Human alfa-fetoprotein: isolation and production of monoclonal antibodies

Biochimie (1990) 72, 369-374 (~ Soci6t~fran~aise de biochimie et biologie mol6culaire/Elsevier, Paris

369

Human alfa-fetoprotein: isolation and production of monoclonal antibodies A Karmali*, C Novo LNETI-DTIQ Bioquimica, Estrada das Palmeiras, Queluz de Baixo 2745, Portugal

(Received 12 February 1990; accepted 28 May 1990)

Summary - Alfa-fetoprotein from human cord serum was purifiedin a single step by hydrophobicinteraction chromatographyon Phenyl Sepha-

rose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6HI0, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purificationfactor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specificfor human alfa-fetoprotem as determined by Western blotting. The affinity constants (K) of these Mab ranged from 106 to 109 l/mol. alfa.fetoprotein / human cord serum / one-step purification/ Phenyl Seplutrose CI.-4B / hybridomatechnology / monoclonal antibodies / hydrophobic interaction chromatography / Protein A-Sepharose CL-4B

Introduction

Alfa-fetoprotein (AFP) is a glycoprotein which plays an important role in fetal development and in cancer [1]. T h e specific determination of A F P in maternal ~ t i m ,,~ w~u as in amniotic fluid during pregnancy is of great interest in the prenatal diagnosis of spina bifida and congenital nephrosis [2]. Moreover, the synthesis of human A F P by germ cell tumors and primary liver cancer is widely used to monitor cancer patients [3]. T h e use of i m p r o v e d immunological assays for measurement of A F P in biological fluids with regard to specificity and sensitivity is therefore of great clinical importance [4]. T h e hybridoma technology described by Milstein and Kohler [5] produces monoelonal antibodies of high specificity and affinity which have been used by others to measure the levels of A F T in biological fluids [ 6 - 8 ] . F u r t h e r m o r e , the fact that A F P exhibits microheterogeneity in biological fluids could be exploited diagnostically which would require the use of specific monoclonal antibodies directed against the different ....

II

polymeric forms of A F P [9, 10]. With regard to this matter, so far, such monoclonai antibodies have not been obtained which could distinguish the different forms of this protein. On the other hand, the hybridoma technology llOllllany l~;ttuH¢;~ the use ~,L a m/~ny purified preparation of antigen for the screening assay of Mabs in culture supernatant [11]. The published purification procedures for human A F P involve the use of several chromatographic techniques such as immunoaffinity, ion-exchange and gel filtration [12-14]. These procedures present some disadvantages such as elution of denatured A F P from immunoaffinity columns, low recoveries and time consuming isolation processes [12]. T h e present work is concerned with a novel one-step isolation scheme of A F P from human cord serum by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B column. This purified preparation was used to raise a panel of Mabs against this protein which were purified on Protein A-Sepharose CL-4B column and some of their properties were studied. .

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Abbreviations: AFT, alfa-fetoprotein; Mab, monoclonal antibody; IRMA, immunoradiometric assay; PBS, phosphate buffered saline;

IEF, isoelectric focusing; A, absorbance; ELISA, enzyme linked immunosoroent assay

370

A Karmali, C Novo

Materials and Methods

Chemicals RPMI 1640, Myoclonc plus (fetal calf serum) and gentamycin were purchased from Gibco Lab, New York. Complete freund adjuvant, PEG 1300-1600, rabbit anti-mouse IgG-alkaline phosphatase conjugate, p-nitrophenyi phosphate, 5-bromo 4-chloro 3-indolyl phosphate, solid phase anti-mouse immunoglobulin antibody, HAT and HT were obtained from Sigma Chemical company (USA). I~l-labelled rabbit anti-mouse light chain was supplied by Amersham, UK. Protein A-Sephmose CL-4B, Sephacryl S-200, ampholine pH range 3.5-10 and Phenyl Sepharose CL-4B were purchased from Pharmacia International (Sweden). A sample of myeloma cell line (Sp2/0Agl4) was from ATCC, USA. A kit based on the ELISA sandwich method for determination of human alfa-fetoprotein was obtained from Abbott, (USA). Membranes (P10) for ultrafiltration units were supplied by Amicon (Ireland), All disposable plasticware were obtained from Costar and all other reagents used were of analytical grade. Animals Female mice from inbred strain Balb/c were obtained from Instituto Gulbenkian de Ci~ncia, Oeiras (Portugal). Methods Protein was determined by the coomassie blue dye binding method [15]. SDS-PAGE and native PAGE were carried out as mentioned previously [16, 17] and stained for protein with coomassie blue. The specific detection of AFP in native PAGE was carried out by staining one haft of the gel for protein with coomassie blue and the other half was used to extract AFP. The unstained gel containing the AFP band was homogenized with PBS pH 7.2 (2 vol), stirred overnight in an end over end mixer at 4°C, centrifuged at 10000 g for 5 rain and the supernatant was assayed for AFP using the AFP kit which is based on the ELISA sandwich method. Immunoglobulin classes and subclasses were determined by Ouchleriony double diffusion analysis using several classes specific antisera such as rabbit anti-mouse immunoglobulin heavy chain (-yl, V2, a and ~) [181. The specificityof Mabs was performed by Western blotting [19]. Briefly,samples of human cord serum containing A F P were anaiyscd by native P A G E (7.5% separating gel) and protein bands were transferred electrophorctically to nitrocellulose paper. Gels wcrc stained with coomassie blue and nitrocellulose blots with amino black. The nitrocelluloseblots were incubated successively with the monoclonal antibody, rabbit anti-mouse IgG alkaline phosphatasc as the 2nd antibody and 5-bromo 4-chloro 3-indolylphosphate as the subslrate according to the conventional ELISA method. The concentration of immunoglobulin (ie IgGl) in monoclonal antibody samples was determined by an immunoradiomctric assay (IRMA) using solidphase anti-mouse immunoglobulin antibody [6]. Briefly,diluted samples and standards of immunoglobulin standards of immunglobulin were incubatcd successively with J~I-labelled rabbit anti-mouse lightchain and solid phase anti-mouse immunoglobulin antibody. After repeated washing, the finalsediment was counted in a gamma counter. The affinityconstant (K) of Mabs was determined using the simple antibody dilution analysis method [20]. Briefly, antibody dilution curves for 5 Mabs were carried out in the presence of a constant concentration of human A F P (I /~g/well) in 96-well microtitre plates and the percentage of bound antigen was plotted against the decreasing antibody concentration for each Mab. The concentration of immunoglobulin was determined at each antibody dilution by IRMA and the affinity constant for these Mabs were simply read off the plot at haft-maximal antigen binding.

Detection of antibody secretion in cult~,re supematants as well as in column eluates was carried out by ELISA using pure AFP (1 Itg/ well) as the antigen, rabbit anti-r~,ouse IgG-alkaline phosphatase conjugate as the 2nd antibody and p-nitrophcnyl phosphate as the substrate [18]. One antibody unit is defined as the amount of Mab required to give a change in absorbance of 1.0 per 30 min at 405 nm due to the action of rabbit anti-mouse lgG-alkaline phosphatase conjugate on p-nitrophenyl phosphate under the standard conditions of ELISA. The concentration of human AFP in samples and column eluates was determined using a AFP kit based on the ELISA sandwich method. Purification of human AFP Human cord serum (supplied by Ginecology Service of (Santa Maria Hospital, Lisbon) (1 ml) was applied to a column (1 x 10 cm) packed with Phenyl Sepharose CL-4B which was previously equilibrated with 10 mM phosphate buffer pH 6.5 containing I M ammonium sulphate. The column was washed with the same buffer system and the concentration of ammonium sulphate was reduced to 0.1 M in the buffer system. The column was washed with this buffer system until A280 was less than 0.03. Human AFP was eluted from the column with 10 mM phosphate buffer pH 6.5 containing 20% (v / v) ethylene glycol and fractions (4 ml) (29-39) containing AFP were pooled and concentrated by pressure dialysis with Pl0 membrane at 4oC. Production of Mab against human AFP Female Balb/c mice (4-wks-old) were immunised on the d fl with purified AFP (5/zg) in complete Freund adjuvant by intraperitoneal injection. Subsequently, immunisations were carried out on the 15th and 30th d with the same amount of antigen in incomplete Freund adjuvant by intraperitoneal injections. Three d after the last immunisation on the 45th d by intravenous route, mice were bled and the titer was determined by ELISA using rabbit anti-mouse IgG-alkaline phosphatase conjugate as the 2nd antibody and p-nitrophenyl phosphateoas the substrate [18]. Subsequently, the spleen cells (3 107) were rased with Sp2/0 Ag 14 (3 107) in the presence of PEG [11]. The selection of hybrids was carried out in HAT medium and positive hybrids were cloned by the limiting dilution method using thymocytes as feeder cells [11]. Monoclunal antibodies (Mabs) were produced from 5 clones (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) either in culture in vitro (RPMI 1640 + 10% (v/v) fetal calf serum) at 37oC and 5% CO 2 or in vivo by ascites fluid [11]. Purification o f Mabs from all five clones on Protein A-Sepharose CL-4B Ascites fluid (1 ml) diluted 1 / 1 with 1.5 M glycine containing 3 M NaCI pH 8.9 was applied to a column (1 x 2 cm) packed with Protein A-Sepharose CL-4B previously equilibrated with 1.5 M glycine containing 3 M NaCi pH 8.9. The column was washed with the same buffer system until A280 was less than 0.03. The Mab was eluted from the column with 0.1 M sodium citrate buffer pH 6.0 and fractions (4 ml) (20-32) containing antibody activity, as determined by the ELISA method at 405 nm, were pooled and concentrated by presure dialysis using a P10 membrane at 4oC.

Results and Discussion A number of isolation schemes for human AFP have b e e n r e p o r t e d in t h e l i t e r a t u r e w h i c h a r e b a s e d o n i m m u n o a f f i n i t y , gel f i l t r a t i o n a n d i o n e x c h a n g e c h r o matographic techniques [12-14]. These purification s c h e m e s p r e s e n t s o m e d i s a d v a n t a g e s a s f a r as i m m u n o -

371

Monoclonal antibodies against human alfa-fetoprotein

chemical studies are concerned since they involve more than one isolation step and in some cases the purified preparation of AFP is partially denatured [12]. The present work reports a novel 1-step purification scheme for AFP from human cord serum by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B column (fig 1). Human AFP was purified with a final recovery of 90% and a purification factor of about 900 (table I). The purified preparation was apparently homogeneous on S D S - P A G E and native PAGE running with a M~ of 72000 (fig 2). The chromatographic behaviour of human AFP on Phenyl Sepharose CLo4B column strongly suggests that this protein is more hydrophobic than serum albumin since the former was only eluted with ethylene glycol whereas serum albumin was easily removed from the column with 0.1 M ammonium sulphate in the buffer system (fig 1). In our laboratory, hydrophobic interaction chromatography has been sucessfully used in the purification of catalase from leaves of Zanthedeschia aethiopica in a single step [21]. O.GO

Table I. One step purification of human a-fetoprotein from

cord serum by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. Purification Total steps protein (mg)

Total AFP (mg)

Specific content of AFP

Recovery Purification (%) factor

(x w-~) (mg / mg protein)

Human cord serum Column eluate

118

0.100

0.11

0.091

0.847

100

827.2

1

91.0

A

976.6

B

Elution With 20%{ Vl V )Ethylene glycol

0.50

A

0.40

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0.20

0.10

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Fig 2. Electrophoretic analysis of purified human AFP. A: Native PAGE of purified sample (10 btg) using a 7.5% 2

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Fig 1. Hydrophobic interaction chromatography of human

cord serum containing AFP on a Phenyl Sepharose CL-4B column. The column was washed with a decreasing gradient of ammonium sulphate (1.0-0.1 M) in 10 mM phosphate buffer pH 6.5. The AFP bound to the column was eluted with 20% (v/v) ethylene glycol in 10 mM phosphate buffer pH 6.5 and fractions (29-39) were pooled and concentrated for electrophoretic analysis.

separating gel. B: SDS-PAGE of purified sample (10/zg) using a 10% separating gel. Margins on the right represent molecular weight markers. Since human AFP and serum albumin run closely in native PAGE, the full identity of the single protein band observed on this gel (fig 2A) was investigated by specific detection of AFP in the native gel using the ELISA sandwich method which confirmed that this homogeneous protein band corresponds to human AFP (result not shown). As far as monoclonal antibody production is concerned, 50% of the total culture wells exhibited hybrid growth and 7% of these (ie 8 culture wells)

372

A Karmali, C Novo

contained anti-AFP activity. In previous reports, 5 - 1 3 culture wells secreting a monoclonal antibody against human AFP were obtained from a single fusion of spleen cells with myeloma cells [4, 8, 22]. In the present work 2 cultures were selected at random for cloning purposes by limiting dilution method and 5 clones (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were used for production, purification and characterisation of Mab. The Mab, produced as ascites fluid from clone 3F6H10 was purified by affinity chromatography on Protein A-Sepharose CL-4B (fig 3) which resulted in a final relative recovery of 80% and a purification factor of about 13 (table II). The purified preparations were apparently homogeneous on S D S - P A G E and native PAGE running with M, values of 25 700 (ie light chain), 57500 (ie heavy chain) and 155000 (ie whole native molecule) respectively (fig 4). The M, value obtained for the purified Mab on native PAGE was also confirmed by gel filtration chromatography on Sephacryl S-200 which was eluted with a Mr value of 150000 (figure not shown). However, the purified preparation of the Mab exhibited a weak contaminating protein band on S D S - P A G E which appeared to be serum albumin. The elution profile of Mabs on Protein A-Sepharose CL-4B column suggests that they are of IgG1 subclass since they w e ~ eluted from the column at pH 6.0 (fig 3). This result was confirmed by Ouchterlony double diffusion analysis of purified Mabs in the presence of rabbit anti-mouse immunoglobulin heavy chains (fig 5B). In previous reports on Mabs against this protein, researchers have reported the synthesis of IgG1 as well as IgA from the hybridoma cultures [4, 8, 22]. With regard to the monoclonality of the antibody, it was analysed by IEF which showed a single family of . . . . . . . . . . . t" values of 6.5-6.9 (fig 5A). Similar band patterns were also obtained with Mabs against human growth hormone as well as with myeloma proteins when they were analysed by IEF [23-25]. This pattern may be due to microheterogeneity in antibody populations which results in charge alteration [23-25]. It must be mentioned that all Mabs from the 5 hybridoma clones were purified and characterised as described in the experimental section although figures have not been presented for all of them. The specificity of Mabs was determined by Western blotting using human cord serum as the source of AFP which revealed a single positive reaction band with AFP on nitrocellulose paper (figure not shown) suggest-ing that it does not cross-react with other serum proteins. The affinity constants (K) for these Mabs against human AFP ranged from 106 to 109 l / m o l by the simple antibody dilution analysis method as shown in table III. In previous reports on Mabs against this protein, researchers also reported K values in the range of 106 and 109 l / m o l [20, 22].

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Fig 3. Affinity chromatography of ascites fluid from hybri-

doma clone 3F6H10 containing Mab against human AFP on a Protein A-Sepharose CL-4B column. The Mab bound to the column was eluted with 0.1 M sodium citrate buffer pH 6.0 and fractions (20-32) were pooled, concentrated and used for further characterization. Since AFP is present in different polymeric forms in human sera [9, 10] which has also been confirmed in the present work (data not shown), it would be of great interest to investigate the immunochemical specificity of these Mabs towards such polymeric forms. Hence, further work is in progress concerning the use of these monoclonal antibodies in the detection and assay of different polymeric forms of AFP in human serum by enzyme immunoassay techniques.

Monoclonal antibodies against human alfa-fetoprotein Table II, Purification of Mabs against human a-fetoprotein by affinity chromatography on Protein A-Sepharose CL-4B. Purification Total steps protein (mg)

Total activity (A units)

Specific content of lgG1 (mg / m g protein)

A

373 B

Recovery Purification (%) factor

6.9 6.5

Ascites fluid

5.85

24.9

Column eluate

1.05

39130

0.068

100

1

32120

0.904

82

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B

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Fig 5. Characterisation of purified Mab from hybridoma clone 3F6H10. A: Isoelectric focusing on polyacrylamide gel (pH range 3 . 5 - 1 0 ) of purified Mab (5 p.g). B: pI markers are represented on the right margins. Ouchtedony double diffusion analysis of purified Mab (100/~g) on agar plates in the presence of rabbit anti-mouse immunoglobulin heavy chains. Purified Mabs are as follows: 1 and 2: rabbit antimouse immunoglobulin y l heavy chain; 3: rabbit antimouse immunoglobulin y2 heavy chain; 4: rabbit antimouse immunoglobulin t~ heavy chain; 5: rabbit anti-mouse immunoglobulin p. heavy chain.

N Itd~ m 57 KDe-43 KDa--

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Acknowledgments We would like to thank P Bajanca and M Marques Pinto for technical ass~.tance.

Fig 4. Electrophoretic analysis of purified Mab from hybridoma clone 3F6H10. A: Native P A G E of purified sample (10 p.g) and ascites fluid (100/~g) using a 7.5% separating gel. B: S D S - P A G E of purified sample (10/zg) and ascites fluid (100 p.g) using a 10% separating gel. Margins represent molecular weight markers.

Table I l l Affinity constants calculated for monoclonal antibodies against human AFP. Mabs from these hybridoma clones were produced as ascites fluid and their affinity constant (K) was calculated using the simple antibody dilution analysis method in the presence of constant concentration of human AFP as described in Materials and Methods. A F P hybridoma clone number

K (1 / mol)

3F6H10 3F6H4 3F6H1 3F6G5 3F6G10

410 9 3 l0 s 2 l0 s 1 107 610 6

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