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Antigenic regions defined by monoclonal antibodies on tumor-associated antigens of bovine leukemia virus-induced lymphosarcoma cells
LeukemiaResearchVol.17,No.2, pp. 187-193,1993. Printedin Great Britain.
ANTIGENIC
REGIONS
0145-2126/93$6.00+ .00
~ 1993 Pergamon Press Lid
DEFINED
ON TUMOR-ASSOCIATED VIRUS-INDUCED
BY MONOCLONAL
ANTIGENS
OF BOVINE
LYMPHOSARCOMA
ANTIBODIES LEUKEMIA
CELLS
Y. AIDA,* K. OKADA,t and M. ONUMA~ *Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305, tDepartment of Veterinary Pathology, Faculty of Agriculture, Iwate University, Morioka 020 and ,Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan
(Received 25 June 1992. Revision accepted 22 September 1992) Abstract--Tumor-associated antigens (TAAs) expressed on enzootic bovine leukosis tumors were divided previously into three types by use of 13 monoclonal antibodies (MAbs): common TAA, partially common TAA and individually distinct TAA. Since MAb-defined epitopes on the common TAA were conserved on both soluble TAA prepared from bovine B-lymphoma cells and untreated viable same cells, all the MAbs that bound to the soluble TAA also bound to untreated viable cells. By contrast, MAb-defined epitopes on the partially common and individually distinct TAAs varied according to the test systems used. Two of seven MAbs were found to bind to both the soluble TAA and viable cells and one MAb bound to the soluble TAA but not to the viable cells.
Key words: Bovine leukemia virus, tumor-associated antigens, monoclonal antibodies, epitope, enzootic bovine leukosis.
INTRODUCTION
with several (but not all) of the EBLs tested; and those in the third group reacted only with homologous tumor cells. Therefore, the T A A may include common T A A , partially common T A A and individually distinct T A A . Although we previously established seven independent MAb-defined epitopes (sites A through G) on the T A A by a competitive binding assay using the soluble TAAs obtained from six individual E B L tumors [1], as shown in Table 1, we did not identify these epitopes on the native tumors. We were, therefore, uncertain whether the epitopes identified on the soluble TAAs were the same as those present on viable tumors. Accordingly, we examined the epitopes of the TAAs on untreated viable tumors. Our initial approach was to perform affinity chromatography of tumor cells on Sepharose 6MB together with immunoelectron microscopy after double staining, in order to determine whether the common, partially common and individually distinct TAAs were present on the same tumors. Moreover, to clarify whether or not the MAb-defined epitopes of the solubilized T A A and the untreated viable tumors were the same, we compared the results of competitive binding assays using untreated viable tumors,
ENZOOTI¢ bovine leukosis (EBL) is a lymphoproliferative disease caused by bovine leukemia virus (BLV) [13]. Tumor cells from cattle with E B L have a tumor-associated antigen (TAA) [8, 10, 14, 18, 19] that is distinct from the BLV-induced antigens that are actively produced by infected cells [14, 19]. In order to clarify the nature of the T A A , we generated 13 monoclonal antibodies (MAbs) against TAAs expressed on tumor cells from cattle with E B L [2]. From the reactivities of the MAbs with individual tumor cells, the 13 MAbs could be divided into three groups. Antibodies in the first group reacted with all the EBLs tested; those in the second group reacted
Abbreviations: EBL, enzootic bovine leukosis; BLV, bovine leukemia virus; TAA, tumor-associated antigen; MAb, monoclonal antibody; FCS, fetal calf serum; DMEM, Dulbecco's minimal essential medium; FA, fluorescent antibody; PBS, phosphate-buffered saline; PLP, peroxidase-lysine-paraformaldehyde; pAg, protein A-colloidal gold; ABC, avidin-biotin-peroxidase complex; ELISA, enzyme-linked immunosorbent assay. Correspondence to: Dr Yoko Aida, Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305, Japan. 187
Y. AIDAet al.
188
TABLE 1. CHARACTERIZATIONOF MONOCLONALANTIBODIESAGAINSTTUMOR-ASSOCIATED ANTIGENS Group
Clone
Immunoglobulin class and subclass
Classification of T A A
Site*
1
e453 c432 c153 c143 885 903 4134 4366 2064 2065 311
IgG2b IgG2b IgG2b IgG2b IgG1 IgG1 IgG2b IgG1 IgG1 IgG1 IgG1
Common Common Common Common Partially common Partially common Partially common Partially common Partially common Partially common Individually distinct
A B B B C D E F NDt ND G
2
3
* Topographical regions were analyzed by a competitive binding assay using partially purified T A A s as antigens. For preparation of TAAs, 6 individual tumors were used. t ND, not determined.
soluble T A A s o b t a i n e d f r o m the same t u m o r s , and other viable tumors. MATERIALS
AND METHODS
MAbs Characterization of the MAbs against TAAs expressed on the EBL tumors was described in previous reports [14, 16]. In the present study, we used 11 MAbs, as listed in Table 1. IgG of MAbs was precipitated from mouse ascitic fluid with ammonium sulfate at 50% saturation and purified by subsequent ion-exchange chromatography on D E A E cellulose [15]. Cells We used two bovine lymphoid cell lines, BLSC-KUI and EBLC-I, which had been established from tumors of cattle with EBL. These cell lines have B-cell surface marker and T A A on the surface of cells and bovine leukemia provirus is integrated in each genome [17]. A lymphoid tumor was obtained from a cow with EBL and fresh tumor cells were collected in Eagle's minimal essential medium supplemented with 10% fetal calf serum (FCS) for affinity chromatography on Sepharose 6MB. Affinity chromatography of tumor cells on Sepharose 6MB Affinity chromatography of tumor cells on Sepharose 6MB was performed as described previously [7]. In brief, IgG of MAb c453 was covalently linked to CNBr-activated Sepharose 6MB (Pharmacia Fine Chemicals, Uppsala, Sweden) as indicated previously [7, 12]. Sepharose 6MB was suspended in Dulbecco's minimal essential medium (DMEM) and poured into a column (1.0cm x 20cm). BLSC-KUI cells (2.5 x 10 7 cells) were loaded onto the column and incubated for 15 min at room temperature. Unbound cells were then washed from the column with DMEM. The specifically bound cells were eluted with rabbit antibodies against mouse IgG as follows. Five ~tl of a solution of rabbit anti-mouse IgG that had been diluted with DMEM were passed through the column. The column was then sealed and incubated for 1 h at 37°C with
occasional gentle stirring of the gel in the upper part of the column with a pipette. The cells were then eluted with 3 ml of rabbit antiserum against mouse IgG and 15 ml of DMEM, both warmed to 37°C. The reactivity of bound and unbound cells with MAbs was examined by an indirect fluorescent antibody (FA) assay of acetone-fixed cells, as described previously [18].
Immunoelectron microscopy A suspension of 2 × 10 6 target cells/ml in phosphatebuffered saline (PBS) was placed in a U-bottomed microtest plate (Nunc, Roskilde, Denmark). The plate was centrifuged at 2000 r.p.m, for 5 min and the supernatant was removed. The cells were fixed in peroxidase-lysineparaformaldehyde (PLP) at 4 °C for 4 h and then washed three times with PBS. For the first staining, the cells were incubated with MAb c453 and stained with protein Acolloidal gold (pAg) by Roth's method [20]. For the second staining, the cells were incubated with either MAb c164 or MAb 885 and stained with avidin-biotin-peroxidase complex (ABC) by Hsu's method [9]. The immunologically stained cells were post-fixed in osmiun tetroxide and embedded in Epon 812. Ultrathin sections were examined under a HS-8 type electron microscope. As negative controis, we used FLK cells (fetal lamb kidney cells persistently infected with BLV) [22] and peripheral blood lymphocytes from BLV-free normal cattle stained by the method described above, as well as EBL tumor cells stained either with normal mouse IgG or with MAb adsorbed with T A A were used. Viable-cell enzyme-linked immunosorbent assay (ELISA) Viable-cell ELISA was carried out as described by Aida et al. [3]. The end-point titre of the ELISA was estimated from a dose-response curve obtained with serial dilutions of MAbs. Normal mouse serum was used as a control, and its absorbance at 405 nm was always less than 0.10. The end-point titre of the ELISA was taken as the reciprocal of the maximum dilution of antibody that gave an absorbance at 405 nm of more than 0.15. Standard ELISA Standard ELISA was carried out as described by Aida
Epitopes on tumor antigens
189
the wells were washed and then an aliquot of peroxidaselabelled IgG, at a dilution of between 1:100 and 1:200 and typically having an absorbance at 405 nm of 0.5, was added. The extent of competitive binding (E) was estimated from the following formula: E = 100 (A-N)/(A-B), where A, B and N are the absorbance at 405 nm in the absences of competitor, in the presence of homologous antibody, and in the presence of competitor, respectively.
RESULTS
FIG. 1. Immunoelectron micrographs of EBLC-I cells. A, unstained cell membrane; B, cell membrane immunostained with MAb c453 and pAg particles (large arrow) and with c164 antibody and ABC (dense band; small arrow); C, cell membrane stained with MAbs c453 (gold particles; large arrows) and 885 (dense band; small arrow), x52,000.
et al. [1] using the TAA (0.5/~g/well) that had been partially purified from BLSC-KUI cells [2]. The end-point titre of the ELISA was estimated as described immediately above. Competitive binding assay
A competitive binding assay was performed by a viablecell ELISA or a standard ELISA, as described by Aida et al. [1] with slight modifications. Peroxidase-labelled MAbs were prepared by the glutaraldehyde method of Avrameas [5]. In brief, for the competitive binding assay by the viablecell ELISA, 50 ~tl of a suspension of 1 × 107 target cells/ ml in PBS-FCS were added to wells that already contained 50 ~tl of serial 10-fold dilutions of each unlabelled competitor IgG. After incubation at 37°C for 1 h, the wells were washed, then an aliquot of peroxidase-labelled IgG, diluted to between 1:100 and 1:200 and typically having an absorbance at 405 nm of 0.8, was added. For competitive binding assays by the standard ELISA, serial 10-fold dilutions of each unlabelled IgG, as competitor, were placed in the wells of microplates that had been absorbed with partially purified TAA. After incubation for 1 h at 37 °C,
In order to determine whether or not the c o m m o n T A A , the partially common T A A and the individually distinct T A A s were present on the same tumor cells, we separated tumor cells from bovine lymphosarcomas by affinity chromatography on Sepharose 6MB. When a suspension of tumor cells, in which about half of the cells reacted with all of the MAbs in Group 1 and with only M A b 4366 among the MAbs in Groups 2 and 3, was applied to Sepharose 6MB coupled with c453 antibody (specific to site A on the common T A A ) , the reactivity of the cells that bound to the c453 antibody showed a 2-fold increase while the cells in the flow-through fraction showed only slight reactivity (Table 2). In the bound cell fraction, a similar increase in reactivity with M A b c432 (specific to site B on the common T A A ) and with MAb 4366 (specific to site F on the partially common T A A ) was observed. Reactivity of the nonadherent cell fraction with MAbs c432 and 4366 was very low. When a suspension of tumor cells was chromatographed on a control column of Sepharose 6MB that had been coupled with IgG from normal mouse serum, 1.8% of cells applied to the column adsorbed to the column (data not shown). Nonspecific adsorption to Sepharose 6MB seemed to be negligible as judged from the very low percentage of tumor cells that bound to the column. These results indicate that the common T A A molecule which was recognized by MAbs c143 and c432, and the partially common T A A molecule which was recognized by MAb 4366 may be present on the same cells. The distribution of antigenic sites on tumor cells was examined, after double staining of tumor cells, by immunoelectron microscopy. When the bovine Blymphoid cell line, EBLC-I, established from leukemic cells of cattle with E B L , was reacted first with MAb c453 and then stained by the pAg m e t h o d and finally by the A B C method after treatment with M A b c164 (specific to site B on the common T A A ) , most of the surface of tumor cells showed double staining. As shown in Fig. 1B, a dense zonal band (small arrow) and gold particles (large arrow) were present on the same tumor cells. Moreover, we also stained cells with MAbs c453 and 885 (specific to site C on the partially common T A A ) by the pAg and A B C
190
Y. AIDA el al. TABLE 2. SEPARATION OF TUMORS BY AFFINITY CHROMATOGRAPHY ON SEPHAROSE 6MB COUPLED WITH IgG OF MONOCLONAL ANTIBODY C143
% of positive cells* against Treatment
c453
c432
4366
% recoveryt
Before chromatography After chromatography: flow-through cells After chromatography: bound cells
51.2 7.6 90.0
65.0 3.9 85.7
43.7 3.7 69.2
34 37
* The reactivity of bound, flow-through and unfractionated cells with monoclonal antibodies (c453, c432 or 4366) was detected by the FA test (see text). t The number of cells (2.5 x 107) applied to the column coupled with IgG of MAb c453 was taken as 100%.
TABLE 3. REACTIVITIES OF MONOCLONAL ANTIBODIES AGAINST TUMOR-ASSOCIATED ANTIGENS WITH VIABLE AND SOLUBILIZED BLSC-KUI TUMOR CELLS
Titre of ELISA using Group cellst 1
2
3
Clone
Solubilized BLSC-KUI cells*
Viable BLSC-KUI
c453 c432 c153 c143 885 903 2064 2065 4134 4366 311
625 3125 3125 3125 625 3125 --$ --625 --
15,625 78,125 78,125 390,625 15,625 ----3125 --
* Standard ELISA was performed using partially purified TAA from disrupted BLSCKUI tumor cells. t Viable-cell ELISA was performed using viable BLSC-KUI tumor cells. ~t Negative.
method, respectively. Again a dense zonal band (small arrow) and gold particles (large arrow) were found on the same tumor cells (Fig. 1C). Thus, the results of immunoelectron microscopy, showing that the common T A A was recognized by MAbs c453 and c164, and the partially c o m m o n T A A was recognized by MAb 885 on the same cells, confirmed directly the results obtained by affinity chromatography on Sepharose 6MB. In order to determine the reactivities with the T A A of MAbs from Groups 1, 2 and 3 by use of different titration systems, we performed a viable-cell E L I S A using untreated viable BLV-induced B-lymphoid cells, namely, B L S C - K U I cells, and a standard E L I S A using partially purified T A A from disrupted BLSC-KUI cells as antigens. As shown in Table 3, all of the Group 1 antibodies bound extensively to the soluble T A A and to untreated viable BLSC-KUI cells. Of the six MAbs in G r o u p 2, MAbs 885 and
4366 were found to bind to untreated viable BLSCKUI cells and to the soluble T A A prepared from the same cells. However, M A b 903 (specific to site D on the partially common T A A ) did not react with untreated viable BLSC-KUI cells but it did react with soluble T A A prepared from the same cells. The other 3 MAbs (2064, 2065 and 4134) in G r o u p 2 did not react with the soluble T A A or with viable BLSCKUI cells. Moreover, antibody 311 (specific to site G on the individually distinct T A A ) from Group 3 did not react with the T A A expressed on BLSC-KUI cells in either titration system. A comparison between the antigenic regions of the T A A expressed on untreated viable BLSC-KUI cells and those of the partially purified T A A from disrupted BLSC-KUI cells was performed by use of the competitive binding assay with the viable-cell E L I S A and the standard E L I S A (Table 4). With MAbs from Group 1, peroxidase-labelled MAb c453 was blocked
191
Epitopes on tumor antigens TABLE 4. RESULTSOF COMPETITIVEBINDINGASSAYSWITHSOLUBLETAA AND VIABLECELLS* Peroxidase-conjugated antibodies Group 1 Antigen Soluble TAA from BLSC-KUI
Viable BLSC-KUI
Viable EBLC-I
Competitor Group 1 c453 c432 c153 c143 Group 2 885 4366 Group 1 c453 c432 c153 c143 Group 2 885 4366 Group 1 c453 c432 c153 c143 Group 2 885 4366
Group 2
c453
c432
c153
c143
+ -
+ + +
+ + +
+ + +
. .
. .
+ . .
. .
+ + + . .
+ . .
. .
. .
+ + + . .
+ + + . .
+ + +
. .
+ + +
4366
m
_
m
-
+ + + . .
885
+
m
+ +
+ +
m
m
m
_
m
-
+
m
* The competitive binding assay was performed by standard ELISA with soluble TAA and by viable-cell ELISA with either viable BLSC-KUI or EBLC-I cells. +, positive inhibition (>50%); - , negative inhibition (<50%).
completely only by the homologous antibody, in both systems, with viable cells and the soluble T A A as antigens. By contrast, peroxidase-labelled MAbs c432, c153 and c143 (specific to site B on the c o m m o n T A A ) were blocked by homologous and other antibodies, with the exception of M A b c453. We found that there were at least two distinct antigenic regions on the c o m m o n T A A that were expressed on both the soluble T A A and untreated viable B L S C - K U I cells, one being recognized only by M A b c453 and the other being recognized by the rest of the antibodies. This topographical analysis gave the same results as those obtained with a mixture of partially purified T A A s from six individual tumors, as shown in Table 1. These results indicate that the MAbdefined antigenic regions on the c o m m o n T A A molecule, expressed on untreated viable B L S C - K U I cells, may correspond to those on the soluble T A A from disrupted B L S C - K U I cells. Since MAbs 885 and 4366 but not other MAbs from Groups 2 and 3 reacted with both disrupted and untreated viable B L S C - K U I cells, we were able to
test only MAbs 885 and 4366 in competitive binding assays. Peroxidase-labelled MAbs 885 and 4366 were each blocked only by the homologous antibody when disrupted B L S C - K U I cells were used. By contrast, peroxidase-labelled MAbs 885 and 4366 were blocked by homologous and heterologous antibodies when untreated viable B L S C - K U I cells were used. These results indicate that MAbs 885 and 4366 bind to independent epitopes on the soluble T A A from disrupted tumor cells and to a single epitope on viable B L S C - K U I cells. In order to compare the antigenic regions of T A A on viable B L S C - K U I cells with those of T A A s on cells from other bovine lymphoid lines, we performed competitive binding assays by the viable-cell E L I S A using E B L C - I cells (Table 4). While peroxidaselabelled M A b c453 was blocked completely only by the homologous antibody, peroxidase-labelled MAbs c432, c153, and c143 were blocked by the homologous antibodies and by other antibodies, with the exception of c453. Thus, the c o m m o n T A A had at least two distinct antigenic regions which were the same in
192
Y. AIDA et al.
both BLSC-KUI and EBLC-I cells. Since peroxidaselabelled MAbs 885 and 4366 were each blocked only by the homologous antibody, MAbs 885 and 4366 may bind to separate and independent antigenic regions on the partially common T A A in EBLC-I cells but they may bind to a single antigenic region on the TAA of BLSC-KUI cells. DISCUSSION In a previous study, using specific MAbs, we identified TAAs expressed on EBL tumor cells as common TAA, partially common T A A and individually distinct TAA, and topographical analysis revealed seven independent antigenic regions, specific for the MAbs, on the three types of T A A [1, 2]. However, we did not determine whether the common, partially common and individually distinct TAAs were present on the same cells. In this study, using affinity chromatography on Sepharose 6MB and immunoelectron microscopy, we showed clearly that the common TAA and the partially common T A A are present on the same tumor cells. All of MAbs from Group 1 bound to the soluble TAA from disrupted tumors and untreated viable cells from tumors. By contrast, although MAb 903, directed against site D on the partially common TAA, bound the soluble T A A from disrupted BLSCI cells, it did not bind to viable BLSC-KUI cells, as shown in Table 3. This phenomenon may have been caused either by structural alteration of the partially common T A A or by a change in the accessibility of the antigenic site during solubilization. It is also possible that a previously hidden antigenic site on the partially common T A A appeared on the surface during solubilization, thereby allowing MAb 903 to bind to the soluble TAA. Similar observations have been made in other systems. Bruck et al. [6] reported that the conformation or accessibility of certain epitopes of the eno glycoprotein gp51 of BLV changed according to the test system used, such as liquidphase radioimmunoassay with plastic-bound gp51 or BLV particles. In the Sindbis virus system, all specific MAbs tested were shown to be inhibited from binding to solid-phase Sindbis virus when premixed with detergent-treated virions, whereas only those having virus-neutralizing activity were competitively inhibited by intact virions [21]. Thus, all cryptic sites on Sindbis virus can be exposed by disruption of virions with a non-ionic detergent. Since the solubilization of the T A A may alter the antigenic regions that are exposed, competitive binding assays were performed with viable and disrupted BLSC-KUI cells in order to characterize the antigenic regions. The antigenic regions (sites A and
B) on the common T A A prepared from disrupted BLSC-KUI cells were conserved on the untreated viable cells. By contrast, MAbs 885 and 4366, which recognized the partially common TAA, bound only to a single epitope on the nontreated viable tumor cells, whereas both MAbs bound to independent antigenic regions on the soluble TAA. This result suggested that, since the soluble T A A prepared from disrupted BLSC-KUI tumors showed greater accessibility with respect to binding to the partially common TAA by MAbs 885 and 4366 than the native TAA expressed on viable tumors, MAbs 885 and 4366 could not be used in competition with each other in a competitive binding assay. Therefore, it is possible that they bind to an independent antigenic region on the disrupted TAA. The exposure of the antigenic region on the partially common T A A may thus be effected by one of the following mechanisms: (a) the antigenic regions of the partially common TAA may become exposed by solubilization but no comformational change occurs that affects the common TAA; or (b) during solubilization, the antigenic regions of the partially common T A A which were previously masked by the common TAA, become exposed as a result of a change in the conformation of the common TAA. However, we found that the avidity of the binding of MAbs to the antigenic regions of the common TAA was not affected by the solubilization, as shown in Table 3. Therefore, the change in reactivity of MAbs from Group 2 with TAA after solubilization may be explained by the first possibility and not by the second possibility. MAbs directed against the common T A A recognized the same epitope on both BLSC-KUI cells and EBLC-I cells. In the case of the partially common TAA, MAbs 885 and 4366 bound to a single epitope on viable BLSC-KUI cells but they bound to independent antigenic regions on EBLC-I cells. Thus, the antigenic determinants defined by the MAbs on the partially common TAA might differ according to the specific tumor, and those of the common T A A might be conserved on all EBL tumor cells. While TAA was expressed constitutively within the membrane of tumor cells, it is known that BLV does not express its gene products at detectable levels in freshly removed lymphocytes or the tumor cells or the infected host, while it often expresses them when these cells are cultured in oitro [11]. Thus, it seems likely that the expression of TAAs plays a key role in BLV-induced leukemogenesis and the maintenance of the tumorous state. Although there is no direct evidence for the possibility, if the antigenic determinants defined by the MAbs on the common TAA are generally conserved between tumor cells from BLV-induced lymphosarcoma cases, they may
Epitopes on tumor antigens themselves be important in BLV-induced leukomogenesis. Further physicoehemical characterization of T A A s and of the distribution of the antigenic determinants that are recognized by the MAbs is currently in progress.
Acknowledgement--We are grateful to Dr Koyama, Kitasato University, for supplying the BLSC-KUI tumor cells.
REFERENCES 1. Aida Y., Onuma M., Mikami T. & Izawa H. (1985) Topographical analysis of tumor-associated antigens on bovine leukemia virus-induced bovine lymphosarcoma. Cancer Res. 45, 1181. 2. Aida Y., Onuma M., Ogawa Y., Mikami T. & Izawa H. (1985) Tumor-associated antigens of bovine leukemia virus-induced bovine lymphosarcoma identified by monoclonal antibodies. Cancer Res. 45, 1174. 3. Aida Y., Onuma M., Kasai N. & Izawa H. (1987) Use of viable-cell ELISA for detection of monoclonal antibodies recognizing tumor-associated antigens on bovine lymphosarcoma cells. Am. J. Vet. Res. 48, 1319. 4. Aida Y., Ochiai K., Ito K., Onuma M., Fujimori F., Fujimoto Y. & Izawa H. (1987) Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens. Am. J. Vet. Res. 48, 1181. 5. Avrameas S. (1969) Coupling enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies, lmmunochem. 6, 43. 6. Bruck C., Portetelle D., Burny A. & Zavada J. (1982) Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions. Virology 122, 353. 7. Chetie V., Mota G. & Sjoquist J. (1978) Separation of cells by affinity chromatography on SpA-Sepharose 6MB. J. Immun. Meth. 21, 133. 8. Hollinshead A. C. & Valli V. E. (1976) Preliminary findings of a tumor-associated antigen in bovine lymphosarcoma. Bibl. Hemat. 43, 369. 9. Hsu S. M., Raine L. & Fanger H. (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577. 10. Jacobs R. M., Valli V. E. O., Wilkie B. N. & Hollinshead A. C. (1981) Partial purification of a common
193
antigen in bovine lymphoma and its use in a lymphocyte blastogenesis assay. Cancer Res. 41, 3000. 11. Kettmann R., Marbaix G., Cleuter Y., Portetelle D., Mammerickx M. & Burny A. (1980) Genomic integration of bovine leukemia provirus and lack of v~ral RNA expression in the target cells of cattle with different responses to BLV infection. Leukemia Res. 4, 509. 12. Manderino G. L., Gooch G. T. & Stavitsky A. B. (1978) Preparation, characterization and functions of rabbit lymph node cell populations. 1. Preparation of KLH primed T and B memory cells with anti-Fab' affinity columns. Cell. Immunol. 41,264. 13. Miller J. M., Miller L. D., Olson C. & Gillette K. G. (1969) Virus-like particles in phytohemagglutininstimulated lymphocytes cultures with reference to bovine lymphosarcoma. J. natn. Cancer Inst. 43, 1297. 14. Okada K., Onuma M., Numakunai S., Kagawa Y., Minamino K., Ito T., Kobayashi Y., Morimoto N., Morita H. & Ohshima K. (1983) Tumor-associated antigen detected by complement-dependent antibody cytotoxicity test and immunofluorescence test in enzootic bovine lymphosarcoma. Jpn. J. Vet. Sci. 45, 195. 15. Onoue K., Yagi Y. & Pressman D. (1964) Multiplicity of antibody proteins in rabbit anti ,-azobenzenearsonate sera. J. Immun. 92, 173. 16. Onuma M., Aida Y., Okada K., Oshima K., Kawakami Y. & Izawa H. (1985) Usefulness of monoclonal antibodies for detection of enzootic bovine leukemia cells. Jpn. J. Cancer Res. (GANN), 76, 959. 17. Onuma M., Koyama H., Aida Y., Obawa Y., Kirisawa R. & Kawakami Y. (1986) Establishment of B-cell lines from tumor of enzootic bovine leukosis. Leukemia Res. 10, 689. 18. Onuma M. & Olson C. (1977) Tumor-associated antigen in bovine and ovine lymphosarcoma. Cancer Res. 37, 3249. 19. Onuma M., Takashima I. & Olson C. (1978) Tumorassociated antigen and cell surface marker in cells of bovine lymphosarcoma. Ann. Rech. Vet. 9, 825. 20. Roth J. (1982) Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J. Histochem. Cytochem. 30, 691. 21. Schamaljohn A. L., Kokubun K. M. & Fole D. G. A. (1983) Protective monoclonal antibodies define maturational and pH-dependent antigenic change in Sindbis virus E1 glycoprotein. Virology 130, 144. 22. Van Der Maaten M. J. & Miller J. M. (1976) Replication of bovine leukemia virus in monolayer cell cultures. Bibl. Haemat. 43, 360.
ANTIGENIC
REGIONS
0145-2126/93$6.00+ .00
~ 1993 Pergamon Press Lid
DEFINED
ON TUMOR-ASSOCIATED VIRUS-INDUCED
BY MONOCLONAL
ANTIGENS
OF BOVINE
LYMPHOSARCOMA
ANTIBODIES LEUKEMIA
CELLS
Y. AIDA,* K. OKADA,t and M. ONUMA~ *Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305, tDepartment of Veterinary Pathology, Faculty of Agriculture, Iwate University, Morioka 020 and ,Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan
(Received 25 June 1992. Revision accepted 22 September 1992) Abstract--Tumor-associated antigens (TAAs) expressed on enzootic bovine leukosis tumors were divided previously into three types by use of 13 monoclonal antibodies (MAbs): common TAA, partially common TAA and individually distinct TAA. Since MAb-defined epitopes on the common TAA were conserved on both soluble TAA prepared from bovine B-lymphoma cells and untreated viable same cells, all the MAbs that bound to the soluble TAA also bound to untreated viable cells. By contrast, MAb-defined epitopes on the partially common and individually distinct TAAs varied according to the test systems used. Two of seven MAbs were found to bind to both the soluble TAA and viable cells and one MAb bound to the soluble TAA but not to the viable cells.
Key words: Bovine leukemia virus, tumor-associated antigens, monoclonal antibodies, epitope, enzootic bovine leukosis.
INTRODUCTION
with several (but not all) of the EBLs tested; and those in the third group reacted only with homologous tumor cells. Therefore, the T A A may include common T A A , partially common T A A and individually distinct T A A . Although we previously established seven independent MAb-defined epitopes (sites A through G) on the T A A by a competitive binding assay using the soluble TAAs obtained from six individual E B L tumors [1], as shown in Table 1, we did not identify these epitopes on the native tumors. We were, therefore, uncertain whether the epitopes identified on the soluble TAAs were the same as those present on viable tumors. Accordingly, we examined the epitopes of the TAAs on untreated viable tumors. Our initial approach was to perform affinity chromatography of tumor cells on Sepharose 6MB together with immunoelectron microscopy after double staining, in order to determine whether the common, partially common and individually distinct TAAs were present on the same tumors. Moreover, to clarify whether or not the MAb-defined epitopes of the solubilized T A A and the untreated viable tumors were the same, we compared the results of competitive binding assays using untreated viable tumors,
ENZOOTI¢ bovine leukosis (EBL) is a lymphoproliferative disease caused by bovine leukemia virus (BLV) [13]. Tumor cells from cattle with E B L have a tumor-associated antigen (TAA) [8, 10, 14, 18, 19] that is distinct from the BLV-induced antigens that are actively produced by infected cells [14, 19]. In order to clarify the nature of the T A A , we generated 13 monoclonal antibodies (MAbs) against TAAs expressed on tumor cells from cattle with E B L [2]. From the reactivities of the MAbs with individual tumor cells, the 13 MAbs could be divided into three groups. Antibodies in the first group reacted with all the EBLs tested; those in the second group reacted
Abbreviations: EBL, enzootic bovine leukosis; BLV, bovine leukemia virus; TAA, tumor-associated antigen; MAb, monoclonal antibody; FCS, fetal calf serum; DMEM, Dulbecco's minimal essential medium; FA, fluorescent antibody; PBS, phosphate-buffered saline; PLP, peroxidase-lysine-paraformaldehyde; pAg, protein A-colloidal gold; ABC, avidin-biotin-peroxidase complex; ELISA, enzyme-linked immunosorbent assay. Correspondence to: Dr Yoko Aida, Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305, Japan. 187
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TABLE 1. CHARACTERIZATIONOF MONOCLONALANTIBODIESAGAINSTTUMOR-ASSOCIATED ANTIGENS Group
Clone
Immunoglobulin class and subclass
Classification of T A A
Site*
1
e453 c432 c153 c143 885 903 4134 4366 2064 2065 311
IgG2b IgG2b IgG2b IgG2b IgG1 IgG1 IgG2b IgG1 IgG1 IgG1 IgG1
Common Common Common Common Partially common Partially common Partially common Partially common Partially common Partially common Individually distinct
A B B B C D E F NDt ND G
2
3
* Topographical regions were analyzed by a competitive binding assay using partially purified T A A s as antigens. For preparation of TAAs, 6 individual tumors were used. t ND, not determined.
soluble T A A s o b t a i n e d f r o m the same t u m o r s , and other viable tumors. MATERIALS
AND METHODS
MAbs Characterization of the MAbs against TAAs expressed on the EBL tumors was described in previous reports [14, 16]. In the present study, we used 11 MAbs, as listed in Table 1. IgG of MAbs was precipitated from mouse ascitic fluid with ammonium sulfate at 50% saturation and purified by subsequent ion-exchange chromatography on D E A E cellulose [15]. Cells We used two bovine lymphoid cell lines, BLSC-KUI and EBLC-I, which had been established from tumors of cattle with EBL. These cell lines have B-cell surface marker and T A A on the surface of cells and bovine leukemia provirus is integrated in each genome [17]. A lymphoid tumor was obtained from a cow with EBL and fresh tumor cells were collected in Eagle's minimal essential medium supplemented with 10% fetal calf serum (FCS) for affinity chromatography on Sepharose 6MB. Affinity chromatography of tumor cells on Sepharose 6MB Affinity chromatography of tumor cells on Sepharose 6MB was performed as described previously [7]. In brief, IgG of MAb c453 was covalently linked to CNBr-activated Sepharose 6MB (Pharmacia Fine Chemicals, Uppsala, Sweden) as indicated previously [7, 12]. Sepharose 6MB was suspended in Dulbecco's minimal essential medium (DMEM) and poured into a column (1.0cm x 20cm). BLSC-KUI cells (2.5 x 10 7 cells) were loaded onto the column and incubated for 15 min at room temperature. Unbound cells were then washed from the column with DMEM. The specifically bound cells were eluted with rabbit antibodies against mouse IgG as follows. Five ~tl of a solution of rabbit anti-mouse IgG that had been diluted with DMEM were passed through the column. The column was then sealed and incubated for 1 h at 37°C with
occasional gentle stirring of the gel in the upper part of the column with a pipette. The cells were then eluted with 3 ml of rabbit antiserum against mouse IgG and 15 ml of DMEM, both warmed to 37°C. The reactivity of bound and unbound cells with MAbs was examined by an indirect fluorescent antibody (FA) assay of acetone-fixed cells, as described previously [18].
Immunoelectron microscopy A suspension of 2 × 10 6 target cells/ml in phosphatebuffered saline (PBS) was placed in a U-bottomed microtest plate (Nunc, Roskilde, Denmark). The plate was centrifuged at 2000 r.p.m, for 5 min and the supernatant was removed. The cells were fixed in peroxidase-lysineparaformaldehyde (PLP) at 4 °C for 4 h and then washed three times with PBS. For the first staining, the cells were incubated with MAb c453 and stained with protein Acolloidal gold (pAg) by Roth's method [20]. For the second staining, the cells were incubated with either MAb c164 or MAb 885 and stained with avidin-biotin-peroxidase complex (ABC) by Hsu's method [9]. The immunologically stained cells were post-fixed in osmiun tetroxide and embedded in Epon 812. Ultrathin sections were examined under a HS-8 type electron microscope. As negative controis, we used FLK cells (fetal lamb kidney cells persistently infected with BLV) [22] and peripheral blood lymphocytes from BLV-free normal cattle stained by the method described above, as well as EBL tumor cells stained either with normal mouse IgG or with MAb adsorbed with T A A were used. Viable-cell enzyme-linked immunosorbent assay (ELISA) Viable-cell ELISA was carried out as described by Aida et al. [3]. The end-point titre of the ELISA was estimated from a dose-response curve obtained with serial dilutions of MAbs. Normal mouse serum was used as a control, and its absorbance at 405 nm was always less than 0.10. The end-point titre of the ELISA was taken as the reciprocal of the maximum dilution of antibody that gave an absorbance at 405 nm of more than 0.15. Standard ELISA Standard ELISA was carried out as described by Aida
Epitopes on tumor antigens
189
the wells were washed and then an aliquot of peroxidaselabelled IgG, at a dilution of between 1:100 and 1:200 and typically having an absorbance at 405 nm of 0.5, was added. The extent of competitive binding (E) was estimated from the following formula: E = 100 (A-N)/(A-B), where A, B and N are the absorbance at 405 nm in the absences of competitor, in the presence of homologous antibody, and in the presence of competitor, respectively.
RESULTS
FIG. 1. Immunoelectron micrographs of EBLC-I cells. A, unstained cell membrane; B, cell membrane immunostained with MAb c453 and pAg particles (large arrow) and with c164 antibody and ABC (dense band; small arrow); C, cell membrane stained with MAbs c453 (gold particles; large arrows) and 885 (dense band; small arrow), x52,000.
et al. [1] using the TAA (0.5/~g/well) that had been partially purified from BLSC-KUI cells [2]. The end-point titre of the ELISA was estimated as described immediately above. Competitive binding assay
A competitive binding assay was performed by a viablecell ELISA or a standard ELISA, as described by Aida et al. [1] with slight modifications. Peroxidase-labelled MAbs were prepared by the glutaraldehyde method of Avrameas [5]. In brief, for the competitive binding assay by the viablecell ELISA, 50 ~tl of a suspension of 1 × 107 target cells/ ml in PBS-FCS were added to wells that already contained 50 ~tl of serial 10-fold dilutions of each unlabelled competitor IgG. After incubation at 37°C for 1 h, the wells were washed, then an aliquot of peroxidase-labelled IgG, diluted to between 1:100 and 1:200 and typically having an absorbance at 405 nm of 0.8, was added. For competitive binding assays by the standard ELISA, serial 10-fold dilutions of each unlabelled IgG, as competitor, were placed in the wells of microplates that had been absorbed with partially purified TAA. After incubation for 1 h at 37 °C,
In order to determine whether or not the c o m m o n T A A , the partially common T A A and the individually distinct T A A s were present on the same tumor cells, we separated tumor cells from bovine lymphosarcomas by affinity chromatography on Sepharose 6MB. When a suspension of tumor cells, in which about half of the cells reacted with all of the MAbs in Group 1 and with only M A b 4366 among the MAbs in Groups 2 and 3, was applied to Sepharose 6MB coupled with c453 antibody (specific to site A on the common T A A ) , the reactivity of the cells that bound to the c453 antibody showed a 2-fold increase while the cells in the flow-through fraction showed only slight reactivity (Table 2). In the bound cell fraction, a similar increase in reactivity with M A b c432 (specific to site B on the common T A A ) and with MAb 4366 (specific to site F on the partially common T A A ) was observed. Reactivity of the nonadherent cell fraction with MAbs c432 and 4366 was very low. When a suspension of tumor cells was chromatographed on a control column of Sepharose 6MB that had been coupled with IgG from normal mouse serum, 1.8% of cells applied to the column adsorbed to the column (data not shown). Nonspecific adsorption to Sepharose 6MB seemed to be negligible as judged from the very low percentage of tumor cells that bound to the column. These results indicate that the common T A A molecule which was recognized by MAbs c143 and c432, and the partially common T A A molecule which was recognized by MAb 4366 may be present on the same cells. The distribution of antigenic sites on tumor cells was examined, after double staining of tumor cells, by immunoelectron microscopy. When the bovine Blymphoid cell line, EBLC-I, established from leukemic cells of cattle with E B L , was reacted first with MAb c453 and then stained by the pAg m e t h o d and finally by the A B C method after treatment with M A b c164 (specific to site B on the common T A A ) , most of the surface of tumor cells showed double staining. As shown in Fig. 1B, a dense zonal band (small arrow) and gold particles (large arrow) were present on the same tumor cells. Moreover, we also stained cells with MAbs c453 and 885 (specific to site C on the partially common T A A ) by the pAg and A B C
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Y. AIDA el al. TABLE 2. SEPARATION OF TUMORS BY AFFINITY CHROMATOGRAPHY ON SEPHAROSE 6MB COUPLED WITH IgG OF MONOCLONAL ANTIBODY C143
% of positive cells* against Treatment
c453
c432
4366
% recoveryt
Before chromatography After chromatography: flow-through cells After chromatography: bound cells
51.2 7.6 90.0
65.0 3.9 85.7
43.7 3.7 69.2
34 37
* The reactivity of bound, flow-through and unfractionated cells with monoclonal antibodies (c453, c432 or 4366) was detected by the FA test (see text). t The number of cells (2.5 x 107) applied to the column coupled with IgG of MAb c453 was taken as 100%.
TABLE 3. REACTIVITIES OF MONOCLONAL ANTIBODIES AGAINST TUMOR-ASSOCIATED ANTIGENS WITH VIABLE AND SOLUBILIZED BLSC-KUI TUMOR CELLS
Titre of ELISA using Group cellst 1
2
3
Clone
Solubilized BLSC-KUI cells*
Viable BLSC-KUI
c453 c432 c153 c143 885 903 2064 2065 4134 4366 311
625 3125 3125 3125 625 3125 --$ --625 --
15,625 78,125 78,125 390,625 15,625 ----3125 --
* Standard ELISA was performed using partially purified TAA from disrupted BLSCKUI tumor cells. t Viable-cell ELISA was performed using viable BLSC-KUI tumor cells. ~t Negative.
method, respectively. Again a dense zonal band (small arrow) and gold particles (large arrow) were found on the same tumor cells (Fig. 1C). Thus, the results of immunoelectron microscopy, showing that the common T A A was recognized by MAbs c453 and c164, and the partially c o m m o n T A A was recognized by MAb 885 on the same cells, confirmed directly the results obtained by affinity chromatography on Sepharose 6MB. In order to determine the reactivities with the T A A of MAbs from Groups 1, 2 and 3 by use of different titration systems, we performed a viable-cell E L I S A using untreated viable BLV-induced B-lymphoid cells, namely, B L S C - K U I cells, and a standard E L I S A using partially purified T A A from disrupted BLSC-KUI cells as antigens. As shown in Table 3, all of the Group 1 antibodies bound extensively to the soluble T A A and to untreated viable BLSC-KUI cells. Of the six MAbs in G r o u p 2, MAbs 885 and
4366 were found to bind to untreated viable BLSCKUI cells and to the soluble T A A prepared from the same cells. However, M A b 903 (specific to site D on the partially common T A A ) did not react with untreated viable BLSC-KUI cells but it did react with soluble T A A prepared from the same cells. The other 3 MAbs (2064, 2065 and 4134) in G r o u p 2 did not react with the soluble T A A or with viable BLSCKUI cells. Moreover, antibody 311 (specific to site G on the individually distinct T A A ) from Group 3 did not react with the T A A expressed on BLSC-KUI cells in either titration system. A comparison between the antigenic regions of the T A A expressed on untreated viable BLSC-KUI cells and those of the partially purified T A A from disrupted BLSC-KUI cells was performed by use of the competitive binding assay with the viable-cell E L I S A and the standard E L I S A (Table 4). With MAbs from Group 1, peroxidase-labelled MAb c453 was blocked
191
Epitopes on tumor antigens TABLE 4. RESULTSOF COMPETITIVEBINDINGASSAYSWITHSOLUBLETAA AND VIABLECELLS* Peroxidase-conjugated antibodies Group 1 Antigen Soluble TAA from BLSC-KUI
Viable BLSC-KUI
Viable EBLC-I
Competitor Group 1 c453 c432 c153 c143 Group 2 885 4366 Group 1 c453 c432 c153 c143 Group 2 885 4366 Group 1 c453 c432 c153 c143 Group 2 885 4366
Group 2
c453
c432
c153
c143
+ -
+ + +
+ + +
+ + +
. .
. .
+ . .
. .
+ + + . .
+ . .
. .
. .
+ + + . .
+ + + . .
+ + +
. .
+ + +
4366
m
_
m
-
+ + + . .
885
+
m
+ +
+ +
m
m
m
_
m
-
+
m
* The competitive binding assay was performed by standard ELISA with soluble TAA and by viable-cell ELISA with either viable BLSC-KUI or EBLC-I cells. +, positive inhibition (>50%); - , negative inhibition (<50%).
completely only by the homologous antibody, in both systems, with viable cells and the soluble T A A as antigens. By contrast, peroxidase-labelled MAbs c432, c153 and c143 (specific to site B on the c o m m o n T A A ) were blocked by homologous and other antibodies, with the exception of M A b c453. We found that there were at least two distinct antigenic regions on the c o m m o n T A A that were expressed on both the soluble T A A and untreated viable B L S C - K U I cells, one being recognized only by M A b c453 and the other being recognized by the rest of the antibodies. This topographical analysis gave the same results as those obtained with a mixture of partially purified T A A s from six individual tumors, as shown in Table 1. These results indicate that the MAbdefined antigenic regions on the c o m m o n T A A molecule, expressed on untreated viable B L S C - K U I cells, may correspond to those on the soluble T A A from disrupted B L S C - K U I cells. Since MAbs 885 and 4366 but not other MAbs from Groups 2 and 3 reacted with both disrupted and untreated viable B L S C - K U I cells, we were able to
test only MAbs 885 and 4366 in competitive binding assays. Peroxidase-labelled MAbs 885 and 4366 were each blocked only by the homologous antibody when disrupted B L S C - K U I cells were used. By contrast, peroxidase-labelled MAbs 885 and 4366 were blocked by homologous and heterologous antibodies when untreated viable B L S C - K U I cells were used. These results indicate that MAbs 885 and 4366 bind to independent epitopes on the soluble T A A from disrupted tumor cells and to a single epitope on viable B L S C - K U I cells. In order to compare the antigenic regions of T A A on viable B L S C - K U I cells with those of T A A s on cells from other bovine lymphoid lines, we performed competitive binding assays by the viable-cell E L I S A using E B L C - I cells (Table 4). While peroxidaselabelled M A b c453 was blocked completely only by the homologous antibody, peroxidase-labelled MAbs c432, c153, and c143 were blocked by the homologous antibodies and by other antibodies, with the exception of c453. Thus, the c o m m o n T A A had at least two distinct antigenic regions which were the same in
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Y. AIDA et al.
both BLSC-KUI and EBLC-I cells. Since peroxidaselabelled MAbs 885 and 4366 were each blocked only by the homologous antibody, MAbs 885 and 4366 may bind to separate and independent antigenic regions on the partially common T A A in EBLC-I cells but they may bind to a single antigenic region on the TAA of BLSC-KUI cells. DISCUSSION In a previous study, using specific MAbs, we identified TAAs expressed on EBL tumor cells as common TAA, partially common T A A and individually distinct TAA, and topographical analysis revealed seven independent antigenic regions, specific for the MAbs, on the three types of T A A [1, 2]. However, we did not determine whether the common, partially common and individually distinct TAAs were present on the same cells. In this study, using affinity chromatography on Sepharose 6MB and immunoelectron microscopy, we showed clearly that the common TAA and the partially common T A A are present on the same tumor cells. All of MAbs from Group 1 bound to the soluble TAA from disrupted tumors and untreated viable cells from tumors. By contrast, although MAb 903, directed against site D on the partially common TAA, bound the soluble T A A from disrupted BLSCI cells, it did not bind to viable BLSC-KUI cells, as shown in Table 3. This phenomenon may have been caused either by structural alteration of the partially common T A A or by a change in the accessibility of the antigenic site during solubilization. It is also possible that a previously hidden antigenic site on the partially common T A A appeared on the surface during solubilization, thereby allowing MAb 903 to bind to the soluble TAA. Similar observations have been made in other systems. Bruck et al. [6] reported that the conformation or accessibility of certain epitopes of the eno glycoprotein gp51 of BLV changed according to the test system used, such as liquidphase radioimmunoassay with plastic-bound gp51 or BLV particles. In the Sindbis virus system, all specific MAbs tested were shown to be inhibited from binding to solid-phase Sindbis virus when premixed with detergent-treated virions, whereas only those having virus-neutralizing activity were competitively inhibited by intact virions [21]. Thus, all cryptic sites on Sindbis virus can be exposed by disruption of virions with a non-ionic detergent. Since the solubilization of the T A A may alter the antigenic regions that are exposed, competitive binding assays were performed with viable and disrupted BLSC-KUI cells in order to characterize the antigenic regions. The antigenic regions (sites A and
B) on the common T A A prepared from disrupted BLSC-KUI cells were conserved on the untreated viable cells. By contrast, MAbs 885 and 4366, which recognized the partially common TAA, bound only to a single epitope on the nontreated viable tumor cells, whereas both MAbs bound to independent antigenic regions on the soluble TAA. This result suggested that, since the soluble T A A prepared from disrupted BLSC-KUI tumors showed greater accessibility with respect to binding to the partially common TAA by MAbs 885 and 4366 than the native TAA expressed on viable tumors, MAbs 885 and 4366 could not be used in competition with each other in a competitive binding assay. Therefore, it is possible that they bind to an independent antigenic region on the disrupted TAA. The exposure of the antigenic region on the partially common T A A may thus be effected by one of the following mechanisms: (a) the antigenic regions of the partially common TAA may become exposed by solubilization but no comformational change occurs that affects the common TAA; or (b) during solubilization, the antigenic regions of the partially common T A A which were previously masked by the common TAA, become exposed as a result of a change in the conformation of the common TAA. However, we found that the avidity of the binding of MAbs to the antigenic regions of the common TAA was not affected by the solubilization, as shown in Table 3. Therefore, the change in reactivity of MAbs from Group 2 with TAA after solubilization may be explained by the first possibility and not by the second possibility. MAbs directed against the common T A A recognized the same epitope on both BLSC-KUI cells and EBLC-I cells. In the case of the partially common TAA, MAbs 885 and 4366 bound to a single epitope on viable BLSC-KUI cells but they bound to independent antigenic regions on EBLC-I cells. Thus, the antigenic determinants defined by the MAbs on the partially common TAA might differ according to the specific tumor, and those of the common T A A might be conserved on all EBL tumor cells. While TAA was expressed constitutively within the membrane of tumor cells, it is known that BLV does not express its gene products at detectable levels in freshly removed lymphocytes or the tumor cells or the infected host, while it often expresses them when these cells are cultured in oitro [11]. Thus, it seems likely that the expression of TAAs plays a key role in BLV-induced leukemogenesis and the maintenance of the tumorous state. Although there is no direct evidence for the possibility, if the antigenic determinants defined by the MAbs on the common TAA are generally conserved between tumor cells from BLV-induced lymphosarcoma cases, they may
Epitopes on tumor antigens themselves be important in BLV-induced leukomogenesis. Further physicoehemical characterization of T A A s and of the distribution of the antigenic determinants that are recognized by the MAbs is currently in progress.
Acknowledgement--We are grateful to Dr Koyama, Kitasato University, for supplying the BLSC-KUI tumor cells.
REFERENCES 1. Aida Y., Onuma M., Mikami T. & Izawa H. (1985) Topographical analysis of tumor-associated antigens on bovine leukemia virus-induced bovine lymphosarcoma. Cancer Res. 45, 1181. 2. Aida Y., Onuma M., Ogawa Y., Mikami T. & Izawa H. (1985) Tumor-associated antigens of bovine leukemia virus-induced bovine lymphosarcoma identified by monoclonal antibodies. Cancer Res. 45, 1174. 3. Aida Y., Onuma M., Kasai N. & Izawa H. (1987) Use of viable-cell ELISA for detection of monoclonal antibodies recognizing tumor-associated antigens on bovine lymphosarcoma cells. Am. J. Vet. Res. 48, 1319. 4. Aida Y., Ochiai K., Ito K., Onuma M., Fujimori F., Fujimoto Y. & Izawa H. (1987) Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens. Am. J. Vet. Res. 48, 1181. 5. Avrameas S. (1969) Coupling enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies, lmmunochem. 6, 43. 6. Bruck C., Portetelle D., Burny A. & Zavada J. (1982) Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions. Virology 122, 353. 7. Chetie V., Mota G. & Sjoquist J. (1978) Separation of cells by affinity chromatography on SpA-Sepharose 6MB. J. Immun. Meth. 21, 133. 8. Hollinshead A. C. & Valli V. E. (1976) Preliminary findings of a tumor-associated antigen in bovine lymphosarcoma. Bibl. Hemat. 43, 369. 9. Hsu S. M., Raine L. & Fanger H. (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577. 10. Jacobs R. M., Valli V. E. O., Wilkie B. N. & Hollinshead A. C. (1981) Partial purification of a common
193
antigen in bovine lymphoma and its use in a lymphocyte blastogenesis assay. Cancer Res. 41, 3000. 11. Kettmann R., Marbaix G., Cleuter Y., Portetelle D., Mammerickx M. & Burny A. (1980) Genomic integration of bovine leukemia provirus and lack of v~ral RNA expression in the target cells of cattle with different responses to BLV infection. Leukemia Res. 4, 509. 12. Manderino G. L., Gooch G. T. & Stavitsky A. B. (1978) Preparation, characterization and functions of rabbit lymph node cell populations. 1. Preparation of KLH primed T and B memory cells with anti-Fab' affinity columns. Cell. Immunol. 41,264. 13. Miller J. M., Miller L. D., Olson C. & Gillette K. G. (1969) Virus-like particles in phytohemagglutininstimulated lymphocytes cultures with reference to bovine lymphosarcoma. J. natn. Cancer Inst. 43, 1297. 14. Okada K., Onuma M., Numakunai S., Kagawa Y., Minamino K., Ito T., Kobayashi Y., Morimoto N., Morita H. & Ohshima K. (1983) Tumor-associated antigen detected by complement-dependent antibody cytotoxicity test and immunofluorescence test in enzootic bovine lymphosarcoma. Jpn. J. Vet. Sci. 45, 195. 15. Onoue K., Yagi Y. & Pressman D. (1964) Multiplicity of antibody proteins in rabbit anti ,-azobenzenearsonate sera. J. Immun. 92, 173. 16. Onuma M., Aida Y., Okada K., Oshima K., Kawakami Y. & Izawa H. (1985) Usefulness of monoclonal antibodies for detection of enzootic bovine leukemia cells. Jpn. J. Cancer Res. (GANN), 76, 959. 17. Onuma M., Koyama H., Aida Y., Obawa Y., Kirisawa R. & Kawakami Y. (1986) Establishment of B-cell lines from tumor of enzootic bovine leukosis. Leukemia Res. 10, 689. 18. Onuma M. & Olson C. (1977) Tumor-associated antigen in bovine and ovine lymphosarcoma. Cancer Res. 37, 3249. 19. Onuma M., Takashima I. & Olson C. (1978) Tumorassociated antigen and cell surface marker in cells of bovine lymphosarcoma. Ann. Rech. Vet. 9, 825. 20. Roth J. (1982) Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J. Histochem. Cytochem. 30, 691. 21. Schamaljohn A. L., Kokubun K. M. & Fole D. G. A. (1983) Protective monoclonal antibodies define maturational and pH-dependent antigenic change in Sindbis virus E1 glycoprotein. Virology 130, 144. 22. Van Der Maaten M. J. & Miller J. M. (1976) Replication of bovine leukemia virus in monolayer cell cultures. Bibl. Haemat. 43, 360.