurita, R., Arai, K., Nakamoto, K., Kato, D., and Niwa, O., Development of electrogenerated chemiluminescence-based enzyme linked immunosorbent assay for sub-pM detection, Anal. Chem., 82, 1692, 2010.
14. Scalia, G., Halonen, P. E., Condorelli, F., Mattila, M. L., and Hierholzer, J. C., Comparison of monoclonal biotin–avidin enzyme immunoassay and monoclonal time-resolved fluoroimmunoassay in detection
of respiratory virus antigens, Clin. Diagn. Virol., 3, 351, 1995.
15. Meyer, H. H., Eisele, K., and Osaso, J., A biotin–streptavidin amplified enzyme immunoassay for
13,14-dihydro-15-keto-PGF2 alpha, Prostaglandins, 38, 375, 1989.
16. Suzuki, Y., Aoyagi, Y., Muramatsu, M., Igarashi, K., Saito, A., Oguro, M., Isemura, M., and Asakura, H.,
A lectin-based monoclonal enzyme immunoassay to distinguish fucosylated and non-fucosylated alphafetoprotein molecular variants, Ann. Clin. Biochem., 27 (Pt 2), 121, 1990.
17. Cullina, M. J. and Greally, J. F., A novel lectin-based enzyme-linked immunosorbent assay for the measurement of IgA1 in serum and secretory IgA1 in secretions, Clin. Chim. Acta, 216, 23, 1993.
18. Finley, P. R., Williams, R. J., and Lichti, D. A., Evaluation of a new homogeneous enzyme inhibitor
immunoassay of serum thyroxine with use of a bichromatic analyzer, Clin. Chem., 26, 1723, 1980.
19. Brown, D. V. and Meyerhoff, M. E., Potentiometric enzyme channeling immunosensor for proteins,
Biosens. Bioelectron., 6, 615, 1991.
20. Gebauer, C. R. and Rechnitz, G. A., Deaminating enzyme labels for potentiometric enzyme immunoassay, Anal. Biochem., 124, 338, 1982.
21. Yin, Z., Liu, Y., Jiang, L. P., and Zhu, J. J., Electrochemical immunosensor of tumor necrosis factor alpha
based on alkaline phosphatase functionalized nanospheres, Biosens. Bioelectron., 26, 1890, 2011.
22. Bratthauer, G. L., Overview of antigen detection through enzymatic activity, Methods Mol. Biol., 588,
23. Fanjul-Bolado, P., González-García, M. B., and Costa-García, A., Voltammetric determination of alkaline phosphatase and horseradish peroxidase activity using 3-indoxyl phosphate as substrate: Application
to enzyme immunoassay, Talanta, 64, 452, 2004.
24. Kaw, C. H., Hefle, S. L., and Taylor, S. L., Sandwich enzyme-linked immunosorbent assay (ELISA) for
the detection of lupine residues in foods, J. Food Sci., 73, T135, 2008.
25. Nanda, S., Muralidhar, K., and Kar, S. K., Thermostable alpha-amylase conjugated antibodies as probes
for immunodetection in ELISA, J. Immunoassay Immunochem., 23, 327, 2002.
26. Shimura, T., Nakamura, T., Kawakami, A., Haga, M., and Kato, Y., A new type of enzyme immunosensor using antigen-bound membrane and multivalent antibody (Fab′-alpha-amylase conjugate), Chem.
Pharm. Bull. (Tokyo), 34, 5020, 1986.
27. Oellerich, M., Enzyme immunoassays in clinical chemistry: Present status and trends, J. Clin. Chem.
Clin. Biochem., 18, 197, 1980.
28. Mattiasson, B., Svensson, K., Borrebaeck, C., Jonsson, S., and Kronvall, G., Non-equilibrium enzyme
immunoassay of gentamicin, Clin. Chem., 24, 1770, 1978.
29. O’Sullivan, M. J., Bridges, J. W., and Marks, V., Enzyme immunoassay: A review, Ann. Clin. Biochem.,
16, 221, 1979.
30. Szucs, J., Pretsch, E., and Gyurcsányi, R. E., Potentiometric enzyme immunoassay using miniaturized
anion-selective electrodes for detection, Analyst, 134, 1601, 2009.
31. Saita, T., Tokunaga, A., Egoshi, M., Tokushima, H., and Fujito, H., Quantification of cibenzoline by
enzyme-linked immunosorbent assay, Yakugaku Zasshi, 127, 1007, 2007.
32. Kopetzki, E., Lehnert, K., and Buckel, P., Enzymes in diagnostics: Achievements and possibilities of
recombinant DNA technology, Clin. Chem., 40, 688, 1994.
Preparation of Immunoconjugates and Immunogens
33. Ko, F. H. and Monbouquette, H. G., Photometric and electrochemical enzyme-multiplied assay techniques using beta-galactosidase as reporter enzyme, Biotechnol. Prog., 22, 860, 2006.
34. Arakawa, H., Maeda, M., and Tsuji, A., Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applications to enzyme immunoassay and DNA probe assay, Anal. Biochem.,
199, 238, 1991.
35. Piao, Y., Lee, D., Lee, J., Hyeon, T., Kim, J., and Kim, H. S., Multiplexed immunoassay using the stabilized enzymes in mesoporous silica, Biosens. Bioelectron., 25, 906, 2009.
36. Krämer, P. M., Weber, C. M., Forster, S., Rauch, P., and Kremmer, E., Analysis of DDT isomers with
enzyme-linked immunosorbent assay and optical immunosensor based on rat monoclonal antibodies as
biological recognition elements, J. AOAC Int., 93, 44, 2010.
37. Kim, B., Park, E. Y., Lee, Y. T., Lee, J. H., and Lee, S. H., Development of homogeneous enzyme immunoassay for the organophosphorus insecticide fenthion, J. Microbiol. Biotechnol., 17, 1002, 2007.
38. Tateishi, K., Yamamoto, H., Ogihara, T., and Hayashi, C., Enzyme immunoassay of serum testosterone,
Steroids, 30, 25, 1977.
39. Litman, D. J., Hanlon, T. M., and Ullman, E. F., Enzyme channeling immunoassay: A new homogeneous
enzyme immunoassay technique, Anal. Biochem., 106, 223, 1980.
40. Dong, T., Sun, J., Liu, B., Zhang, Y., Song, Y., and Wang, S., Development of a sensitivity-improved
immunoassay for the determination of carbaryl in food samples, J. Sci. Food Agric., 90, 1106, 2010.
41. Peuravuori, H. and Korpela, T., Pyrophosphatase-based enzyme-linked immunosorbent assay of total IgE
in serum, Clin. Chem., 39, 846, 1993.
42. Baykov, A. A., Kasho, V. N., and Avaeva, S. M., Inorganic pyrophosphatase as a label in heterogeneous
enzyme immunoassay, Anal. Biochem., 171, 271, 1988.
43. Tsuji, A., Maeda, M., Arakawa, H., Shimizu, S., Ikegami, T., Sudo, Y., Hosoda, H., and Nambara, T.,
Fluorescence and chemiluminescence enzyme immunoassays of 17 alpha-hydroxyprogesterone in dried
blood spotted on filter paper, J. Steroid Biochem., 27, 33, 1987.
44. Terouanne, B., Nicolas, J. C., Descomps, B., and Crastes de Paulet, A., Coupling of delta 5,3-ketosteroid
isomerase to human placental lactogen with intermolecular disulfide bond formation. Use of this conjugate for a sensitive enzyme immunoassay, J. Immunol. Methods, 35, 267, 1980.
45. Casu, A. and Avrameas, S., Conjugation of lactic-dehydrogenase with proteins by the use of glutaraldehyde: Detection of antigens in the immunocompetent cells by means of the conjugates, Ital. J. Biochem.,
18, 166, 1969.
46. Wu, C., Irie, S., Yamamoto, S., and Ohmiya, Y., A bioluminescent enzyme immunoassay for prostaglandin E(2) using Cypridina luciferase, Luminescence, 24, 131, 2009.
47. Wu, C., Kawasaki, K., Ogawa, Y., Yoshida, Y., Ohgiya, S., and Ohmiya, Y., Preparation of biotinylated
cypridina luciferase and its use in bioluminescent enzyme immunoassay, Anal. Chem., 79, 1634, 2007.
48. Samanta, A. K. and Ali, E., Homogeneous enzyme immunoassay of estriol using lysozyme, Indian J.
Med. Res., 87, 615, 1988.
49. Dhar, T. K., Samanta, A. K., and Ali, E., Homogeneous enzyme immunoassay of estradiol using estradiol-3-O-carboxymethyl ether as hapten, Steroids, 51, 519, 1988.
50. Rodgers, R., Crowl, C. P., Eimstad, W. M., Hu, M. W., Kam, J. K., Ronald, R. C., Rowley, G. L., and
Ullman, E. F., Homogeneous enzyme immunoassay for cannabinoids in urine, Clin. Chem., 24, 95, 1978.
51. Paknejad, M., Javad Rasaee, M., Mohammadnejad, J., Pouramir, M., Rajabibazl, M., and Kakhki, M.,
Development and characterization of enzyme-linked immunosorbent assay for aflatoxin B1 measurement
in urine sample using penicillinase as label, J. Toxicol. Sci., 33, 565, 2008.
52. Venkatesa Perumal, S., Umapathi, V., Ambwani, T., and Lakhchaura, B. D., A competitive dipstick
enzyme immunoassay for diagnosis of early pregnancy in bovine, Reprod. Domest. Anim., 43, 744, 2008.
53. Kim, C. K. and Lim, S. J., Liposome immunoassays using phospholipase C or alkaline phosphatase,
Methods Enzymol., 373, 260, 2003.
54. Fromell, K., Hulting, G., Ilichev, A., Larsson, A., and Caldwell, K. D., Particulate platform for bioluminescent immunosensing, Anal. Chem., 79, 8601, 2007.
55. Chandler, H. M., Cox, J. C., Healey, K., MacGregor, A., Premier, R. R., and Hurrell, J. G., An investigation of the use of urease-antibody conjugates in enzyme immunoassays, J. Immunol. Methods, 53, 187,
56. Goding, J. W., Methods useful in antibody purification, in Monoclonal Antibodies: Principles and
Practice, 2nd edn., Academic Press, New York, 1986, p. 1011.
57. Menozzi, F. D., Vanderpoorten, P., Dejaiffe, C., and Miller, A. O. A., One-step purification of mouse
monoclonal antibodies by mass ion exchange chromatography on zetaprep, J. Immunol. Methods, 99,
Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
58. Lindmark, R., Thoren-Tolling, K., and Sjoquist, J., Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera, J. Immunol. Methods, 62, 1, 1983.
59. Björck, L. and Kronvall, G., Purification and some properties of streptococcal protein G, a novel IgG
binding reagent, J. Immunol., 133, 969, 1984.
60. Fassina, G., Ruvo, M., Palombo, G., Verdoliva, A., and Marino, M., Novel ligands for the affinity-chromatographic purification of antibodies, J. Biochem. Biophys. Methods, 49, 481, 2001.
61. Eliasson, M., Andersson, R., Olsson, A., Wigzell, H., and Uhlén M., Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG, J. Immunol., 142,
62. Andrew, S. M. and Titus, J. A., Purification of immunoglobulin G, Curr. Protoc. Immunol., Chapter 2,
Unit 2.7, 2001.
63. Andrew, S. M., Titus, J. A., Coico, R., and Amin, A., Purification of immunoglobulin M and immunoglobulin D, Curr. Protoc. Immunol., Chapter 2, Unit 2.9, 2001.
64. Pack, T. D., Purification of human IgA, Curr. Protoc. Immunol., Chapter 2, Unit 2.10B, 2001.
65. Zhao, Y., Gutshall, L., Jiang, H., Baker, A., Beil, E., Obmolova, G., Carton, J., Taudte, S., and Amegadzie,
B., Two routes for production and purification of Fab fragments in biopharmaceutical discovery research:
Papain digestion of mAb and transient expression in mammalian cells, Protein Expr. Purif., 67, 182, 2009.
66. Andrew, S. M. and Titus, J. A., Fragmentation of immunoglobulin G, Curr. Protoc. Cell Biol., Chapter
16, Unit 16.4, 2003.
67. Jones, R. G. and Landon, J., Enhanced pepsin digestion: A novel process for purifying antibody F(ab′)(2)
fragments in high yield from serum, J. Immunol. Methods, 263, 57, 2002.
68. Rousseaux, J., Rousseaux-Prévost, R., and Bazin, H., Optimal conditions for the preparation of Fab and
F(ab′)2 fragments from monoclonal IgG of different rat IgG subclasses, J. Immunol. Methods, 64, 141, 1983.
69. Rousseaux, J., Rousseaux-Prevost, R., and Bazin, H., Optimal conditions for the preparation of proteolytic fragments from monoclonal IgG of different rat IgG subclasses, Methods Enzymol., 121, 663, 1986.
70. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T., Enzyme-labeling
of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining,
J. Immunoassay, 4, 209, 1983.
71. Ngo, T. T. (ed.), Nonisotopic Immunoassay, Plenum Press, New York, 1988.
72. Hong, J., Lee, A., Han, H., and Kim, J., Structural characterization of immunoglobulin G using timedependent disulfide bond reduction, Anal. Biochem., 384, 368, 2009.
73. Palmer, J. L. and Nisonoff, A., Dissociation of rabbit gamma-globulin into half molecules after reduction
of one labile disulfide bond, Biochemistry, 3, 863, 1964.
74. Grassetti, D. R. and Murray, J. F., Jr., Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiopyridine, Arch. Biochem. Biophys., 103, 1132, 1967.
75. Yoshitake, S., Hamaguchi, Y., and Ishikawa, E., Efficient conjugation of rabbit Fab′ with β-D-galactosidase
from Escherichia coli, Scand. J. Immunol., 10, 81, 1979.
76. Cherkaoui, S., Bettinger, T., Hauwel, M., Navetat, S., Allémann, E., and Schneider, M., Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface,
J. Pharm. Biomed. Anal., 53, 172, 2010.
77. Penefsky, H. S., A centrifuged-column procedure for the measurement of ligand binding by beef heart
F1, Methods Enzymol., 56, 527, 1979.
78. Boorsma, D. M. and Kalsbeek, G. L., A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method, J. Histochem. Cytochem., 23, 200, 1975.
79. Avrameas, S. and Ternynck, T., Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunochemistry, 8, 1175, 1971.
80. Boorsma, D. M. and Streefkerk, J. G., Peroxidase-conjugate chromatography isolation of conjugates
prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel, J. Histochem. Cytochem.,
24, 481, 1976.
81. Boorsma, D. M. and Streefkerk, J. G., Periodate or glutaraldehyde for preparing peroxidase conjugates?
J. Immunol. Methods, 30, 245, 1979.
82. Tijssen, P. and Kurstak, E., Highly efficient and simple methods for the preparation of peroxidase and
active peroxidase–antibody conjugates for enzyme immunoassays, Anal. Biochem., 136, 451, 1984.
83. Nakane, P. K., Recent progress in the peroxidase-labeled antibody method, Ann. N. Y. Acad. Sci., 254,
84. Simons, B., Kaplan, H., and Hefford, M. A., Novel cross-linked enzyme–antibody conjugates for Western
blot and ELISA, J. Immunol. Methods, 315, 88, 2006.
Preparation of Immunoconjugates and Immunogens
85. Modesto, R. R. and Pesce, A. J., The reaction of 4,4′-difluoro-3,3′-dinitro-diphenyl sulfone with gammaglobulin and horseradish peroxidase, Biochim. Biophys. Acta, 229, 384, 1971.
86. Abuknesha, R. A., Luk, C. Y., Griffith, H. H. M., Maragkou, A., and Iakovaki, D., Efficient labelling
of antibodies with horseradish peroxidase using cyanuric chloride, J. Immunol. Methods, 306, 211,
87. Modesto, R. R. and Pesce, A. J., Use of tolylene diisocyanate for the preparation of a peroxidase-labelled
antibody conjugate. Quantitation of the amount of diisocyanate bound, Biochim. Biophys. Acta, 295, 283,
88. Clyne, D. H., Norris, S. H., Modesto, R. R., Pesce, A. J., and Pollak, V. E., Antibody enzyme conjugates:
The preparation of intermolecular conjugates of horseradish peroxidase and antibody and their use in
immunohistology of renal cortex, J. Histochem. Cytochem., 21, 233, 1973.
89. Ternynck, T. and Avrameas, S., Conjugation of p-benzoquinone treated enzymes with antibodies and Fab
fragments, Immunochemistry, 14, 767, 1977.
90. Jeanson, A., Cloes, J. M., Bouchet, M., and Rentier, B., Preparation of reproducible alkaline phosphataseantibody conjugates for enzyme immunoassay using a heterobifunctional linking agent, Anal. Biochem.,
172, 392, 1988.
91. Ishikawa, E., Hashida, S., Kohno, T., and Ranaka, K., Methods for enzyme-labeling of antigens, antibodies
and their fragments, in Nonisotopic Immunoassay, Ngo, T. T. (ed.), Plenum Press, New York, 1988, p. 27.
92. Ford, D. J., Radin, R., and Pesce, A. J., Characterization of glutaraldehyde coupled alkaline phosphataseantibody and lactoperoxidase–antibody conjugates, Immunochemistry, 15, 237, 1978.
93. Falini, B. and Taylor, C. R., New developments in immunoperoxidase techniques and their application,
Arch. Pathol. Lab. Med., 107, 105, 1983.
94. Liu, Z., Gurlo, T., and von Grafenstein, H., Cell-ELISA using beta-galactosidase conjugated antibodies,
J. Immunol. Methods, 234, 153, 2000.
95. Hashida, S., Imagawa, M., Inoue, S., Ruan, K. H., and Ishikawa, E., More useful maleimide compounds
for the conjugation of Fab′ to horseradish peroxidase through thiol groups in the hinge, J. Appl. Biochem.,
6, 56, 1984.
96. Kurstak, E., Enzyme Immunodiagnosis, Academic Press, Orlando, FL, 1986, p. 20.
97. Fujiwara, K., Saita, T., and Kitagawa, T., The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a coupling agent in the preparation of enzyme–antibody conjugates, J. Immunol. Methods, 110, 47, 1988.
98. Deelder, A. M. and de Water, R., A comparative study on the preparation of immunoglobulin–galactosidase conjugates, J. Histochem. Cytochem., 29, 1273, 1981.
99. Guesdon, J.-L., Amplification systems for enzyme immunoassay, in Nonisotopic Immunoassay, Ngo, T. T.
(ed.), Plenum Press, New York, 1988, p. 85.
100. Avrameas, S., Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies, Immunochemistry, 6, 43, 1969.
101. Engvall, E. and Perlmann, P., Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of
immunoglobulin G, Immunochemistry, 8, 871, 1971.
102. Muzykantov, V. R., Sakharov, D. V., Sinitsyn, V. V., Domogatsky, S. P., Goncharov, N. V., and Danilov,
S. M., Specific killing of human endothelial cells by antibody-conjugated glucose oxidase, Anal.
Biochem., 169, 383, 1988.
103. Avrameas, S., Ternynck, T., and Guesdon, J.-L., Coupling of enzyme to antibodies and antigens, in
Quantitative Enzyme Immunoassay, Engvall, E. and Pesce, A. J. (eds.), Blackwell Scientific, Oxford,
U.K., 1978, p. 7.
104. Van der Waart, M. and Schuurs, A. H. M. W., Towards the development of a radioenzyme-immunoassay
(REIA), J. Anal. Chem., 279, 142, 1976.
105. Pene, J., Rousseau, V., and Stanislawski, M., In-vitro cytolysis of myeloma tumor cells with glucose
oxidase and lactoperoxidase antibody conjugates, Biochem. Int., 13, 233, 1986.
106. Ishikawa, E., Enzyme immunoassay of insulin by fluorimetry of the insulin-glucoamylase complex,
J. Biochem., 73, 1319, 1973.
107. Herrmann, J. E. and Morse, S. A., Conjugation of enzymes to anti-poliovirus globulin: Effect of enzyme
molecular weight on virus neutralization capacity, Immunochemistry, 11, 79, 1974.
108. Wei, R. and Riebe, S., Preparation of a phospholipase C-antihuman IgG conjugate, and inhibition of its
enzymatic activity by human IgG, Clin. Chem., 23, 1386, 1977.
109. Lal, R. B., Brown, E. M., Seligmann, B. E., Edison, L. J., and Chused, T. M., Selective elimination of
lymphocyte subpopulations by monoclonal antibody-enzyme conjugates, J. Immunol. Methods, 79, 307,
Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
110. Guesdon, J. L., Jouanne, C., and Avrameas, S., An amplification system using BSA-antibody conjugate
for sensitive enzyme immunoassay, J. Immunol. Methods, 58, 133, 1983.
111. Rauterberg, E. W., Schieck, C., Kreft, H., and Römer, W., Optimal conditions for the preparation of ferritin-labeled antibodies defined by binding to their antigen in an ELISA, Immunobiology, 166, 439, 1984.
112. Shapiro, H. M., Glazer, A. N., Christenson, L., Williams, J. M., and Strom, T. B., Immunofluorescence
measurement in a flow cytometer using low-power helium-neon laser excitation, Cytometry, 4, 276,
113. Kronick, M. N. and Grossman, P. D., Immunoassay techniques with fluorescent phycobiliprotein conjugates, Clin. Chem., 29, 1582, 1983.
114. Triantafilou, K., Triantafilou, M., and Wilson, K. M., Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging, Cytometry, 41, 226, 2000.
115. Oi, V. T., Glazer, A. N., and Stryer, L., Fluorescent phycobiliprotein conjugates for analyses of cells and
molecules, J. Cell Biol., 93, 981, 1982.
116. Hardy, R. R., Purification and coupling of fluorescent proteins for use in flow cytometry, in Handbook
of Experimental Immunology, 4th edn., Vol. 1, Weir, D. M. (ed.), Blackwell Scientific, Edinburgh, U.K.,
1986, Chapter 13.
117. Tawde, S. S. and Ram, J. S., Conjugation of antibody to ferritin by means of p,p′-difluoro-m,
m′-dinitrodiphenylsulphone, Arch. Biochem. Biophys., 97, 429, 1962.
118. Rauterberg, E. W. and Schieck, C., Ferritin-labeling of antibodies by glutaraldehyde. Comparison of conjugates prepared at different antibody: Ferritin: glutaraldehyde ratios, Immunobiology, 159, 307, 1981.
119. Rudick, R. A., Bloechl, E. K., and Knutson, D. W., Preparation of monoclonal antibody-ferritin conjugates of high specific activity, Histochemistry, 80, 269, 1984.
120. Singer, S. J. and Schick, A. F., The properties of specific stains for electron microscopy prepared by the
conjugation of antibody molecules with ferritin, J. Biophys. Biochem. Cytol., 9, 519, 1961.
121. van Gijlswijk, R. P., van Gijlswijk-Janssen, D. J., Raap, A. K., Daha, M. R., and Tanke, H. J., Enzymelabelled antibody-avidin conjugates: New flexible and sensitive immunochemical reagents, J. Immunol.
Methods, 189, 117, 1996.
122. Boorsma, D. M., Van Bommel, J., and Vanden Heuvel, J., Avidin-HRP conjugates in biotin–avidin immunoenzyme cytochemistry, Histochemistry, 84, 333, 1986.
123. Engvall, E., Preparation of enzyme-labelled staphylococcal protein A and its use for detection of antibodies, in Quantitative Enzyme Immunoassay, Engvall, E. and Pesce, A. J. (eds.), Blackwell Scientific,
Oxford, U.K., 1978, p. 25.
124. Denisov, V. N. and Metelitsa, D. I., Catalytic and immunochemical properties of ferritin conjugates with
horseradish peroxidase, Biokhimiia, 52, 1248, 1987.
125. Khanna, P. L., Dworschack, R. T., Manning, W. B., and Harris, J. D., A new homogeneous enzyme immunoassay using recombinant enzyme fragments, Clin. Chim. Acta, 185, 231, 1989.
126. Fujiwara, K. and Saita, T., The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a heterobifunctional
agent in developing enzyme immunoassay for neurotensin, Anal. Biochem., 161, 157, 1987.
127. Kato, K., Hamaguchi, Y., Fukui, H., and Ishikawa, E., Enzyme-linked immunoassay. I. Novel method for
synthesis of the insulin-beta-D-galactosidase conjugate and its applicability for insulin assay, J. Biochem.,
78, 235, 1975.
128. Pradelles, P., Grassi, J., Chabardes, D., and Guiso, N., Enzyme immunoassays of adenosine cyclic
3′,5′-monophosphate and guanosine cyclic 3′,5′-monophosphate using acetylcholinesterase, Anal.
Chem., 61, 447, 1989.
129. Butz, S., Rawer, S., Rapp, W., and Birsner, U., Immunization and affinity purification of antibodies using
resin-immobilized lysine-branched synthetic peptides, Pept. Res., 7, 20, 1994.
130. De Silva, B. S., Egodage, K. L., and Wilson, G. S., Purified protein derivative (PPD) as an immunogen
carrier elicits high antigen specificity to haptens, Bioconjug. Chem., 10, 496, 1999.
131. Romestand, B., Rolland, J. L., Commeyras, A., Coussot, G., Desvignes, I., Pascal, R., and VandenabeeleTrambouze, O., Dendrigraft poly-L-lysine: A non-immunogenic synthetic carrier for antibody production, Biomacromolecules, 11, 1169, 2010.
132. Zhang, Q., Wang, L., Ahn, K. C., Sun, Q., Hu, B., Wang, J., and Liu, F., Hapten heterology for a specific
and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion,
Anal. Chim. Acta, 596, 303, 2007.
133. Zhang, Q., Wu, Y., Wang, L., Hu, B., Li, P., and Liu, F., Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb, Anal. Chim.
Acta, 625, 87, 2008.
Preparation of Immunoconjugates and Immunogens
134. Zhang, Q., Zhang, W., Wang, X., and Li, P., Immunoassay development for the class-specific assay for
types I and II pyrethroid insecticides in water samples, Molecules, 15, 164, 2010.
135. Basu, A., Nara, S., Chaube, S. K., Rangari, K., Kariya, K. P., and Shrivastav, T. G., The influence of
spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten, Clin. Chim. Acta, 366, 287, 2006.
136. Chen, Y., Wang, X., Wang, J., and Tang, S., Preparation of acetochlor antibody and its application on
immunoaffinity chromatography cleanup for residue determination in peanuts, J. Agric. Food Chem., 57,
137. Xu, T., Shao, X. L., Li, Q. X., Keum, Y. S., Jing, H. Y., Sheng, W., and Li, J., Development of an enzymelinked immunosorbent assay for the detection of pentachloronitrobenzene residues in environmental
samples, J. Agric. Food Chem., 55, 3764, 2007.
138. Gandhi, S., Sharma, P., Capalash, N., Verma, R. S., and Suri, C. R., Group-selective antibodies based
fluorescence immunoassay for monitoring opiate drugs, Anal. Bioanal. Chem., 392, 215, 2008.
139. Tanaka, H., Yan, S., Miura, N., and Shoyama, Y., Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies, Cytotechnology, 42, 101, 2003.
140. Shinkaruk, S., Lamothe, V., Schmitter, J. M., Fructus, A., Sauvant, P., Vergne, S., Degueil, M., Babin, P.,
Bennetau, B., and Bennetau-Pelissero, C., Synthesis of haptens and conjugates for ELISA of glycitein:
Development and validation of an immunological test, J. Agric. Food Chem., 56, 6809, 2008.
141. Shinkaruk, S., Bennetau, B., Babin, P., Schmitter, J. M., Lamothe, V., Bennetau-Pelissero, C., and Urdaci,
M. C., Original preparation of conjugates for antibody production against Amicoumacin-related antimicrobial agents, Bioorg. Med. Chem., 16, 9383, 2008.
142. Sathe, M., Derveni, M., Allen, M., and Cullen, D. C., Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten-protein conjugate for antibody development, Bioorg. Med. Chem. Lett., 20, 1792, 2010.
143. Hegde, V. L. and Venkatesh, Y. P., Generation of antibodies specific to D-mannitol, a unique haptenic
allergen, using reductively aminated D-mannose-bovine serum albumin conjugate as the immunogen,
Immunobiology, 212, 119, 2007.
144. Sreenath, K. and Venkatesh, Y. P., Reductively aminated D-xylose-albumin conjugate as the immunogen
for generation of IgG and IgE antibodies specific to D-xylitol, a haptenic allergen, Bioconjug. Chem., 18,
145. Bergeron, R. J., Bharti, N., Singh, S., McManis, J. S., Wiegand, J., and Green, L. G., Vibriobactin antibodies: A vaccine strategy, J. Med. Chem., 52, 3801, 2009.
146. Shim, J. Y., Kim, Y. A., Lee, E. H., Lee, Y. T., and Lee, H. S., Development of enzyme-linked immunosorbent assays for the organophosphorus insecticide EPN, J. Agric. Food Chem., 56, 11551, 2008.
147. Lee, J. K., Ahn, K. C., Stoutamire, D. W., Gee, S. J., and Hammock, B. D., Development of an enzymelinked immunosorbent assay for the detection of the organophosphorus insecticide acephate, J. Agric.
Food Chem., 51, 3695, 2003.
148. Krämer, P. M., Forster, S., and Kremmer, E., Enzyme-linked immunosorbent assays for the sensitive
analysis of 2,4-dinitroaniline and 2,6-dinitroaniline in water and soil, Anal. Bioanal. Chem., 391, 1821,
149. Lee, J. K., Park, S. H., Lee, E. Y., Kim. Y. J., and Kyung, K. S., Development of an enzyme-linked
immunosorbent assay for the detection of the fungicide fenarimol, J. Agric. Food Chem., 52, 7206,
150. Ramón-Azcón, J., Sánchez-Baeza, F., Sanvicens, N., and Marco, M.-P., Development of an enzymelinked immunosorbent assay for determination of the miticide bromopropylate, J. Agric. Food Chem., 57,
151. Ahn, K. C., Watanabe, T., Gee, S. J., and Hammock, B. D., Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin,
J. Agric. Food Chem., 52, 4583, 2004.
152. Imagawa, M., Hashida, S., Ishikawa, E., and Freytag, J. W., Preparation of a monomeric 2,4-dinitrophenyl Fab′-beta-D-galactosidase conjugate for immunoenzymometric assay, J. Biochem., 96, 1727,
153. Beyzavi, K., Hampton, S., Kwasowski, P., Fickling, S., Marks, V., and Clift, R., Comparison of horseradish peroxidase and alkaline phosphatase-labelled antibodies in enzyme immunoassays, Ann. Clin.
Biochem., 24 (Pt 2), 145, 1987.
154. Montoya, A. and Castell, J. V., Long-term storage of peroxidase-labelled immunoglobulins for use in
enzyme immunoassay, J. Immunol. Methods, 99, 13, 1987.
Application of Chemical
Conjugation for the
Preparation of Immunotoxins
and Other Drug Conjugates
for Targeting Therapeutics
Drug conjugates developed as a result of systemic pharmacotherapy are target-specific cytotoxic
agents.1 The underlying concept involves coupling a therapeutic agent to a delivery molecule with
specificity for a defined target-cell population. Antibodies with high affinity for antigens are natural choices for targeting agents.2–5 With the availability of high-affinity monoclonal antibodies
(mAbs) and their fragments, the prospects of antibody-targeting therapeutics have become promising. Toxic substances that have been conjugated to mAbs include protein toxins, low-molecularweight drugs, biological response modifiers, and radionuclides.6 Antibody–toxin conjugates are
frequently termed immunotoxins, whereas immunoconjugates consisting of antibodies and lowmolecular-weight drugs such as methotrexate and adriamycin are called chemoimmunoconjugates.
Immunomodulators contain biological response modifiers that are known to have regulatory functions such as lymphokines, growth factors, and complement-activating cobra venom factor (CVF).
Radioimmunoconjugates consist of radioactive isotopes, which may be used as therapeutics to kill
cells with radiation or used for imaging.
In addition to antibodies, other molecules that have specific receptors or binding sites on target
cells have also been used as targeting agents. These include transferrin, α2-macroglobulin, epidermal growth factor (EGF), and hormones. When hormones are used as the targeting agents, the term
hormonotoxin is frequently used. Toxins have also been conjugated to antigens to selectively kill
antigen-responsive B cells.7
The coupling of targeting agents with toxic moieties is most commonly performed with heterobifunctional cross-linking reagents. Earlier attempts to conjugate toxins and antibodies by homobifunctional reagents generated nonspecific cross-linking products. More specific and efficient
cross-linking techniques have been developed.8 This chapter will review the procedures that have
been used to prepare immunotoxins and other cytotoxic drug conjugates. Theoretically, a crosslinking agent used to couple one toxin is applicable to other toxins. In fact, such is the case for most
of the toxins described.
13.2 TARGETING AGENTS AND TOXINS
13.2.1 Choice of Targeting Agents
In almost all cases, the cytotoxic chemotherapeutic agents, such as paclitaxel, cisplatin, and doxorubicin (DOX), cannot distinguish cancer cells from normal cells. Consequently, in the development or choice of tumor-specific delivery systems for anticancer agents, recognizing the intrinsic
Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
differences between normal and tumor cells is the most important success factor for efficacious
cancer chemotherapy. While new drug delivery systems continue to evolve, there are many choices
of targeting agents as listed below. Understanding the specificity of these agents facilitates the
development of tumor-directed drug conjugates.
As mentioned above, the toxin carriers in immunotoxins are antibodies.5,9 One of the most important aspects of targeting therapy is the specificity of the carrier agents toward the target.8 Among
all antibodies, mAbs raised against specific markers on the surface of tumor cells are the most
highly selective. Although these mAbs may have therapeutic value on their own, their cytotoxicities
are greatly enhanced by conjugating them to highly cytotoxic drugs, which may be too toxic to be
used alone. Thus, to achieve the full potential of immunotoxins, the antibody should be carefully
selected such that it binds selectively to the target tissue with high affinity and has little crossreactivity with healthy tissues. Ideally, the antibodies should be raised against those antigens that
are highly expressed on the cell surface for maximum therapeutic potential. To avoid immunogenicity of these antibodies, nonimmunogenic humanized forms of antibodies should be used. These
considerations are very important as demonstrated by the fact that a KS1/4 antibody–methotrexate
conjugate for nonsmall cell lung cancer10 and an antibody–DOX conjugate, BR96-Dox, for gastric
adenocarcinoma11 and metastatic breast cancer12 were all found to lack therapeutic benefit. On the
other hand, the antibody-maytansinoid conjugates seemed to provide superior antitumor activity.8
Also, chimeric antitransferrin receptor antibodies in which the constant region of the antibody is
substituted by a human-constant-region-inhibited proliferation and directly induced apoptosis in
hematopoietic-derived cell lines.13
126.96.36.199 Other Naturally Occurring Molecules
In addition to antibodies, many other naturally occurring biopolymers and substances have been
used as toxin carriers. These include vitamins, fatty acids, carbohydrates, transferrin, lectin, inulin,
and regulatory and signaling molecules. Chen et al.14 have developed a mechanism-based tumortargeting drug delivery system based on tumor-specific vitamin-receptor-mediated endocytosis.
In this case, biotin (a vitamin) is linked to cytotoxic agents such as taxoid through a disulfide
linkage attached to a phenyl group. Such a disulfide bond can be cleaved upon endocytosis by
endogenous thiols, for example, glutathione (GSH) and thioredoxin, to generate the desirable thiophenolate or sulfhydrylphenyl species and the free toxin. The synthesis involves pyridine disulfide
and N-hydroxysuccinimide. Other essential vitamins that have overexpressed receptors on cancer cell surface have also been used. These include folic acid,15 vitamin B12,16 and riboflavin.17
Unfortunately, in most cases, the synthesis of the linker between the vitamin derivatives and the
toxin is complicated involving many steps.
Another naturally occurring substance used as a drug carrier based on endocytosis is transferrin.13
Transferrins are iron-binding proteins. The human transferrin consists of two domains, homologous to each other with 679 amino acids. Iron-bound transferrin is internalized after binding to
transferrin-specific receptors by receptor-mediated endocytosis. Since the transferrin receptor
is expressed in inflammation and in proliferating malignant cells, transferrin may be used as a
drug carrier for therapy. Several drugs have been conjugated to transferrin and tested for efficacy against cancer cells. These drugs include adriamycin, cisplatin, chlorambucil, daunorubicin, protein synthesis inhibitors, plant-derived toxins, and many others.18 Adriamycin–transferrin
conjugate has been shown to be toxic to human leukemia, erythroleukemia, colorectal carcinoma, breast adenocarcinoma, mesothelioma, liver carcinoma, and cervical adenocarcinoma cell
lines.19 Cisplatin-transferrin inhibits the growth of the human epidermoid carcinoma cell line.20
The drugs chlorambucil and daunorubicin conjugated with transferrin show increased cytotoxicity in human breast cancer cell and small cell lung carcinoma compared with unconjugated
drug.21,22 The effect of other drug-transferrin conjugates has been reported.18 Many methods have
Preparation of Immunotoxins and Other Drug Conjugates
been used to prepare the transferring-drug conjugates such as Schiff base formation,18 acidsensitive maleimide derivatives, and glutaraldehyde.18–22
Polyunsaturated fatty acids (PUFAs) have also been used as tumor-specific molecules.
Representative naturally occurring PUFAs include linolenic acid (LNA), linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Kuznetsova
et al.23 prepared the conjugates of DHA, LNA, and LA with second-generation taxoids and studied their efficacy in vivo against ovarian and colon tumor xenografts. The coupling procedure for
esterification uses N,N′-dicyclohexylcarbodiimide (DCC) as a coupling agent, methylene chloride
as a solvent system, and dimethylaminopyridine (DMAP) as a catalyst. The authors demonstrated
that two of the PUFA-taxoid conjugates show total regression of drug-resistant and drug-sensitive
tumors in animal models.
Hyaluronic acid (HA, also called hyaluronan) is a polymer of disaccharides, composed of
d-glucuronic acid and d-N-acetylglucosamine, linked together via alternating β-1,4 and β-1,3 glycosidic bonds. HA can contain up to 25,000 disaccharide repeats in length with molecular weights
ranging from 5,000 to 20,000,000 Da. It is distributed widely throughout connective, epithelial,
and neural tissues. There are three main groups of cell receptors for HA: CD44, RHAMM (receptor for HA-mediated motility) and ICAM-1 (intracellular adhesion molecule-1). Because of these
receptors, HAs have been employed as tumor-specific modules to construct tumor-targeting drug
conjugates.24 Akima et al.25 coupled mitomycin C and epirubicin to HA by carbodiimide chemistry
and found that the former adduct was selectively taken up by, and was toxic to, a lung carcinoma
xenograft. Luo and Prestwich26 covalently attached taxol with N-hydroxysuccinimide to HA modified with adipic. The HA-taxol conjugates were shown to have selective toxicity toward the human
breast, colon, and ovarian cancer cell lines that overexpress HA receptors. Saravanakumar et al.27
modified HA with an amine-terminated hydrotropic N,N-diethylnicotinamide (DENA) oligomer to
synthesize hydrotropic HA (HydroHA) derivatives. Paclitaxel (PTX), a highly hydrophobic chemotherapeutic agent, was coupled to a HydroHA using carbodiimide chemistry. The HydroHA-PTX
conjugates were selectively taken up by the cancer cell line with overexpressing CD44. Luo et al.28
also prepared a modified HA with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and
conjugated to DOX. The HPMA-HA-DOX conjugate was found to have higher cytotoxicity against
human breast cancer, ovarian cancer, and colon cancer.
Lectins are sugar-binding proteins that are highly specific for the structures of sugar moieties. Lectin wheat germ agglutinin (WGA), for example, binds to N-acetyl-d-glucosamine and
sialic acid of carbohydrates. It has a high binding rate to intestinal cell lines of human origin,
human colonocytes, and prostate cancer cells. Moreover, it is also taken up into the cytoplasm
of enterocyte-like Caco-2 cells.29 Gabor et al.30 conjugated fluorescein-bovine serum albumin
(F-BSA) to lectin WGA using a homobifunctional cross-linker, divinyl sulfone, to react with the
amino groups of both proteins. The F-BSA-WGA conjugate were bound specifically to Caco-2
cells and exhibited uptake into the cells. The authors concluded that WGA-mediated drug delivery is a promising strategy. With another lectin, concanavalin A (Con-A), which binds specifically to mannose, Anande et al.31 prepared mucoadhesive microspheres of diloxanide furoate
(DF) for the effective treatment of amoebiasis. The carboxyl groups of Eudragit microspheres
of DF were linked to Con-A using ethyl-3,3-(dimethylaminopropyl) carbodiimide (EDC) and
N-hydroxysuccinimide. The conjugate increased the mucoadhesiveness and provided controlled
release of DF in simulated GI fluids.
Heparin is a highly sulfated glycosaminoglycan that has been conjugated to deoxycholic acid
(DOCA) for drug delivery.32 Park et al.32,33 prepared heparin-DOCA (HD) conjugates by covalently bonding N-(2-aminoethyl)deoxycholylamide to heparin via amide formation with EDC.
The HD conjugates were loaded with DOX through heparin nanoparticle formation. These
DOX-loaded heparin nanoparticles displayed a sustained drug release pattern and enhanced
therapeutic effect against squamous cell carcinoma and B16F10 melanoma. Wang et al.34 also
prepared various heparin conjugates by activating heparin to a mixed anhydride intermediate to
Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
which paclitaxel and amino acid-paclitaxel derivatives have been linked. These conjugates were
shown to arrest MCF-7 cells in the G2/M phase of cell cycle.
Gellan gum, also known commercially as Phytagel or Gelrite, is a water-soluble polysaccharide
produced by bacterium, Sphingomonas elodea. The repeating unit of the polymer is a tetrasaccharide, which consists of two residues of d-glucose along with one residue of l-rhamnose and
one residue of d-glucuronic acid connected through an (α1 → 3) glycosidic bond. Krauland et al.35
conjugated l-cysteine to deacetylated gellan gum (DGG) by a carbodiimide. The DGG-cysteine
conjugate was capable of forming inter- and/or intramolecular disulfide bonds in aqueous solution.
The authors suggested that the conjugated polymer represents a promising novel excipient for various drug delivery systems.
188.8.131.52 Synthetic Peptides and Nucleotides
Many peptides, polypeptides, and proteins have been shown to posses the ability to traverse biological membranes. These matrices have been successfully used for the intracellular delivery of many
therapeutic agents including small molecules, proteins, peptides, oligonucleotides, plasmids, and
nanoparticles.36 Unbiased biopanning of phage-displayed peptide libraries has generated a suite of
cancer targeting peptidic ligands.37 Among these, cell-penetrating peptides, which are 9–35 mer
cationic and/or amphipathic peptides, can be linked to a variety of anticancer therapeutics, making
them an efficient, effective, and nontoxic mechanism for drug delivery.38
Specifically, receptors for certain peptide hormones are expressed in a relatively high concentration on a variety of cancer cells. These peptide hormones can serve as carriers for a local delivery of
cytotoxic agents or radiopharmaceuticals to the tumors. The most widely investigated of these peptide hormones is the hypothalamic hormone, somatostatin.39 The short plasma half-life of the native
forms of somatostatin prompted an avid search for more stable and more potent synthetic analogs
as demonstrated by the successful clinical use of radiolabeled somatostatin analog, Octreoscan, for
the detection and treatment of some somatostatin receptor-positive tumors. In recent years, a series
of other cytotoxic peptide hormone conjugates based on derivatives of hypothalamic hormones
such as luteinizing hormone-releasing hormone (LHRH) and the brain-gut hormone bombesin were
prepared. A derivative of DOX, 2-pyrrolino-DOX, which is 500–1000 times more active than its
parent compound was coupled to somatostatin octapeptide, to LHRH analogue, [D-Lys6]LHRH,
and to bombesin-like peptide. These conjugates were investigated for their effectiveness in various
cancers including ovarian and breast cancers,39 hepatocellular carcinoma,40 and prostate cancer.41
Oligonucleotides have also been used as targeting agent for drug delivery. Of particular importance are the aptamers, which are short single-stranded nucleic acids with a defined three-dimensional shape that allows them to interact with high affinity with a target molecule.42,43 They have
been used to target distinct cell subtypes and tissues. The most established and best characterized
aptamer in this regard is the prostate-specific membrane antigen (PSMA)-binding nucleic acid molecule A10. This aptamer has been conjugated with cytotoxic molecules, thereby allowing their selective delivery to cancer cells. For example, Farokzhad et al.44 have attached chemotherapeutics, such
as docetaxel, to the 5′-amino end of the aptamer by an NHS/EDC approach and showed the efficacy of such an aptamer-drug conjugate in an in vivo xenograft rat tumor model system. The same
group also directly complexed aptamer A10 with DOX and demonstrated that treatment of prostate
cancer cells with the complex resulted in a significant reduction of tumor-cell proliferation.45 Chu
et al.46 also conjugated the aptamer to the toxin gelonin, a ribosome-inactivating protein (RIP), with
N-succinimidyl-3-(2-pyridylodithio)propionate (SPDP). The aptamer conjugate not only promoted
uptake of gelonin into target cells but also decreased the toxicity of gelonin in nontarget cells.
In addition to chemotherapeutics, siRNA molecules have also been coupled to aptamer
A10 either directly by nucleotidic extensions or indirectly through the assembly of tetrameric
streptavidin–biotin complexes.47,48 Several other aptamers have also been used as aptamer-based