Immunochemistry of Hybridomas
Part of the
NATO ASI Series
book series (NSSA, volume 152)
The attraction of monoclonal antibodies lies in the potential availability of an unlimited supply of material, homogeneity, reproducibility, and the feasibility of manipulation for specific purposes. Their use, however, is limited because of structural and physicochemical idiosyncrasies, occasionally restrictive factors influencing affinity and avidity, unique cross-reactivities, lack of multivalency compared to polyclonal antibodies, and restricted Fc region-specific functions. Approaches to the isolation, purification and fragmentation of monoclonal antibodies derive from basic concepts of the four chain and domain structure of immunoglobulins that have developed from the study of human M-components and a large number of well-characterized mouse myeloma proteins. The specific antibody activity used to generate the hybridoma provides an additional property that can be utilized to advantage in selecting clones and purifying material. Each monoclonal antibody is unique and presents its own set of problems affecting isolation, stability and suitability for chemical modification and radiolabeling. Isotypic determinants correlate with biological activities such as binding to staphylococcal protein A, Clq or specific Fc receptors, that may be important for purification and therapy. Analysis of fragmentation patterns obtained with various proteolytic enzymes [F(ab′)2, Fab, Fab′, Fab/c] can be related to accumulating protein and DNA sequence data. Fragments generated need to be defined for each hybridoma and may present additional idiosyncrasies. These include instability in standard solvents, poor reconstitutability following reduction, and loss of affinity for antigen. To some extent, idiosyncrasies reflect the idiotypic specificities of the antibody. Structural correlates of idiotypy include the binding site for a specific antigen (hypervariable regions), framework determinants, and conformational antigens that may be lost on varying pH, exposure to denaturants, or separation of heavy and light chains. In spite of these problems, however, fragments are more interesting therapeutically, as well as for the design of specific assays, because of loss of effector functions and the potential for artificial constructs. These properties have been exploited in the use of somatic mutants, transfectomas and hybrid molecules. Injection of such fragments to another species may bypass most (though not necessarily all) of the host response to isotypic and allotypic determinants, and still permit various anti-idiotypic antibodies to develop, only some of which may in fact interfere with antibody binding.
KeywordsLight Chain Effector Function Caprylic Acid Antigen Binding Site Primary Amino Acid Sequence
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
D. Pressman, The development and use of radiolabeled anti-tumor antibodies, Cancer Res.
40:2960–2964 (1980).PubMedGoogle Scholar
S. W. Burchiel, N. L. Warner, Immunological considerations relating to the radioimmunochemical detection of cancer, in:
“Tumor Imaging,” S. W. Burchiel, B. A. Rhodes, and B. E. Friedman, eds., Masson Publ., USA, pp. 27–37 (1982).Google Scholar
A. R. Bradwell, D. S. Fairweather, P. W. Dykes, A. Keeling, A. Vaughan, J. Taylor, Limiting factors in the localization of tumors with radiolabeled antibodies, Immunology Today
6:163–170 (1985).CrossRefGoogle Scholar
S. Ghosh, A. M. Campbell, Multispecific monoclonal antibodies, Immunology Today
7:217–222 (1986).CrossRefGoogle Scholar
D. L. Shawler, R. M. Bartholomew, L. M. Smith, R.O. Dillman, Human immune response to multiple injections of murine monoclonal IgG, J. Immunol.
135:1530–1535 (1985).PubMedGoogle Scholar
C. A. Bona, B. Pernis, Idiotypic networks, in:
“Fundamental Immunology,” W. E. Paul, ed., Raven Press, pp 577–592 (1984).Google Scholar
H. Koprowski, D. Herlyn, M. Lubeck, E. Degreitas, H. F. Sears, Human anti-idiotype antibodies in cancer patients: Is the modulation of the immune response beneficial for the patient?, Proc. Natl. Acad. Sci.
2:216–219 (1984).CrossRefGoogle Scholar
J. J. Cebra, J. L. Komisar, P. A. Schweitzer, CH
isotype “switching” during normal B lymphocyte development, Ann. Rev. Immunol.
2:493–548 (1984).CrossRefGoogle Scholar
H. Sakano, J. H. Rogers, K. Huppi, C. Brack, A. Traunecker, R. Maki, R. Wall, S. Tonegawa, Domains and the hinge region of an immunoglobulin heavy chain are encoded in separate DNA segments, Nature
2–7:627–633 (1979).CrossRefGoogle Scholar
A. Feinstein, N. Richardson, M. J. Taussig, Immunoglobulin flexibility in complement activation, Immunology Today
7:169–174 (1986).CrossRefGoogle Scholar
J. W. Goding, “Monoclonal Antibodies: Principles and Practice,” Academic Press, New York, 2nd Edition (1986).Google Scholar
M. Potter, Immunoglobulins and immunoglobulin genes, in:
“The Mouse in Biomedical Research,” H. L. Foster et al, eds., Vol. 3, Academic Press, New York, pp. 347–380 (1984).Google Scholar
S. Natsumme-Sakai, K. Motonishi, S. Migita, Quantitative estimations of five classes of immunoglobulin in inbred mouse strains, Immunology
32:861–870 (1977).Google Scholar
J. L. Fahey, S. Sell, The immunoglobulins of mice V. The metabolic (catabolic) properties of five immunoglobulin classes, J. Exp. Med.
122:41–58 (1965).PubMedCrossRefGoogle Scholar
C. De Imeval, J. R. L. Pink, C. Milstein, Interchain bridges of mouse IgG2a and IgG2b, Nature
228:930–932 (1970).CrossRefGoogle Scholar
J. Svasti, C. Milstein, The disulfide bridges of a mouse immunoglobulin G1 protein, Biochem. J.
126:837–850 (1972).PubMedGoogle Scholar
P. L. Ey, S. J. Prowse, C. R. Jenkins, Isolation of pure IgG1, IgG2a, IgG2b immunoglobulins from mouse serum using protein A — Sepharose, Immunochemistry
15:429–436 (1978).PubMedCrossRefGoogle Scholar
I. Seppala, H. Sarvas, F. Peterix, O. Makela, The four subclasses of IgG can be isolated from mouse serum by using protein A — Sepharose, Scand. J. Immunol.
14:335–342 (1983).CrossRefGoogle Scholar
P. Ralph, I. Nakonig, B. Diamond, D. Yelton, All classes of murine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes, J. Immunol.
125:1885–1888 (1980).PubMedGoogle Scholar
B. Diamond, D. E. Yelton, A new Fc receptor on mouse macrophages binding IgG3, J. Exp. Med.
153:514–519 (1981).PubMedCrossRefGoogle Scholar
M. S. Neuberger, K. Plasensky, Activation of mouse complement by monoclonal mouse antibodies, Eur. J. Immunol.
11:1012–1016 (1981).PubMedCrossRefGoogle Scholar
V. T. Oi, T. M. Vvong, R. Hardy, J. Reidler, J. Dangle, L. A. Herzenberg, L. Stayer, Correlation between segmental flexibility and effector function of antibodies, Nature
307:136–139 (1984).PubMedCrossRefGoogle Scholar
J. C. Unkeless, H. Fleit, I. S. Mellman, Structural aspects and heterogeneity of immunoglobulin Fc receptors, Adv. Immunol.
31:247–270 (1981).PubMedCrossRefGoogle Scholar
C. L. Anderson, R. J. Looney, Human leukocyte IgG Fc receptors, Immunology Today
7:264:266 (1986).CrossRefGoogle Scholar
H. M. Grey, A. Sher, N. Shalitin, The subunit structure of mouse IgA, J. Immunol.
105:75–84 (1970).PubMedGoogle Scholar
H. Bazin, A. Beckers, P. Querinjean, Three classes and four subclasses of rat immunoglobulins : IgM, IgA, IgE, and IgG1, IgG2a, IgG2b, IgG2c, Eur. J. Immunol.
4:44–48 (1974).PubMedCrossRefGoogle Scholar
G. A. Medgyesi, G. Fust, J. Gergely, H. Bazin, Classes and subclasses of rat immunoglobulins: interaction with the complement system and with staphylococcal Protein A, Immunochemistry
15:125–129 (1978).PubMedCrossRefGoogle Scholar
J. Rousseaux, M. T. Picque, H. Bazin, G. Biserte, Rat IgG subclasses: differences in affinity to protein A-Sepharose, Molec. Immunol.
18:639–645 (1981).CrossRefGoogle Scholar
R. Nilsson, E. Myhre, G. Kronvall, H. O. Sjogren, Fractionation of rat IgG subclasses and screening for IgG Fc-binding to bacteria, Molec. Immunology
19:119–126 (1982).CrossRefGoogle Scholar
P. B. Carter, H. Bazin, Immunology, in:
“The Laboratory Rat,” H. J. Baker, J. R. Linsey, S. H. Weisbrodt, eds., Academic Press, Vol.2, pp. 182–212 (1980).Google Scholar
Hughes-Jones, B. D. Gorisk, J. C. Howard, The mechanism of synergistic complement-mediated lysis of rat red cells by monoclonal IgG antibodies, Eur. J. Immunol.
13:635–641 (1983).PubMedCrossRefGoogle Scholar
G. Hale, M. Clark, H. Waldmann, Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes, J. Immunol.
134:3056–3061 (1985).PubMedGoogle Scholar
S. J. Smith-Gill, F. D. Finkelman, M. Potter, Plasmacytomas and murine immunoglobulins, Methods Enzymol.
116:121–145 (1985).PubMedCrossRefGoogle Scholar
H. Bazin, F. Cormont, L. De Clercq, Purification of rat monoclonal antibodies, Meth. Enzymol.
121:638–652 (1986).PubMedCrossRefGoogle Scholar
S. Dissanayake, F. C. Hay, Isolation of pure normal IgG1 from mouse serum, Immunochemistry
12:101–103 (1975).PubMedCrossRefGoogle Scholar
C. D. Bruck, D. Portelle, C. Glineur, A. Bollen, One step purification of mouse monoclonal antibodies from ascites fluid by DEAE affigel blue chromatography, J. Immunol. Methods.
53:313–320 (1982).PubMedCrossRefGoogle Scholar
G. Russo, L. Callegaro, E. Lanza, S. Ferrone, Purification of IgG monoclonal antibody by caprylic acid precipitation, J. Immunol. Meth.
65:269–271 (1983).CrossRefGoogle Scholar
F. Franek, Purification of IgG monoclonal antibodies from ascites fluid based on rivanol precipitation, Methods Enzymol.
121:631–638 (1986).PubMedCrossRefGoogle Scholar
J. R. Deschamps, J. E. K. Hildreth, D. Derr, J. T. August, A high-performance liquid chromatographic procedure for the purification of mouse monoclonal antibodies, Anal. Biochem.
147:451–454 (1985).PubMedCrossRefGoogle Scholar
S. W. Burchiel, Purification and analysis of monoclonal antibodies by high-performance liquid chromatography, Meth. Enzymol.
121:451–454 (1986).Google Scholar
J. W. Goding, Use of staphylococcal protein A as an immunological reagent, J. Immunol. Meth.
20:241–253 (1978).CrossRefGoogle Scholar
J. J. Langone, Protein A of a staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumococci, Adv. Immunol.
32:157–252 (1982).PubMedCrossRefGoogle Scholar
S. W. Burchiel, B. A. Khaw, B. A. Rhodes, T. W. Smith, E. Haber, Immunopharmacokinetics of radiolabeled antibodies and their fragments, in:
“Tumor Imaging,” S. W. Burchiel, B. A. Rhodes, B. E. Friedman, eds., pp. 125–139 (1982).Google Scholar
P. D. Gorevic, F. C. Prelli, B. Frangione, Immunoglobulin G (IgG), Methods Enzymol.
116:3–25 (1985).PubMedCrossRefGoogle Scholar
G. Gorini, G. A. Megyesi, G. Doria, Heterogeneity of mouse G globulins as revealed by enzymatic proteolysis, J. Immunol.
103:1132–1142 (1969).PubMedGoogle Scholar
P. Parham, M. J. Androlewicz, F. M. Brodsky, N. J. Holmes, J. P. Ways, Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHC antigens, J. Immunol. Methods
53:133–173 (1982).PubMedCrossRefGoogle Scholar
J. Rousseaux, G. Biserte, H. Bazin, The differential enzyme sensitivity of rat immunoglobulin G subclasses to papain and pepsin, Molec. Immunol.
17:469–482 (1980).CrossRefGoogle Scholar
J. Rousseaux, R. Rousseaux-Prevost, H. Bazin, Optimal conditions for the preparation of proteolytic fragments from monoclonal IgG of different rat IgG subclasses, Methods Enzymol.
121:663–669 (1986).PubMedCrossRefGoogle Scholar
E. Lamoyi, A. Nisonoff, Preparation of F(ab′)2
fragments from mouse IgG of various subclasses, J. Immunol. Meth.
56:235–243 (1983).CrossRefGoogle Scholar
P. Parham, On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from Balb/c mice, J. Immunol.
131:2895–2902 (1985).Google Scholar
P. Parham, Preparation and purification of active fragments of mouse monoclonal antibodies, in:
“Handbook of Experimental Immunology,” D. M. Weir, ed., Blackwell, 4th Edition, Vol.1, Chapter 14 (1986).Google Scholar
S. Dissanayake, F. C. Hay, Pepsin digestion of mouse IgG immunoglobulins : subfragments of the Fc regions, Immunochemistry
12:373–378 (1975).PubMedCrossRefGoogle Scholar
M. J. Shulman, M. J. C. Heusser, C. Filkin, G. Kohler, Mutations affecting the structure and function of immunoglobulins M, Molec. Cell. Biol.
2:1033–1043 (1982).PubMedGoogle Scholar
W. D. Mathews, L. F. Reichardt, Development and application of an efficient procedure for converting mouse IgM into small active-fragments, J. Immunol. Methods
50:239–253 (1982).CrossRefGoogle Scholar
J. M. Bidlack, P. C. Mabie, Preparation of Fab fragments from a mouse monoclonal IgM, J. Immunol. Methods
91:157–162 (1986).PubMedCrossRefGoogle Scholar
C. P. Milstein, N. E. Richardson, E. V. Deverson, A. Feinstein, Interchain disulfide bridges of mouse immunoglobulin M, Biochem. J.
151:615–624 (1975).PubMedGoogle Scholar
M. Kehry, C. Sibley, J. Fuhrman, J. Schrling, L. Hood, Amino acid sequence of a mouse immunoglobulin u chain, Proc. Natl. Acad. Sci.
76:2932–2936 (1979).PubMedCrossRefGoogle Scholar
R. A. DePinho, L. B. Feldman, M. D. Scharff, Tailor-made monoclonal antibodies, Ann. Int. Med.
104:225–233 (1986).PubMedGoogle Scholar
V. T. Oi, S. L. Morrison, Chimeric antibodies, Biotechniques
4:214–221 (1986).Google Scholar
S. L. Morrison, Transfectomas provide novel chimeric antibodies, Science
229:1202–1207 (1985).PubMedCrossRefGoogle Scholar
T. J. Kipps, Switching the isotype of monoclonal antibodies, in:
“Hybridoma Technology in the Biosciences and Medicine,” T. A. Springer, ed., Plenum Press, New York, pp. 89–101 (1985).CrossRefGoogle Scholar
W. D. Cook, S. Rudikoff, A. M. Guisti, M. D. Scharff, Somatic mutation in a cultured mouse myeloma cell affects antigen binding, Proc. Natl. Acad. Sci.
79:1240–1244 (1982).PubMedCrossRefGoogle Scholar
D. E. Yelton, M. D. Scharff, Mutant monoclonal antibodies with alterations in biological functions, J. Exp. Med.
156:1131–1148 (1982).PubMedCrossRefGoogle Scholar
M. J. Glennie, G. T. Stevenson, Univalent antibodies kill tumor cells in vitro and in vivo, Nature
295:712–714 (1982).PubMedCrossRefGoogle Scholar
S. P. Cobbold, H. Waldmann, Therapeutic potential of monovalent monoclonal antibodies, Nature
308:460–462 (1984).PubMedCrossRefGoogle Scholar
M. Brennan, A chemical technique for the preparation of bispecific antibodies from Fab′ fragments of mouse monoclonal IgG1, Biotechniques
4:424–427 (1986).Google Scholar
G. T. Stevenson, M. J. Glennie, F. E. Par, F. K. Stevenson, H. F. Watts, P. Wyeth, Preparation and properties of Fab IgG, a chimeric univalent antibody designed to attack tumor cells, Biosci. Reports
5:991–998 (1985).CrossRefGoogle Scholar
M. S. Neuberger, G. T. Williams, R. D. Fox, Recombinant antibodies possessing novel effector functions, Nature
312:604–608 (1984).PubMedCrossRefGoogle Scholar
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