The Application of Labeled Antibody Technics in Studying Cell Antigens1

[CANCER RESEARCH 28, 1372-1381,July 1968]

Robert C. Mellors The Hospital for Special Surgery, Affiliated with The New York Hospital, Cornell University Medical College, and the Department of Pathology, Cornell University Medical College, New York, New York

INTRODUCTION by the direct (one-step, one-layer) procedure (12), that is, direct staining with fluorescent antibody, or by the indirect The underlying principles of the immunofluorescence method, (multiple step, "sandwich") procedure (100), in which un originated and developed by A. H. Coons and his associates (10- labeled antibody reacts with antigen and is followed by, and 12), are these: in turn stained by fluorescent antibody against the deposited (a) Antibodies can be conjugated with chemical compounds y-globulins. Antibody (Chart lÃ²) is usually localized by the (32), including colored dyes (57), without destroying the ca indirect procedure (13), using a layer of antigen followed by pacity of the antibody to react specifically with its antigen. fluorescent antibody against this antigen. However, labeled (6) Antibodies can be conjugated with fluorescent dyes antigens of high molecular weight and labeled antigen-excess (fluorescent antibodies) and used as immunospecific stains for soluble immune complexes (preformed "sandwich") can be the histochemical detection of antigens (11, 12). used for the detection of antibody by the direct procedure (63- (c) Antigens used indirectly as unlabeled intermediaries 65). A provisional or presumptive identification of antigen- (followed by fluorescent antibodies) or directly when labeled antibody complex (Chart le) and/or denatured or aggregated (fluorescent antigens) serve as immunospecific reagents for the â€¢y-globulinismade by the indirect procedure using a layer of histochemical detection of antibodies (13). guinea pig serum complement (28, 48) or human yMrheumatoid Immunofluorescence stainingâ€”like precipitation, agglutina factor (50, 55), followed in turn by the corresponding fluo tion, and complement-fixationâ€”is a means of visualizing an rescent antibody. tigen-antibody reactions. It belongs to and must be controlled The details of immunofluorescence technics will be found in by the general body of analytical immunologie methods (43), the publications previously cited, and only a brief outline of which includes two others of recent origin and wide usageâ€”im- procedures will be given here. munodiffusion and immunoelectrophoresis (30). Fluorescent 1. Methods of preparing antigens and schedules for im labels can be used also for in vivo tracing of proteins and other munization are discussed by Kabat and Mayer (43). In our organic substances (69, 94). standard procedure for immunizing rabbits, soluble antigen is An advantage of the immunofluorescence technic is that it incorporated in Freund's complete adjuvant (Difco, Detroit, is a nondestructive (in situ) method, a fruitful union of mor Michigan) and injected intramuscularly (10 mg protein per phology and immunology (68), with the inherent specificity injection) on two or more occasions, at weekly intervals, fol of immunochemical reactions and with localizing sensitivity at lowed by test-bleeding at 3 weeks after the last injection, a the limit of resolution of the light (or fluorescence) microscope. booster dose if indicated at that time, and a final bleeding 3 A disadvantage is that the method does not lend itself readily weeks thereafter. Antiserums containing high titers of anti to quantitation. bodies are desired because the layer thickness, rapidity of de The applications of the immunofluorescence method include position, and closeness of packing of the antibodies on the anti the detection of infectious agents, foreign antigens, endogenous gen are probably enhanced by high concentration of antibodies. antigens, and specific antibodies, the study of immunobiology 2. The antibody-active fraction of the antiserum is separated and immunopathology, and the investigation of diseases of from the immunologically indifferent proteins (albumin, Â«-, unknown etiology. Reviews and extended articles include those /3-gIobulins) before, or in some instances after, conjugation by Coons (8, 9), Mellors (61), Cherry et al. (6), and Beutner with the fluorescent dye. Fractionation procedures include: cold (2), and the excellent monograph by Nairn (68). alcohol precipitation (16), ammonium sulfate precipitation, DEAE-celluIose chromatography (Fig. 1) (15, 20, 26, 52, 56, TECHNIC 78, 88), and gel filtration through Sephadex (A. B. Pharmacia, Uppsala, Sweden) (29). The reagents and the technics of immunofluorescence are 3. The antibody-active fraction, preferably the yG-globulin symbolized in Chart 1. Antigen (Chart la) is localized either fraction (Fig. 1), if prepared from rabbit antiserum, is conju gated with a fluorescent dye. Most used is fluorescein iso- 1The author's experimental work has been supported by grants thiocyanate (Chart 2) (89), with apple-green fluorescence, from the NIH, USPHS, and from the American Cancer Society. crystalline form, Chromatographie purity (18, 31), and which

1372 CANCER RESEARCH VOL. 28

MICROSCOPICDEMONSTRATION REAGENTS

a) Antigen x@ Fluorescent label

p C Specific antibody

Antibodytoy-globulins

(indirect! in-vitro "sandwich")

/ \l/.

cjp Specificantigen

Iâ€”I |â€”i Aggregatedorhigh- ^ r1-! p molecularweightantigen

Antigen-excesssoluble ' immunecomplex

(direct; preformed "sandwich")

c) Antigen-anti body (jZjjHfi\ complex or denatured njÃ¼lgj or aggregated y-globulin JL

Guineapigcomplementor human y^ rheumatoidfactor (indirect; provisional)

Chart 1. Symbolic representation of the immunofluorescence method for the microscopic localization of antigen, antibody, and antigen-antibody complex (provisional). is a successor to fluorescein isocyanate (12). Lissamine rhoda- 4. The optimum conditions for conjugating antibodies with mine B (RB200) (5) with red-orange fluorescence, 1-dimethyl- fluorescein isothiocyanate introduce about two or three mole aminonaphthalene-5-sulphonic acid (DANS) (7, 59), rhodamine cules of fluorescein per molecule of antibody (12, 28), a re B isothiocyanate (89), and tetramethylrhodamine isocyanate sult obtained in our laboratory with dye Â¡protein (rabbit yG- (37) and isothiocyanate (18) are also used, sometimes also globulin) weight ratios of 0.01-0.015 and overnight conjugations with fluorescein isothiocyanate to provide contrasting labels at pH 9.0 and 4Â°C.Fluorescent conjugates are freed of un- on two antibodies with differing specificities (96) or simulta reacted fluorescent materials by dialysis, absorption with neous labels on the antigen and the antibody components of charcoal, or gel filtration through Sephadex G-25 (19, 29), and soluble immune complexes (63). they are used in staining procedures at a protein concentration

JULY 1968 1373

less sensitive than in vivo methods, such as the induction of O (HsC2>2N N(C2H5>2 transplantation immunity (67).

APPLICATIONS

COOH Antigenic differences between tumor and host have been demonstrated by in vivo induction of transplantation immunity and by in vitro methods, such as cytotoxicity, complement N=C=S S03Na fixation, and other serologie procedures. My brief remarks will be confined mainly to applications of labeled antibody methods Fluorescein isothiocyanate I Lissamine rhodamine B200 in the study of cell antigens of animal tumors or human tu mors and will focus on the detection of specific tumor antigens Chart 2. Chemical formulas of fluorescein isothiocyanate (isomer in situ. I) and lissamine rhodamine B (RB200). Cell Antigens Induced by Viruses of 0.5-1.0 mg/ml, with or without further purification, in DNA Viruses. A number of viruses containing DNA, among cluding absorption with tissue powders (12). An optimally them, mouse polyoma virus (97), simian virus 40 (SV40) (17, labeled antibody-fluorescein conjugate has in agar gel about 22), and some human adenoviruses (41, 51, 77, 99) induce the same electrophoretic mobility as the native antibody (Fig. tumor formation when inoculated into newborn hamsters and 1) ; over-labeled antibody molecules have faster mobility mice. While infectious virus is not recoverable from the re (more electronegativity) and produce troublesome nonspecific sulting tumors, the tumor cells often contain a virus-induced, staining reactions with tissue sections (21, 26, 31); under- but structurally nonvirion, new antigen, called tumor (T) labeled antibody molecules inhibit specific staining (27). antigen, detectable by complement fixation (41, 42, 92) and 5. Cryostat sections of unfixed tissue (12, 14, 53, 76), freeze- by immunofluorescence (SO, 82). drying (58), freeze-substitution (1), and gelatin embedding In the immunofluorescence studies of Pope and Rowe (79) (4) procedures, paraffin sections for the study of microbial and of Kapp et al. (82), all cells transformed by SV40 in vivo, polysaccharides (34, 45), and protein antigens (93), minute such as hamster tumors (Fig. 3) or in vitro, such as cells of needle biopsies (3), smears, brush preparations, cells in sus hamster (Fig. 4) and human origin, were found to contain pension (66), and tissue cultures have all been used in im- SV40 tumor antigen localized exclusively in the nucleus in par- munofluorescence studies, the first mentioned most frequently. ticulate form with sparing of the nucleolus. The results (82) 6. An essential requirement is a decisive demonstration of obtained by immunofluorescence and complement-fixation the immunospecificity of the fluorescent antibody (Fig. 2) and methods appeared to measure the same or similar antigens, of the staining reaction obtained with it. In general it must since only animals bearing large tumors had circulating anti be shown (12) that: (a) Prior treatment of a section with bodies capable of reacting in both tests. Negative control unlabeled homologous antiserum or antibody (but not homol serums included those obtained from normal hamsters and ogous normal serum) abolishes or markedly decreases the from hamsters immunized with live SV40 which proved to be specific staining reaction; or that unlabeled homologous anti- resistant to SV40 transformed cells. In these studies, the cells serum in mixture with homologous fluorescent antibody in were fixed in acetone for only 5 minutes, since longer periods suitable proportions inhibits the specific staining, whereas nor of fixation caused a diminution or even complete loss of the mal or heterologous serum does not (25) ; and/or that (6) immunofluorescence reaction. absorption of fluorescent antibody with homologous antigen In cells transformed by adenovirus Type 12 in vivo (ham abolishes the staining. In addition, it is useful to demonstrate ster and mouse tumors) or in vitro (cells of hamster and hu that: (c) Conjugates prepared from heterologous antiserums man origin), adenovirus Type 12 tumor antigen was visualized or normal serum do not stain the tissue section, (d) Chemical by immunofluorescence mainly in particulate form in the fixatives, such as 30 to 40 percent formaldehyde which destroys cytoplasm and less frequently as nuclear flecks or homogeneous protein antigens, also abolish specific staining, (e) Normal tis staining of nucleus and cytoplasm (80). sues, except when endogenous tissue antigens are the object of A defective SV40-adenovirus hybrid population was recently study, and pathologic tissues with appropriate control lesions discovered and found to contain, in addition to normal adeno are not stained. virus, a virus containing a defective SV40 genome encased in 7. Fluorescence microscopes with a very high intensity of an adenocapsid (40, 85, 91). The new virus has been named ultraviolet and blue-violet illumination are used for the ex PARA ("particle aiding replication of adenovirus") by Rapp amination of immunofluorescence preparations. et d. (83). The defective SV40 genome in PARA can be trans 8. The minimum antigen concentration detectable by im ferred from one adenovirus to another by a process called munofluorescence is estimated to be about 10~16 gm per transcapsidation (83, 90). By means of this process, a non- square micron (8, 81). When compared with other technics oncogenic population of viruses can be converted into an for the detection of mouse isoantigens, the immunofluorescence oncogenic one. For example, an oncogenic PARA virus with method is the most sensitive serologie one, followed by absorp Type 7 coat can undergo transcapsidation with Type 2 adeno tion, hemagglutination, hemolysis, and cytotoxicity, but it is virus, itself nononcogenic, to yield oncogenic PARA virus with

1374 CANCER RESEARCH VOL. 28

Type 2 coat (86). Thus, a fundamental question is whether variety of procedures, among them the membrane immuno the defective SV40 genome, the adenovirus genome, or both fluorescence reaction with living target cells (46). are responsible for tumor induction (86). A study of tumors MÃ¶ller (66) first used living cell suspensions in a study of induced in newborn hamsters by PARA-adenovirus Type 7 murine histocompatibility isoantigens and described the typical and its transcapsidant, PARA-adenovirus Type 2, indicated ring-like pattern of surface membrane immunofluorescence. that most of the tumors contained the SV40 tumor antigen Pinocytosis and cell injury led to partÃculate and diffuse non and that the serum of the animals contained the corresponding specific staining reactions that were morphologically distin antibody. Antibody against adenovirus tumor antigen (of ap guishable from the "specific" ring reaction. As far as the H-2 propriate type) was also present in some of the serums. Thus, (histocompatibility-2) system was concerned, membrane (and the results indicated that both the adenovirus genome and the cytoplasmic) localization could also be demonstrated in his PARA (defective SV40) genome persisted in the tumor cells. tologie sections. A similar distribution of H-2 isoantigens ca Aside from intranuclear tumor antigen, another new antigen pable of provoking immunologie enhancement was demonstrated has been detected by Tevethia et al. (98) in the cytoplasm of in our laboratory in work carried out with Drs. Nathan Kaliss cells transformed by SV40. The antigen was localized at the and Eleanor Lappano (unpublished data), in sections of ascites surface of cultured cells tested in monolayers, and when cells tumor cell-blocks fixed with cold alcohol or methanol and ex were reacted in suspension with the immunofluorescence rea posed to specific mouse-immune serum. However, the intensity gents, a bead-like circumferential distribution was often ob of specific apple-green fluorescence was low in comparison served (Fig. 5). The surface antigen was detected by using with the blue autofluorescence of the control sections, and the serum from hamsters previously immunized with SV40 and true microscopic picture was reproducible only in full color subsequently rejecting challenge by SV40 transformed cells. (Figs. 6-8) and not in black and white. The surface antigen differed from intranuclear tumor antigen Klein and Klein (46), in a comprehensive study of the anti- in the following respects (84) : (a) Serums from hamsters genie properties of murine lymphomas induced by Moloney bearing large tumors induced by SV40 reacted with the tumor virus, found that the establishment of transplantation resist antigen but not with the surface antigen of the transformed ance was paralleled by the appearance of circulating antibodies cells. (6) Serums from hamsters resisting the transplantation detectable by immunofluorescence and cytotoxicity tests. Ex of transformed cells following vaccination with SV40 reacted posure of the lymphoma cells to specific mouse-immune serum with the surface antigen but not with the tumor antigen. It followed by incubation with fluorescent rabbit antibodies is to be noted that, as a control, inoculation of hamsters with against mouse globulins resulted in cellular fluorescence that cells not transformed by SV40 failed to produce antibodies considerably exceeded that of the control cells in respect to against either the nuclear or the surface antigen. Rapp et al. the number of cells stained and the pattern of staining (mul (87) have recently shown that the SV40 determinants carried tiple dots and sectors of different sizes on the cell membrane). by PARA include those responsible for conferring transplanta tion resistance and for inducing the intranuclear tumor antigen, Other Cell Antigens but this is not to conclude that the surface antigen is a trans There is evidence, including observations by the immuno plantation (histocompatibility) antigen. fluorescence method, that tumor cells may either lose antigens Recently, ferritin-labeled antibody has been used in the found in the normal cells of origin, gain new ones, or possibly electron microscopic localization of adenovirus Type 12 tumor revert to the production of antigens typical of earlier stages antigen (44) and SV40 tumor antigen (75) in virus-trans of development. Using the immunofluorescence method and formed cells. In adenovirus Type 12-transformed human organ-specific antiserums against hamster kidney and against amnion cells, adenovirus Type 12 tumor antigen was found rat liver, Weiler (101, 102) found that these antiserums did (44) to be associated with at least two different intranuclear not react with chemically induced carcinomas of hamster kid components, namely, bundles of fibers and patches of fibro- ney and rat liver. Comparable results were obtained with com granular material, but not apparently with the nucleolus, plement fixation tests (103), and the immunofluorescence nuclear chromatin, or viral coat. In SV40-transformed hamster findings were later confirmed and extended by others (36, 72). cells, SV40 tumor antigen was localized in aggregates of various Employing organ-specific antiserum against normal human size scattered at random in the nuclear matrix (75), but it is skin, Nairn et cd. (72) showed that skin-specific antigens were not apparently associated with fibrous material. lacking in squamous cell, basal cell, and sweat gland carci In summary, the demonstration of tumor antigen or of nomas of human skin. Adenomatous tumors (malignant and antibody against this antigen seems to be the most sensitive benign) of the human colon (54, 70, 71), carcinomas of the method presently available for detecting virus activity after human uterine cervix (35), and testicular neoplasms (38) were the disappearance of infectious virus (86). It is also appro shown similarly by the immunofluorescence method to have priate to comment that the concept of "defectiveness" of an lost antigens characteristic of the homologous normal organs. oncogenic DNA virus in another respect, namely, failure of Hillemans (35) also found that epidermoid carcinoma of synthesis of coat antigens of the complete virus, was first sug human uterine cervix contained an antigen lacking in the nor gested some ten years ago by immunofluorescence studies of mal cervical epithelium. Multiple methods (immunodiffusion, Shope papilloma viral antigens (60, 62, 73, 95). immunoelectrophoresis, hemagglutination, passive cutaneous RNA Viruses. Murine lymphomas and leukemias induced by anaphylaxis, and immunofluorescence) were used by Gold and RNA viruses contain specific tumor antigens as shown by a Freedman (23) to demonstrate what was thought to be a spe-

JULY 1968 1375

cific tumor antigen in adenocarcinomas of human colon, but Fink and Cowles (C. J. D. Zarafonetis (ed.), International subsequently the same antigen was also found in embryologie Conference on Leukemia/Lymphoma. Philadelphia: Lea & tissues (24). Hiramoto et al. (39), using paired fluorescent Febiger, in press). labels on antibodies against myosin and against connective tis sue, found that sarcomatous (and cultured normal) muscle cells contained either one antigen or the other and that the REFERENCES cultured normal cells sometimes contained both of them, 1. Balfour, B. Immunological Studies on a Freeze-Substitution apparently depending upon the state of cell maturation. Method of Preparing Tissue for Fluorescent Antibody Stain In summary, while antigenic differences between normal and ing. Immunology, 4: 206-218, 1961. malignant cells exist, it is often difficult to determine whether 2. Beutner, E. H. Immunofluorescent Staining: The Fluorescent these antigens are unique to the cell or represent quantitative Antibody Method. Bacteriol. Rev., 25: 49-76, 1961. elevations of normal components that are usually present in 3. Bonomo, L., Tursi, A., and Del Zotti, G. An Embedding very small amounts (49). Technic for Immunofluorescent Studies of Minute Specimens Two studies relating to the presence of specific antigens in of Tissue Collected with a Needle. Am. J. Clin. Pathol., 43: Burkitt lymphomas have been reported recently (33, 47). 555-556, 1964. Klein et al. (47) tested the serums of Burkitt lymphoma pa 4. Burkholder, P. M. Complement Fixation in Diseased Tissues. tients in indirect immunofluorescence reactions against living I. Fixation of Guinea Pig Complement in Sections of Kidney Burkitt lymphoma cells derived from fresh biopsy material of from Humans with Membranous Glomerulonephritis and Rats Injected with Anti-Rat Kidney Serum. J. Exptl. Med., allogeneic and autologous source. The serums of certain of the 114: 605-616, 1961. Burkitt lymphoma patients gave a high incidence of positive 5. Chadwick, C. S., McEntegart, M. G., and Nairn, R. C. reactions (membrane immunofluorescence) against Burkitt Fluorescent Protein Tracers. A Trial of New Fluorochromes lymphoma cells while some did not. As control material, nor and the Development of an Alternative to Fluorescein. Im mal lymph node cells or leukemia cells of Swedish patients and munology, /: 315-327, 1958. erythrocytes or normal bone marrow cells of Burkitt lymphoma 6. Cherry, W. B., Goldman, M., and Carski, T. R. Fluorescent patients were used. The Burkitt-positive serums showed no Antibody Techniques in the Diagnosis of Communicable reactivity with these control cells. A small number of positive Diseases. Washington, D. C.: U. S. Govt. Printing Office, 1960. reactions with Burkitt lymphoma cells was given by serums 7. Clayton, R. M. Localization of Embryonic Antigens by Anti- obtained from hospitalized African patients with nonmalignant Sera Labelled with Fluorescent Dyes. Nature, 174: 1059, 1954. diseases or with malignant diseases other than Burkitt lym 8. Coons, A. H. Histochemistry with Labelled Antibody. Intern. Rev. Cytol., 5: 1-23, 1956. phoma and from healthy blood relatives of the Burkitt pa 9. Coons, A. H. Fluorescent Antibody Methods. In: J. F. tients. Henle and Henle (33) have found by indirect immuno Danielli (ed.), General Cytochemical Methods, pp. 399-422. fluorescence tests that serums from Burkitt lymphoma pa New York: Academic Press, 1958. tients elicited brilliant nuclear staining in a small proportion 10. Coons, A. H. The Beginnings of Immunofluorescence. J. of the lymphoblasts in lymphoma cell lines derived from Immunol., 67: 499-503, 1961. Burkitt lymphomas. However, this reaction was not restricted 11. Coons, A. H., Creech, H. J., Jones, R. M., and Berliner, E. to these serums; many control serums from African, as well as The Demonstration of Pneumococcal Antigen in Tissues by American, individuals also gave positive tests, although of the Use of Fluorescent Antibody. J. Immunol., 46: 159-170, lower fluorescence intensity, as did also fluorescein-labeled 1942. pooled human y-globulin of American origin. Additional work 12. Coons, A. H., and Kaplan, M. H. Localization of Antigen in made it fairly certain that the immunofluorescence technic Tissue Cells. II. Improvements in a Method for the Detec tion of Antigen by Means of Fluorescent Antibody. J. Exptl. detected those cultured Burkitt cells which harbored infection Med., 91: 1-13,1950. with herpes-like virus and that this virus, or a close relative 13. Coons, A. H., Leduc, E. H., and Connolly, J. M. Studies on of it, was widely distributed not only in Africa but also in the Antibody Production. I. A Method for the Histochcmical United States and probably elsewhere. Recently, Old et al. Demonstration of Specific Antibody and Its Application to a (74) have demonstrated a high incidence of precipitating anti Study of the Hyperimmune Rabbit. J. Exptl. Med., 103: 49- body to an antigen present in cultured Burkitt lymphoma cells 60, 1955. in human serums obtained from African patients with Burkitt 14. Coons, A. H., Leduc, E. H., anil Kaplan, M. H. Localization lymphoma and African and American patients with carcinoma of Antigen in Tissue Cells. VI. The Fate of Injected Foreign of the postnasal space and a lower incidence in serums ob Proteins in the Mouse. J. Exptl. Med., 93: 173-188, 1951. tained from normal donors, patients with malignant neoplastic 15. Curtain, C. C. The Chromatographie Purification of Fluores- cein-Antibody. J. Histochem. Cytochem., 9: 484^186, 1961. diseases of other types, and patients with nonneoplastic dis 16. Deutsch, H. F. Separation of Antibody-active Proteins from eases. Whether or not the precipitating antibody is directed against an antigenic component of the herpes-like virus found Various Animal Sera by Ethanol Fractionation Techniques. Methods Med. Res., 5: 284-300, 1952. in cultured Burkitt lymphoma cells is presently unknown. 17. Eddy, B. E., Borman, G. S., Grubbs, G. E., and Young, R. D. Fink and her coworkers have used a variety of immuno Identification of the Oncogenic Substance in Rhesus Monkey logie technics, including immunofluorescence, in the study of Kidney Cell Culture as Simian Virus 40. Virology, n': 65-75, mainly viral antigens in human leukemia, and this work ex 1962. tending over several years has been summarized recently by 18. Felton, L. C., and McMillion, C. R. Chromatographically

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Pure Fluorcsccin and Tetrametliylrhodamine Isothiocyanates. D. Immunohistochemical Staining Properties of the N-2FAA Anal. Biochem., 2: 178-180, 1961. Rat Hepatoma. Cancer Res., 21: 1372-1376, 1961. 19. Fothergill, J. E., and Nairn, R. C. Purification of Fluorescent 37. Hiramoto, R., Engel, K., and Pressman, D. Tetramethyl- Protein Conjugates: Comparison of Charcoal and "Sepha- rhodamine as Immunohistochemical Fluorescent Label in the dex." Nature, 19-2: 1073-1074, 1961. Study of Chronic Thyroiditis. Proc. Soc. Exptl. Biol. Med., 20. Frommhagen, L. H., and Martins, M. J. A Comparison of 97: 611-614, 1958. Fluorescein-laboled 7-Globulins Purified by Rivanol and 38. Hiramoto, R., Jurand, J., Bernecky, J., and Pressman, D. DEAE Chromatography. J. Immunol., 90: 116-120, 1963. Lack of Staining of Testicular Tumors by Anti-Sperm and 21. Frommhagen, L. H., and Spendlove, R. S. The Staining Prop Anti-Testis Antibodies. Proc. Soc. Exptl. Biol. Med., Ill: erties of Human Serum Proteins Conjugated with Purified 505-507, 1962. Fluorescein Isothiocyanate. J. Immunol., 89: 124-131, 1962. 39. Hiramoto, R., Jurandowsky, J., Bernecky, J., and Pressman, 22. Girardi, A. J., Sweet, B. H., Slotnick, V. B., and Hilleman, D. Immunochemical Differentiation of Rhabdomyosarcomas. M. R. Development of Tumors in Hamsters Inoculated in Cancer Res., el: 383-386, 1961. the Neonatal Period with Vacuolating Virus, SV-40. Proc. 40. Huebnor, R. J., Chanock, R. M., Rubin, B. A., and Casey, Soc. Exptl. Biol. Med., 109: 649-660, 1962. M. J. Induction by Adenovirus Type 7 of Tumors in Ham 23. Gold, P., and Freedman, S. 0. Demonstration of Tumor- sters Having the Antigenic Characteristics of SV 40 Virus. Specific Antigens in Human Colonie Carcinomata by Im- Proc. Nati. Acad. Sei. U. S., 62: 1333-1340, 1964. munological Tolerance and Absorption Techniques. J. Exptl. 41. Hucbner, R. J., Rowe, W. P., and Lane, W. T. Oncogenic Med., lÃ¨i: 439-462, 1965. Effects in Hamsters of Human Adenovirus Types 12 and 18. 24. Gold, P., and Freedman, S. 0. Specific Antigenic Similarity Proc. Nati. Acad. Sei. U. S., 48: 2051-2058, 1962. between Malignant Adult and Normal Fetal Tissues of the 42. Huebner, R. J., Rowe, W. P., Turner, H. C., and Lane, W. T. Human Digestive System. J. Clin. Invest., 44: 1051-1052, Specific Adenovirus Complement-fixing Antigens in Virus- 1965. free Hamster and Rat Tumors. Proc. Nat!. Acad. Sei. U. S., 25. Goldman, M. Staining Toxoplasma Condii with Fluorescein- 50: 379-389, 1963. labelled Antibody. II. A New Serologie Test for Antibodies to 43. Kabat, E. A., and Mayer, M. M. Experimental Immuno- Toxoplasma Based upon Inhibition of Specific Staining. J. chemistry, Ed. 2. Springfield, Illinois: Charles C Thomas, Exptl. Med., /0-5: 557-573, 1957. Publisher, 1961. 26. Goldstein, G., Slizys, I. S., and Chase, M. W. Studies on 44. Kalnins, V. L, Stich, H. F., and Yohn, D. S. Electron Micro Fluorescent Antibody Staining. I. Non-specific Fluorescence scopic Localization of Virus-associated Antigens in Human with Fluorescein-coupled Sheep Anti-rabbit Globulins. J. Amnion Cells (AV-3) Infected with Human Adeno-Virus, Exptl. Med., 114: 89-110, 1961. Type 12. Virology, 28: 751-754, 1966. 27. Goldstein, G., Spalding, B. H., and Hunt, Jr., W. B. Studies 45. Kaplan, M. H., Coons, A. H., and Deane, H. W. Localization on Fluorescent Antibody Staining. II. Inhibition by Sub- of Antigen in Tissue Cells. III. Cellular Distribution of optimally Conjugated Antibody Globulins. Proc. Soc. Exptl. Pneumococcal Polysaceharides Types II and III in the Biol. Med., Ill: 416-421, 1962. Mouse. J. Exptl. Med., 91: 15-30, 1950. 28. Goldwasser, R. A., and Shepard, C. C. Fluorescent Antibody 46. Klein, E., and Klein, G. Antigenic Properties of Lymphomas Methods in the Differentiation of Murine and Epidemic Induced by the Moloney Agent. J. Nati. Cancer Inst., SÃ©: Typhus Sera; Specificity Changes Resulting from Previous 547-568, 1964. Immunization. J. Immunol., 82: 373-380, 1959. 47. Klein, G., Clifford, P., Klein, E., and StjernswÃ¤rd, J. Search for Tumor-specific Immune Reactions in Burkitt Lymphoma 29. Gordon, M. A., Edwards, M. R., and Tompkins, V. N. Re finement of Fluorescent Antibody by Gel Filtration. Proc. Patients by the Membrane Immunofluorescence Reaction. Proc. Nati. Acad. Sei. U. S., 55: 1628-1635, 1966. Soc. Exptl. Biol. Med., 709: 96-99, 1962. 48. Klein, P., and Burkholder, P. M. Die Darstellung von 30. Grabar, P., and Burtin, P., (ed.). Immunoelectrophoretic fixierten Komplement mit markierten Antikomplement. Analysis. New York: Elsevier Publishing Co., 1964. Schweiz. Z. Allgem. Pathol., SÃ:729-731, 1959. 31. Griffin, C. W., Carski, T. R., and Warner, G. S. Labeling 49. Korngold.L. Do Specific Cancer Antigens Exist? In: R. J. C. Procedures Employing Crystalline Fluorescein Isothiocyanate. Harris (ed.), Specific Tumor Antigens, UICC Monograph J. Bacteriol., 8S: 534-537, 1961. Series, Vol. 2, pp. 13-19. Copenhagen: Munksgaard, 1967. 32. Heidelberger, M., Kendall, F. E., and Soo Hoo, C. M. Quanti 50. Lachmann, P. J. MÃ¼ller-Eberhard, H. J., Kunkel, H. G., and tative Studies on the Precipitin Reaction. Antibody Produc Paronetto, F. The Localization of in Vivo Bound Comple tion in Rabbits Injected with an Azo-Protein. J. Exptl. Med., ment in Tissue Section. J. Exptl. Med., 115: 63-82, 1962. 68: 137-152, 1933. 51. Larson, V. M., Girardi, A. J., Hilleman, M. R., and Zwickey, 33. Henle, G., and Henle, W. Immunofluorescence in Cells De R. E. Studies on Oncogenicity of Adenovirus Type 7 Viruses rived from Burkitt's Lymphoma. J. Bacteriol., 91: 1248-1256, in Hamsters. Proc. Soc. Exptl. Biol. Med., IIS: 15-24, 1965. 1966. 52. Levy, H. B., and Sober, H. A. A Simple Chromatographie 34. Hill, A. G. S., Deane, H. W., and Coons, A. H. Localization Method for Preparation of Gamma Globulin. Proc. Soc. of Antigen in Tissue Cells. VI. Capsular Polysaccharide of Exptl. Biol. Med., 103: 250-252, 1960. Friedlander Bacillus, Type B, in the Mouse. J. Exptl. Med., 53. Linderstr0m-Lange, K., and Mogensen, K. R. Studies on En 92: 35-44, 1950. zymatic Histochemistry. XXXI. Histological Control of His- 35. Hillemanns, H. G. Serologische und immunhistologische tochemical Investigations. C.R. Lab. Carlsberg, Ser. Chini., Untersuchungen zur Entstehung des Portiokrebses. Z. Â£3:27-35, 1938. Naturforsch., 17b: 240-261, 1962. 54. Lord, M. D. Large-bowel Cancer: An Immunological Study. 36. Hiramoto, R., Bernecky, J., Jurandowsky, J., and Pressman, Lancet, 2: 811-812, 1962.

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55. McCormick, J. M. Use of Fluorescein-Iabelled Rheumatoid Localization of SV 40-induced Neoantigen with Ferritin- Factor for Locating Sites of Antibody Fixation in Tissues. Labeled Antibody. Virology, 31: 183-186, 1967. Nature, 194: 302-303, 1962. 76. Pearse, A. G. E. Histochemistryâ€”Theoretical and Applied, 56. McDevitt, H. O., Peters, J. H., Pollard, L. W., Harter, J. G., Ed. 2, pp. 137-153 and 722-740. London: J. & A. Churchill, and Coons, A. H. Purification and Analysis of Fluorescein- Ltd., 1960. labeled Antisera by Column Chromatography. J. Immunol., 77. Pereira, M. S., Pereira, H. G., and Clarke, S. K. R. Human 00: 634-642, 1963. Adenovirus Type 31. A New Serotype with Oncogenic Prop 57. Mai-rack, J. Nature of Antibodies. Nature, 133: 292-293, erties. Lancet, /: 21-23, 1965. 1934. 78. Peterson, E. A., and Sober, H. A. In: S. P. Colwick and 58. Marshall, J. M. Localization of Adrenocorticotropic Hormone N. O. Kaplan (eds.), Methods in Enzymology, Vol. 5, pp. by Histochemical and Immunochemical Methods. J. Exptl. 3-27. New York: Academic Press, 1962. Med., 94: 21-29, 1951. 79. Pope, J. H., and Rowe, W. P. Detection of Specific Antigen 59. Mayersbach, H. Immunohistologische Methoden. II. Ein in SV40-transformed Cells by Immunofluorescence. J. Exptl. weiterer Markierungsfarbstoff: Dimethyl-1-Naphthylamin- Med., ISO: 121-128, 1964. sulfonsÃ¤ure-5. Acta Histochem., 6: 351-368, 1958. 80. Pope, J. H., and Rowe, W. P. Immunofluorescent Studies of 60. Mellors, R. C. Viruses, Genes and Cancer. Federation Proc., Adenovirus 12 Tumors and of Cells Transformed or Infected 17: 714-723, 1958. by Adenoviruses. J. Exptl. Med., ISO: 577-588, 1964. 61. Mellors, R. C. Fluorescent-antibody Method. In: R. C. 81. Pressman, D., Yagi, Y., and Hiramoto, R. A Comparison of Mellors (ed.), Analytical Cytology, Ed. 2. New York: Fluoresccin and I131 as Labels for Determining in Vivo Lo McGraw-Hill Co., Inc., 1959. calization of Anti-tissue Antibodies. Intern. Arch. Allergy, 62. Mellors, R. C. Tumor cell Localization of the Antigens of 12: 125-136, 1958. the Shope Papilloma Virus and the Rous Sarcoma Virus. Can 82. Rapp, F., Butel, J. S., and Melnick, J. L. Virus-induced Intra cer Res., 20: 744-746, 1960. nuclear Antigen in Cells Transformed by Papovavirus SV40. 63. Mellors, R. C., and Brzosko, W. J. Studies in Molecular Proc. Soc. Exptl. Biol. Med., 116: 1131-1135, 1964. Pathology. I. Localization and Pathogenic Role of Heterol- 83. Rapp, F., Butel, J. S., and Melnick. J. L. SV40-adenovirus ogous Immune Complexes. J. Exptl. Med., 115: 891-902, 1962. "Hybrid" Populations: Transfer of SV40 Determinants from 64. Mellors, R. C., Heimer, R., Coreos, J., and Korngold, L. One Type of Adenovirus to Another. Proc. Nati. Acad. Sci., Cellular Origin of Rheumatoid Factor. J. Exptl. Med., 110: 54: 717-724, 1965. 875-886, 1959. 84. Rapp, F., and Melnick, J. L. Papovavirus SV40, Adenovirus 65. Mellors, R. C., Nowoslawski, A., Korngold, L., and Sengson, and their Hybrids: Transformation, Complementation, and B. L. Rheumatoid Factor and the Pathogenesis of Rheuma Transcapsidation. Progr. Med. Virol., 8: 349-399, 1966. toid Arthritis. J. Exptl. Med., 113: 475-484, 1961. 85. Rapp, F., Melnick, J. L., Butel, J. S., and Kitahara, T. The 66. MÃ¶ller,G. Demonstration of Mouse Isoantigens at the Cellu Incorporation of SV40 Genetic Material into Adenovirus 7 lar Level by the Fluorescent Antibody Technique. J. Exptl. as Measured by Intranuclear Synthesis of SV40 Tumor An Med., 114: 415-434, 1961. tigen. Proc. Nati. Acad. Sci., SÃ©:1348-1352, 1964. 67. MÃ¶ller, G. Fluorescent Antibody Technique for Demonstra 86. Rapp, F., Melnick, J. L., and Levy, B. Correlation of Im tion of Isoantigens in Mice. Methods Med. Res., 10: 58-69, munology and Histopathology of Tumors Induced by De fective SV40-Adenovirus Hybrids. Am. J. Pathol., 60: 849- 1964. 859, 1967. 68. Nairn, R. C., (ed.). Fluorescent Protein Tracing, Ed. 2. 87. Rapp, F., Tevethia, S. S., and Melnick, J. L. Papovavirus London: E. & S. Livingstone Ltd., 1964. SV40 Transplantation Immunity Conferred by an Adeno- 69. Nairn, R. C., Chadwick, C. S., and McEntegart, M. G. virus-SV40 Hybrid. J. Nati. Cancer Inst., 36: 703-708, 1966. Fluorescent Protein Tracers in the Study of Experimental 88. Riggs, J. L., Loh, P. C., and Eveland, W. C. A Simple Frac- Liver Damage. J. Pathol. Bacteriol., 76: 143-153, 1958. tionation Method for Preparation of Fluorescein-labeled 70. Nairn, R. C., Fothergill, J. E., McEntegart, M. G., and Gamma Globulin. Proc. Soc. Exptl. Biol. Med., 105: 655-658, Richmond, H. G., Loss of Gastro-intestinal-specific Antigen 1960. in Neoplasia. Brit. Med. J., /: 1791-1793, 1962. 89. Riggs, J. L., Seiwald, R. J., Burckhalter, J. H., Downs, C. M., 71. Nairn, R. C., McEntegart, M. G., Fothergill, J. E., and and Metcalf, T. G. Isothioeyanate Compounds as Fluorescent Porteous, I. B. Specific Antibody Against Gastrointestinal Labeling Agents for Immune Serum. Am. J. Pathol., 34: Mucosa. Lancet, e: 109, 1961. 1081-1097, 1958. 72. Nairn, R. C., Richmond, H. G., McEntegart, M. G., and 90. Rowe, W. P., Studies of Adenovirus-SV40 Hybrid Viruses. Fothergill, J. E. Immunological Differences between Normal III. Transfer of SV40 Gene between Adenovirus Types. Proc. and Malignant Cells. Brit. Med. J., 2: 1335-1340, 1960. Nati. Acad. Sci., 64: 711-717, 1965. 73. Noyes, W. F., and Mellors, R. C. Fluorescent Antibody De 91. Rowe, W. P., and Baum, S. G. Evidence for a Possible tection of the Antigens of the Shope Papilloma Virus in Genetic Hybrid between Adenovirus Type 7 and SV40 Papillomas of the Wild and Domestic Rabbit. J. Exptl. Med., Viruses. Proc. Nati. Acad. Sci., 62: 1340-1347, 1964. 106: 555-562, 1957. 92. Sabin, A. B., and Koch, M. A. Evidence of Continuous Trans 74. Old, L. J., Boyse, E. A., Oettgen, H. F., de Harven, E., mission of Noninfectious SV 40 Viral Genome in Most or Geering, G., Williamson, B., and Clifford, P. Precipitating All SV 40 Hamster Tumor Cells. Proc. Nati. Acad. Sci., 49: Antibody in Human Serum to an Antigen Present in Cul 304-311, 1963. tured Burkitt's Lymphoma Cells. Proc. Nati. Acad. Sci., 66: 93. Sainte-Marie, G. A Paraffin Embedding Technique for 1699-1704, 1966. Studies Employing Immunofluorescence. J. Histochem. Cyto- 75. Oshiro, L. S., Rose, H. M., Morgan, C., and Hsu, K. C. The chem., 10: 250-256, 1962.

1378 CANCER RESEARCH VOL. 28

94. Schiller, A. A., Schayer, R. W., and Hess, E. L. Fluorescein- 99. Trentin, J. J., Yabe, Y., and Taylor, G. The Quest for Human conjugated Bovine Albumin. Physical and Biological Proper Cancer Viruses. Science, 737: 835-841, 1962. ties. J. Gen. Physiol., 36: 489-505, 1953. 100. Weller, T. H., and Coons, A. H. Fluorescent Antibody Studies 95. Shope, R. E. Are Animal Tumor Viruses Always Virus-like? with Agents of Varicella and Herpes Zoster Propagated in J. Gen. Physiol., IÃŸ:143-154, 1962. Vitro. Proc. Soc. Exptl. Biol. Med., 86: 789-794, 1954. 96. Silverstein, A. M. Contrasting Fluorescent Labels for Two 101. Weiler, E. HistolÃ³gica! Demonstration by Means of Fluores- Antibodies. J. Histochem. Cytochem., 5: 94, 1957. cing Antibodies. Brit. J. Cancer, 10: 560-563, 1956. 97. Stewart, S. E., Eddy, B. E., Gochhenour, A. M., Borgese, N. 102. Weiler, E. Die Ã„nderung der serologischen SpezifitÃ¤t von G., and Grubbs, G. E. The Induction of Neoplasms with a Leberzellen der Ratte wÃ¤hrendder Cancerogenese durch p-Di- Substance Released from Mouse Tumors by Tissue Culture. methyl-aminoazobenzol. Z. Naturforsch., lib: 31, 1956. Virology, 3: 380-400, 1957. 103. Weiler, E. Antigenic Differences Between Normal Hamster 98. Tevethia, S. S., Katz, M., and Rapp, F. New Surface Antigen Kidney and Stilboestrol Induced Kidney Carcinoma: Com in Cells Transformed by Simian Papovavirus SV40. Proc. plement Fixation Reactions with Cytoplasmic Particles. Brit. Soc. Exptl. Biol. Med., 119: 896-901, 1965. J. Cancer, 10: 553-559, 1956.

Fig. 1. Immunoelectrophoresis analysis of fluorescent antibodies, prepared by DEAE-cellulose chromatography of rabbit antiserums and compared (as antigens) with whole rabbit antiserum (lop) and purified native rabbit 7G-globulins (bottom). The antiserum trough contains in each instance sheep antiserum against rabbit serum globulins. The fluorescent antibodies (middle four pairs of arcs) have fluorescein-protein molecular ratios of 2.7, 3.7, 4.6, and 6.7 and show progressingly increased mobilities (electrophoretic migration towards the left). Fig. 2. Immunoelectrophoresis analysis of a fluorescent antibody. Two (upper) antiserum troughs contain rabbit antiserum against mouse serum proteins; two (lower) troughs contain fluorescein-labeled rabbit antibody against mouse -y-globulins. Two (upper) anti gen wells contain whole mouse serum; the third (lower) well contains purified native mouse -y-globulins. Migration is toward the left. Fig. 3. SV40 tumor antigen in hamster cells transformed in vivo by SV40. The cells are in a tissue culture line originally established from a hamster tumor induced by SV40. All of the cells contain intranuclear tumor antigen. Immunofluorescence. X 500. (Courtesy of Dr. Fred Rapp.) Fig. 4. SV40 tumor antigen in hamster cells transformed in vitro by SV40. All of the cells contain intranuclear tumor antigen. Im munofluorescence. X 500. (Courtesy of Dr. Fred Rapp.) Fig. 5. Surface antigen of SV40-transformed cells reacted in suspension. Immunofluorescence. X 400. (Courtesy of Dr. Fred Rapp.) Figs. 6-8. Murine histocompatibility antigens demonstrated in 4-fi paraffin sections of strain A/Ks sarcoma I ascites tumor cells by an indirect immunofluorescence procedure using specific mouse (C57BL/Ks) immune serum (Fig. 6). Negative controls include use of mouse (C57BL/Ks) nonimmune serum in the indirect procedure (Fig. 7) and buffered saline in the direct procedure (Fig. 8). Cell blocks were fixed in cold ethanol or cold methanol. Fluorescein-labeled rabbit antibody against mouse immunoglobulins with specificity shown in Fig. 2. X 340.

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Robert C. Mellors

Cancer Res 1968;28:1372-1381.

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