Immunology Letters, 32 (1992) 91 -96

Elsevier IMLET 01763

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens H i d e y u k i A s a o k a I , Shigeyuki N i s h i n a k a 2, N o b u t a k a W a k a m i y a 3, H a r u o M a t s u d a I and M a s a y o s h i Murata 1 1Department of Microbiology and Hygiene, Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, HigashiHiroshima; 2Biotechnology Development Center, Engineering, Research and Development Division, NKK Corporation, Minamiwatarida-cho Kawasaki-ku, Kawasaki; and 3Department of Pathology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

(Received 19 September 1991; revisionreceived 18 December 1991; accepted 18 December 1991) 1. Summary Two chicken monoclonal antibodies (MAbs), H U / C h 2 - 7 and H U / C h 6 - 1 , against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGca LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb H U / C h 2 - 7 (IgG) reacted strongly with HD3 and IV3NeuGco~-nLcaCer (HD5) and weakly with VI3NeuGcot-nLc6Cer (HD7) and 4-0acetyl-HD3. H U / C h 6 - 1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against H D antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing Key words: Chicken monoclonal antibody; Hanganutziu-

Deicher antigen; N-glycolylneuraminicacid (NeuGc) Correspondence to: H. Matsuda, Department of Microbiology and Hygiene,Faculty of Applied BiologicalScience, Hiroshima University, Kagamiyama, Higashi-Hiroshima724, Japan. Abbreviations: HD, Hanganutziu-Deicher; NeuGc, N-glyco-

lylneuraminicacid; NeuAc, N-acetylneuraminicacid; GSL, glycosphingolipid.

gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc. 2. Introduction Heterophil HD antigens [1, 2] were first extracted and purified as N-glycolylneuraminic acid (NeuGc)-containing glycosphingolipids (GSLs) from equine and bovine erythrocytes by Higashi et al. [3] and Merrick et al. [4]. NeuGc is widely distributed in mammalian species with the exceptions of humans and chickens. However, the H D antigen has been found in colon cancers [5], melanomas [6], retinoblastoma cell lines [7] and a gastric cancer cell line [8] of humans and in Marek's disease (MD) tumor cells and MD-derived cell lines of chickens [9, 10]. Anti-HD antibody is useful for immunological detection of glycoconjugates containing NeuGc. In fact, human HD antibodies from sera of patients with various diseases including cancer [11] and chicken HD antibodies prepared from chickens hyr per-immunized with the antigen have been used for detection of NeuGc in various human cancers and for immunological analysis of the antigen [12]. Recently, Usuba et al. [13] established a human monoclonal antibody (MAb), 1F42E31G7 (IgM) that recognized HD antigen. The hybridoma producing this MAb was developed by fusion of a human hybrid myeloma cell line and human spleen cells primed in vitro with particulate HD3 antigen.

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However, Usuba et al. [13] pointed out that they could not rule out the possibility that the MAb was established by chance. In our previous studies [14, 15] we established a few chicken MAbs against defined antigens by cell fusions using thymidine kinase (TK)-deficient chicken B cell lines. On the other hand, a high titer of H D antibody is known to be produced in chickens immunized with purified H D antigen-active GSLs. To obtain a suitable reagent for detection of H D antigen, therefore, we attempted to fuse a chicken TK-deficient B cell line with spleen cells from chickens hyper-immunized with HD antigen. The present paper describes the establishment of two chicken MAbs against H D antigen and their immunological specificities. 3. Materials and Methods

3.1. Glycosphingolipids and immunizations The glycosphingolipids (GSLs) used in this study are listed in Table 1. Four-week-old White Leghorn chickens were immunized intramuscularly and intracutaneously with 0.1 mg HD3 mixed with an equal amount of methylated bovine serum albumin (BSA) and Freund's complete adjuvant. After four weeks, the chickens were given a second intravenous (i.v.) injection of 0.1 mg of HD3 mixed with methylated BSA. A final i.v. injection of the same antigen was given 14 days later.

3.2. Cell lines A chicken B cell line, R27H4 [15], lacking thymidine kinase (TK) was used as a parental cell line in fusion experiments. The cells are resistant to 10 ~g/ml of trifluorothymidine (TFT) but die within five days in HAT medium. R27H4 produces IgM of unknown specificity. The R27H4 cells were routinely maintained in RPMI 1640 or Iscove's modified Dulbecco's medium (IMDM) supplemented with 10°70 fetal bovine serum (FBS) and 10/~g/ml of T F T at 38.5 °C under 5°7o CO 2 in air. 3.3. Cell fusion and screening R27H4 cells were fused with spleen cells from HD3-immunized chickens as described previously [15], and after cultivation of the fused cells in HAT medium for 1 0 - 14 days, the culture supernatants of growing ceils were used for identification of H D antibody-secreting hybridomas by enzyme-linked immunosorbent assay (ELISA) with HD3 in the solid phase as described previously [12]. 3.4. Cell cloning As cell cloning by a soft agar culture method [15] in this study was unsuccessful, it was performed by a newly developed intercup method (manuscript in preparation). Briefly, growing hybridoma cells were suspended in RPMI 1640 supplemented with

TABLE 1 Structures of glycosphingolipids. H D 3 w a s p u r i f i e d f r o m e q u i n e e r y t h r o c y t e s [3]. H D 5 , H D 7 , 4 - O - a c e t y l - H D 3 a n d G M 1 were k i n d l y p r o v i d e d b y Dr. N a i k i . G M 3 w a s p u r c h a s e d f r o m B i o - C a r b C h e m i c a l s ( L u n d , S w e d e n ) . G a l , D - g a l a c t o s e , G l u , D-glucose; G l u N A c , N - a c e t y l - D - g l u c o s a m i n e G a l N A c , N - a c e t y l - o - g a l a c t o s a m i n e ; N e u G c , N - g l y c o l y l n e u r a m i n i c a c i d ; N e u A c , N - a c e t y l n e u r a m i n i c a c i d ; Cer, c e r a m i d e . Abbreviation

Chemical structure

HD3 HD5 HD7 4-O-acetyl-HD3 GM3 GM1

NeuGcc~2 - 3Gal/31 - 4G1c/31 - 1 ' C e r NeuGcc~2 - 3Gal/31 - 4GIuNAc/31 - 3Gal/31 - 4G1c/31 - 1' C e r N e u G c ~ 2 - 3Gal/31 - 4 ( G l u N A c / 3 1 - 3 G a l ~ 1 - 4)2Glc/31 - 1' C e r 4 - O - a c e t y l - N e u G c c ~ 2 - 3Gal/31 - 4Glcj31 - 1' C e r N e u A c c ~ 2 - 3Gal/31 - 4 G 1 c / 3 1 - 1 ' C e r Gal/31 - 3 G a l N A c ~ 1 - 4Gal/31 - 4G1c/31 - 1' C e r 1~2-3 NeuAc


10070 FBS and plated into 24-well culture plates in a total volume of 0.5 ml/well at a density of 1 cell/ ml. Then intercups (Sanko-Junyaku Co., Ltd. Japan) were slowly introduced into each well of the plates and 0.5 ml of the same hybridoma cell suspension (2 × 105 -106 cells/ml) was put into each cup. The plates were then incubated at 38.5 °C for 14-20 days in a CO2-incubator. Cells from each well in which a single colony was visible were then propagated to a large scale culture. 3.5. Antibody titration quantification

and immunoglobulin

3.7. Neuraminidase treatment Samples of 50/zl of HD3 (0.5 mg/ml) in PBS containing 0.05070 taurodeoxycholic acid (TDC) (Sigma Chemical Co., USA) were incubated with 10 /~1 of a solution of 50 mUnits of streptococcal neuraminidase (Seikagaku Kogyo Co., Tokyo) or 10/~1 of PBS at 37 °C for 2 h. Then the solution was replaced by methanol and dot-blotted (5 #1) onto TLC plates. After drying, the antigenicity of the materials was determined by the immunostaining method described previously. 4. Results

Specific antibody titer of MAbs against HD3 antigen was determined by ELISA using 96-well ELISA plates coated with HD3 antigen (100 ng/ well). Serially diluted culture supernatants (50 #1) for antibody titration were added to each well and incubated for 1 h at 37 °C. The wells were then washed and 50/~1 of peroxidase-conjugated goat anti-chicken IgG (H + L) (Kirkegaard and Perry Laboratories, MD) was added. Color development and measurement were done as described previously [12]. The antibody titer was expressed as the reciprocal of the highest dilution giving a positive reaction. The IgG contents secreted into the culture supernatants from hybridomas were determined by ELISA as described previously [15].

3.6. Thin-layer chromatography/immunostaining Thin-layer chromatography (TLC)/immunostaining was performed as described previously [16].

A TK-deficient B cell line, R27H4, was fused with spleen cells from HD3-immunized chickens and the hybridomas were screened for antigen-specific antibodies. The results of three fusions are summarized in Table 2. In these separated fusions, growing hybridomas were observed in 7.9 - 47.4070 of the total cultures and the highest fusion efficiency was observed using IMDM. In wells with the growing cells, a total of 72 hybridomas produced HD antibodies. However, only two of the hybridomas showed stable antibody production. The abilities of the remaining cells to produce antibodies were lost within 30 days after fusion. These hybridomas were named HU/Ch2 and HU/Ch6, respectively. Cloning of these two hybridomas by the soft agar method was difficult and so cloning was achieved by a new method using intercups. By this method, the cloning of these hybridomas HU/Ch2 and HU/ Ch6 were successful. Totals of 14 and 8 clones of

TABLE 2 Summary of generations of hybridomas. Fusion experiment


Exp. 1 Exp. 2 Exp. 3

RPMI 1640 RPMI 1640 IMDM b

Hybridoma/ total culture (°7o) 68/864 (7.9) 102/912 (11.1) 273/576 (47.4)

Specific hybridomaa/ total hybridoma (070) 12/68 (17.6) 19/102 (18.6) 41/273 (15.0)

a Number of hybridomas positive for HD3 determined by ELISA as described in Materials and Methods. b Iscove's modified Dulbecco's medium.


TABLE 3 Properties of two chicken MAbs. MAb


Ig class a

lg concentration (/.tg/ml)

Antibody titer to HD3

HU/Ch2- 7 HU/Ch6 - 1

R27H4 R27H4


1.01 _+ 0.08 b 1.96 _+ 0.15

80 320

a Immunoglobulin class o f M A b was determined by a Western blotting analysis as described previously [15]. b Mean _+ SD for triplicated samples.

hybridomas H U / C h 2 and H U / C h 6 were obtained. The immunological properties of antibodies secreted from a HU/Ch2-derived clone ( H U / C h 2 - 7 ) and a HU/Ch6-derived clone ( H U - C h 6 - 1 ) were examined by ELISA and Western blotting analysis, and results are shown in Table 3. The antibodies secreted from these hybridomas were of the IgG class. The concentrations of IgG secreted into the culture media were determined 90 days after fusion to be 1.01 +0.08 #g/ml ( H U / C h 2 - 7 ) and 1.96 + 0.15 t~g/ml ( H U / C h 6 - 1), respectively. The antibody titers to HD3 antigen of these MAbs were determined by ELISA to be 1:80 and 1:320, respectively. The reactivities of the two antibodies against several gangliosides including H D antigens were examined by a TLC/immunostaining method. H U / C h 2 - 7 reacted strongly with HD3 and HD5 and

Fig. 2. Reactivities of MAbs with neuraminidase-treated HD3. NCS, normal chicken serum; Anti-HD3 Poly Ab, chicken antiserum against HD3.

weakly with HD7 and 4-O-acetyl-HD3, whereas H U / C h 6 - 1 reacted with only HD3 and HD5 (Fig. 1). These two antibodies did not react with N-acetylneuraminic acid (NeuAc)-containing gangliosides (GM1 and GM3). To confirm the relationship between NeuGc-con-



1 2 3 4 5 6




1 2 3 4 8 6

Fig. 1. T L C / i m m u n o s t a i n i n g with H U / C h 2 - 7 and H U / C h 6 - 1 of several gangliosides. A control T L C plate was stained with 25% H2SO 4 reagent. Lane 1, HD3; Lane 2, 4-O-acetyl-HD3; Lane 3, HD5; Lane 4, HD7; Lane 5, GM3; Lane 6, GM1.


taining gangliosides and the specificities of the two antibodies, we examined the reactivities of the two MAbs with HD3 treated with neuraminidase on TLC plates by the immunostaining method. The reactivities of these MAbs with HD3 antigen were greatly reduced by treatment of the antigen (Fig. 2).

5. Discussion In this study, we established two chicken hybridomas, H U / C h 2 - 7 and H U / C h 6 - 1, producing MAb against H D antigens. These hybridomas showed stable antibody production and cell growth and their clonings were successful. Two clones, H U / C h 2 - 7 and H U / C h 6 - 1, separated from H U / C h 2 and H U / C h 6 produced only specific IgG antibody, though the parental R27H4 cells produced non-specific IgM. In a previous fusion study using R27H4 cells [15], we obtained several hybridomas producing anti-KLH MAb reconstructed with specific IgG and non-specific IgM. Recently, we found that although most R27H4 cells produced IgM, few produced Ig chains (unpublished data). Therefore, the present results suggest that H U / C h 2 - 7 and H U / C h 6 - 1 might have been obtained by fusion of immune spleen cells and Ignon-productive R27H4 cells. The anti-HD human MAbs, recently established in two laboratories [13, 17], were both IgM-type antibodies. Therefore, the MAbs to H D antigens obtained in this study are the first reported IgG type anti-HD MAbs. By the intercup method for cell cloning of chicken hybridomas, a total of 22 clones were obtained from H U / C h 2 and H U / C h 6 hybridomas and their clonings were reproducible, The chemical structures of HD3 and GM3 are the same except in the terminal sialic acid of the carbohydrate chain (Table 1). MAbs H U / C h 2 - 7 and H U / C h 6 - 1 reacted with HD3, which has NeuGc at its non-reducing terminal, on TLC-immunostaining, but did not react with GM3 having NeuAc at its terminal. These results and the decrease of HD-antigenicity on neuraminidase treatment (Fig. 2) indicate that these MAbs may recognize NeuGc as a major antigenic epitope. In fact, H U / C h 2 - 7 reacted with other NeuGc-containing gangliosides (HD5, HD7 and 4-O-acetyl-HD3) besides HD3, whereas the reactivity of H U / C h 6 - 1 was restricted to HD3 and HD5. H U / C h 2 - 7 ,

which is highly reactive with HD3 and HD5 as well as H U / C h 6 - 1, was weakly reactive with H D 7 and 4-O-acetyl-HD3. Chicken antisera raised against HD3 antigen recognized not only HD3, HD5 and HD7, but also 4-O-acetyl-HD3, which has the composition of natural GSLs [18]. A human MAb 2 - 3 9 M (IgM) specific for HD antigens established by Yamaguchi et al. [17] reacts with three kinds of HD gangliosides, HD3, HD5 and HD7 [19]. These facts suggest that H U / C h 2 - 7 recognizes the same antigenic determinant. A human MAb 1F42E31G7 (IgM), recently established by Usuba et al. [13], reacted strongly with HD3 and HD5 but not with HD7, indicating that it was directed precisely toward the terminal NeuGc and whole structure of HD3 and HD5 like H U / C h 6 - 1. Therefore, the difference in the specificities of H U / C h 2 - 7 and H U / C h 6 - 1 to H D antigens might be related to the whole structures, especially the hydroxy radical at position four of the NeuGc residue and the number of saccharides in their carbohydrate chains. Further immunological and biochemical studies on the specificities of the two MAbs described here may supply valuable information on H D antigens.

Acknowledgements We thank Dr. M. Naiki, National Institute of Health, Japan, for providing several GSLs, and Dr. H. Higashi, Mitsubishi Kasei Institute of Life Sciences, for helpful discussion. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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[8] Fukui, Y., Maru, M., Ohkawara, K., Miyake, T., Wang, D., Ito, T., Higashi, H., Naiki, M., Wakamiya, N. and Kato, S. (1989) Biochem. Biophys. Res. Commun. 160, 1149.

[9] lkuta, K., Kitamoto, N., Saito, C. and Kato, S. (1980) Biken J. 23, 57. [10] Ikuta, K., Kitamoto, N., Shoji, H., Kato, S. and Naiki, M. (1981) Biken J. 24, 23. [11] Kasukawa, R., Kano, K., Bloom, M. L. and Milgrom, F. (1976) Clin. Exp. Immunol. 25, 122. [12] Higashi, H., Ikuta, K., Ueda, S., Kato, S., Hirabayashi, Y., Matumoto, M. and Naiki, M. (1984) J. Biochem. 95, 785. [13] Usuba, O., Fujii, Y., Miyoshi, I., Naiki, M. and Sendo, F. (1988) Jpn. J. Cancer Res. (Gann) 79, 1340.


[14] Nishinaka, S., Matsuda, H. and Murata, M. (1989) Int. Arch. Allergy Appl. Immunol. 89, 416. [15] Nishinaka, S., Suzuki, T., Matsuda, H. and Murata, M. (1991) J. Immunol. Methods 139, 217. [16] Higashi, H., Fukui, Y., Ueda, S., Kato, S., Hirabayashi, Y., Matumoto, M. and Naiki, M. (1984) J. Biochem. 95, 1517. [17] Yamaguchi, H., Furukawa, K., Fortunato, S. R., Livingston, P. O., Lloyd, K. O., Oettgen, H. F. and Old, L. J. (1987) Proc. Natl. Acad. Sci. USA 84, 2416. [18] Fujii, Y., Higashi, H., Ikuta, K., Kato, S. and Naiki, M. (1982) Mol. Immunol. 19, 87. [19] Furukawa, K., Yamaguchi, H., Oettgen, H. F., Old, L. J. and Lloyd, K. O. (1988) J. Biol. Chem. 263, 18507.

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (...
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