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The Application of Labeled Antibody Technics in Studying Cell Antigens1

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The Application of Labeled Antibody Technics in Studying Cell Antigens1 [CANCER RESEARCH 28, 1372-1381,July 1968] The Application of Labeled Antibody Technics in Studying Cell Antigens1 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 lò) 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 •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 staining—like precipitation, agglutina factor (50, 55), followed in turn by the corresponding fluo tion, and complement-fixation—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 usage—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, «-, 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 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. Labeled Antibodies and Cell Antigens MICROSCOPICDEMONSTRATION REAGENTS a) Antigen x@ Fluorescent label p C Specific antibody Antibodytoy-globulins (indirect! in-vitro "sandwich") (\ / \l/. cjp Specificantigen I—I |—i Aggregatedorhigh- ^ r1-! p molecularweightantigen Antigen-excesssoluble ' immunecomplex (direct; preformed "sandwich") c) Antigen-anti body (jZjjHfi\ complex or denatured njülgj 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 ¡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 4°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 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. Robert C. Mellors 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
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