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Purification and Characterization of Monoclonal Antibodies Against the Free α-Subunit of Human Chorionic Gonadotrophin Carlos Novo,2 Ana Domingos,2 and Amin Karmali1,*
Abstract Monoclonal 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 culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free α-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies antiαhCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free α-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free α-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary 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 pituitary hormones; hybridoma technology; monoclonal antibodies; TSH; FSH; LH.
1. Introduction Human chorionic gonadotropin (hCG) is a glycoprotein hormone, which is synthesized and secreted by the trophoblasts of the placenta (1). This hormone plays an important role in the production of progesterone by the corpus luteum, which is required for the maintenance of early pregnancy in humans. This protein consists of two dissimilar and noncovalently bonded subunits α and β, which contain 92 and 145 amino acid residues, respectively (2). From the structural point of view, the α-subunit of hCG is identical to other pituitary hor-
mones (i.e., follicle-stimulating hormone [FSH], luteinizing hormone [LH], and thyroid-stimulating hormone [TSH]) (3). On the other hand, the β-subunits vary in size from 114 amino acid residues in LH to 145 residues in hCG, which contains six disulfide bridges (3). There is a high degree of homology in the amino acid sequence of hCG and other hormones in the first 114 amino acid residues (LH 85%; FSH 36%; TSH 46%) (3). Human chorionic gonadotropin hormone is a potential marker in the diagnosis of pregnancy and a variety of diseases such as ectopic pregnancy, choriocarcinoma, and testicular cancer
*Author to whom all correspondence and reprint requests should be addressed: 1Laboratório de Engenharia Bioquímica do Departamento de Engenharia 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. Molecular Biotechnology 2001 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2001/17:2/119–128/$12.50
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120 (4). Hence, the specific determination of hCG in urine and other biological fluids is of great interest from the diagnostic point of view (5). However, in certain tumors, some workers have observed high levels of free α- and β-subunits of hCG in circulation (6). The use of improved immunochemical assays for measurement of either free αor β-subunit in the presence of relatively high concentrations of other pituitary hormones in biological fluids as regards to specificity and sensitivity is therefore of great clinical importance in cancer diagnosis (6). The hybridoma technology described by Milstein and Kohler (7) has been used in previous reports to obtain monoclonal antibodies against hCG, which were highly specific tor determination of this glycoprotein in biological fluids (8,9). As regards to specific assays for free α- and β-subunits, a number of monoclonal antibodies (Mabs) have been isolated that detect either of these free subunits in the presence of relatively high concentration of hCG (10–12). However, in the case of free α-subunit, the Mabs isolated crossreact with other intact pituitary glycoprotein hormones (i.e., TSH and LH) as well as with their corresponding free α-subunit (11). On the other hand, monoclonal antibodies play an important role in basic research because they can be used as powerful tools to study the topology of protein molecules (12). Such an approach has been sucessfully applied for detection of local conformation in several proteins (12). The present work is concerned with production of monoclonal antibodies against the free α-subunit of hCG by hybridoma technology. Subsequently, the Mab was purified by affinity chromatography on Protein A-Sepharose CL4B column and some of its properties were studied.
2. Materials and Methods 2.1. 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–alkaline phosphatase conjugate, p-nitrophenyl phosphate, solid phase anti-mouse innunoglobulin antibody, MOLECULAR BIOTECHNOLOGY
Novo, Domingos, and Karmali peroxidase, hypoxantine aminoptorin thymidine (HAT), and (HT) were obtained from Sigma Chemical Company. I125-labeled rabbit anti-mouse light chain was supplied by Amersham, U.K. Protein A Sepharose CL-4B, Sephacryl S-200, and ampholine pH range 5–8 were purchased from Pharmacia International (Sweden). A sample of myeloma cell line (Sp2/0Ag14) was from ATCC. Membranes (P10) for ultrafiltration units were supplied by Amicon, Ireland. Free subunits and intact molecules of pituitary hormones (hCG, TSH, FSH, and LH) were supplied by U.C.B. Bioproducts, Belgium. All disposable plasticware were obtained from Costar and all other reagents used were analytical grade.
2.2. Animals Female mice from inbred strain Balb/c were obtained from Instituto Gulbenkian de Ciência, Oeiras, Portugal.
2.3. Methods 2.3.1. Protein Assay Protein concentration was determined by the Coomassie blue dye binding method (13).
2.3.2. Electrophoretic Analysis SDS-PAGE and native PAGE were carried out as mentioned previously (14,15) and stained for protein with silver nitrate (16). 2.3.3. Immunoglobulin Class and Subclass Immunoglobulin class and subclass were determined by Ouchterlony double diffusion analysis using several class specific antisera such as rabbit anti-mouse immunoglobulin heavy chain (γ1, γ2, α, and µ) (17). 2.3.4. Concentration of Immunoglobulins The concentration of immunoglobulin (i.e., IgG1) in monoclonal antibody samples was determined by an immunoradiometric assay (IRMA) using solid-phase anti-mouse immunoglobulin antibody (18). Briefly, diluted samples and standards of immunoglobulin were incubated successively with I125-labeled rabbit anti-mouse light chain and solid-phase anti-mouse immunoglobulin antibody. After repeated washings, the final sediment was counted in a gamma counter. Volume 17, 2001
Monoclonal Antibodies Against the Free α-Subunit of hCG 2.3.5. Determination of Affinity Constant The affinity constant (K) of Mab was determined using the simple antibody dilution analysis method (19). Briefly, an antibody dilution curve for this Mab was carried out in the presence of a constant concentration of free α-subunit of hCG (0.2 µg/well) in 96-well microtiter plates and the percentage of bound antigen was plotted against the decreasing antibody concentration for this Mab. The concentration of immunoglobulin was determined at each antibody dilution by IRMA and the affinity constant for this Mab was simply read off the plot at half-maximal antigen binding. 2.3.6. Enzyme-Linked Immunosorbent Assay (ELISA) Detection of antibody secretion in culture supernatants as well as in column eluates was carried out by ELISA using hCG, αhCG, and βhCG as the antigens (16 ng/well), rabbit anti-mouse IgG– alkaline phosphatase conjugate as the second antibody and p-nitrophenyl phosphate as the substrate (17). 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 IgG–alkaline phosphatase conjugate on p-nitrophenyl phosphate under the standard conditions of ELISA. 2.3.7. Production of Mabs Against hCG Female Balb/c mice (4-wk-old) were immunized on d 0 with native hCG (50 µg) in complete Freund adjuvant by subcutaneous injection. Subsequently, four immunizations were carried out at one month intervals with the same amount of antigen in phosphate-buffered saline (PBS) by intraperitoneal injections. Three days after the last immunization, mice were bled and the titer was determined by ELISA using rabbit anti-mouse IgG–alkaline phosphatase conjugate as the second antibody and p-nitrophenyl phosphate as the substrate (17). Subsequently, the spleen cells (8 × 107) were fused with Sp2/0 Ag 14 (2 × 107) in the presence of PEG (20). 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 (20). Mabs were produced from seven clones (2A9, 3G6, 1E2, 2B10, MOLECULAR BIOTECHNOLOGY
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2B3, 2A4, and 1C4) either in culture in vitro (RPMI 1640 + 10% [v/v] fetal calf serum) at 37ºC and 5% CO2 or in vivo as ascites fluid (20).
2.3.8. Purification of Mab from Hybridoma Clone 1C4 on Protein A-Sepharose CL-4B Ascites fluid (1 mL) diluted 1:2 with 1.5 M glycine buffer containing 3 M NaCl pH 8.9 was applied to a column (1 × 2 cm) packed with Protein A-Sepharose CL-4B previously equilibrated with 1.5 M glycine buffer containing 3 M NaCl 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) (13–23) containing antibody activity as determined by ELISA method at 405 nm were pooled and concentrated by pressure dialysis using a P10 membrane at 4°C. 2.3.9. Isoelectric Focusing Isoelectric focusing (IEF) of purified samples of Mabs was carried out in polyacrylamide gels using an ampholine pH range of 5–8 (21) and the protein bands were stained with silver nitrate. 2.3.10. Determination of Crossreactivity of Mabs with Other Pituitary Hormones The binding of Mab to pituitary hormones was investigated by a two-step competitive enzyme immunoassay using a Mab-coated filter-paper disc and peroxidase–αhCG conjugate as the labeled hormone (1). The bound enzyme–hormone conjugate was detected by using 1,2-phenylenediamine and hydrogen peroxide as substrates and the formation of product was followed at 492 nm (1). The crossreactivity of pituitary hormones in this two-step competitive enzyme immunoassay was determined by displacement of labeled α-hCG by crossreacting hormone. The percentage crossreactivity was calculated from the amount of hormone causing half displacement of the labeled hormone on a weight-ratio basis (11). The following glycoprotein hormones were used as the source of antigen: hCG, FSH, LH, TSH, αhCG, αFSH, αLH, αTSH, βhCG, βFSH, βLH, and βTSH. Briefly, this assay was carried out with filterpaper discs to which Mabs against hCG were Volume 17, 2001
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chemically attached. The concentration of pituitary hormones was varied from 0 to 10,000 µg/L in this assay system, which was incubated with the Mab-coated disc as well as peroxidase–αhCG conjugate. The bound peroxidase–αhCG was detected by incubation with 1,2-phenylenediamine and hydrogen peroxide as substrates at 37°C for 15 min. The reaction was stopped by the addition of H 2SO4 and the color developed was read at 492 nm (1).
3. Results and Discussion 3.1. Production and Purification of Mabs Monoclonal antibodies have been raised against hCG in this work using the hybridoma technology described by Kohler and Milstein (7). Hybrid growth was observed in 65% of the total culture wells and 8% of the total wells (i.e., 13 cultures) contained anti-hCG activity. In previous reports, 3 to 37 culture wells secreting antibodies against hCG (i.e., either to free subunits or to the intact molecule) were obtained from a single fusion of spleen cells with myeloma cells (22,23). In the present work, 12 positive hybrid cultures secreting antibodies recognized both free α and β subunits of hCG, whereas only one hybrid cell line (B6) reacted specifically to the free α-subunit of this hormone. Hence, the positive hybrid cell line B6 secreting antibodies anti-αhCG was selected for cloning purposes by limiting dilution method because it did not crossreact with the free β-subunit of hCG. The cloning of hybridoma B6 resulted in 84 clones that secreted Mabs against the free α-subunit of hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free α-subunit of hCG was selected for purification and characterization purposes. The Mab produced as ascites fluid from clone 1C4 was purified by affinity chromatography on Protein A-Sepharose CL-4B (Fig. 1) that resulted in a final relative recovery of antibody activity of 75% and a purification factor of 12 (Table 1). Other research workers have purified Mabs against hCG using two steps that involve DEAE-Affi-gel Blue and affinity chromatography with a final recovery of antibody activity of 15% and a purification factor of about 20 (10,23). MOLECULAR BIOTECHNOLOGY
Fig. 1. Affinity chromatography of ascites fluid from hybridoma clone 1C4 containing Mab against free α-subunit of hCG 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 (13–23) were pooled, concentrated and used for further characterization.
3.2. Characterization and Crossreactivities of Mabs The purified preparation of Mab was analyzed by SDS-PAGE, which revealed two protein bands (Fig. 2A) with Mr values of 27,500 (i.e., light chain) and 60,000 (i.e., heavy chain). However, the purified preparation apparently exhibited a single protein band on native PAGE with an Mr of 151,000 Dalton, which represents the whole native molecule (Fig. 2B). The Mr value obtained for the purified Mab on native PAGE was also confirmed by gel filtration chromatography on Sephacryl S-200, which was eluted with an Mr value of 155,000 Dalton (figure not shown). The elution profile of Mabs from seven clones (2A9, 3G6, 1E2, 2B10, 2B3, 2A4, and 1C4) on Protein A-Sepharose CL-4B column suggests that they are of IgG1 subclasse since they were eluted from the column at pH 6.0 (Fig. 1). This result was confirmed by Ouchterlony double diffusion analysis of purified Mabs in the presence of rabbit anti-mouse immunoglobulins heavy chains (figure not shown). In previous reports on Mabs against this glycoproVolume 17, 2001
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Table 1 Purification of Mab Against Free α-Subunit of hCG by Affinity Chromtography on Protein A-Sepharose CL-4B Purification steps
Total Protein (mg)
Total Mab Activity (A units)
Specific content of IgG1 (mg/mg protein)
Recovery (%)
1. Ascites fluid 2. Column eluate
41.51 2.58
11 344 8 454
0.067 0.813
100 74.5
Purification factor 1 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) and ascites 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 are represented pI markers.
tein hormone, research workers have reported the synthesis of IgG1, IgG2, and IgM from the hybridoma cultures (10–12). As regards to the monoclonality of the antibody, it was analyzed by IEF, which revealed a MOLECULAR BIOTECHNOLOGY
single family of bands with pI values in the range of 6.7–7.0 (Fig. 2C). Similar band patterns were also obtained with Mabs against human growth hormone as well as with myeloma proteins when they were analyzed by IEF (24–26). This band Volume 17, 2001
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Novo, Domingos, and Karmali Table 2 Some Properties of Mabs Against the Free-α-Subunit of Human Chorionic Gonadotrophin Published in the Literature
Mabs
Affinity constant (L/mol)
Mab 75
6.6 ×
Mab 71
3.4 × 106
Mab 3 Mab α-subunit Mab AHT20 Mab
n.d.a 1.1 × 1010 5 × 108 n.d.a
a n.d
107
Crossreactivity (%)
Reference
hCG 1.2%; FSH 6.7%; TSH 1.6%; LH 4%; Free-αhCG 100%; Free-αFSH 23%; Free-αLH 32%. hCG 5%; FSH 50%;TSH 4%; LH 14%; Free-αhCG 100%. Free-αhCG 100%; hCG 3%; LH 33%. hCG 100%; FSH 100%; TSH 100%; LH 100%. Free-αhCG 100%; Free-αLH (41%.); hCG (n.d.). hCG 0.1%; FSH 2.0%; TSH 0.014%; LH 0.8% Free-αhCG100%; Free-αFSH 77%; Free-αLH 64% Free-αTSH 84%.
(11) (11) (27) (28) (29)
(30)
– indicates not determined
Fig. 3. (this and opposite page) Specificity and crossreactivity of Mab to pituitary hormones. The binding of Mab to pituitary hormones was determined by a two-step competitive enzyme immunoassay method. Varying amounts of the appropriate unlabeled hormones were competed with peroxidase-αhCG for binding to purified 1C4 Mab. The bound peroxidase-αhCG was detected by incubation with 1,2-phenylenediamine and hydrogen peroxide as substrates at 37°C for 15 min. The reaction was stopped by the addition of H2SO4 and the color developed was read at 492 nm as described in Materials and Methods. (A) - Free α-subunit of hCG; - Intact FSH; - Intact LH; - Intact TSH; - Intact hCG; (B) - Free α-subunit of hCG; - βFSH; - βLH; - βTSH; (C) - Free α-subunit of hCG; - αFSH; - αLH; - αTSH.
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pattern may be explained on the basis of microheterogeneity in antibody populations, which results in charge alteration (24–26). The affinity constant (K) for this Mab against the free α-subunit of hCG was determined to be MOLECULAR BIOTECHNOLOGY
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1.5 × 107L/mol by the simple antibody dilution analysis method. In previous reports on Mabs against the free α-subunit of hCG (Table 2), research workers have reported K values in the range of 3.4 × 106 to 1.1 × 1010 L/mol (11,28,29). Volume 17, 2001
126 The binding of this Mab to pituitary hormones (hCG, TSH, FSH, and LH) either as free subunits or intact molecules was investigated by a two-step competitive enzyme immunoassay. In the present work, crossreactions (by mass) of pituitary glycoprotein hormones with free α-subunit of hCG for Mab 1C4 were 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 hybridoma clone 1C4 secreted a monoclonal antibody that recognized specifically the free α-subunit of hCG because it exhibited a low crossreactivity toward the intact molecule and the free β-subunit (Figs. 3A and B). There are two possible explanations for this result: 1. The Mab recognizes an epitope on the free α-subunit of hCG, which is masked in the intact hCG. 2. The Mab recognizes an epitope on the free α-subunit, which is not available in the intact molecule due to conformational changes in the native molecule.
In addition to this, the Mab apparently did not react with the free β-subunit of FSH, LH, and TSH suggesting that it recognizes an epitope on the free α-subunit of hCG (Fig. 3B). The data in Fig. 3C also show that the Mab reacts with the free α-subunit of all pituitary hormones, which strongly supports the idea that it recognizes an epitope on the free α-subunit. This is in agreement with the structural homology of α-subunits of all pituitary hormones (3). Furthermore, the data in Fig. 3 revealed that the Mab reacted differently with the free α-subunit of all pituitary hormones suggesting that the epitope recognized by the Mab is present in a different conformation in the free α-TSH, FSH, and LH subunits (5). In fact, some research workers have found differences in the carbohydrate moieties of the common α-subunits of hCG, LH, TSH, and FSH that affect their conformation (5,32,33). This difference in α-subunit of pituitary hormones could alter the binding of Mab to the altered conformation of the epitope (5). Other research workers have also obtained different crossreactivity of Mabs with free α-subMOLECULAR BIOTECHNOLOGY
Novo, Domingos, and Karmali unit of pituitary hormones (Table 2), which can be explained on the basis of conformational changes in the epitope (11,29,30). But on the other hand, the data presented in this work revealed that TSH exhibits apparently the highest crossreactivity among the intact hormones toward this Mab (Fig. 3A). These results may be explained by the fact that the α epitope detected by this Mab is apparently more exposed on the α-subunit in intact TSH than in hCG (31). The β-subunit in TSH obviously folds in a different manner from hCG, which makes the epitope on this intact molecule partially available to the antibody. However, the data in Fig. 3A and 3B revealed that the glycoprotein hormones did not crossreact with Mab in the range of 0–200 µg/L in this assay system. In previous reports (Table 2), similar Mabs were also isolated that recognized epitopes on α-subunit of hCG whereas they did not react with intact hCG (11,27,29,30). However, one of the Mabs crossreacted significantly with native FSH and TSH, which suggests that the α epitope detected by this Mab is apparently more exposed on the α-subunit in intact FSH and TSH than in hCG (10,11,31). On the other hand, some research workers have also isolated monoclonal antibodies that recognize epitopes on the free β-subunit of hCG but they do not crossreact with the intact molecule suggesting that these Mabs bind to epitopes on the free β-subunit of hCG (12,22). The Mabs presented in this work exhibit some advantages over the Mabs published in the literature (Table 2). The current Mab could be purified in a one-step purification procedure involving Protein A-affinity chromatography with a high recovery of antibody activity (Table 1). Although the present Mab is a low- or intermediate-affinity antibody, it was used in a two-step competitive enzyme immunoassay of high sensitivity for determination of α-subunit of hCG in biological fluids that could detect as little as 2 µg/L of α-subunit (data not shown). On the other hand, this Mab exhibits low levels of crossreactivity (Fig. 3) toward other pituitary hormones compared with other Mabs (Table 2) (11,27,28). Furthermore, the present Mab can be used to devise a one-step isolation scheme for α-subunit of hCG by immunoaffinity chromatogVolume 17, 2001
Monoclonal Antibodies Against the Free α-Subunit of hCG raphy (data not shown). Since this Mab is a low- or intermediate-affinity antibody (34), the antigen (α-subunit) can be eluted from the immunoaffinity column under mild elution conditions (data not shown) as opposed to the use of drastic elution conditions required in immunoaffinity chromatography with high-affinity antibodies (34). Further work is in progress concerning the topology of the intact hCG as well as the α-subunit using a panel of monoclonal antibodies and synthetic peptides.
Acknowledgments We would like to thank the technical assistance of Isabel Pereira and João Pedroso. The authors acknowledge a research grant from PEDIP, Portugal. References 1. Talwar, G. P., Gaur, A. (1986) Human chorionic gonadotrophin, hCG, in Methods in Enzymatic Analysis, vol. 11, (Bergmeyer, H. U., Bergmeyer, J., Grable, N., eds.), 3rd ed., VHC Veinheim, pp. 419–439. 2. Tegoni, M., Spinelli, S., Verhoeyen, M., Davis, P., and Cambillau, C. (1999) Crystal structure of a ternary complex between human chorionic gonadotrophin (hCG) and two Fv fragments specific for the α and β-subunits. J. Mol. Biol. 289, 1375–1385. 3. Lapthorn, A. J. , Harris, D. C., Littlejohn, A., et al. (1994) Crystal structure of human chorionic gonadotrophin. Nature 369, 455–461. 4. Alfthan, H. and Stenman, U. H. (1996) Pathophysiological importance of various molecular forms of human choriogonadotrophin. Mol. Cell. Endocrinol. 125, 107–120. 5. Madersbacher, S. and Berger, P. (2000) Antibodies and Immunoassays. Methods 21, 41–50. 6. Grossmann, M., Tarutmann, M. E., Poertl, S., et al. (1994) Alpha-subunit and human chorionic gonadotrophin-beta immunoreactivity in patients with malignant endocrine gastroenteropancreatic tumours. Eur. J. Clin. Invest. 24, 131–136. 7. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256, 495–497. 8. Norman, R. J., Poulton, T., Gard, T., and Chard, T. (1985) Monoclonal antibodies to human chorionic gonadotropin: implications for antigenic mapping, immunoradiometric assays and clinical applications. J. Clin. Endocrinol. Metab. 61, 1031–1038. 9. Armstrong, E. G., Ehrlich, P. H., and Birken, S. (1984) Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal, non-pregnant and pregnant individuals. J. Clin. Endocrinol. Metab. 59, 867–874.
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10. Thotakura, N. R. and Bahl, O. P. (1985) Highly specific and sensitive hybridoma antibodies against the α-subunit of human glycoprotein hormones. Endocrinology 117, 1300–1308. 11. Norman, R. J., Haneef, R., Buck, R. H., and Joubert, S. M. (1987) Measurement of the free alpha subunit of human glycoprotein hormones by a monoclonal antibody-based immunoradiometric assay and further exploration of antigenic sites on the choriogonadotropin molecule. Clin. Chem. 33, 1147–1151. 12. Bidart, J-M., Troalen, F., Salesse, R., Housfield, G. R., Bohuon, C. J., and Bellet, D. H. (1987) Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin β-subunit. J. Biol.Chem. 262, 8551–8556. 13. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 14. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 15. Hames, B. D. (1981) An introduction to polyacrylamide gel electrophoresis, in Gel Electrophoresis of Proteins (Hames, B. D. and Rickwood, D., eds.) IRL, pp. 1–86. 16. Wray, W., Boulikes, T., Wray, V. P., and Hancock, R. (1981) Silver stain of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–202. 17. Johnstone, A. and Thorpe, R. (1987) lmmunoassays, in Immunochemistry in Practice (Johnstone, A. and Thorpe, R., eds.) Chapter 11, Blackwell Scientific Publications, Oxford, pp. 257–260. 18. Hunter, W. M., Bennie, J. G., Brock, D. J. H., and Heyningen, V. V. (1982) Monoclonal antibodies for use in an immunoradiometric assay for alfafetoprotein. J. Immunol. Methods 50, 133–137. 19. Heyningen, V., Brock, D. S. H., and Heyningen, S. (1983) A simple method for ranking the affinities of monoclonal antibodies. J. Immunol. Methods 62, 147–152. 20. Brown, G. and Ling, N. R. (1988) Murine monoclonal antibodies, in Antibodies, vol. 1 (Rickwood, D. and Hames, B. D., eds.) IRL, Oxford, pp. 81–89. 21. Campbell, A. M. (1991) Protocols for PAGE, in Monoclonal Antibody Technology. The production and characterization of rodent and human hybridoma. (Burdon, R. H. and Knippenberg, P. H., eds.) Elsevier, New York, pp. 240–241. 22. Nordblom, G. D., Kabza, G. A., and Beierwaltes, V. H. (1981) Development and characterization of a monoclonal antibody which distinguishes the β-subunit of human chorionic gonadotropin (βhCG) in the presence of hCG. Endocrinology 109, 1290–1292. 23. Thotakura, N. R. and Bahl, O. P. (1986) Purification and properties of a monoclonal antibody specific to the free β-subunit of human chorionic gonadotropin
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Volume 17, 2001