Antigenic determinants on human choriogonadotropin alpha-subunit. I. Characterization of topographic sites recognized by monoclonal antibodies.

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigenic determinants on human choriogonadotropin alpha-subunit. II. Immunochemical mapping by a monoclonal antipeptide antibody.

In order to study antigenic site(s) present in the carboxyl-terminal part of the alpha-subunit of human choriogonadotropin (hCG-alpha), we attempted to produce site-specific antibodies directed against a 34-residue synthetic peptide analogous to region 59-92 of hCG-alpha. From a fusion experiment performed with a mouse injected with hCG-alpha-(59-92)-peptide conjugated to tetanus toxoid as immunogen, we selected a monoclonal antipeptide antibody (designated FA36) which has high binding activity for 125I-hCG-alpha but not for 125I-hCG in a radioimmunoassay. This antibody is of the IgG1 subclass and displays an affinity constant for 125I-hCG-alpha of 3.1 x 10(8) M-1. Hapten inhibition experiments performed by either radioimmunoassay or enzyme-linked immunosorbent assay with synthetic peptides spanning different portions of the region (59-92) demonstrated that the binding site of FA36 resides on (minimally) the six COOH-terminal amino acids of hCG-alpha, namely Cys-Tyr-Tyr-His-Lys-Ser, and that FA36 binds preferentially to peptides containing a carboxyl group on the COOH-terminal residue. Monoclonal immunoradiometric assays were established to determine the location of antigenic regions recognized by FA36, by antibody AHT20 (which binds only to hCG-alpha), and by antibody HT13 (which binds to both hCG and hCG-alpha). FA36 has the capacity to bind to hCG-alpha bound to either AHT20 or HT13, demonstrating that both AHT20 and HT13 antibodies are directed against antigenic regions distinct from the epitope of FA36. Monoclonal immunoradiometric assays were also carried out to study the binding of FA36 to hCG, the ovine and equine lutropin alpha-subunit, or hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core). Whereas significant binding of 125I-FA36 was observed with the ovine lutropin alpha-subunit, no binding was found with the equine lutropin alpha-subunit. As expected, FA36 did not bind to hCG-alpha core. Binding was also not detected with hCG, confirming that FA36 is specific for free hCG-alpha and that the COOH-terminal part of hCG-alpha is either weakly or (more likely) not at all accessible in the alpha/beta-dimer for antibody binding. Finally, immunoblots performed on hCG-alpha-(59-62)-peptide and various denatured alpha-subunits indicated that, with the exception of the equine lutropin alpha-subunit, FA36 detected various denatured alpha-subunits and particularly the alpha-subunit of carp gonadotropin-thyrotropin. This latter observation suggests a high degree of homology between the COOH-terminal regions of the alpha-subunits of fish gonadotropin and analogous mammalian hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical mapping of a specific domain on human choriogonadotropin using anti-protein and anti-peptide monoclonal antibodies.

In an attempt to localize topographic domains specific to native human chorionic gonadotropin (hCG), we studied the discontinuous antigenic regions recognized by a monoclonal anti-hCG antibody designated as C8 which binds only to hCG and does not cross-react with either the free hCG-alpha and hCG-beta subunits or other glycoprotein hormones. Using two-site monoclonal immunoradiometric assays (M-IRMAs), we found that C8 antibody and an anti-peptide antibody (FB12) directed to residues 110-116 of hCG-beta did not bind simultaneously to hCG. This observation suggested that C8 binds to residues of hCG-beta included either in the antibody-binding region of FB12 or in close proximity to amino acids 110-116. To further delineate the regions of hCG-beta recognized by C8, we carried out hapten inhibition experiments with synthetic peptides corresponding to various regions of hCG-beta. The peptide corresponding to residues 109-122 and subpeptides (111-122 or 112-122) inhibited the binding of 125I-hCG to C8, whereas weak inhibition was observed with subpeptide 113-122. By studying the binding of C8 to the 1-112 disulfide-bonded part of hCG-beta (hCG-beta core) recombined with hCG-alpha, we were able to confirm that C8 binds to a region including or near to Asp112. M-IRMAs showed that C8 does not bind to the recombinant molecule lacking residues 113-145 of hCG. Taken together, these results indicate that a limited number of residues located on hCG-beta near to Asp112, and most likely the sequence Asp111-Asp112-Pro113, are included in the discontinuous antigenic region recognized by C8. We then attempted to localize residues of hCG-alpha that constitute another part of the determinant which bound to C8. Six synthetic peptides corresponding to various regions of hCG-alpha did not inhibit binding of 125I-hCG to C8. In contrast, M-IRMAs demonstrated that C8 is capable of binding recombinant products composed of the hCG-beta subunit and the alpha subunits from human, equine, and porcine species. These results indicate that C8 recognizes a region of the alpha subunit highly conserved in these three species. Finally, we determined that the discontinuous regions recognized by C8 are partially accessible on the CG/LH-receptor complex.

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit.

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.

The immune response to a synthetic peptide analogous to the 109-145 beta hCG carboxyl-terminus is directed against two major and two minor regions.

The immune response to a 37-amino acid synthetic peptide analogous to the carboxyl-terminal part (109-145) of the human chorionic gonadotropin beta subunit (beta hCG) was studied with monoclonal antibodies selected from 31 cell fusion experiments. Analysis of the immunogenic determinants borne on the synthetic peptide (CTP) showed a prevailing response to two immunodominant regions. The first was located on the 110-116 amino acid sequence of the CTP which is also the most hydrophilic region: 50% of anti-CTP antibodies selected for their high binding to 125I beta hCG were directed to this sequence. A second immunodominant portion was recognized by four antibodies, and comprised amino acids 134 to 139, representing a highly O-glycosylated region on the native protein. Moreover, a unique antibody designated FB13 bound to a region located on the last seven amino acids (139-145) of beta hCG. Finally, a hypothetical conformational determinant was recognized by antibody FB02 within the 121-145 region. Thus, the immune response to CTP was directed against two major and two minor regions. These antigenic determinants were demonstrated to be accessible for antibody binding on both the hCG molecule and its beta subunit. Localization of these epitopes suggests a relationship between the hydrophilicity and the immunological potency of different CTP regions.

Sensitive and specific assay for human chorionic gonadotropin (hCG) based on anti-peptide and anti-hCG monoclonal antibodies: construction and clinical implications.

We developed a highly sensitive and specific assay for hCG using monoclonal antibodies (Mabs) directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl-terminus (CTP) of beta hCG. Five antibodies that varied by either their affinity for beta hCG or their specificity for epitopes on CTP were investigated. To measure hormone levels, we used as the radiolabeled indicator an alpha-subunit-reactive Mab. The monoclonal-immunoradiometric assay had a lower limit of sensitivity of 0.05 ng/ml. Serum levels of hCG or hCG-like material with CTP structure were measured in 229 healthy blood donors; 1.1% of healthy men and 4.6% of nonpregnant women younger than 50 yr had serum values varying between 0.05 and 0.23 ng/ml. Moreover, 6 to 7 healthy women older than 50 yr had detectable levels in the 0.05-0.20 ng/ml range. To study the disappearance rates in normal women, we followed serum hCG serum levels of 6 women who had previously received a single im injection of the hormone. These individuals failed to develop a pregnancy after in vitro fertilization; hCG declined from 0.5 to 0.05 ng/ml within 2 weeks. These results were in contrast to the findings in 12 patients with hCG-producing tumors. In 9 patients without any evidence of recurrent disease, hCG levels became undetectable within 5 months. However, 3 others had levels consistently above 0.05 but below 0.5 ng/ml. In 2 of these three patients, subsequent increasing hCG levels were associated with tumor recurrence. We conclude that this hCG assay based on both anti-peptide and anti-hCG Mabs may be useful in tumor monitoring.

Identification of epitopes associated with hCG and the beta hCG carboxyl terminus by monoclonal antibodies produced against a synthetic peptide.

We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.

Serum alpha-fetoprotein levels in human disease: perspective from a highly specific monoclonal radioimmunoassay.

A rapid multisite radioimmunoassay for measurement of human alpha-fetoprotein (AFP) that uses two high-affinity monoclonal antibodies directed against distinct and separate determinants on the protein was developed and designated M-RIA. The sensitivity of the "simultaneous-sandwich" M-RIA is approximately equal to 0.5 ng/ml of serum after a 1-hr incubation period. Serum AFP levels have been measured in 1747 individuals with hepatocellular carcinoma (HCC), acute and chronic hepatitis B virus infection, chronic hepatitis B surface antigen (HBsAg)-carrier states, cirrhosis, other malignant tumors, and normal and disease controls to determine the specificity of the assay. Eighty percent (68/85) of patients with HBsAg-positive HCC had AFP levels of greater than 200 ng/ml (range, 260 to greater than 200,000 ng/ml). In contrast, all 450 normal subjects and 477 chronic HBsAg-positive carriers had levels of less than 20 ng/ml. More importantly, in acute and chronic hepatitis B, cirrhosis, and other malignant tumors and in the remaining disease controls, AFP levels were less than 20 ng/ml in 99.3% of the subjects, the great majority (greater than 96%) being less than 5 ng/ml. Indeed only two of 1635 individuals, one with acute hepatitis and the other with carcinoma of the esophagus had AFP levels of greater than 100 ng/ml. These observations are at variance with previous studies with conventional polyvalent RIAs of AFP levels of greater than 20 ng/ml in approximately equal to 40% of acute and chronic hepatitis and in 30% of cirrhosis. This striking specificity of the M-RIA is probably due in part to recognition of epitopes unique to AFP and suggest that such an assay may be used in the detection, early identification, and monitoring of AFP-producing tumors in high-risk populations.