Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

MOLECULAR BIOTECHNOLOGY Volume 17, 2001

Monoclonal Antibodies Against the Free α-Subunit of hCG 119

119

Molecular Biotechnology 2001 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2001/17:2/119–128/$12.50

*Author to whom all correspondence and reprint requests should be addressed: 1Laboratório de Engenharia Bioquímica do Departamento deEngenharia Química do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1900 Lisboa – Portugal. E-mail:[email protected]. 2Serviço de Bioquímica II do Departamento de Biotecnologia e Química Fina do INETI. Estrada do Paço do Lumiar,1699 Lisboa Codex.

RESEARCH

AbstractMonoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by

hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culturewells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secretinghybrids. A positive hybrid cell line secreting antibodies against the free α-subunit of hCG was cloned twiceby limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-αhCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free α-subunit ofhCG was selected for purification and characterization purposes. This hybridoma cell line secreted mono-clonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A SepharoseCL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of thisMab revealed that it recognized specifically an epitope on the free α-subunits of hCG, FSH, LH, and TSH asdetermined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with otherpituitary hormones either as free subunits or intact molecules as follows: αhCG 100%; intact hCG 1.8%;βhCG 0.14%; αFSH 24.5%; intact FSH 0.8%; βFSH 0.09%; αLH 20.5%; intact LH 0.9%; βLH 0.08%;αTSH 50.5%; intact TSH 3.7%; βTSH 0.07%;

The affinity constant (K) of this Mab with respect to free α-subunit of hCG was found to be 1.5 × 107

I/mol as determined by the simple antibody dilution analysis method.Index Entries: Chorionic gonadotropin hormone (hCG); free α-subunit of hCG; free α-subunits of pitu-

itary hormones; hybridoma technology; monoclonal antibodies; TSH; FSH; LH.

1. IntroductionHuman chorionic gonadotropin (hCG) is a gly-

coprotein hormone, which is synthesized andsecreted by the trophoblasts of the placenta (1).This hormone plays an important role in the pro-duction of progesterone by the corpus luteum,which is required for the maintenance of earlypregnancy in humans. This protein consists of twodissimilar and noncovalently bonded subunits αand β, which contain 92 and 145 amino acid resi-dues, respectively (2).

From the structural point of view, the α-sub-unit of hCG is identical to other pituitary hor-

mones (i.e., follicle-stimulating hormone [FSH],luteinizing hormone [LH], and thyroid-stimulat-ing hormone [TSH]) (3). On the other hand, theβ-subunits vary in size from 114 amino acid resi-dues in LH to 145 residues in hCG, which con-tains six disulfide bridges (3). There is a highdegree of homology in the amino acid sequenceof hCG and other hormones in the first 114 aminoacid residues (LH 85%; FSH 36%; TSH 46%) (3).

Human chorionic gonadotropin hormone is apotential marker in the diagnosis of pregnancyand a variety of diseases such as ectopic preg-nancy, choriocarcinoma, and testicular cancer

Purification and Characterization of Monoclonal Antibodies Againstthe Free α-Subunit of Human Chorionic Gonadotrophin

Carlos Novo,2 Ana Domingos,2 and Amin Karmali1,*


120 Novo, Domingos, and Karmali

(4). Hence, the specific determination of hCG inurine and other biological fluids is of great inter-est from the diagnostic point of view (5). However,in certain tumors, some workers have observedhigh levels of free α- and β-subunits of hCG incirculation (6). The use of improved immuno-chemical assays for measurement of either free α-or β-subunit in the presence of relatively high con-centrations of other pituitary hormones in biologi-cal fluids as regards to specificity and sensitivityis therefore of great clinical importance in cancerdiagnosis (6).

The hybridoma technology described byMilstein and Kohler (7) has been used in previousreports to obtain monoclonal antibodies againsthCG, which were highly specific tor determina-tion of this glycoprotein in biological fluids (8,9).As regards to specific assays for free α- andβ-subunits, a number of monoclonal antibodies(Mabs) have been isolated that detect either ofthese free subunits in the presence of relativelyhigh concentration of hCG (10–12). However, inthe case of free α-subunit, the Mabs isolatedcrossreact with other intact pituitary glycoproteinhormones (i.e., TSH and LH) as well as with theircorresponding free α-subunit (11).

On the other hand, monoclonal antibodies playan important role in basic research because theycan be used as powerful tools to study the topol-ogy of protein molecules (12). Such an approachhas been sucessfully applied for detection of localconformation in several proteins (12). The presentwork is concerned with production of monoclonalantibodies against the free α-subunit of hCG byhybridoma technology. Subsequently, the Mabwas purified by affinity chromatography on Pro-tein A-Sepharose CL4B column and some of itsproperties were studied.

2. Materials and Methods2.1. Chemicals

RPMI 1640, Myoclone plus (fetal calf serum),and gentamycin were purchased from Gibco Lab.Grand Island, N.Y. Complete Freund adjuvant,PEG 1300-1600, rabbit anti-mouse lgG–alkalinephosphatase conjugate, p-nitrophenyl phosphate,solid phase anti-mouse innunoglobulin antibody,

peroxidase, hypoxantine aminoptorin thymidine(HAT), and (HT) were obtained from SigmaChemical Company. I125-labeled rabbit anti-mouselight chain was supplied by Amersham, U.K. Pro-tein A Sepharose CL-4B, Sephacryl S-200, andampholine pH range 5–8 were purchased fromPharmacia International (Sweden). A sample ofmyeloma cell line (Sp2/0Ag14) was from ATCC.Membranes (P10) for ultrafiltration units weresupplied by Amicon, Ireland.

Free subunits and intact molecules of pituitaryhormones (hCG, TSH, FSH, and LH) were sup-plied by U.C.B. Bioproducts, Belgium. All dis-posable plasticware were obtained from Costarand all other reagents used were analytical grade.

2.2. AnimalsFemale mice from inbred strain Balb/c were

obtained from Instituto Gulbenkian de Ciência,Oeiras, Portugal.

2.3. Methods2.3.1. Protein Assay

Protein concentration was determined by theCoomassie blue dye binding method (13).

2.3.2. Electrophoretic AnalysisSDS-PAGE and native PAGE were carried out

as mentioned previously (14,15) and stained forprotein with silver nitrate (16).

2.3.3. Immunoglobulin Class and SubclassImmunoglobulin class and subclass were deter-

mined by Ouchterlony double diffusion analysisusing several class specific antisera such as rabbitanti-mouse immunoglobulin heavy chain (γ1, γ2,α, and µ) (17).

2.3.4. Concentration of ImmunoglobulinsThe concentration of immunoglobulin (i.e.,

IgG1) in monoclonal antibody samples was deter-mined by an immunoradiometric assay (IRMA)using solid-phase anti-mouse immunoglobulinantibody (18). Briefly, diluted samples and stan-dards of immunoglobulin were incubated succes-sively with I125-labeled rabbit anti-mouse lightchain and solid-phase anti-mouse immunoglobu-lin antibody. After repeated washings, the finalsediment was counted in a gamma counter.



2.3.5. Determination of Affinity ConstantThe affinity constant (K) of Mab was deter-

mined using the simple antibody dilution analysismethod (19). Briefly, an antibody dilution curvefor this Mab was carried out in the presence of aconstant concentration of free α-subunit of hCG(0.2 µg/well) in 96-well microtiter plates and thepercentage of bound antigen was plotted againstthe decreasing antibody concentration for thisMab. The concentration of immunoglobulin wasdetermined at each antibody dilution by IRMAand the affinity constant for this Mab was simplyread off the plot at half-maximal antigen binding.

2.3.6. Enzyme-Linked ImmunosorbentAssay (ELISA)

Detection of antibody secretion in culture super-natants as well as in column eluates was carriedout by ELISA using hCG, αhCG, and βhCG asthe antigens (16 ng/well), rabbit anti-mouse IgG–alkaline phosphatase conjugate as the secondantibody and p-nitrophenyl phosphate as the sub-strate (17). One antibody unit is defined as theamount of Mab required to give a change inabsorbance of 1.0 per 30 min at 405 nm due to theaction of rabbit anti-mouse IgG–alkaline phos-phatase conjugate on p-nitrophenyl phosphateunder the standard conditions of ELISA.

2.3.7. Production of Mabs Against hCGFemale Balb/c mice (4-wk-old) were immu-

nized on d 0 with native hCG (50 µg) in completeFreund adjuvant by subcutaneous injection. Sub-sequently, four immunizations were carried out atone month intervals with the same amount of anti-gen in phosphate-buffered saline (PBS) by intra-peritoneal injections. Three days after the lastimmunization, mice were bled and the titer wasdetermined by ELISA using rabbit anti-mouseIgG–alkaline phosphatase conjugate as the secondantibody and p-nitrophenyl phosphate as the sub-strate (17). Subsequently, the spleen cells (8 × 107)were fused with Sp2/0 Ag 14 (2 × 107) in the pres-ence of PEG (20). The selection of hybrids wascarried out in HAT medium, and positive hybridswere cloned by the limiting dilution method usingthymocytes as feeder cells (20). Mabs were pro-duced from seven clones (2A9, 3G6, 1E2, 2B10,

2B3, 2A4, and 1C4) either in culture in vitro(RPMI 1640 + 10% [v/v] fetal calf serum) at 37ºCand 5% CO2 or in vivo as ascites fluid (20).

2.3.8. Purification of Mab from HybridomaClone 1C4 on Protein A-Sepharose CL-4B

Ascites fluid (1 mL) diluted 1:2 with 1.5 M gly-cine buffer containing 3 M NaCl pH 8.9 wasapplied to a column (1 × 2 cm) packed with Pro-tein A-Sepharose CL-4B previously equilibratedwith 1.5 M glycine buffer containing 3 M NaClpH 8.9. The column was washed with the samebuffer system until A280 was less than 0.03. TheMab was eluted from the column with 0.1 Msodium citrate buffer pH 6.0 and fractions (4 mL)(13–23) containing antibody activity as deter-mined by ELISA method at 405 nm were pooledand concentrated by pressure dialysis using a P10membrane at 4°C.

2.3.9. Isoelectric FocusingIsoelectric focusing (IEF) of purified samples

of Mabs was carried out in polyacrylamide gelsusing an ampholine pH range of 5–8 (21) and theprotein bands were stained with silver nitrate.

2.3.10. Determination of Crossreactivity ofMabs with Other Pituitary Hormones

The binding of Mab to pituitary hormones wasinvestigated by a two-step competitive enzymeimmunoassay using a Mab-coated filter-paperdisc and peroxidase–αhCG conjugate as thelabeled hormone (1). The bound enzyme–hormoneconjugate was detected by using 1,2-phenylene-diamine and hydrogen peroxide as substrates andthe formation of product was followed at 492 nm(1). The crossreactivity of pituitary hormones inthis two-step competitive enzyme immunoassaywas determined by displacement of labeled α-hCGby crossreacting hormone. The percentage cross-reactivity was calculated from the amount of hor-mone causing half displacement of the labeledhormone on a weight-ratio basis (11). The follow-ing glycoprotein hormones were used as the sourceof antigen: hCG, FSH, LH, TSH, αhCG, αFSH,αLH, αTSH, βhCG, βFSH, βLH, and βTSH.

Briefly, this assay was carried out with filter-paper discs to which Mabs against hCG were



chemically attached. The concentration of pitu-itary hormones was varied from 0 to 10,000 µg/Lin this assay system, which was incubated withthe Mab-coated disc as well as peroxidase–αhCGconjugate. The bound peroxidase–αhCG wasdetected by incubation with 1,2-phenylenedi-amine and hydrogen peroxide as substrates at37°C for 15 min. The reaction was stopped by theaddition of H2SO4 and the color developed wasread at 492 nm (1).

3. Results and Discussion3.1. Production and Purification of Mabs

Monoclonal antibodies have been raised againsthCG in this work using the hybridoma technologydescribed by Kohler and Milstein (7). Hybrid growthwas observed in 65% of the total culture wells and8% of the total wells (i.e., 13 cultures) containedanti-hCG activity. In previous reports, 3 to 37 cul-ture wells secreting antibodies against hCG (i.e.,either to free subunits or to the intact molecule)were obtained from a single fusion of spleen cellswith myeloma cells (22,23). In the present work,12 positive hybrid cultures secreting antibodiesrecognized both free α and β subunits of hCG,whereas only one hybrid cell line (B6) reactedspecifically to the free α-subunit of this hormone.

Hence, the positive hybrid cell line B6 secret-ing antibodies anti-αhCG was selected for clon-ing purposes by limiting dilution method becauseit did not crossreact with the free β-subunit ofhCG. The cloning of hybridoma B6 resulted in 84clones that secreted Mabs against the free α-sub-unit of hCG. One of these hybridoma clones (1C4)secreting monoclonal antibodies against the freeα-subunit of hCG was selected for purificationand characterization purposes.

The Mab produced as ascites fluid from clone1C4 was purified by affinity chromatography onProtein A-Sepharose CL-4B (Fig. 1) that resultedin a final relative recovery of antibody activity of75% and a purification factor of 12 (Table 1).Other research workers have purified Mabs againsthCG using two steps that involve DEAE-Affi-gelBlue and affinity chromatography with a finalrecovery of antibody activity of 15% and a purifi-cation factor of about 20 (10,23).

Fig. 1. Affinity chromatography of ascites fluidfrom hybridoma clone 1C4 containing Mab againstfree α-subunit of hCG on a Protein A-SepharoseCL-4B column. The Mab bound to the column waseluted with 0.1 M sodium citrate buffer pH 6.0 andfractions (13–23) were pooled, concentrated and usedfor further characterization.

3.2. Characterization and Crossreactivitiesof Mabs

The purified preparation of Mab was analyzedby SDS-PAGE, which revealed two proteinbands (Fig. 2A) with Mr values of 27,500 (i.e.,light chain) and 60,000 (i.e., heavy chain). How-ever, the purified preparation apparently exhib-ited a single protein band on native PAGE withan Mr of 151,000 Dalton, which represents thewhole native molecule (Fig. 2B). The Mr valueobtained for the purified Mab on native PAGEwas also confirmed by gel filtration chromatog-raphy on Sephacryl S-200, which was elutedwith an Mr value of 155,000 Dalton (figure notshown). The elution profile of Mabs from sevenclones (2A9, 3G6, 1E2, 2B10, 2B3, 2A4, and1C4) on Protein A-Sepharose CL-4B columnsuggests that they are of IgG1 subclasse sincethey were eluted from the column at pH 6.0(Fig. 1). This result was confirmed by Ouch-terlony double diffusion analysis of purifiedMabs in the presence of rabbit anti-mouse immu-noglobulins heavy chains (figure not shown). Inprevious reports on Mabs against this glycopro-



Table 1Purification of Mab Against Free α-Subunit of hCG by Affinity Chromtography

on Protein A-Sepharose CL-4B

Total Total Mab Specific contentPurification Protein Activity of IgG1 Recovery Purification

steps (mg) (A units) (mg/mg protein) (%) factor

1. Ascites fluid 41.51 11 344 0.067 100 12. Column eluate 2.58 8 454 0.813 74.5 12.1

Fig. 2. Electrophoretic analysis of purified Mab from hybridoma clone 1C4. (A) SDS-PAGE of purified sample(10 µg) and ascites fluid (100 µg) using a gradient gel of 8–25%, (B) Native PAGE of purified sample (10 µg) andascites fluid (100 µg) using a gradient gel of 8–25%. On the margins are represented molecular weight markers,(C) Isoelectric focusing on polyacrylamide gel (pH range 5.0–8.0) of purified Mab (5 µg). On the left margin arerepresented pI markers.

tein hormone, research workers have reported thesynthesis of IgG1, IgG2, and IgM from thehybridoma cultures (10–12).

As regards to the monoclonality of the anti-body, it was analyzed by IEF, which revealed a

single family of bands with pI values in the rangeof 6.7–7.0 (Fig. 2C). Similar band patterns werealso obtained with Mabs against human growthhormone as well as with myeloma proteins whenthey were analyzed by IEF (24–26). This band



Table 2Some Properties of Mabs Against the Free-α-Subunit of Human Chorionic Gonadotrophin

Published in the Literature

Affinity constant CrossreactivityMabs (L/mol) (%) Reference

Mab 75 6.6 × 107 hCG 1.2%; FSH 6.7%; TSH 1.6%; LH 4%;Free-αhCG 100%; Free-αFSH 23%;Free-αLH 32%. (11)

Mab 71 3.4 × 106 hCG 5%; FSH 50%;TSH 4%; LH 14%;Free-αhCG 100%. (11)

Mab 3 n.d.a Free-αhCG 100%; hCG 3%; LH 33%. (27)Mab α-subunit 1.1 × 1010 hCG 100%; FSH 100%; TSH 100%; LH 100%. (28)Mab AHT20 5 × 108 Free-αhCG 100%; Free-αLH (41%.); hCG (n.d.). (29)Mab n.d.a hCG 0.1%; FSH 2.0%; TSH 0.014%; LH 0.8%

Free-αhCG100%; Free-αFSH 77%; Free-αLH 64%Free-αTSH 84%. (30)

an.d – indicates not determined

Fig. 3. (this and opposite page) Specificity and crossreactivity of Mab to pituitary hormones. The binding ofMab to pituitary hormones was determined by a two-step competitive enzyme immunoassay method. Varyingamounts of the appropriate unlabeled hormones were competed with peroxidase-αhCG for binding to purified 1C4Mab. The bound peroxidase-αhCG was detected by incubation with 1,2-phenylenediamine and hydrogen peroxideas substrates at 37°C for 15 min. The reaction was stopped by the addition of H2SO4 and the color developed wasread at 492 nm as described in Materials and Methods. (A) � - Free α-subunit of hCG; � - Intact FSH; � - IntactLH; � - Intact TSH; � - Intact hCG; (B) � - Free α-subunit of hCG; � - βFSH; � - βLH; � - βTSH; (C) � - Freeα-subunit of hCG; � - αFSH; �- αLH; � - αTSH.



pattern may be explained on the basis of micro-heterogeneity in antibody populations, whichresults in charge alteration (24–26).

The affinity constant (K) for this Mab againstthe free α-subunit of hCG was determined to be

1.5 × 107L/mol by the simple antibody dilutionanalysis method. In previous reports on Mabsagainst the free α-subunit of hCG (Table 2),research workers have reported K values in therange of 3.4 × 106 to 1.1 × 1010 L/mol (11,28,29).



The binding of this Mab to pituitary hormones(hCG, TSH, FSH, and LH) either as free subunitsor intact molecules was investigated by a two-stepcompetitive enzyme immunoassay. In the presentwork, crossreactions (by mass) of pituitary glyco-protein hormones with free α-subunit of hCG forMab 1C4 were as follows: αhCG 100%; intacthCG 1.8%; βhCG 0.14%; αFSH 24.5%; intactFSH 0.8%; βFSH 0.09%; αLH 20.5%; intact LH0.9%; βLH 0.08%; αTSH 50.5%; intact TSH3.7%; βTSH 0.07%;

The hybridoma clone 1C4 secreted a mono-clonal antibody that recognized specifically thefree α-subunit of hCG because it exhibited a lowcrossreactivity toward the intact molecule and thefree β-subunit (Figs. 3A and B). There are twopossible explanations for this result:

1. The Mab recognizes an epitope on the free α-sub-unit of hCG, which is masked in the intact hCG.

2. The Mab recognizes an epitope on the freeα-subunit, which is not available in the intactmolecule due to conformational changes in thenative molecule.

In addition to this, the Mab apparently did notreact with the free β-subunit of FSH, LH, and TSHsuggesting that it recognizes an epitope on the freeα-subunit of hCG (Fig. 3B). The data in Fig. 3Calso show that the Mab reacts with the free α-sub-unit of all pituitary hormones, which stronglysupports the idea that it recognizes an epitope onthe free α-subunit. This is in agreement with thestructural homology of α-subunits of all pituitaryhormones (3). Furthermore, the data in Fig. 3revealed that the Mab reacted differently with thefree α-subunit of all pituitary hormones suggest-ing that the epitope recognized by the Mab ispresent in a different conformation in the freeα-TSH, FSH, and LH subunits (5). In fact, someresearch workers have found differences in thecarbohydrate moieties of the common α-subunitsof hCG, LH, TSH, and FSH that affect their con-formation (5,32,33). This difference in α-subunitof pituitary hormones could alter the binding ofMab to the altered conformation of the epitope(5). Other research workers have also obtaineddifferent crossreactivity of Mabs with free α-sub-

unit of pituitary hormones (Table 2), which canbe explained on the basis of conformational changesin the epitope (11,29,30). But on the other hand,the data presented in this work revealed that TSHexhibits apparently the highest crossreactivityamong the intact hormones toward this Mab(Fig. 3A). These results may be explained by thefact that the α epitope detected by this Mab isapparently more exposed on the α-subunit inintact TSH than in hCG (31). The β-subunit inTSH obviously folds in a different manner fromhCG, which makes the epitope on this intact mol-ecule partially available to the antibody. However,the data in Fig. 3A and 3B revealed that the gly-coprotein hormones did not crossreact with Mabin the range of 0–200 µg/L in this assay system. Inprevious reports (Table 2), similar Mabs werealso isolated that recognized epitopes on α-sub-unit of hCG whereas they did not react with intacthCG (11,27,29,30). However, one of the Mabscrossreacted significantly with native FSH andTSH, which suggests that the α epitope detectedby this Mab is apparently more exposed on theα-subunit in intact FSH and TSH than in hCG(10,11,31). On the other hand, some researchworkers have also isolated monoclonal antibodiesthat recognize epitopes on the free β-subunit ofhCG but they do not crossreact with the intactmolecule suggesting that these Mabs bind toepitopes on the free β-subunit of hCG (12,22).

The Mabs presented in this work exhibit someadvantages over the Mabs published in the litera-ture (Table 2). The current Mab could be purifiedin a one-step purification procedure involving Pro-tein A-affinity chromatography with a high recov-ery of antibody activity (Table 1). Although thepresent Mab is a low- or intermediate-affinity anti-body, it was used in a two-step competitive enzymeimmunoassay of high sensitivity for determinationof α-subunit of hCG in biological fluids that coulddetect as little as 2 µg/L of α-subunit (data notshown). On the other hand, this Mab exhibits lowlevels of crossreactivity (Fig. 3) toward other pitui-tary hormones compared with other Mabs (Table 2)(11,27,28). Furthermore, the present Mab can beused to devise a one-step isolation scheme forα-subunit of hCG by immunoaffinity chromatog-



raphy (data not shown). Since this Mab is a low- orintermediate-affinity antibody (34), the antigen(α-subunit) can be eluted from the immunoaffinitycolumn under mild elution conditions (data notshown) as opposed to the use of drastic elution con-ditions required in immunoaffinity chromatogra-phy with high-affinity antibodies (34).

Further work is in progress concerning thetopology of the intact hCG as well as the α-sub-unit using a panel of monoclonal antibodies andsynthetic peptides.

AcknowledgmentsWe would like to thank the technical assis-

tance of Isabel Pereira and João Pedroso. Theauthors acknowledge a research grant fromPEDIP, Portugal.

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Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin