Journal of Virological Methods. 31 (1992) l-12 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00

VIRMET 01270

Comparison of an EBV transformed cell line and an EBV hybridoma cell line producing the same human anti-HBs monoclonal antibody A. Sa’adu”, H. Walkerb, M. Locniskara, D. Bidwell”, C. Howarda, K.P.W.J. McAdam” and A. Voller”* ’ aDefartment of Clinical Sciences, London School of Hygiene and Tropical Medicine, London, (UK), Department of Haematology. University College Hospital, Gower Street, London, (UK) and ‘Institute of Zoology, Zoological Society of London, RegentS Park, London, (UK)

(Accepted 2 September



A stable human-mouse heterohybridoma secreting human anti-HBs monoclonal antibody in continuous culture for 12 months was generated. It grew faster than the parent EBV transformed lymphoblastoid cell line (LCL) but produced the same level of specific antibody. The LCL was positive for the Epstein-Barr Virus Nuclear Antigen (EBNA), human CD 23 and contained a diploid number of human chromosomes. The heterohybridoma was negative for EBNA, CD 23 and mouse Ly-1 mouse, despite retaining a full complement of diploid mouse chromosomes and a limited number of human chromosomes. EBV transformed cell line; EBV hybridoma; Human anti-HBs monoclonal antibody


The technique of initial EBV transformation of lymphocytes prior to fusion to malignant cells has been termed the ‘EBV hybridoma’ technique (Kozbor et al., 1982). Kozbor and Roder (1984) reported that transforming cells with EBV 2-8 wk before fusion increased the fusion frequency from 20 x 10m6 to over 100 x 10w7. This was attributed to superior activation and proliferation of B cells by EBV. Correspondence to: A. Sa’adu, Dept. of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, UK.


The malignant fusion partner may be human or mouse but must be ouabainresistant to allow selection of EBV hybridoma clones_ Human monoclonal antibodies have been so produced to tetanus toxoid (Kozbor et al., 1982); carcinoma cells (Kozbor et al., 1984); Mycobacterium leprae (Foung et al., 1985); endotoxin (Teng et al., 1985); prostatic acid phosphatase (Yamaura et al., 1985); and the Rhesus D antigen (Thompson et al., 1986). A stable LCL producing an IgGi, kappa human anti-HBs monoclonal antibody was successfully cloned (Sa’adu et al., in press (a)). It secreted specific antibody in continuous culture for over 18 months and was successfully fused with a mouse myeloma using polyethylene glycol (PEG). A stable heterohybridoma producing the same IgG,, kappa human anti-HBs monoclonal antibody, was successfully cloned. Comparison of the A2(N) LCL and the A2(N).X63 heterohybridoma forms the basis of this report.

Materials and Methods Tissue culture A2(N) cells (42 x 106) were fused to P3-X63-Ag8.653 cells (4 x 106) in the presence of PEG according to the method of Thompson et al., (1986). Established cell lines were then cloned by limiting dilution on 96 well tissue culture plates using feeder cells of mouse peritoneal cells (1 x lo5 cells per well). Cells were cultured in medium consisting of RPM1 1640 supplemented with 15% fetal bovine serum (FBS), 4% non-essential amino acids, 2 mM Lglutamine, 1 mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (pg/ ml); (Gibco-Biocult, Paisley, Renfrewshire, U.K.). Cell count and viability scores were performed, and supernatant collected periodically and stored at 4°C until IgG levels were determined. IgG assays were performed in unison to prevent inter-assay variations. Immunoassays Indirect ELISA Immulon 2 microtitre plates (Dynatech Laboratories Inc., Alexandria, VA, U.S.A.) were coated with goat anti-human IgG antibody (Tago Inc., Burlingame, CA, U.S.A.) and incubated with tissue culture supernatants. The plates were incubated with goat anti-human IgG peroxidase conjugate (Tago) and the ortho-phenylenediamine hydrochloride (OPD; Sigma Chemical Co. Ltd., Poole, Dorset, U.K.) substrate added. The enzyme reactions were stopped with acid and Optical densities (OD) measured. Competitive inhibition ELISA The A2(N), A2(N).X63 and an irrelevant human anti-HBs monoclonal antibody were incubated on two Immulon 1 plates (Dynatech) coated with recombinant yeast-derived HBsAg (Merck


Sham and Dohme Ltd., Hoddesdon, Herts, U.K.). Each plate was incubated conjugated to the horseradish with-either the A2(N) or A2(N).X63’antibodies peroxidase enzyme using the periodate method (Voller and Bidwell, 1986). Substrate addition and OD measurements were performed as above. Immunojluorescent


A double fluorescent technique involving red fluorescence from tetrarhodamine isothiocyanate (TRIC; Sigma) and green fluorescence of fluorescein isothiocyanate (FITC; Sigma) was used. Cell membrane antigens (CD 23 and Ly-1) were detected using an indirect immunofluorescent technique in which mouse monoclonal antibodies directed as these antigens were detected using a goat anti-mouse IgG TRIC (Sigma). Nuclear antigen (EBNA) was detected using the indirect immunofluorescent assay of Reedman and Klein (1973), in which complement fixing polyclonal human serum directed against EBNA was detected using a goat anti-human C3 FITC (Sigma). After serially combining these two fluorescent assays the double fluorescence was photographed. Relative DNA content of cell lines

The DNA content Ormeod (1990). Chromosomal

of the cell lines was assessed according

to the method


analysis of cell lines

Cell cultures were Garson (1983) and membranes burst and (Seabright, 1971) and

synchronized according to the method of Webber and cells harvested. The cell nuclei were fixed, nuclear chromosomes banded (Summer et al., 1971) trypsinised Geimsa stained.

Results Growth rates of cell lines

The P3-X63-Ag8.653 mouse myeloma grew rapidly with a doubling time of about 48 h. In contrast, the A2(N) human LCL had a doubling time of 6-7 days. The A2(N).X63 heterohybridoma was intermediate in growth rate, with a doubling time of 224 days. The rate of immunoglobulin (Ig) production was similar for the LCL and heterohybridoma, the maximum levels were 10 pg/ml. Cellular markers

Results of the double fluorescent staining of the cell lines are shown in Fig. 1. The LCL is positive for EBNA and CD 23 (Fig. la). The mouse myeloma is


Fig. 1. Fluorescent staining of cell lines. The LCL is positive for EBNA and CD 23 (a). The orange cell surface fluorescent is produced by the mouse anti-CD 23 monoclonal antibody and the goat anti-mouse IgG TRIC. The green nuclear fluorescence is produced by the anti-EBNA complement fixing polyclonal human sera and the goat anti-human C3 FITC. The mouse myeloma is EBNA negative but positive for LyI (b). The cell surface orange fluorescent was produced by the mouse anti-Ly-I monoclonal antibody and the goat anti-mouse IgG TRIC. There is no nuclear staining because mouse myeloma cells are EBNA negative. The heterohybridoma is negative for EBNA, human CD 23, mouse Ly-1 (c).

EBNA negative but Ly-1 positive (Fig. lb). The heterohybridoma for EBNA, human CD 23 and mouse Ly-1 (Fig. lc).

is negative

Relative DNA content of cells The LCL has a relative DNA content of 225 (Fig. 2A) which is 10% higher than the normal diploid cell of 200, although within the normal range. The mouse myeloma had a relative DNA content of 267 (Fig. 2B). However, the EBV hybridoma, A2(N).X63, had a bimodal distribution with two small peaks, one at 200 and the second at 500 (Fig. 2C). Competitive


The percentage

of inhibition

of the A2(N),


and the irrelevant


A 225



B 1



0 1






Fluorescencearea Fig. 2. Relative DNA content of cell lines. Tissue culture cells were harvested and fixed in 70% ethanol at 4°C for 30 min. After adding RNase, they were stained in propridium iodide at 37°C for 30 min. The relative DNA content was determined on a fluorescent activated cell sorter gated on area times width. The A2(N) LCL (A) has a single peak at 225; the P3-X63-Ag8.653 mouse myeloma (B) has a single peak at 267, whilst the AZ(N).X63 heterohybridoma (C) has two peaks at 200 and 500.

human anti-HBs monoclonal antibody against the A2(N) conjugate is shown in Fig. 3a. The percentage inhibition of these antibodies against the A2(N).X63


iercentag e Inhibition







+ *


* 4









0 ie

Antibody Titre

b) Percentage




__ __-







OJC * 10










x 80







x x 0 1280 2560 b40118

Antibody Titre Fig. 3. Competitive inhibition ELISA. The ability of both the A2(N) I-0-l and the A2(N).X63 [-*-I human anti-HBs monoclonal antibodies to inhibit the binding of the A2(N) peroxidase-labelled conjugate is shown in (a) and that of the A2(N).X63 peroxidase-labelled conjugate is shown in (b). An irrelevant human antiHBs monoclonal antibody [-x-l failed to demonstrate any inhibition of either conjugated antibody.



Cell type Cultures Nuclei Centromeres Chromosome

Cell lines




LCL Clumps Small Submetacentric 36, Human

Myeloma Monolayer Large Acrocentric 40, Mouse

Heterohybridoma Heaped up Large Mixture 40, mouse with Human No. 2, 3, 7, 14 and 22

conjugate is shown in Fig. 3b. Both antibodies are able to inhibit each conjugate, but the irrelevant human anti-HBs monoclonal antibody demonstrates no inhibition. Chromosomal

analysis of cell lines

Table I summarises the phenotypic and genotypic characteristics of the three cell lines. A2(N) is a human LCL which grows in clumps, has small nuclei and 36 submetacentric human chromosomes. P3-X63-Ag8.653 is a mouse myeloma which grows as a monolayer, has large nuclei and 40 acrocentric (i.e. a chromosome with a terminally placed centromere) mouse chromosomes. A2(N).X63 is a heterohybridoma which grows in heaped up layers, has large nuclei and contains 40 mouse chromosomes but only a limited number of human chromosomes (numbers 2, 3, 7, 14 and 22).

Discussion A stable heterohybridoma was cloned which secreted human anti-HBs monoclonal antibody in continuous culture for 12 months. In summary it is an IgG,, kappa antibody which recognises the ‘a’ group determinant of HBsAg and inhibits the binding of the parent antibody (A2(N)) (Sa’adu et al., in press a, b). The claim of Kozbor et al. (1984) that heterohybridomas produce higher levels of specific antibodies compared to LCL conflicts with our finding of similar Ig secretion which is in agreement with those of Cote et al. (1983) and Houghton et al. (1983). The levels of specific antibodies from our clones compare favourably with those of Burnett et al. (1985) and Desgranges (1987) and his co-workers. The CLB-Hu-HBsAg-1 cell line of Stricker et al. (1985) generated by ‘cell driven’ EBV transformation (co-culturing with a lymphoblastoid cell line) secreted higher levels of antibody than those cell lines generated by ‘viral’ EBV transformation (culturing with EBV viral particles). Croce et al. (1980) have suggested that the preferential loss (segregation) of human chromosomes, especially chromosome 2 coding for kappa light chains,


is responsible for the loss of human Ig secretion by human-mouse heterohybridomas. More recently, however, Thompson et al. (1986) have suggested that the genetic instability of human-mouse heterohybridomas is not greater than that of mouse-mouse hybridomas. Chromosomal study of the heterohybridoma allowed the demonstration of the mixture of human and mouse chromosomes. A full set of 40 mouse chromosomes appeared to be present, however, only a limited number of human chromosomes were retained. The retention of human chromosomes 2 and 14 is consistent with its ability to secrete the IgG,, kappa human anti-HBs monoclonal antibody. Taylor et al. (1988) have reported that the loss of expression of parental products in inter-species hybridoma made by fusing cells of different lineages, termed extinction, is quite a common occurrence. Extinction of EBNA and CD 23 by the heterohybridoma may result from segregation of human chromosomes. However, the loss of the Ly-1 is not readily explained as the full compliment of mouse chromosomes was retained. Extinction of surface markers on B cell heterohybridomas generated for the sole purpose of producing human monoclonal antibodies may be of only academic interest. It is, however, of fundamental importance in the generation of T cell hybridomas whose cell surface antigens/receptor systems are of vital importance in their immune interactions. Occasionally the induction of antigen expression in hybridomas derived from negative parent cell lines has been reported. Rettig et al. (1987) found that heterohybridomas generated by fusing Thy- 1 negative human peripheral lymphocytes or LCL and Thy-l negative Chinese hamster fibroblasts begin to express human Thy-l. Similarly, Taylor et al. (1988) demonstrated human CD 4 expression by two human-mouse heterohybridomas generated following fusion of parent cell lines negative for CD 4. These workers have postulated that the phenomena of extinction and induction of antigens can be explained by chromosome segregation or by positive and negative ‘trans-acting regulatory signals’ on the transcription or translation of nuclear information. However, it is possible that changes in conformation and insertion of normally transcribed and translated antigens into the cell membrane are responsible. The relative DNA content of the LCL and the mouse myeloma fusion partner confirmed their diploid nature. The pattern of the chromosomal content for the heterohybridoma suggests that these cells contain a varying number of chromosomes and is similar to the pattern produced by the MHG7 heterohybridoma of Glassy et al. (1985) and may be characteristic of all human-mouse heterohybridomas. Our data suggest that the heterohybridoma does not confer any advantage over the LCL, but the fusion was carried out long after the transformation of the original B cells using a stable cloned LCL. Normally, the advantages conferred by fusing EBV transformed cells with mouse myeloma cells comes when fusions are performed within 334 wk of transformation, resulting in ‘rescue’ of unstable LCL and higher antibody yields (Kozbor et al., 1982;


Kozbor and Roder, 1984). However, if our heterohybridoma can be adapted to grow in mouse ascites as others have (Insel, 1984; Tiebout et al., 1984; Yamaura et al., 1985), this would provide a cheap, simple and rapid method of producing large quantities of its human monoclonal antibody.

Acknowledgements A.S. was supported by grants from the Williams Medical Research Fellowship and the Overseas Students’ Research Fund. The laboratories acknowledge support from the Lawson Trade Trust, the Overseas Development Administration, the Rockefeller Foundation and the Wellcome Trust. We are grateful to Dr. Michael O’Hare for performing the relative DNA analysis on selected cell lines. Finally, we are grateful to Mrs. Amy Davey for preparation of the manuscript.

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Comparison of an EBV transformed cell line and an EBV hybridoma cell line producing the same human anti-HBs monoclonal antibody.

A stable human-mouse heterohybridoma secreting human anti-HBs monoclonal antibody in continuous culture for 12 months was generated. It grew faster th...
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