Methods for Enzyme-Labeling of Antigens Antibodies and their Fragments

  • Eiji Ishikawa
  • Seiichi Hashida
  • Takeyuki Kohno
  • Koichiro Tanaka


A number of reagents have been used for enzyme-labeling of antigens and antibodies during the past two decades. However, most of them suffer from serious disadvantages, and only a few reagents are in current use, including glutaraldehyde, periodate, maleimide compounds and pyridyl disulfide compounds (Ishikawa et al., 1983a).


Sodium Phosphate Buffer Pepsin Digestion Conjugation Efficiency Maleimide Group Calf Intestine 


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  1. Avrameas, S., and Ternynck, T., 1971, Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunochemistry, 8: 1175.PubMedCrossRefGoogle Scholar
  2. Baumann, G., and Abramson, E. C., 1983, Urinary growth hormone in man: Evidence for multiple molecular forms, J. Clin. Endocrinol. Metab., 56: 305.PubMedCrossRefGoogle Scholar
  3. Beaucamp, K., Bergmeyer, H. U., and Beutler, H-O., 1974, Coenzymes, metabolites, and other biochemical reagents, in: “Methods of Enzymatic Analysis” Bergmeyer, H. U., ed., Academic Press, New York, p. 545.Google Scholar
  4. Borkowski, A., Puttaert, V., Gyling, M., Muquardt, C., and Body, J. J., 1984, Human chorionic gonadotropin-like substrance in plasma of normal nonpregnant subjects and women with breast cancer, J. Clin. Endocrinol. Metab., 58: 1171.PubMedCrossRefGoogle Scholar
  5. Craven, G. R., Steers, Jr., E., and Anfinsen, C. B., 1965, Purification, composition, and molecular weight of the ß-galactosidase of Escherichia coli K12, J. Biol. Chem., 240: 2468.PubMedGoogle Scholar
  6. Dissanayake, S., and Hay, F. C., 1975, Pepsin digestion of mouse IgG immunoglobulins subfragments of the Fc region, Immunochemistry, 12: 373.PubMedCrossRefGoogle Scholar
  7. Dorrington, K. J., and Tanford, C., 1970, Molecular size and conformation of immunoglobulins, Adv. Immunol., 12: 333.PubMedCrossRefGoogle Scholar
  8. Drobny, E. C., Amburn, K., and Baumann, G., 1983, Circadian variation of basal plasma growth hormone in man, J. Clin. Endocrinol. Metab., 57: 524.PubMedCrossRefGoogle Scholar
  9. Engström, L., 1961, Studies on calf-intestinal alkaline phosphatase. I. Chromatographic purification, microheterogeneity and some other properties of the purified enzyme, Biochim. Biophys. Acta, 52: 36.PubMedCrossRefGoogle Scholar
  10. Ey, P. L., Prowse, S. J., and Jenkin, C. R., 1978, Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose, Immunochemistry, 15: 429.PubMedCrossRefGoogle Scholar
  11. Gorini, G., Medgyesi, G. A., and Doria, G., 1969, Heterogeneity of mouse myeloma γG globulins as revealed by enzymatic proteolysis, J. Immunol., 103: 1132.PubMedGoogle Scholar
  12. Grassetti, D. R., and Murray, Jr., J. F., 1967, Determination of sulfhydryl groups with 2,2′-or 4,4′-dithiodipyridine, Arch. Biochem. Biophys., 119: 41.PubMedCrossRefGoogle Scholar
  13. Guesdon, J.-L., Ternynck, T., and Avrameas, S., 1979, The use of avidinbiotin interaction in immunoenzymatic techniques, J. Histochem. Cytochem., 27: 1131.PubMedCrossRefGoogle Scholar
  14. Hashida, S., Nakagawa, K., Imagawa, M., Inoue, S., Yoshitake, S., Ishikawa, E., Endo, Y., Ohtaki, S., Ichioka, Y., and Nakajima, K., 1983, Use of inorganic salts to minimize serum interference in a sandwich enzyme immunoassay for human growth hormone using Fab’-horseradish peroxidase conjugate, Clin. Chim. Acta, 135: 263.PubMedCrossRefGoogle Scholar
  15. Hashida, S., Imagawa, M., Inoue, S., Ruan, K-h., and Ishikawa, E., 1984, More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge, J. Appl. Biochem., 6: 56.PubMedGoogle Scholar
  16. Hashida, S., Nakagawa, K., Ishikawa, E., and Ohtaki, S., 1985a, Basal level of human growth hormone (hGH) in normal serum, Clin. Chim. Acta, 151: 185.PubMedCrossRefGoogle Scholar
  17. Hashida, S., Ishikawa, E., Nakagawa, K., Ohtaki, S., Ichioka, T., and Nakajima, K., 1985b, Demonstration of human growth hormone in normal urine by a highly specific and sensitive sandwich enzyme immunoassay, Anal. Lett., 18(B13): 1623.CrossRefGoogle Scholar
  18. Hashida, S., Imagawa, M., Ishikawa, E., and Freytag, J. W., 1985c, A simple method for the conjugation of affinity-purified Fab’ to horseradish peroxidase and ß-D-galactosidase from Escherichia coli, J. Immunoassay, 6: 111.PubMedCrossRefGoogle Scholar
  19. Hashida, S., and Ishikawa, E., 1985d, Use of normal IgG and its fragments to lower the non-specific binding of Fab’-enzyme conjugates in sandwich enzyme immunoassay, Anal. Lett., 18(B9): 1143.CrossRefGoogle Scholar
  20. Imagawa, M., Yoshitake, S., Hamaguchi, Y., Ishikawa, E., Niitsu, Y., Urushizaki, I., Kanazawa, R., Tachibana, S., Nakazawa, N., and Ogawa, H., 1982a, Characteristics and evaluation of antibody-horseradish peroxidase conjugates prepared by using a maleimide compound, glutaraldehyde, and periodate, J. Appl. Biochem. 4: 41.Google Scholar
  21. Imagawa, M., Hashida, S., Ishikawa, E., and Sumiyoshi, A., 1982b, Evaluation of Fab’-horseradish peroxidase conjugates prepared using pyridyl disulfide compounds, J. Appl. Biochem., 4: 400.Google Scholar
  22. Imagawa, M., Yoshitake, S., Ishikawa, E., Niitsu, Y., Urushizaki, I., Kanazawa, R., Tachibana, S., Nakazawa, N., and Ogawa, H., 1982c, Development of a highly sensitive sandwich enzyme imraunoassay for human ferritin using affinity-purified anti-ferritin labelled with ß-D-galactosidase from Escherichia coli, Clin. Chim. Acta, 121: 277.PubMedCrossRefGoogle Scholar
  23. Imagawa, M., Hashida, S., Ishikawa, E., and Freytag, J. W., 1984a, Preparation of a monomeric 2,4-dinitrophenyl Fab’-ß-D-galactosidase conjugate for immunoenzymometric assay, J. Biochem., 96: 1727.PubMedGoogle Scholar
  24. Imagawa, M., Hashida, S., Ishikawa, E., Niitsu, Y., Urushizaki, I., Kanazawa, R., Tachibana, S., Nakazawa, N., and Ogawa, H., 1984b, Comparison of ß-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab’ by sandwich enzyme immunoassay technique, J. Biochem., 96: 659.PubMedGoogle Scholar
  25. Imagawa, M., Yoshitake, S., Ishikawa, E., Kanoh, E., Tsunetoshi, Y., Iwasa, S., Konishi, E., Kondo, K., Ichioka, Y., and Nakajima, K., 1984c, Level of human chorionic gonadotropin-like substance in serum of normal subjects, Anal. Lett., 17(B7): 575.CrossRefGoogle Scholar
  26. Inoue, S., Hashida, S., Tanaka, K., Imagawa, M., and Ishikawa, E., 1985, Preparation of monomeric affinity-purified Fab’-ß-D-galactosidase conjugate for immunoenzymometric assay, Anal. Lett., 18(B11): 1331.CrossRefGoogle Scholar
  27. Inoue, S., Hashida, S., Ishikawa, E., Mori, T., Imura, H., Ogawa, H., Ichioka, T., and Nakajima, K., 1986, Highly sensitive sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum using monoclonal anti-hTSH ß-subunit IgG1-coated polystyrene balls and polyclonal anti-human chorionic gonadotropin Fab’-horseradish peroxidase conjugate, Anal. Lett., in press.Google Scholar
  28. Ishikawa, E., and Kato, K., Ultrasensitive enzyme immunoassay, 1978, Scand. J. Immunol. 8 (Suppl. 7): 43.CrossRefGoogle Scholar
  29. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T., 1983a, Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining, J. Immunoassay, 4: 209.PubMedCrossRefGoogle Scholar
  30. Ishikawa, E., Imagawa, M., and Hashida, S., 1983b, Ultrasensitive enzyme immunoassay using fluorogenic, luminogenic, radioactive and related substrates and factors to limit the sensitivity, Develop. Immunol., 18: 219.Google Scholar
  31. Ishikawa, E., Yoshitake, S., Imagawa, M., and Sumiyoshi, A., 1983c, Preparation of monomeric Fab’-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining, Ann. New York Acad. Sci., 420: 74.CrossRefGoogle Scholar
  32. Jaquet, H., and Cebra, J. J., 1965, Comparison of two precipitaing derivatives of rabbit antibody: Fragment I dimer and the product of pepsin digestion, Biochemistry, 4: 954.PubMedCrossRefGoogle Scholar
  33. Johannsson, A., Stanley, C. J., and Self, C. H., 1985, A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry, Clin. Chim. Acta, 148, 119.PubMedCrossRefGoogle Scholar
  34. Keilin, D., and Hartree, E. F., 1951, Purification of horse-radish peroxidase and comparison of its properties with those of catalase and methaemoglobin, Biochem. J., 49: 88.PubMedGoogle Scholar
  35. Kondo, K., Imagawa, M., Iwasa, S., Kitada, C., Konishi, E., Suzuki, N., Yamoto, M., Nakano, R., Yoshitake, S., and Ishikawa, E., 1984, A specific and sensitive sandwich enzyme immunoassay for human chorionic gonadotropin using antibodies against the carboxy-terminal portion of the ß-subunit, Clin. Chim. Acta, 138: 229.PubMedCrossRefGoogle Scholar
  36. Lamoyi, E., and Nisonoff, A., 1983, Preparation of F(ab’)2 fragments from mouse IgG of various subclasses, J. Immunol. Methods, 56: 235.PubMedCrossRefGoogle Scholar
  37. Lindmark, R., Thorèn-Tolling, K., and Sjöquist, J., 1983, Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera, J. Immunol. Methods, 62: 1.PubMedCrossRefGoogle Scholar
  38. Mandy, W. J., and Nisonoff, A., 1963, Effect of reduction of several disulfide bonds on the properties and recombination of univalent fragments of rabbit antibody, J. Biol. Chem., 238: 206.Google Scholar
  39. Morton, R. K., 1955, Some properties of alkaline phosphatase of cow’s milk and calf intestinal mucosa, Biochem. J., 60: 573.PubMedGoogle Scholar
  40. Olive, C., and Levy, H. R., 1971, Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides, J. Biol. Chem., 246: 2043.Google Scholar
  41. Palmer, J. L., and Nisonoff, A., 1964, Dissociation of rabbit γ-globulin into half molecules after reduction of one labile disulfide bond, Biochemistry, 3: 863.PubMedCrossRefGoogle Scholar
  42. Parham, P., 1983, On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice, J. Immunol., 131: 2895.PubMedGoogle Scholar
  43. Penefsky, H. S., 1979, A centrifuged-column procedure for the measurement of ligand binding by beef heart F1. in: “Methods in Enzymology” Colowick, S. P., and Kaplan, N. O., eds., Vol. LVI, Academic Press, New York, p. 527.Google Scholar
  44. Ruan, K-h., Imagawa, M., Hashida, S., and Ishikawa, E., 1984, An improved method for the conjugation of Fab’ to ß-D-galactosidase from Escherichia coli, Anal. Lett., 17(B7): 539.CrossRefGoogle Scholar
  45. Ruan, K-h., Hashida, S., Yoshitake, S., Ishikawa, E., Wakisaka, O., Yamamoto, Y., Ichioka, T., and Nakajima, K., 1985a, A micro-scale affinity-purification of Fab’-horseradish peroxidase conjugates and its use for sandwich enzyme immunoassay of insulin in human serum, Clin. Chim. Acta, 147: 167.PubMedCrossRefGoogle Scholar
  46. Ruan, K-h., Hashida, S., Yoshitake, S., Ishikawa, E., Wakisaka, O., Yamamoto, Y., Ichioka, T., and Nakajima, K., 1985b, A more sensitive and less time-consuming sandwich enzyme immunoassay for insulin in human serum with less serum interference, Ann. Clin. Biochem., in press.Google Scholar
  47. Seppälä, I., Sarvas, H., Pèterfy, F., and Mäkelä, O., 1981, The four subclasses of IgG can be isolated from mouse serum by using protein A-Sepharose, Scand. J. Immunol., 14: 335.PubMedCrossRefGoogle Scholar
  48. Stuchbury, T., Shipton, M., Norris, R., Malthouse, J. P. G., and Brocklehurst, K., 1975, A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety, Biochem. J., 151: 417.PubMedGoogle Scholar
  49. Svasti, J., and Milstein, C., 1972, The disulphide bridges of a mouse immunoglobulin G1 protein, Biochem. J., 126: 837.PubMedGoogle Scholar
  50. Tanaka, K., and Ishikawa, E., 1984, Highly sensitive bioluminescent assay of dehydrogenases using NAD(P)H: FMN oxidoreductase and luciferase from Photobacterium fischeri, Anal. Lett., 17(B18): 2025.CrossRefGoogle Scholar
  51. Udenfriend, S., Stein, S., Böhlen, P., Dairman, W., Leimgruber, W., and Weigele, M., 1972, Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range, Science, 178: 871.PubMedCrossRefGoogle Scholar
  52. Utsumi, S., and Karush, F., 1965, Peptic fragmentation rabbit γG-immunoglobulin, Biochemistry, 4: 1766.CrossRefGoogle Scholar
  53. Wilson, M. B., and Nakane, P. K., 1978, Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies, in: “Immunofluorescence and Related Staining Techniques” Knapp, W., Holubar, K., and G. Wick, eds., Elsevier/North-Holland Biomedical Press, Amsterdam, p. 215.Google Scholar
  54. Yoshitake, S., Hamaguchi, Y., and Ishikawa, E., 1979, Efficient conjugation of rabbit Fab’ with ß-D-galactosidase from Escherichia coli, Scand. J. Immunol., 10: 81.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Eiji Ishikawa
    • 1
  • Seiichi Hashida
    • 1
  • Takeyuki Kohno
    • 1
  • Koichiro Tanaka
    • 1
  1. 1.Department of BiochemistryMedical College of MiyazakiKiyotake, MiyazakiJapan

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