Production of monoclonal antibodies to dehydroepiandrosterone-sulphate after immunization of mouse with dehydroepiandrosterone-bovine serum albumin conjugate

J. steroid Biochem. Vol. 32, No. 4, pp. 553-558,

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0022-473 l/89 53.00 + 0.00

1989

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reserved

PRODUCTION OF MONOCLONAL ANTIBODIES TO DEHYDROEPIANDROSTERONE-SULPHATE AFTER IMMUNIZATION OF MOUSE WITH DEHYDROEPIANDROSTERONE-BOVINE SERUM ALBUMIN CONJUGATE P. PARVAZ,* B. MAWIAN, M. C. PATRICOT,I. GARCIA, A. REVOL,E. MAPPUS,’ C. GRENOT’and C. Y. CUILLERON’ Laboratoire de Biochimie, Hopital Ste. Eugtnie, Centre Hospitalier, Lyon-Sud, 69310 Pierre-E&rite, and ‘Unite de Recherches Endocriniennes et Metaboliques chez l’Enfant, I.N.S.E.R.M. U.34, Hopital Debrousse, 29, rue Soeur-Bouvier, 69322 Lyon Cedex 1, France (Received 31 May 1988; received for publication 22 November 1988)

SummPry-Monoclonal antibodies with a much higher specificity for DHA-S than for DHA were obtained from a BALB/c mouse immunized with a non-sulphated DHA-‘ICMO-BSA antigen. An improved fusion technique using PEG containing 10% DMSO instead of PEG alone increased the number of positive hybridomas. One of the five monoclonal antibodies obtained, showed a high affinity for DHA-S (K, = 1OroM-r) and very low cross-reactions with androsterone (0.62%) and androsterone sulphate (0.83%) which made it potentially useful for direct quantitation of DHA-S in human serum.

binding characteristics for direct quantitation of serum DHA-S. The present work describes some attempts to obtain mouse anti-DHA and anti-DHA-S monoclonal antibodies using either the DHA-7CMO-or the DHA-15ethylthiocarboxylic (DHA-lSETC)-haptens, which both have a side chain remote from the 3-OH antigenic determinant. In order to increase the antibody response of mice [7,8], three carrier proteins, bovine serum albumin (BSA), bovine thyroglobulin (BTG) and keyhole limpet hemocyanin (KLH) were tested as well as the use of long term immunization.

INTRODUCTION Immunization of BALB/c mice with dehydroepiandrosterone-7(0_carboxymethyl)oxime-bovine serum albumin (DHA-‘ICMO-BSA) antigen has been reported to allow access to monoclonal antibodies showing a very high cross-reaction with DHA sulphate (DHA-S) [1,2]. This cross-reaction with DHA-S is rather surprising since highly specific anti-DHA polyclonal rabbit antibodies have previously been obtained using a similar DHA-7CMO hapten which was linked to the BSA carrier through the C-7 position remote from the free 3-OH group [l]. On the other hand, DHA-3-hemisuccinate haptens are known to produce ewe and rabbit anti-DHA polyclonal antibodies showing a high cross-reaction with DHA-S [3-S], whereas 17a-hydroxyprogesterone (17a -OHP)-4-carboxyethylthio haptens led to mouse anti- 17~7-0HP monoclonal antibodies, with up to 25 1% cross-reactions with 17a -hydroxypregnenolone-3-sulphate [6]. In these two examples, the cross-reactions with 3-sulphated analogues could be attributed to the masking of the C-3 position. The possibility of obtaining anti-DHA-S monoclonal antibodies without the constraints of the synthesis of antigens bearing totally sulphated DHA haptens [ 1,2], might considerably facilitate access to monoclonal antibodies with the appropriate

MATERIALSAND METHODS Synthesis of DHA antigens

Two haptens, DHA-7CM0 and DHA-ISETC were synthesized using methods previously described [9-l 1] and were converted to the corresponding activated N-hydroxysuccinimide (NHS) esters. The activated ester of DHA-7CM0 was coupled to BSA, BTG and KLH whereas the activated ester of DHA15ETC was coupled to KLH only. The coupling efficiency was systematically controlled by thin-layer chromatography on silica plates (Merck GF 254) developed either with ethyl acetate (solvent 1) or with a chloroform-acetone-acetic acid 7: 2: 1 mixture (solvent 2). Detection was performed by spraying with 9 M H2S0, and heating at 150°C. These conjugates were separated from uncoupled haptens by dialysis followed by a filtration on Sepharose 4B [12]. The number of steroid residues bound per molecule

*To whom correspondence and reprint requests should be sent. 553

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of protein was measured by U.V. adsorption at 280nm in the cases of DHA-7CMO-BSA and of DHA-‘ICMO-BTG conjugates. Immunization Female, 6 week-old BALB/c mice (IFFA-CREDO, L’Arbresle, France) were divided into two groups of 8 and one group of 4 animals. Each mouse received an intraperitoneal (i.p.) injection of antigen (100 rg) dissolved in 40 ~1 of phosphate buffer saline (PBS), pH 7.4 and emulsified in an equal volume of complete Freund’s adjuvant (Behring). Booster injections were performed at Cweek intervals using incomplete Freund’s adjuvant. The mice were bled through the retro-orbital sinus 20 days after each injection. Sera were stored at -20°C after centrifugation (Microfuge, 5 min, 20°C). Three days before fusion, the mice received an i.p. injection of antigen (3OOkg) in PBS (100 pl). Binding activity measurements (a) Antibody titration.Diluted serum or ascites fluid (in PBS, pH = 7.4, BSA O.l%, NaN, 0.1%: PBSBSA) or culture supematants (100 ~1 samples), were incubated for 2 h at 4”C, with 20,000 dpm of tritiated tracer [( 1,2,6,7-‘HI-DHA, 90 Ci/mmol or [7-“HIDHA-S ammonium salt, 25 Ci/mmol, Amersham) in the same buffer (100 ~1). The unbound steroid was then separated after incubation for 10 min at +4”C with 0.5 ml of charcoal-dextran suspension (100 mg activated charcoal (Sigma) 1OOmg dextran T-70 (Pharmacia) in 50 ml PBS pH = 7.4) followed by centrifugation (3OOOg, 5 min, +4”C). Radioactivity of 0.5 ml of supematant was measured in 4ml of scintillation liquid (Pica-Fluor 15 Packard, Packard 4450 counter). Antibody titres were determined as the dilution showing 50% binding of the added tracer. (6) Test of monoclonal antibody no. 161 for direct RIA of DI-IA4. The monoclonal antibody no. 161 from ascites fluid diluted in PBS-BSA (1: 10,000) was employed to establish a standard curve by incubation (see conditions above) with [3H]DHA-S and 8 concentrations ranging from 78 to 10,008 pg of DHA-S per tube (total volume = 300 ~1). Plasma samples were diluted (1: 200 or 1: 500) in PBS-BSA before measurement. (c) Specificity and associationconstants.The crossreactions of monoclonal anti-DHA-S antibodies were measured according to the method of Abraham [ 131. The association constants were determined by Scatchard plots [14]. Cell fusion (a) Preparation of myeloma cells. SP2/0 myeloma cells [15] were cultured (37”C, humidified incubator, 5% CO,) in RPMI-1640 medium (Flow) supplemented with 3 g/l glucose, 2mM L-glutamin (bioMerieux), 1 mM sodium pyruvate (Flow), 100 iu/ml penicillin and 50 p g/ml streptomycin

(bioMerieux), 10% heat-inactivated fetal calf serum (FCS) (Flow) and containing 20 FM I-axaguanine (Sigma). Just before fusion, cells were washed twice (8min centrifugation at lOOg, 20°C) with RPMI1640 alone and resuspended in the same medium. (b) Preparation of fee&r cells. One day before fusion, a non-immunized BALB/c mouse was sacrificed. The spleen cells were washed and resuspended at a concentration of l@cells/ml, in the above-mentioned RPMI- 1640 supplemented medium without 8-axaguanine but containing 50 PM hypoxanthine (Sigma), 10 PM axaserine (Sigma) and 20% FCS. The cell suspension was distributed (100 ~1 per well) into 96 microwell plates (Flow) which were incubated as above. (c) Preparation of immunized spleen cells. Just before fusion, the selected donor mouse was sacrificed. The spleen cells were washed and resuspended in RPMI-1640 medium alone. (d) Fusion. Two fusogens were prepared: (i) Polyethyleneglycol (PEG) 4000 38% (1.25 g PEG 4000-Merck-in 2 ml of RPMI-1640 alone), (ii) PEG 4000 with 10% dimethylsulphoxide (DMSO) (1 .Og PEG 4000, 0.1 ml DMSG-Merck-in 1 ml PBS, pH = 7.0) [16]. Fusogens were then sterilized by filtration (Millex 0.22 pm). Fusion was performed by slow addition (=45 s) of 1 ml of fusogen to the cell pellet obtained after centrifugation (lOOg, 8 min) of a 5: 1 mixture of immunized mouse spleen cells and SP2/0 myeloma cells in RPMI-1640. When the PEG fusion was used, the cell fusion mixture after 1 min incubation at 20°C was diluted slowly (# 1 min) with lOm1 of RPMI-1640 medium. The cells were then washed twice with the same medium and resuspended in the culture medium described above for the feeder cells at a concentration of lo6 cells per ml. In the case of the PEG-IO% DMSO fusogen, the cell fusion mixture was diluted immediately by slow addition of 50 ml of RPMI- 1640 medium. After 8 min incubation at 20°C and 2min incubation at 37°C [16], the cells were centrifuged and resuspended as mentioned for PEG fusogen. The fused cell suspensions were distributed (100 ~1 per well) in the 96 microwell plates previously seeded with feeder cells which were then incubated as described before. Half of the cell culture supernatant in each well was replaced by fresh medium 5 days later. After 20% of conIIuence was reached, aliquots of hybridoma supematants (100 ~1) were tested by RIA to detect anti-DHA or antiDHA-S antibodies. The selected antibody secreting hybridomas were cultured without using feeder cells. Two weeks after fusion, azaserine was totally removed from the added culture medium and hypoxanthine was progressively eliminated while the concentration of FCS was also gradually reduced to 10%. Cloning Cloning of selected hybridomas was performed by the limited dilution technique, using a suspension of

Monoclonal antibodies to DHA-S about 3 cells per ml of supplemented RPMI-1640 medium containing 10% FCS which was distributed (100 ~1 per well) in 96 microwell plates seeded with feeder cells. Half of the culture medium in each well was renewed every 5 days. The plates were checked in order to make sure that no more than one clone was formed in each well. Screening of supematants for anti-DHA and anti-DHA-S antibodies was performed as above. IdentiJication of immunoglobulin classes

The immunoglobulin classes of monoclonal antibodies from culture supematants were determined by the Ouchterlony double diffusion assay using a commercial anti-mouse immunoglobulin kit (Serotec, England). Obtention of ascites j?uidF

Adult BALB/c mice were injected i.p. with 0.5 ml of pristane (2,6,lO,lCtetramethylpentadecane_Sigma). After 10 days the mice were injected i.p. with lo5 to lo6 cells of cloned hybridomas suspended in 0.2ml of RPMI-1640 medium and the ascites fluids were drained l&15 days later. After centrifugation (5 min, 2500 g, 20°C) the supematants were kept frozen at -20°C. RESULTS

Characterization of DHA antigens

The number of DHA-7CM0 residues bound per molecule of carrier protein was estimated by U.V. adsorption to be * 20 mol steroid per mol BSA and

555

k: 90 mol steroid per mol BTG. The turbidity of KLH conjugates and the absence of chromophore on the DHA-15 ETC hapten did not allow U.V. measurements. Therefore, merely qualitative evidence that covalent coupling had occurred was provided by thin-layer chromatography on silica gel plates of aliquots of the coupling mixtures. Comparison with standards showed that in all cases most of the activated ester was coupled to the carrier protein at the bottom of the silica plate (solvent 1). A spot was also present at the Rr of the corresponding carboxylic acid (solvent 2), thus indicating a concomitant partial hydrolysis of the activated ester during the coupling reaction. However, this spot was much less intense than that observed in the absence of coupling reaction when no activation of the hapten was performed. Immunization of mice and characterization of serum antibodies

The BALB/c mice were divided into three groups. Group 1 (8 mice) received the DHA-7CMO-BSA antigen, group 2 (8 mice) received DHA-7CMO-BTG and group 3 (4 mice) was injected with a 1: 1 mixture of DHA-7CMO-KLH and DHA-ISETC-KLH. The evolution with time of serum antibody titres was followed by measuring the binding capacities for [‘HI-DHA at the beginning, as well as the binding activities for [3H]DHA-S after the eighth injection (Table 1). Repeated immunizations (7-8 booster injections) were found to increase substantially the relatively low antibody titres observed initially either with DHA7CMO-BSA or with DHA-7CMO-BTG antigens

Table I. The evolution with time of immunization of mouse serum antibody titres for DHA and DHA-S Injections

2nd

Anti-DHA/Anti-DHA-S antibody titres 5th 8th

9th

Groupl(DHA-7CMO-RSA) Mouse No. :

5

3 4 5 6 I 8

5 < 5’ 15 15 c5 15 Group 2 (DHA-7Ckl;;BTG) Injections 9 30 10 10 II
150 SO

25O/looO 200/800

250/1000

100

800/2000 -

1000/3ooo. -

2s(r 200’ 2ooc 25P

5th 8th 200 lOO/500 15b 50 200/750 100 200/750 100 1000/2000~ 50 2001750 + D~~I~ETC-ICLH) 200/750

2nd 25’ 10 25 25 fusion.

“Mice used for other experiments.

5th 50 25 1W

6th 50b 10 -

9th 150/1000 500/2ooo’ 250/15W 250/15@ 200/750b 7th (1 yr later) 1000/25b -

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which both gave similar responses. The use of a KLH carrier failed in our hands to give higher titres than the two BSA or BTG carriers. In all mice immunized with DHA-‘ICMO-BSA and DHA-7CMO-BTG, much higher antibody titres were always observed for DHA-S than for DHA. The highest titres for both DHA and DHA-S were found in the case of mouse No. 3 (group 1) after the ninth injection (DHAS, 1: 3000; DHA, 1: 1000). Only one mouse of group 3 (mouse No. 19), immunized with the DHA-KLH antigen mixture, survived up to the sixth injection and was tested one year later, after a seventh injection with the DHA-lSETC-KLH antigen alone. The serum of this mouse showed a much higher antibody titre for DHA (1: 1000) than for DHA-S (1:25) but the animal died before fusion could be performed. Fusion

Fusion was performed with the spleen cells of the six donor mice showing the highest serum antibody titres in groups 1 and 3 (Table l), either for DHA (mice No. $6, 8, 17 and 20) or both DHA and DHA-S (mouse No. 3). Mice of group 2 were kept for other experiments (not described in this paper). The spleen cells were fused with non-secreting SP2/0 myeloma cells [ 151 using PEG as fusogen. A modified fusion technique using PEG and 10% DMSO [16] was also employed in the case of mouse No. 3 (DHA-7CMO-BSA) starting from the same spleen cell suspension in order to compare the efficiency of the two methods. The technique using PEG alone gave 133 hybridomas out of 960 seeded wells with only one antibody-secreting hybridoma (clone 203) whereas the protocol using PEG + DMSO gave 205 hybridomas out of 960 wells among which 4 antibody-secreting hybridomas were isolated (clones No. 68,73, 115 and 161). In order to avoid possible interferences in the detection of secreted anti-DHA and anti-DHA-S antibodies, the FCS employed for cell culture was carefully selected for its very low inhibition of the Table 2. Characteristics

of monoclonal

binding activity of [HIDHA and [‘HIDHAS with 2 corresponding commercial rabbit antisera. The first fusion attempts performed up to the fifth injection with donor mice showing low serum antiDHA antibody titres (1: 2501, led to few slightly positive clones. The screening of hybridoma supernatants was first performed on 100~1 samples by measuring the binding capacity for 20,000 dpm of [‘HIDHA. No supernatant showed more than 50% binding, therefore no cloning was attempted. Fusions performed with spleen cells of mouse No. 3 which showed much higher serum antibody titres after the ninth injection (1: 1000 for DHA and 1: 3000 for DHA-S) allowed strongly positive clones to be obtained. A test using a mixture of [3H]DHA and [3H]DHA-S (20,000 dpm of each) was performed on the fused cells of mouse No. 3. In this last fusion the use of the two PEG and PEG 10% DMSO fusogens led respectively to 1 and 4 hybridomas which produced antibodies showing more than 50% binding of the mixed tracers and therefore were selected for cloning. Further titration of supematants of the corresponding hybridomas with [3H]DHA or [‘HIDHAS employed separately, indicated a much higher binding activity for DHA-S than for DHA. Cloning and ascites production

The 5 selected hybridomas from mouse No. 3 were cloned by the limited dilution method. The binding activities of supematants of all the clones were measured separately with [3H]DHA and [‘HIDHAS. In all cases the binding activity for DHA was much lower than for DHA-S (Table 2). For each hybridoma, only the 3 clones showing the highest binding activities for DHA and DHA-S were selected and cultured. All these clones were found to be stable with time. The binding characteristics of monoclonal antibodies with [‘HIDHAS were determined in ascites fluids (Table 2). Similar values were obtained with the corresponding culture supematants (not shown). The association constants ranged from 0.08 to 10 x 109M-‘. The lowest cross-reactions with androsterone (0.62%) and with androsterone sulphate

antibodies secreted by five clones from mouse No. 3 immunized with DHA-‘ICMO-BSA

Clone no. Affinity constant’ (IO9M-‘) Immunogtobulin classb

68 2.5 Wl

Dehydroepiandrosterone-sulphate Dehydroepiandrostcrone Dehydroepiandrosterone-acetate Androsterone sulphate Androsterone Epi-androsterone Aetio-cholanolone A’-androatenediol Cortisol A’-pregnenolone

100 9.3 16.1 41.7 28.6 13.3 3.5 0.01 0.01 1.3

‘Measured on ascites fluids. bMeasured on culture supematants. CAll steroids were purchased from Steraloids.

73 0.12 kM.

115 5.3 WZbK Cm!la reaetioa (Y.)‘” 100 100 10.1 12.9 14.6 0.9 164 4.1 118 1.9 60 4.8 50 0.7 0.09 0.02 1.2 co.01 9.6 co.01

161 10.2 f&K+ 100 18.5 2.1 0.83 0.62 4.5 0.7 co.01
203 0.08 W, 100 4.3 8.2 11.9 10.6 3.2 8.8 0.09 1.2 3.4

Monoclonal antibodies to DHA-S Table 3. Comparative determination of DHA-S concentrationsin 3 plasma pools, using monoclonal antibody 161 and a commercial antiserum

No. of tests Pool 1 Pool 2 Pool 3

5 5 5

Monoclonal antibody 161 Mean (lmol/l) SD 1.31 3.92 9.04

0.12 0.08 0.26

Anti-DHA-S antiserum’ Mean SD (rmolll) 1.97 4.40 9.73

0.08 0.25 0.39

‘Antiserum from a bioMCrieux DHA-S Kit.

(0.83%) were observed for the monoclonal antibody No. 161 which also had the highest affinity for DHA-S. The 3 clones selected from each of the 5 initial hybridomas always belonged to the same immunoglobulin class (Table 2) and showed similar binding characteristics, thus suggesting that they derive from the same clone. Evaluation of the performance of monoclonal antibody No. 161 for the direct radioimmunoassay of DHA -S

The ascites fluid containing monoclonal antibody no. 161 was employed with a rH]DHA-S tracer for the direct measurement of DHA-S concentrations in diluted samples of three human plasma pools having low, medium and high DHA-S levels. The concentrations of DHA-S were found to be close to values obtained with a commercial rabbit antiserum for the same plasma samples, thus confirming that monoclonal antibody no. 161 has enough specificity for direct assay of DHA-S in plasma (Table 3). DISCUSSION

The two DHA-7CM0 and DHA-15ETC haptens were chosen in order to obtain the best possible immunological recognition of the two characteristic A5 3-OH and 17-ketone groups. The DHA-7CM0 derivative had indeed been found to produce specific rabbit antibodies [ 1] despite the conjugation of its A5 double-bound to the 7-oxime chain which might have altered the antigenic character of the A5 3-OH group. This cannot occur with the DHA-15ETC hapten. This last structure has, however, the potential disadvantage of the proximity between the 15s chain and the 17position which might decrease the specific recognition of the 1Fketone group. Immunization of BALB/c mice performed in this study with DHA-7CMO-BSA or DHA-7CMO-BTG antigens always produced serum antibodies that were more specific for DHA-S than for DHA. This was also true for the 5 monoclonal antibodies obtained from one of the donor mice. This observation corroborates the previously described cross-reactivity with DHA-S of monoclonal antibodies obtained from mice immunized with a non-sulphated DHA-7CMOBSA antigen [1,2]. However, the only surviving mouse, immunized with a mixture of DHA-7CM0 and DHA-ISETC coupled to KLH, produced serum

551

antibodies more specific for DHA than for DHA-S. Since specific rabbit anti-DHA antibodies have been obtained from similar DHA-7CMO-BSA antigens [I], it seems very improbable that the enhanced cross-reactivity with DHA-S of mouse antibodies could result from a structural alteration of the antigen before injection. This much higher specificity for DHA-S than for DHA differs from the crossreactivity between 3’-OH and 3’ sulphate esters which occurs when the carrier is linked to the steroid hapten through either the C-3 [3-S] or the C-4 positions [6] as a result of low immunological recognition of the C-3 site. Recently, the transformation of DHA into DHA-S has been reported in mouse either after oral ingestion or after in vitro incubation with liver cells when very low doses of DHA had been used. In the same experiment, acetylation of the 3-OH group was also reported with higher doses of DHA [17]. In this study, all the monoclonal, antibodies which were selected, showed much lower cross-reactions with DHA acetate than with DHA-S. Therefore, one can speculate whether the above unexpected DHA-S cross-reactivity might result from a rapid in vitro sulphatation of the injected hapten in some mice but no direct evidence could be found in the literature concerning in vivo sulphatation of DHA haptens. The use of the more immunogenic BTG or KLH carrier proteins [7,8] instead of BSA, did not improve the immune response to DHA antigen. Long-term immunization with high doses of antigens (100 pg) and 4-week intervals between booster injections led to relatively good antibody responses to DHA haptens in mice. Previous studies [18] have shown that the immunization protocol may influence the monoclonal antibody characteristics. The substitution of the classical fusion technique using PEG by a modified protocol employing PEG containing 10% DMSO [16] increased the number of hybridomas obtained from the same cell suspension as well as the number of antibody-secreting clones. As only one spleen was available, no further experiments could be undertaken in order to establish whether this improvement was a consequence of the once controversed potentiating effect of DMSO on PEG fusing action [19]. The hypothesis of a decrease of the surface potential and of charge neutralization of cell membrane by both DMSO and PEG has been put forward as an explanation for this potentiating effect [20]. Fusion experiments with PEG alone starting from mouse with low serum anti-DHA antibody titres failed to produce clones with sufficient binding activities. However, a recent report (211 has shown that monoclonal anti-aldosterone antibodies can be produced from mice with low antibody titres. Fusion performed with spleen cells of mouse No. 3, for which high antibody response could be. reached by long-term immunization, allowed only one clone to be obtained with high binding activities for DHA and DHA-S, using the PEG fusogen. The modified

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PEG-DMSO method proved to be much more efficient since it led to 4 highly positive clones from the same cell mixture. After cloning by the limited dilution method, it was not possible to detect any monoclonal antibody more specific for DHA than for DHA-S. It has been reported that in similar conditions both anti-DHA and anti-DHA-S monoclonal antibodies could be obtained with the same DHA-7CMO-BSA antigen 111.In the present study, mouse No. 19, immunized with a mixture of DHA-7CMO-KLH and DHAlSETC-KLH, was the only animal showing serum antibodies more specific for DHA than for DHA-S but no monoclonal antibody could be obtained from this mouse which died before fusion. One of these five anti-DHA-S monoclonal antibodies (No. 161) was specific enough to allow the direct measurement of DHA-S in three reference plasma pools. Further studies are in progress in order to develop a reliable direct assay for DHA-S using this monoclonal antibody and an appropriate [‘251]DHA-S tracer. Acknowledgements-The authors wish to acknowledge the skilful assistance of Mrs Aline Gagne. They also kindly thank Dr M. J. Carew for reviewing the final English manuscript version.

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