Development and characterization of a new, highly specific antibody to the human chorionic gonadotropin-beta fragment.

0013-7227/91/1283-1255$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 3 Printed in U.S.A.

Development and Characterization of a New, Highly Specific Antibody to the Human Chorionic Gonadotropin-/? Fragment* ALEXANDER KRICHEVSKY, STEVEN BIRKEN, JOHN O'CONNOR, KAREN BIKEL, JOHN SCHLATTERER, CHEN YI, GLADYS AGOSTO, AND ROBERT CANFIELD Departments of Pathology and Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

in both liquid and solid phase immunoassay formats. We have used this new monoclonal antibody in immunoradiometric assays to measure specifically the hCG/3 fragment in urine throughout pregnancy as well as in the sera of two individuals with cancers producing the hCG /3-subunit. We discovered that the hCG/3 fragment can bind three monoclonal antibodies simultaneously, indicating that although the epitope for antibody B210 is a new determinant exposed on the hCG/3 fragment and not on intact hCG or on free hCG )3-subunit, the hCG/3 fragment retains at least two other hCG/3-related epitopes intact, i.e. those that bind monoclonal antibodies B108 and B201. (Endocrinology 128: 1255-1264,1991)

ABSTRACT. In addition to high concentrations of hCG, pregnancy urine contains even higher concentrations of a fragment of the hCG (8-subunit. This biologically inactive material complicates immunological measurement of hCG, since it crossreacts with many polyclonal and monoclonal antibodies to the hCG /3-subunit that are employed for assays of hCG in urine. Although we and others have developed antibodies to this fragment, specific measurement of the fragment in the presence of free hCG/3 has remained difficult due to intrinsic cross-reactivity of these antibodies with the intact hCG/3. Rather than attempt to increase specificity by assay optimization, we developed a new, highly specific monoclonal antibody, designated B210, which cross-reacts less than 0.1% with the free hCG /3-subunit

I

T HAS been established that pregnancy urine, especially during the first trimester, contains large quantities of hCG. Measurement of this hormone formed the basis of the early immunological and bioassay tests for pregnancy (1). During the past decade, evidence has emerged indicating that human pregnancy urine also contains large quantities of a fragment of the hCG 0subunit as well as the individual hormone subunits (2, 3). This was demonstrated by gel filtration followed by immunoassay of the fractions using antisera directed to the /3-subunit of hCG. The presence of this material introduced significant errors in the immunological measurement of intact hCG using subunit antisera because of cross-reactivity with most such antisera. This fragment, which lacked the COOH-terminal portion of hCG/3, was also found to be present in the urine of patients with various cancers (4, 5). hCG assays have been used to monitor the course of therapy for successful treatment

of certain cancers (1), a n d t h e hCG/3 fragment molecule

Received July 2, 1990. Address requests for reprints to: Dr. A. Krichevsky, Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. * These results were presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990. This work was supported by NIH Grants HD-15454 and RR-00645.

presented another possible tumor marker (6, 7). Accurate quantitation of the hCG fragment was difficult because antibodies to the intact hCG /3-subunit generally cross-reacted completely with the fragment. Thus, the separation of components by molecular size was required before accurate quantitation of the relative amounts of intact hormone, hCG /3-subunit, and hCG/3 fragment was possible. Several groups, including ours, have developed immunological measurement systems that can assay the hCG/3 fragment without the need for gel filtration. For example, Akar et al. (8) developed polyclonal immunoassay systems to measure the hCG/3 fragment. Earlier, we reported the primary structure of the hCG/3 fragment (9), the preparation of monoclonal antibodies to the hCGjS fragment, as well as the development of immunoradiometric assay (IRMA) systems for this molecule (10). However, these systems, despite optimization of specificity by manipulation of the concentration of capture antibody, remain intrinsically limited by the specificity of the hCG/3 fragment antibody, i.e. its intrinsic cross-reactivity with the hCG j8-subunit. We, therefore, searched for an antibody to an epitope unique to the hCGjS fragment rather than attempted to

1255

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 June 2015. at 20:18 For personal use only. No other uses without permission. . All rights reserved.

SPECIFIC ANTIBODY TO hCG/3 FRAGMENT

1256

further optimize existent assays. We illustrate the unique specificity of our new measurement system by evaluation of both standard solutions and clinical specimens. We have also used this new antibody to investigate the number of epitopes displayed by the hCG/3 fragment. Solid phase immunological experiments have provided evidence that as many as three different antibodies can bind simultaneously to this 10,000 mol wt two-chain polypeptide. Materials and Methods Immunoassay methods Methods for both solid and liquid phase RIAs were described previously (10, 11). Radiolabeling of antigens and antibodies (10-30 /xg) was performed with 1 mCi Na125I (Amersham, Arlington Heights, IL) using Iodogen (Pierce Chemical Co., Rockford, IL) according to the manufacturer's information. Timed resolved fluoroimmunometric assays Europium-based time-resolved immunofluorometric assays (IFMA) have been described previously (12, 13) and were performed with the following modifications. The microtiter wells employed in the IFMA were white opaque Removawell Strips (Dynatech Laboratories, Inc., Chantilly, VA). The assay buffer was 50 mM Tris (pH 7.8), 0.9% NaCl, 1% BSA, and 0.05% NaN3. The plate-washing solution (wash buffer) was normal saline containing 0.05% Tween-20 (Fisher Biotech, Pittsburgh, PA). Antibodies were biotinylated employing the NHS-LC-Biotin reagent (obtained from Pierce). Briefly, the antibody, in 1 M sodium carbonate buffer (pH 9.1), was reacted with the Pierce reagent (dissolved in dimethylsulfoxide; 2 mg reagent/mg antibody) by mixing at room temperature. After a 1-h reaction time, the reaction mixture was dialyzed at 4 C twice against 0.1 M bicarbonate buffer with 0.05% NaN3. The biotinylated antibody was diluted in assay buffer containing mouse serum, and this stock solution was stored in the refrigerator. The detection system consists of a europium-containing chelate, 4,7 bis-(chlorosulfophenyl)l,10-phenanthroline 2,9-dicarboxylic acid (Eurofluor, Cyberfluor, Toronto, Ontario, Canada), linked to thyroglobulin, which, in turn, is conjugated to streptavidin. The signal is generated by pulsed excitation by laser at 337.1 nm and measurement of the delayed europium fluorescence at 615 nm with a CyberFluor 615 fluorometer. Method of evaluation of simultaneous binding of three antibodies The assay procedure involved coating the wells of microtiter plates with a solution of monoclonal antibody B201 (50 Mg/ml) in sodium carbonate buffer (0.2 M; pH 9.5) for 24 h at 4 C, washing the wells (four times), blocking with 1% BSA (300 nV well; 24 h at 4 C), and again washing (four times). hCG 0subunit fragment standards in phosphate buffer (0.01 M NaHPO4, 0.15 M NaCl, 0.01 M EDTA, 0.1% NaN3, and 0.1% bovine 7-globulin, pH 7.4) were then added to the wells. The standards (50 jul/well) plus biotinylated B108 (50 /zl/well) were

Endo«1991 Voll28-No3

mixed on a shaker for 2 min and incubated at 37 C for 1 h. The wells were aspirated, washed (four times), and then incubated at 37 C for 45 min with the europium tracer solution. The wells were again aspirated, washed six times with wash buffer, dried for 10 min in a strip dryer, and read in the Cyberfluor 615 Fluorometer (Cyberfluor, Toronto, Ontario, Canada) to obtain the B108 europium standard curve (see Fig. 7). To determine binding of the third antibody to hCG/3 fragment, the wells were incubated with a solution of 125I-labeled B210 (120,000 cpm) and incubated overnight. The tubes were aspirated, washed (six times), and counted in the 7-counter to obtain the radiolabeled antibody curve. An alternative procedure, involving simultaneous addition of the standards and labeled antibodies, also produced standard curves for both labeled antibodies. A control experiment, in which the immobilized capture antibody B 201 was omitted, and the wells were coated with just BSA, failed to produce binding curves. Other combinations of capture and trace antibodies produced similar results.

Gel filtration by FPLC columns Samples were gel filtered, as indicated, on two Superose 12 columns (Pharmacia Fine Chemicals, Piscataway, NJ) in tandem, using 0.1 M ammonium bicarbonate as the buffer (9). The columns were used on a Waters HPLC (Milford, MA), with unions purchased from Pharmacia. The flow rate was 0.5 ml/ min, and 1-min fractions were collected. Recovery and assay validation study using Superose 12 columns Pregnancy urine specimens as well as hCG/3 fragment-spiked male urine samples were gel filtered (1-ml samples) on Superose 12, as described above, except that a protein carrier (0.1 mg/ml bovine 7-globulin) was included in the column buffer to prevent adsorption to surfaces. The B210-based IRMA was used to measure the content of hCG/? fragment in the raw urine specimen and then used to measure each gel-filtered fraction in the elution position expected for standard /? fragment. The quantities of hCG# fragment found in the fractions were summed and compared to the total obtained for the raw urine sample. Cloning of B210 The new hCG/3 fragment antibody was produced by selection of a clone from the same fusion that produced the earlier hCG/? fragment antibodies B201 and B204 (10). All of the original wells from that fusion v/ere rescreened by evaluating the binding of five radiolabeled antigens: hCG, hCG /?-subunit, hLH, hLH /3-subunit, and hCG/3 fragment. Two supernatants of 28 saved bound only labeled hCG/3 fragment and none of the other tracers. The cells from 1 original well with the highest supernatant titer to the hCG/3 fragment were cloned to obtain the cell line that produces the antibody B210. The cloning was accomplished by plating 600 cells into 60 wells at 10 cells/well and 200 cells into 200 wells at 1 cell/well. No positives were found in the 1 cell/well seeding. However, 2 wells of the 10 cells/well seeding were positive. From one of those 2 wells, the B210 antibody was cloned. After cloning of the cell line producing antibody B210, we compared its behavior in both liquid and solid phase assays and found that it displayed selectivity


SPECIFIC ANTIBODY TO hCG/3 FRAGMENT for hCG/3 fragment in both formats. Thus, it had unique properties in its specificity toward hCG/3 fragment and loss of reactivity to hCG /3-subunit.

Results The new B210 antibody was produced by the cloning of a rare cell, which is estimated to occur approximately 1 of 400 cells (see Materials and Methods). The antibody was produced in sufficient quantity to facilitate its characterization and application. Figure 1 (A, B, and C) shows a comparison of three dose-response studies (liquid phase RIAs) which illustrates the progression of increased specificity in our development of monoclonal antibodies to the /3 fragment of hCG. B201, our first monoclonal antibody raised against the hCG/3 fragment, was found to bind to a different site than most hCG/3 antibodies, but was not significantly selective for the hCG/3 fragment over the

10 !

1257

free hCG /3-subunit. B204, our most specific antibody before the one described in the current report, is shown to cross-react on the order of 5-10% with free hCG /3subunit. Antibody B210 appears to cross-react less than 0.5% with hCG /3-subunit in a liquid phase RIA. When different preparations of hCG /3-subunit were assayed with antibody B210 in a similar fashion, we found different cross-reactivities, i.e. if the ED50 for CR123 /3 is considered to be 100%, CR129 and CR117 hCG /3-subunit displayed lower cross-reactions of approximately 50%, while CR125 was higher at 300%. This indicates that much or all of the cross-reaction of antibody B210 with hCG /3-subunit is due to the presence of small quantities of hCG/3 fragment contamination within the purified hCG /3-subunit sample (see Discussion). The comparative affinity constants for the hCG /3subunit and hCG/3 fragment for each of the antibodies we have generated against the hCG/3 fragment are shown in Table 1. This information shows that although each of the antibodies displays approximately the same affinity toward the hCG/3 fragment (10~10 M/liter), the B210 antibody displays 3 orders of magnitude lower affinity to the free hCG /3-subunit compared to B201 and 2 orders lower affinity than B204. Because we had demonstrated different cross-reactivities for different preparations of hCG /3-subunit with antibody B210, we needed to separate all hCG/3 fragment contamination from hCG /3-subunit to define the true cross-reactivity of hCG /3-subunit with B210. Therefore, we gel filtered purified hCG fragment (Fig. 2A) and purified hCG /3-subunit (CR119; Fig. 2B) separately on FPLC Superose 12. The column fractions were analyzed by our assays for hCG/3 fragment as well as for hCG /3subunit (6). Figure 2A shows that hCG/3 fragment elutes as 90% (fractions 60-63) monomer and 10% dimer (fractions 54-56), as reported previously (8). Figure 2B shows the standard elution pattern of the hCG /J-subunit. Each fraction was assayed with a hCG /3-subunit assay as well as with our hCG/3 fragment assay. The y-axis scale of the hCGjS fragment assay is expanded in the inset of Fig. 2B to show that the hCG/3 fragment assay displays 0.1% true cross-reactivity with the hCG /3-subunit. This is calculated from fraction 53, in which 105 pmol/ml are detected by the hCG /3-subunit assay for the peak of hCG /3-subunit material (Fig. 2B), while only 0.1 pmol/ml are

Concentration of Standard (moles/liter) FlG. 1. Relative immunoreactivity of three j8 fragment antibodies with the /3 fragment and free 0-subunit. The three panels show dose-response curves from liquid phase RIAs, using radiolabeled P fragment as tracer with cold displacing standards of /3 fragment and CR123 /3-subunit. All assays were conducted at the same time with the same tracer. If other preparations of purified j8-subunit were employed (see text), different /? cross-reactivities were found using antibody B210, showing that each /3-subunit preparation contained different amounts of /3 fragment as a contaminant.

TABLE 1. The affinity constants of each of the three antibodies to the hCG /J-subunit and fragment (/Sfrag) Competitors Antibody

Tracer 0frag

B201 B204 B210

(Sfrag 0frag /3frag

hCG/3 10

5 x 10 5 x 1010 6 X 1010

5 X 1010 2X10° 3X 107


Endo«1991 Voll28-No3

SPECIFIC ANTIBODY TO hCG/? FRAGMENT

1258 100-

45

50

0»gg8oQooooQOo88goggggggggggggggggggg

55

45

45

faction FlG. 2. Determination of intrinsic cross-reactivity of antibody B210 with the hCG /3-subunit. Panels show immunoassay of eluent fractions from Superose 12 gel permeation chromatography. A is a separation of the hCG/3 fragment on gel permeation, while B is /3-subunit. Fractions shown in A were assayed by B210 for hCG/3 fragment (O-O); fractions in B were assayed for intact hCG /3-subunit (D-Q). Each fraction in B was also assayed with antibody B210 for /3 fragment (O-O). The /3 fragment assay was expanded in the inset by 1000-fold to show that the authentic cross-reactivity with fractions containing /3-subunit free of /3 fragment is approximately 0.1%.

detected by the hCG0 fragment assay of the same fraction (Fig. 2B, inset). The authentic hCG/3 fragment is separated from the hCG /3-subunit on this column and appears in the elution positions of the hCG/3 fragment dimer and monomer whose positions are defined in Fig. 2A. This observation that antibody B210 has authentic cross-reactivity with free hCG /3-subunit was further substantiated by the finding that significant quantities of intact subunit are absorbed to a B210 immunoaffinity column (data not shown). A similar gel filtration strategy can be used to assess the authentic cross-reactivity of the hCG/3 fragment assay with whole hCG and the a-subunit. In both cases, the contamination of hCG/3 fragment in these preparations is also easily visualized by assaying the identical fractions for their major component, i.e. hCG or free

55

60

65

70

75

80

Fraction FlG. 3. Determination of intrinsic cross-reactivity of antibody B210 with hCG (A) and hCGa (B). The experiment was conducted as described in Fig 2. A, Gol permeation of hCG. V-V, Assay for intact hCG; O-O, /3 fragment assay. The inset shows /3 fragment assay of each fraction in A expanded 300-fold. The /3 fragment assay indicates 0.1% cross-reaction under the hCG position (fraction 50) and the presence of 0.3% /3 fragment (as /3 fragment monomer) in the hCG preparation (fraction 62); hCG/3 fragment appears as a dimer at fraction 55. B, Gel permeation separation of hCGa. A-A, Assay for a-subunit; O-O, /? fragment assay. The /3 fragment assay was expanded 100-fold (inset) to show the absence of authentic /3 fragment activity under the a peak (fractions 55-60; /3 fragment dimer) and the presence of 1.5% ft fragment contamination (peaking at fraction 62) in the a preparation.

hCG 0-subunit as well as the hCG/3 fragment. Figure 3A depicts gel filtration of intact hCG on tandem Superose 12 columns (see Materials and Methods). Each fraction was assayed for intact hCG and hCG/? fragment, the latter being displayed with an expanded ordinate in the inset of Fig. 3. This experiment shows that the hCG/3 fragment assay also cross-reacts 0.1% with intact hCG, and that this hCG preparation contains about 0.3% contamination with hCG/3 fragment. Figure 3B shows the gel filtration pattern of purified a-subunit assayed with an a assay, while the inset shows the results of assays of the same fractions with the B210-based hCG/3 fragment assay in i;he expanded scale. Figure 3B demonstrates that this preparation of a-subunit contains about 1.5% contamination with the hCG/3 fragment and no cross-reactivity with free a-subunit. The measure-


SPECIFIC ANTIBODY TO hCGjS FRAGMENT ment of hCG/5 fragment on the ascending limb of the asubunit can be accounted for by the presence of hCG/3 fragment dimer. We next examined the sera of two patients with tumors producing hCG /3-subunit (an ovarian seminoma and an ovarian choriocarcinoma) using the same gel filtration separation scheme. The gel filtration serves to define the sizes of the immunoreactive species being measured in sera. Figure 4, A and B, are gel filtration profiles of the sera from two cancer patients from Dr. D. Bellet, who determined that the hCG free 0-subunit was the predominant circulating form of hCG. In the case of the patient with an ovarian seminoma shown in Fig. 4A, as much as half of the immunoreactivity measured as free hCG /3subunit by hCG /3-subunit assays is shown to be circulating hCG jS-subunit fragment. In the case of the patient with the ovarian choriocarcinoma whose serum gel filtration profile is shown in Fig. 4B, only about 10% of the (3 immunoreactivity can be attributed to the hCG/3 frag-

1259

ment, and the majority is due to the hCG /?-subunit. With validation of the specificity of our hCGjS fragment assay, we returned to reexamine data on pregnancy reported by other laboratories, specifically the relative quantities of hCG, free hCG /3-subunit, and hCG/3 fragment found in the urine of a pregnant woman during each trimester of pregnancy. Results from a single individual, whom we sampled twice weekly, are shown in Fig. 5. The concentrations of hCG/3 fragment, free hCG /3subunit, and hCG were compared. At each time point, it was found that the molar concentration of hCG/? fragment exceeded that of hCG. When the level of hCG fell, so did that of the hCG/3 fragment. In another ongoing study of pregnancy, we have confirmed the results of Marshall et al. (14) that the concentration of hCG in urine reflects the average concentration in serum during pregnancy (serum/urine ratio of 1.1-1.2:1). Weekly analysis displayed considerable variations of this ratio, but the average during the entire pregnancy was close to 1.0. Thus, the concentrations of hCG in urine are reflective of values present in serum. The ratio of hCG/3 fragment concentration to hCG concentration was highest during the first trimester, but the concentrations of each were similar during much of the second and third trimesters. Free hCG /3-subunit was measurable only during the first 12 weeks of this pregnancy. Again, in order to validate the molecular species we were measuring, one representative sample was selected from each trimester from the patient shown in Fig. 5, each urine specimen was gel filtered, and the eluent fractions were assayed for hCG, free a-subunit, and hCGjS fragment. These results are shown in Fig. 6. Each trimester sample displayed the same immunological profile of molecular species, that is hCGjS fragment eluted in the position expected from studies with standard urinary material, and, likewise, hCG and a-subunit appeared where expected. Validation that the quantity of hCG /3-subunit 600

00000000000009

55

60

65

70

75

80

FRACTION FIG. 4. Gel permeation chromatography of two sera from patients with malignancies containing material with j8-subunit immunoreactivity material. Assay of the fractions from gel size separation of the sera from the patient with ovarian seminoma shown in A for hCG/3 (D-D) and hCGjS fragment (O-O) show that 50% of the hCG/3-type immunoreactivity is actually due to circulating /? fragment. The serum of the patient with ovarian choriocarcinoma shown in B has only about 10% the molar ratio of 0 fragment relative to hCG/3.

10

15

20 25 30 WEEK OF PREGNANCY

35

FIG. 5. Twice weekly analysis of the urine from a pregnant woman for hCG (V-V), hCG/3 (D-D), and /3-fragment (0-0). The assays are expressed as picomoles per mg creatinine to correct for relative concentrations of analytes in the urine specimens.


SPECIFIC ANTIBODY TO hCG,3 FRAGMENT

1260

Endo • 1991 Voll28«No3

ues similar to those in the patient specimens. Thus, the epitope we are measuring in the raw urine sample behaves similar or identical to the standard preparation of hCG/3 fragment, and it is not necessary to gel filter patient urine samples to confirm correct measurement of the fragment. We and others have reported that the urine of postmenopausal women contained hCG/3 fragment. Using the B210-based IRMA we have confirmed this observation. In an ongoing study we have found that postmenopausal women displayed a range of hCG/3 fragment excretion of 0.005-0.029 pmol/mg creatinine in urine, while the values of normal women ranged between 0.002-0.012 pmol/ mg creatinine, and those of men were between 0.0010.005 pmol/mg creatinine. Identification of multiple epitobes on hCGfi fragment 40

45

50

55

55

60

65

60

65

70

75

80

FRACTION

FiG. 6. Gel permeation chromatography of three urine specimens selected from each of the trimesters from the patient shown in Fig. 5. A, First trimester; B, second trimester; C, third trimester. Chromatography was as described in Fig. 2. V-V, hCG; A-A, a-subunit; O-O, /3 fragment assay. TABLE 2. Recovery study of /3 fragment from Superose 12 column (B210-B108*) Cone, in Fractions fractions summed (pmol) Pregnancy urine 1 Pregnancy urine 2 Spike urine 1 Spike urine 2

55-69 55-69 55-69 55-69

130 39 153 51

The hCG/? fragment, which represents less than half the mass of the intact hCG /3-subunit, was assessed for the number of antibodies that could bind simultaneously by using purified monoclonal antibodies labeled with different detection probes. For this assay one antibody is used as a capture (B201), a second as an 125I-labeled detection antibody (B210), and a third as an europiumlabeled detection antibody (B108). Figure 7 illustrates that both the europium-labeled antibody (B108) and the 125 I-labeled antibody (B210) bind simultaneously to the hCGjS fragment whem it is bound to the B201 antibody, demonstrating that three antibodies can bind simultaneously to the hCG/3 fragment. A topological cartoon is shown as Fig. 8, which indicates this finding in the context of the other antibody-binding regions mapped previously (10) on the surface of hCG. 100T

Load urine Recovery (filtered; rate pmol) 159 46 156 62

82 85 98 82

fragment is accurately measured in the raw urine samples is shown in Table 2. Two samples of pregnancy urine at 4-fold different concentrations and two samples of hCG/3 fragment-spiked male urine samples of similar concentrations were measured by the B210-based IRMA and then gel filtered on the Superose system. The fractions in the expected elution position of hCG/3 fragment were measured by the same IRMA, and the total hCG/? fragment recovered was calculated. Recoveries ranged between 82-98%. Spiked standards displayed recovery val-

10

100 1000 DOSE FMOLE/ML

10 000

FiG. 7. Simultaneous binding of three monoclonal antibodies to hCG fragment. Monoclonal antibody B201 was immobilized on the surface of a microtiter well, serving to capture hCG/3 fragment. Monoclonal antibodies B210 and B108, labeled, respectively, with 126I ( • - • ) and Eu+3 (O-O). Fluorescence measurement and 125I radioactivity indicate simultaneous binding of the two differently labeled antibodies. Similar curves were obtained whether the two tracers were incubated sequentially or simultaneously. No binding curves were obtained if the capture antibody was not coated onto the well.


SPECIFIC ANTIBODY TO hCG/? FRAGMENT B2IO

1261

completely inhibited the binding of the iodinated antibody, while similar wells, to which no blocking antibody was added, bound trace [125I]B201 at a level of 44%. These results, shown in Table 3, support these dual label studies described above and further indicate the presence of at least three epitopes on the /3 fragment molecule.

B2OI

Discussion

B2OI B2O2 B204/205

B203 B206

BI09 FlG. 8. A schematic drawing of antigenic regions and binding sites for the hCG jS-subunit-related monoclonal antibodies. Enlargement represents (dashed line on cartoon of /3-subunit) hCG /3-subunit fragment with B108 epitope common for intact hCG/3, hCG /3-subunit, and /3subunit fragment; new B210 epitope is specific only for /3-subunit fragment. The B201 epitope is present on both the /3-subunit and the /3 fragment.

Further confirmation of the existence of three epitopes on the hCG/3 fragment molecule was sought through blocking studies with a series of unlabeled liquid phase antibodies mapped to site II, the SB-6-type site on hCG /3-subunit (Fig. 8), an epitope conserved in the hCG/3 fragment molecule. In these experiments, monoclonal antibody B210 was employed as the capture. The EI210 capture- [125I]B201 detection system is an IRMA for hCG/3 fragment, i.e. both antibodies bind simultaneously to the hCG/3 fragment. In the experiment described, an excess of unlabeled B201 (1 /ug/well) was added to the wells in the presence of one of three different radioiodinated site II antibodies (B203, B206, or B108). Each of the three different site II antibodies bound to the B210hCG/3 fragment complex regardless of whether unlabeled B201 was present in the well. Addition of cold B201 to wells in which [125I]B201 was employed as trace almost

The fragment of the /3-subunit of hCG, first described over a decade ago in the urine of pregnant women as well as in the urine of certain cancer patients, has become an important consideration in recent years in reproductive research, cancer detection, and the monitoring of anticancer therapy. The fragment may arise as the result of different routes of production: either direct secretion from trophoblastic or other tissues (4, 15), or peripheral degradation of hCG in the circulation or kidneys (5, 16, 17). Wehmann et al. (18) performed studies of the halflife in humans of injected hCG/3 fragment and found it to be cleared very rapidly from the circulation, leading to the conclusion that secretion of the fragment directly by tissues into the blood is unlikely to account for a significant quantity of hCG/3 fragment in the urine. Whatever the origins of the large concentrations of hCG/3 fragment in pregnancy urine or the urine of cancer patients, the accurate measurement of this molecule is difficult because it contains the most common conformational epitopes present on intact hCG /3-subunit (Fig. 8, site II) and, therefore, is highly cross-reactive with most monoclonal antibodies and polyclonal antisera to the hCG /3-subunit. Akar et al. (8) developed polyclonal antibodies to the hCG/3 fragment, which exhibited relatively low crossreactivity with the intact hCG /3-subunit. However, monoclonal antibodies offer the advantage of an abundant supply and consistent specificity and affinity as well as the opportunity to develop immunoassays of enhanced selectivity and sensitivity by use of IRMA methodology. The challenge in the development of monoclonal antibodies is the selection of those with high affinities, high specificities, and the capability of binding simultaneously with other desirable monoclonal antibodies so that multisite assay systems can be established. This publication TABLE 3. Tabulation of binding parameters to wells blocked with liquid phase B201 % 125I trace antibody Blocking antibody B201 B203 None

B201

B203

B206

B108

1 34 44

46 6 48

25 NT NT

18 NT NT

B210 is the solid phase capture antibody; hCG/3 fragment is the standard. NT, Not tested.


1262

SPECIFIC ANTIBODY TO hCG/? FRAGMENT

describes selection of such an antibody by the cloning of a relatively rare cell secreting an antibody with less than 0.1% cross-reactivity to intact hCG 0-subunit and with a high affinity (in the range of 6 X 1010 M/liter). This antibody, designated B210, has been characterized and subsequently used to examine clinical specimens. The first question to be addressed in characterization of this hCG/3 fragment monoclonal antibody was its cross-reaction with intact hCG 0-subunit. Liquid phase RIA using hCG /3-subunit standards and hCG/5 fragment standards showed that antibody B210 was much more specific than its predecessors, B201 and B204, developed previously (10). Different preparations of hCG /3-subunit were shown to exhibit differing cross-reactivity in B210 assays due to varying contaminations with hCG/3 fragment. Wehmann et al. (19) have shown previously that a-subunit and hCG contain minute contaminating quantities of hCG/? fragment, and it now appears that preparations of hCG /3-subunit may contain some as well. This possibility inhibited accurate assessment of the true cross-reactivity of B210 with intact /3-subunit. Consequently, we performed the gel filtration-immunoassay studies shown in Figs. 2 and 3. Previously we had shown that the gel permeation FPLC columns Superose 12, connected in tandem, proved efficient in separating hCG/3 fragment monomeric and dimeric forms from intact hCG /3-subunit (9). We found that the hCG/3 fragment separates well from hCG /3-subunit under these conditions by mixing various quantities of subunit and hCG/3 fragment standard (unpublished results). Therefore, by measuring the quantity of intact hCG /3-subunit in fractions throughout the separation from the Superose columns and comparing these values with those measured in each of the same fractions with the B210 assay system, we calculated that the authentic cross-reactivity of B210 with the hCG 0-subunit was no more than 0.1%. These assays were performed using the IRMA format, as described previously for antibody B204 (9,10). Using the same gel filtration-assay technique, it is clear that intact hCG and the free a-subunit display negligible crossreactivity with B210, although each has somewhat more hCG/3 fragment contamination compared with the hCG 0-subunit than was reported by Wehmann et al. (19), who used similar techniques with a polyclonal antiserum liquid phase RIA system. The purpose of the generation of a highly specific hCG/3 fragment antibody was to permit IRMA measurement of this molecule in unprocessed urine specimens without the need for gel filtration. Although serum usually contains very little measurable hCG/3 fragment, some cancers have been observed to result in high levels of free /3-subunit concentration in serum. The question that we sought to address was whether the free /3-subunit measured by standard immunoassays was due in part to

Endo«1991 Voll28«No3

the presence of high concentrations of hCG/3 fragment. Therefore, we measured hCG/3 fragment content in the raw serum of two cancer patients found to have high concentrations of free hCG /?-subunit. Those sera were then gel filtered to check our estimate of hCG/3 fragment content compared to hCG /3-subunit concentration. Figure 4 shows that one sample contained significant hCG/3 fragment, while the other contained mainly free hCG /?subunit. The B210 IRMA had approximated the correct hCG/3 fragment content of the serum specimen shown in Fig. 4A, but the results in Fig. 4B raise an intriguing question. Figure 4B indicates that more hCG/3 fragment activity appears under the hCG /?-subunit peak than was expected from cross-reactivity calculated from urinary standards in Fig. 2B. We have observed this occasionally with serum specimens, as shown in Fig. 4B, but generally not with urine samples, as shown in Fig. 6, leading to the hypothesis that B210-like epitopes may appear at low levels in molecules of different sizes from that of the urinary hCG/3 fragment in serum specimens. Such observations warrant additional investigation of serum samples as well as addit ional studies of urine samples from such individuals displaying serum anomalies. We used pregnancy urine samples to validate our specific assay system in urine. Urine samples from a pregnant woman were measured twice weekly for concentrations of intact hCG a- and 0-subunits and hCG /3-subunit fragments. Table 2 presents data showing that the measurement of hCG/? fragment at two different ranges in pregnancy urine closely agrees with the total hCG/3 fragment measured at the expected mol wt position of the material after gel filtration of the urine specimens. In addition, male urine spiked with authentic hCG/3 fragment standards behaved in a very similar fashion. Thus, the B210-based assay system has been validated for use with clinical specimens, since the content of hCG/3 fragment measured in raw urine is in agreement with the quantity of authentic hCG/? fragment observed after gel filtration of these sa.mples. Preliminary studies of postmenopausal women with the B210-based IRMA showed that approximately half were positive for the hCG/3 fragment in urine. The fragment could also be detected at very low levels in normal nonpregnant women as well as in men. The pattern of hC!Gj8 fragment concentration in urine collected serially during an entire pregnancy warrants comment. Kato and Braunstein performed a similar study (3), using gel filtration and RIA of individual chromatography fractions using the hCG /3-subunit antiserum SB-6 (20). These investigators did not monitor pregnancies twice weekly, nor did they monitor the entire course of each pregnancy because of the labor involved in gel filtration and assay of each fraction (3). Instead, single urine samples were obtained from 15 women at



different stages of pregnancy. Despite the differences in sample collection, their results are very similar to the serial samples of our current study. The molar concentration of the hCG/3 fragment exceeds the concentration of hCG during most of pregnancy. Differences dependent upon individual variations would be expected. In fact, data from an ongoing study indicate that some pregnant women excrete less hCG/3 fragment during pregnancy. Additional studies are warrented to explore individual anomalies from the prevalent patterns of excretion of the hCG/3 fragment. Figure 8 is a cartoon showing a map of antibodybinding regions based on steric exclusion, that is antibodies that cannot bind at the same time are portrayed as binding to the same shaded area. The /3 fragment region is expanded to show that 3 antibodies can bind simultaneously to this molecule of approximately 10,000 mol wt. This finding was documented by direct binding assays using 2 detection antibodies with 2 different labels, one a radiolabel and the second an europium-based fluorometric label. In addition, antibody-blocking experiments were performed to provide further evidence that 3 antibodies could bind to the /? fragment at the same time. This observation can be employed as a basis for increased selectivity and sensitivity in the measurement of the hCG/3 fragment. The epitope for B210 is one that is exposed on the hCG/3 fragment, but not on the intact hCG /3-subunit. Despite the exposure of a new epitope, the hCG|3 fragment has retained the B108 and B201 epitopes present on the free hCG /3-subunit, showing that those areas of the molecule have not been perturbed even though more than half of the hCG /3-subunit structure is absent in the hCG/3 fragment. How can three antibodies bind to this small molecule? Recent studies of the F(ab) antibody fragments crystallized in combination with their ligands showed that the antibody-binding site on the surface of the ligand may be quite large (750 A2) (21). Such findings indicate the limitations of immunological mapping by steric hindrance experiments due to the broad area of antibody interaction. For example, as many as 17 amino acids from different regions of both lysozyme (22) and neuraminidase (23) may be in contact with the complementarity-determining regions of the F(ab)s. The latter study purported to show that the antibody may induce alterations in folding of the ligand upon binding. If it was necessary to alter the folding pattern of the hCG/3 fragment to allow binding of 3 antibodies simultaneously, we would not have expected to find that each of the antibodies could bind to the hCGjS fragment alone. Schwartz et at. (24) speculated that as many as 12 epitopes may theoretically fit on the surface of hCG, which has an 8000-A2 surface area. Since the hCG/3 fragment represent one fourth the mass of intact hCG, the finding of at least 3 epitopes may be theoretically possible even

1263

without significant antibody-deforming stress on the molecule.

References 1. Hussa RO 1987 The Clinical Marker hCG. Praeger, New York 2. Schroeder HR, Halter CM 1983 Specificity of human /3-choriogonadotropin assays for the hormone and for immunoreactive fragment present in urine during normal pregnancy. Clin Chem 29:667671 3. Kato Y, Braunstein GD 1988 /3-Core fragment is a major form of immunoreactive urinary chorionic gonadotropin in human pregnancy. J Clin Endocrinol Metab 66:1197-1201 4. Masure HR, Jaffee WL, Sickel MA, Birken S, Canfield RE, Vaitukaitis JL 1981 Characterization of a small molecular size urinary immunoreactive human chorionic gonadotropin (hCG)-like substance produced by normal placenta and, by hCG-secreting neoplasms J Clin Endocrinol Metab 53:1014-1020 5. Papapetrou PD, Sakarelou NP, Braouzi H, Fessas PH 1980 The value of measurement of immunoreactive hCG in the urine as a screening procedure. Cancer 45:2583-2592 6. O'Connor JF, Schlatterer JP, Birken S, Krichevsky A, Armstrong EG, McMahon D, Canfield RE 1988 Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its j8-subunit, and /? core fragment in urine application to malignancies. Cancer Res 48:1361-1366 7. Cole LA, Wang Y, Elliot M, Latif M, Chambers JT, Setsuko K, Schwartz PE 1988 Urinary human chorionic gonadotropin free 0subunit and /3-core fragment: a new marker of gynecological cancers. Cancer Res 48:1356-1360 8. Akar AH, Wehmann RE, Blithe DL, Blacker C, Nisula BC 1988 A radioimmunoassay for the /3-core fragment of human choriogonadotropin. J Clin Endocrinol Metab 66:538-545 9. Birken S, Armstrong EG, Gawinowicz Kolks MA, Cole LA, Agosto GM, Krichevsky A, Vaitukaitis JL, Canfield RE 1988 Structure of the human chorionic gonadotropin /? subunit fragment from pregnancy urine. Endocrinology 123:572-583 10. Krichevsky A, Armstrong EG, Schlatterer J, Birken S, O'Connor J, Bikel K, Silverberg S, Lustbader JW, Canfield RE 1988 Preparation and characterization of antibodies to the urinary fragment of the human chorionic gonadotropin /?-subunit. Endocrinology 123:584-593 11. Ehrlich PH, Moustafa ZA, Krichevsky A, Birken S, Armstrong EG, Canfield RE 1985 Characterization and relative orientation of epitopes for monoclonal antibodies and antisera to human chorionic gonadotropin. Am J Reprod Immunol Microbiol 8:48-54 12. Hemmila I, Dakubu S, Mukkala VM, Siitari H 1984 Europium as a label in time-resolved immunofluorometric assays. Anal Biochem 137:335-343 13. Pettersson K, Siitari H, Hemmila I, Lovgren EST, Hanninen V, Pirjo Tanner F, Stenman UH 1983 Time-resolved fluoroimmunoassay of human choriogonadotropin. Clin Chem 29:60-64 14. Marshall JR, Hammond CB, Ross GT, Jacobson A, Rayford P, Odell WD 1968 Plasma and urinary chorionic gonadotropin during early human pregnancy. Obstet Gynecol 32:760-764 15. Cole LA, Birken S 1988 Origin and occurrence of human chorionic gonadotropin /3-subunit core fragment. Mol Endocrinol 2:825-830 16. Papapetrou PD, Nicopoulou SC 1986 The origin of a human chorionic gonadotropin /3-subunit-core fragment excreted in the urine of patients with cancer. Acta Endocrinol (Copenh) 112:415422 17. LeFort GP, Stolk JM, Nisula BC 1986 Renal metabolism of the /?subunit of human choriogonadotropin in the rat. Endocrinology 119:924-931 18. Wehmann RE, Blithe DL, Flack MR, Nisula B 1989 Metabolic clearance rate and urinary clearance of purified /3-core. J Clin Endocrinol Metab 69:510-517 19. Wehmann RE, Blithe DL, Akar AH, Nisula BC 1988 0-Core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotropin and its asubunit. J Endocrinol 117:147-152


1264


20. Vaitukaitis JL, Braunstein GD, Ross GT 1972 A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 113:751-758 21. Bently GA, Alzari PM, Amit AG, Boulot C, Guillan-Chitarra V, Fischmann T, Lascombe MB, Marivzza RA, Poljak RJ, Riottat MM, Saul FA, Souchan H, Tello D 1989 Studies of structure and specificity of some antigen-antibody complexes Phil Trans R Soc Lond [B] 323:487-494

Endo • 1991 Vol 128 • No 3

22. Amit AG, Mariuzza RA, Phillips SEV, Poljak RJ 1986 Three dimensional structure of an antigen antibody complex at 2.8 A resolution. Science

A highly immunoreactive peptide fragment of human luteinizing hormone alpha subunit, discerned with a new, "sequence-specific" radioimmunoassay.

Antibodies to the carboxyl-terminal fragment of human chorionic gonadotropin beta-subunit: characterization of antibody recognition sites using synthetic peptide analogues.

Characterization of a monoclonal antibody specific for human active renin.

Development and characterization of a new human hepatic cell line.

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.

Comparison of specific immunoassays for detection of the beta-core human chorionic gonadotrophin fragment in body fluids.

A rapid, sensitive and specific radioimmunoassay for human chorionic gonadotrophin.

Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua.

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor.

Distribution of the beta-core human chorionic gonadotrophin fragment in human body fluids.

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39.

Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

Characterization of a new monoclonal anti-glypican-3 antibody specific to the hepatocellular carcinoma cell line, HepG2.

Carbohydrate structures of beta-core fragment of human chorionic gonadotropin isolated from a pregnant individual.

Development, manufacturing and characterization of a highly purified, liquid immunoglobulin g preparation from human plasma.

Establishment and characterization of a new highly metastatic human osteosarcoma cell line derived from Saos2.

Development and characterization of a novel GHR antibody antagonist, GF185.

Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

Purification and characterization of a highly specific polyclonal antibody against human extracellular signal-regulated kinase 8 and its detection in lung cancer.

A new, highly sensitive and specific assay for chymotrypsin.

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

Site-specific modification of a fragment of a chimeric monoclonal antibody using reverse proteolysis.