Native versus deglycosylated IgM in anti-MAG neuropathy: Correlation with clinical status - Study of 10 cases.

BACKGROUND/PURPOSE: In anti-myelin associated glycoprotein (anti-MAG) neuropathies, there is evidence that anti-MAG antibodies are pathogenic but numerous studies report the absence or a weak correlation between the titers of these antibodies and disease course. In this study we assessed the relationships between MAG and glycosylated moieties located on Fc fragment of IgM anti-MAG. MATERIAL AND METHODS: IgM were extracted from the serum of 8 patients with anti-MAG neuropathy and in 2 patients with anti-MAG antibodies without anti-MAG neuropathy. Anti-MAG activity was performed with pre- and post-deglycosylated IgM extracts using indirect immunofluorescence (IIF) and ELISA. Sera from 49 patients with IgM monoclonal gammopathy without neurological disease were tested as control group (CG). Results were compared to clinical scores. For 4 patients the affinity constant of IgM with MAG was analyzed pre- and post-deglycosylated, using surface plasmon resonance technology (SPR). RESULTS: The relationships between MAG and glycosylated moieties of IgM anti-MAG were confirmed by kinetic and immunological assays. Deglycosylation resulted in a decrease in anti-MAG titers. Post-deglycosylation anti-MAG titers trended with changes in IgM titers and allowed quantifying anti-MAG antibodies without a saturation of the testing method. After deglycosylation, the titers better represented pathogenic activity and help to follow a given patient's clinical status prospectively. Six patients from CG (12.2%) had anti-MAG antibody titers over positive threshold: 1000 Bühlmann-Titer-Units (BTU) supporting the hypothesis of neutral intermolecular interactions between IgM and MAG. Deglycosylation allowed distinguishing infra clinical forms from neutral relationships forms, when the titers are weak but this assay remains essentially a diagnostic tool.

Antimyeloperoxidase antibodies are a useful marker of disease activity in antineutrophil cytoplasmic antibody-associated vasculitides.

OBJECTIVE: To evaluate the relevance of monitoring antimyeloperoxidase antibody levels in the management of antimyeloperoxidase-associated vasculitides. METHODS: Thirty-eight patients with antimyeloperoxidase-associated vasculitides were included: microscopic polyangiitis (n = 18), Wegener's granulomatosis (n = 15) and Churg-Strauss syndrome (n = 5). Baseline characteristics and outcomes were recorded. Serial measurements of antimyeloperoxidase antibody levels were performed (ELISA, positive > or = 20 IU/ml). RESULTS: All patients achieved vasculitis remission after a mean time of 2.0 months (SD 0.9), with a significant decrease in the mean antimyeloperoxidase antibody level at remission (478 vs 41 IU/ml (SD 598 vs 100); p<0.001). Twenty-eight (74%) patients became antimyeloperoxidase antibody negative. After a mean follow-up of 54 months (SD 38), 12 cases of clinical relapse occurred in 11/38 (29%) patients. Relapses were associated with an increase in antimyeloperoxidase antibody levels in 10/11 (91%) patients (34 vs 199 IU/ml (88 vs 314); p = 0.002). The reappearance of antimyeloperoxidase antibodies after achieving negative levels was significantly associated with relapse (odds ratio 117; 95% CI 9.4 to 1450; p<0.001). Antimyeloperoxidase antibodies showed a positive predictive value of 90% and a negative predictive value of 94% for relapse of vasculitis. Up to 60% of cases of relapse occurred less than 12 months after the reappearance of antimyeloperoxidase antibodies. Relapse-free survival was significantly worse for patients who exhibited a reappearance of antimyeloperoxidase antibodies than in those with persistent negative antimyeloperoxidase antibodies (p<0.001). The antimyeloperoxidase antibodies serum level was strongly correlated with the Birmingham vasculitis activity score and the disease extent index (r = +0.49; p = 0.002). CONCLUSION: Through monitoring, antimyeloperoxidase antibodies are a useful marker of disease activity and a good predictor of relapse in antimyeloperoxidase-associated vasculitides.

Peptide immunogen mimicry of a protein-specific structural epitope on human choriogonadotropin.

It is a challenge to construct synthetic immunogens that elicit antibodies (Abs) both directed to conformational epitopes and specific for a complex protein like human choriogonadotropin (hCG). A monoclonal antibody specific for hCG bound to regions around Lys45 of the alpha subunit (hCG alpha) and Asp112 of the beta subunit (hCG beta). A peptide comprising residues 46 to 55 of hCG alpha and residues 106 to 116 of hCG beta elicited Abs in rabbits that were directed to a discontinuous epitope and were specific for hCG. These Abs inhibited the binding of hCG to its receptor. Thus, a synthetic immunogen can mimic a conformational-specific epitope and can be useful for vaccine development.

Evaluation of the LIAISON ANA screen assay for antinuclear antibody testing in autoimmune diseases.

Antinuclear antibodies (ANA) are widely detected by immunofluorescence on HEp-2 cells in patients with connective tissue diseases and other pathological conditions. We evaluated the first-automated chemiluminescence immunoassay for the detection of ANA (LIAISON ANA screen, DiaSorin). This study was carried out simultaneously in two laboratories by testing 327 patient samples with clinically defined connective diseases, 273 routine samples for ANA screening, and 300 blood donors. A total of 268 out of 337 IIF-positive sera were positive with LIAISON ANA screen (79.5% of agreement) and 240 out of 263 IIF-negative sera were negative with LIAISON ANA screen (91.2% of agreement). After resolution of discrepant results, the concordance reached, respectively, 94.9% and 98.8%. The specificity was 99.3% and the sensitivity was 94%. Unlike results obtained by other ANA screening assays, we observed acceptable sensitivity and specificity. Despite the presence of HEp-2 cell extract, we failed to detect some antibodies as antinucleolar, antinuclear envelope, and antiproliferating cell nuclear antigen. This automated assay allows quick process to results and exhibits satisfactory sensitivity for the detection of the main ANA specificities of connective tissue diseases.

Genetic heterogeneity of the hypervariable region I of Hepatitis C virus and lymphoproliferative disorders.

B-cell lymphoproliferative disorders (BCLD) have been associated with chronic hepatitis C virus (HCV) infection. The HCV glycoprotein E2 (gpE2) hypervariable region I (HVR-I) may be a potential antigenic candidate to promote B-cell proliferation. The purpose of this study was to analyze the influence of HVR-I sequence variability in the development of BCLD. HVR-I sequences were studied in 29 chronically HCV-infected patients with (n=15) or without (n=14) BCLD. After PCR amplification of the gpE2 region, analysis of the 81 bp HVR-I encoding fragment was performed on 7-18 clones per patient. HVR-I sequence complexity was slightly lower in patients with BCLD (mean 0.347) than without (0.468) (P=0.2), though, sequence diversities were similar (0.0370 vs 0.0954, P=0.239). Phylogenetic analysis did not reveal any BCLD-associated clustering. In our population, neither the recently described insertion between positions 1 and 2 of HVR-I nor residues at positions 4 and 13 were particularly linked to BCLD. As previously described, we confirm the high degree of conservation of HVR-I residues T-2, G-6 and G-23 in our patients. Contrary to recent findings, our analysis based on multiple clones per patient analysis did not reveal any particular motif associated with BCLD.

Identification and measurement of calcitonin precursors in serum of patients with malignant diseases.

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

Free human chorionic gonadotropin beta subunit in gonadal and nongonadal neoplasms.

The diagnostic value of elevated human chorionic gonadotropin (hCG) and its free alpha (hCG alpha) and beta (hCG beta) subunit serum levels as specific tumor markers for nongonadal malignancies is controversial. In the present report, different monoclonal based immunoradiometric assays specific for hCG and its free hCG alpha and hCG beta subunits have been used to reevaluate the presence of these molecules in the serum of patients with a wide variety of tumors. Serum samples from patients with newly diagnosed, persistent, or recurrent malignancies of either known (n = 717) or unknown (n = 32) primary site, healthy blood donors (n = 309), and nonmalignant disease controls (n = 86) were studied using four highly specific and sensitive monoclonal based immunoradiometric assays to hCG and its free subunits. Low level hCG elevations (less than 1000 pg/ml) were found to be common in cancer patients, normal subjects, and disease controls. However, serum levels greater than 1000 pg/ml were highly diagnostic of gonadal tumors and specifically identified nonseminomatous testicular tumors. Significant serum elevations of free hCG alpha subunit (as high as 3000 pg/ml) were found in approximately 96% of cancer patients, normal individuals, and disease controls. In contrast, free hCG beta subunit levels (greater than or equal to 100 pg/ml) were detected in 70 and 50% of patients with nonseminomatous and seminomatous testicular cancers, respectively, and in 47% of bladder, 32% of pancreatic, and 30% of cervical carcinomas. All normal subjects and disease controls had free hCG beta levels less than 100 pg/ml. Thus, the detection of the free hCG beta subunit in serum of nonpregnant subjects was highly diagnostic of malignancy in general and specifically defines a subgroup of aggressive nongonadal malignancies.

Specific immunostaining for CCP II, a novel calcitonin carboxyl terminal peptide encoded by the calcitonin/CGRP gene.

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way.

Variations of serum IgG subclass levels in hepatitis C virus infection during interferon-alpha therapy.

Serum IgG1 levels are selectively increased in patients with chronic hepatitis C virus (HCV) infection. In 15 patients who received interferon (IFN)-alpha therapy, serum levels of immunoglobulin classes and IgG subclasses were measured during treatment and after it was discontinued. In spite of important individual variations, mean IgG, IgG1, IgA and IgM levels decreased during therapy and tended to return to pre-treatment levels afterwards, with no detectable correlation with clinical and biological parameters. These results suggest an effect of IFN-alpha on in vivo immunoglobulin production, in HCV carriers.

Transfusion-associated TT virus co-infection in patients with hepatitis C virus is associated with type II mixed cryoglobulinemia but not with B-cell non-Hodgkin lymphoma.

OBJECTIVE: To assess the prevalence of TT virus (TMV) infection in a series of patients with chronic hepatitis C virus (HCV) infection, with or without benign (mixed cryoglobulinemia) or malignant (B-cell non-Hodgkin lymphoma (B-NHL)) lymphoproliferative disease. METHODS: Sixty-six HCV patients were studied, including patients with mixed cryoglobulinemia (n=30), B-NHL (n=15), and no mixed cryoglobulinemia or B-NHL (n=21). All HCV patients had increased transaminase levels and were HCV RNA positive. Patients were considered to have mixed cryoglobulinemia if two successive determinations of their serum cryoglobulin level were above 0.05 g/L. Mixed cryoglobulinemia-negative patients never had mixed cryoglobulins in their serum on multiple determinations. Subjects without HCV infection included 79 patients with histologically proven B-NHL, and 50 healthy blood donors. Serum samples were analyzed for TTV DNA by nested polymerase chain reaction, with two couples of primers in different regions of the genome, in two independent laboratories. RESULTS: In the group of HCV-positive patients, TTV DNA was found in one of 15 (6.7%) patients with B-NHL, and in nine of 51 (17.6%, P = 0.43) of those without B-NHL. Among HCV-positive patients without B-NHL, TTV DNA was more frequently found in those with type II mixed cryoglobulinemia vasculitis than in those without it (six of 16 (37.5%) versus two of 21 (9.5%), P = 0.05). In subjects without HCV infection, TTV DNA was present in 10 of 79 (12.7%) patients with B-NHL and in seven of 50 (14.0%, P = 0.82) blood donors. CONCLUSION: In patients chronically infected with HCV, TTV co-infection: (1) is not associated with the presence of B-NHL; and (2) is more frequently found in patients presenting a type II mixed cryoglobulinemia vasculitis.

Phase I clinical trial of monoclonal anti-human chorionic gonadotropin antibody in women with an ectopic pregnancy.

A phase-I clinical trial of a mouse monoclonal anti-human chorionic gonadotropin (hCG) antibody designated as HT13 was conducted in three patients treated for ectopic pregnancy. Doses of 5 and 25 mg of purified antibody were injected into patients. Monoclonal antibody serum levels dropped to 0 within 1 to 5 days. Human antibodies directed against mouse immunoglobulins were not detected at up to 41 days after injection. In one patient, the tubal pregnancy resolved, as confirmed by hCG levels of less than 10 mUl/mL and by tubal patency at hysterosalpingography. The time of resolution was 30 days. In two patients, salpingectomy was performed because of persistence of elevated hCG levels, whereas HT13 had a striking effect on progesterone (P) and estradiol (E2) serum levels. The injection of anti-hCG antibody did not appear to interfere with the subsequent fertility of patients, and two out of three patients later developed a successful pregnancy. While the precise role of antibody injection in the interruption of ectopic pregnancy remains speculative, the injection of monoclonal anti-hCG antibody appears to induce a dramatic decrease in the production of both P and E2.

Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies.

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.

Detection of a 45-kilodalton antigen overexpressed in a cisplatin-resistant human ovarian carcinoma cell line.

To characterize the membrane changes associated with cisplatin resistance, we raised monoclonal antibodies (MAbs) against a cisplatin-resistant subline (OV1/DDP) derived from a human ovarian carcinoma cell line (OV1/p). An MAb, designated OCP02, was selected for its particularly high affinity for the resistant cell line. It bound 3.1-fold higher to OV1/DDP cells than to OV1/p cells and recognized an M(r) 45K antigen. This antigen appeared to be present in several normal and tumorous tissues. Its distribution in normal tissues was mainly detected in tissues involved in secretory processes, suggesting that this antigen could be related to a transport mechanism in normal cells as well as in drug-resistant cells.

Routine use of Zenit RA, a novel chemiluminescent immunoanalyzer in autoimmune disease diagnosis.

The detection of antibodies is useful to diagnose and/or to classify autoimmune diseases as connective tissue diseases and vasculitis. Zenit RA is a fully automated immunoanalyzer. The aim of this study was to compare the predictive and discriminative performance of the Zenit RA anti-cyclic citrullinated peptide (CCP), anti-cardiolipin (aCL) and anti-ß 2 glycoprotein 1 (aB2GP1) tests to conventional ELISAs on clinically well-defined groups of patients and to daily evaluate the determination of anti-extractable nuclear antigen (ENA), anti-double stranded DNA (dsDNA), anti-myeloperoxidase (MPO) and anti-proteinase 3 (PR3) antibodies in a hospital laboratory. Reagents available on Zenit RA analyzer exhibit good diagnostic performances, regarding sensitivity, specificity, positive and negative predictive values. Global agreements between Zenit RA and conventional tests were from 90 to 98 % (Kappa-values ranging 0.56-0.94): 96 % for anti-CCP, 90-94 % for aCL and aB2GP1, 94 % for anti-dsDNA, 97 % for anti-ENA, 98 % for anti-MPO and 95 % for anti-PR3 antibodies. Zenit RA analyzer is easy to rapidly detect the most common autoantibodies in autoimmune diseases. This system has a potential to provide clinically useful data within a short time. Because of the flexibility of its work modalities, it is well adapted to determine antigenic specificities in daily practice.

Clinical significance of anti-Ro52 (TRIM21) antibodies non-associated with anti-SSA 60kDa antibodies: results of a multicentric study.

Ro52 antigen has recently been identified as TRIM21 protein, but the clinical significance of anti-Ro52/TRIM21 antibodies remains controversial. The aim of this multicentric study was to investigate the significance of anti-Ro52 antibodies without anti-SSA/Ro60 antibodies in various connective diseases. Sera were selected by each laboratory using its own method (ELISA, immunodot or Luminex technology), and then performed with ANA Screen BioPlex™ reagent (BIO-RAD). Among the 247 screened sera, 155/247 (63%) were confirmed as anti-Ro52 positive and anti-SSA/Ro60 negative. These sera were analyzed for the detection of other antibodies in relation with clinical settings. Isolated anti-Ro52 antibodies were detected in 89/155 (57%) sera. For the remaining sera (66/155), the main antibodies associations were Sm/SmRNP or Chromatin (n=38; 57%), Jo1 (n=17; 26%) and CenpB (n=9; 14%). Clinical data from the 155 patients showed high prevalence in autoimmune diseases (73%) including myositis or dermatomyositis (n=30), lupus (n=23); Sjögren and/or sicca syndrome (n=27); CREST or Systemic sclerosis (n=11) and autoimmune hepatitis (n=11). We found that pulmonary manifestations were often associated with the presence of anti-Ro52 antibodies (n=34, 22%), in addition with anti-tRNA synthetases, anti-SRP or anti-Ku antibodies (18/34) or isolated in half of cases (16/34). Separate detection of anti-Ro52 antibodies might be useful in related antisynthetase syndrome diagnosis. The presence of anti-Ro52 antibodies should probably precede development of autoimmune disease and must induce sequential follow-up of positive patients, particularly in interstitial lung disease progression.

Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene.

We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.

Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies.

To study antigenic sites present in the beta-subunit of human thyrotropin (hTSH), we produced site-specific antibodies directed against synthetic peptides analogous to the 1-18, 44-59, and 85-112 regions of the thyrotropin beta-subunit. The hTSH beta(1-18) peptide-carrier conjugate elicited antisera capable of binding to both radiolabeled hTSH and its beta-subunit whereas antibodies elicited against the hTSH beta(44-59) peptide-carrier conjugate bound only to the peptide. Thus, the NH2-terminal region of hTSH beta appears to be accessible at the surface of the hormone whereas the hTSH beta(44-59) region may be poorly accessible. Two monoclonal antipeptide antibodies that bound to 125I-hTSH beta, designated as TS01 and TS02, were selected after immunization with the hTSH beta(85-112) peptide-carrier conjugate. The antigenic site recognized by TS01 was located on the eight COOH-terminal(105-112) amino acid residues. TS02 antibody bound to an antigenic region included within Cys95 and Cys105. Both antigenic sites appeared to be more accessible on the free hTSH beta than on the hormone. Immunoblots performed on various preparations containing TSH revealed that TS02 antibody detected the beta-subunit from both the human and bovine species but not the rat TSH beta. Under reducing conditions, a low molecular weight material was identified in hTSH beta, likely caused by intrachain nicking.

Effect of 51p1-related gene copy number (V1-69 locus) on production of hepatitis C-associated cryoglobulins.

Monoclonal IgM in type II mixed cryoglobulins (MC) preferentially use 51p1-related immunoglobulin VH genes. In normal preimmune B lymphocytes, 51p1-related gene expression is proportional to the germ-line gene dosage, which can be 0-4. To determine whether 51p1-related gene dosage influences the occurrence of type II MC or the VH gene bias in cryoglobulin IgM, we studied 47 patients chronically infected with hepatitis C virus (HCV), 24 MC+, 23 MC-. By Western analysis, 11 cryoprecipitate IgM (46%) were detected by G6 (a marker for 51p1-related gene products), eight (33%) by Staphylococcal Protein A (a VH3 family marker), and five (21%) by neither, indicating a 23-fold bias favouring 51p1-related genes. All 11 MC+, G6+ patients possessed > or = 1 copy of a 51p1-related gene; nine of the 36 others had none. The mean copy number of 51p1-related genes was greater in MC+ than MC- patients, and in MC+, G6+ patients versus the 36 others (P < 0.04), but significant differences were not seen in analyses restricted to patients with > or = 1 copy of a 51p1-related gene. We conclude that when a 51p1-related gene is present, a strong bias favours G6+ IgM in HCV-associated type II MC, but this bias is not greatly increased by a high dosage of 51p1-related genes. Furthermore, patients lacking 51p1-related genes also produce MC, but with G6- IgM.

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)

Distribution of VRA09, an ovarian tumor-associated antigen, distinguishing borderline serous tumors.

We recently reported the characterization of an antigen designated VRA09, identified by a monoclonal antibody and overexpressed on the surface of vincristine-resistant human ovarian carcinoma cells. In the present study, we analyze the distribution of this antigen in normal and tumor tissues. Its pattern of expression appears to differ from that described for other drug-resistance- and/or tumor-associated antigens. In normal tissues, the antigen has a restricted histological distribution and appears to be localized in mesoderm-derived tissues. In tumor tissues, VRA09 expression was mainly detected in serous ovarian tumors. Indeed, VRA09 is strongly expressed in papillary serous cystadenocarcinomas and their metastases, and more specifically in the basement membranes of serous tumors of borderline malignancy. In contrast, no immunostaining was observed in normal ovarian tissue or benign tumors. The detection of this antigen may help to identify serous ovarian tumors by distinguishing tumors of low malignancy from cystadenocarcinomas.