PubMed Health⌕ Search

Biomedical subjects

J Hilgers

Publications and source records attributed to J Hilgers.

At least 19 recordsLinked to original sources
PubMed2001

Antibody-dependent cell-mediated cytotoxicity can be induced by MUC1 peptide vaccination of breast cancer patients.

Human polymorphic epithelial mucin (PEM, MUC1) is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of glandular epithelium and is over-expressed and hypo-glycosylated in adenocarcinomas. The extracellular part of the molecule consists mainly of a variable number of 20 amino acid repeats that contain cryptic epitopes exposed in malignancy. The objective of our study was to determine whether humanized MUC1 MAbs and Abs induced by vaccination of breast cancer patients with MUC1 peptides can effect an antibody-dependent cell-mediated cytotoxicity (ADCC). An in vitro assay has been set up in which the breast tumor cell line ZR-75-1 is used as target and PBMC of healthy donors as effector cells. Different target and effector cells, as well as various MUC1 MAbs were tested to optimize the efficacy of the in vitro assay. The humanized MAb HuHMFG-1, which recognizes the PDTR sequence in the MUC1 tandem repeat, induced a strong cell-mediated cytotoxicity. Nine MUC1-expressing tumor cell lines, including 3 bone marrow-derived cell lines, as well as 2 MUC1-transfected cell lines were susceptible to different extent to MUC1 Ab-dependent killing. Large variations in the killing capacity of PBMC from healthy donors were found. The NK cells were the essential effector cells for the MUC1 Ab-dependent killing. Plasma samples with induced high levels of MUC1 Ab were obtained from breast cancer patients repeatedly immunized with a KLH-conjugated 33-mer or 106-mer MUC1 tandem repeat. Pre- and post-vaccinated plasma samples of these patients were compared in the ADCC assay and it could be clearly demonstrated that the induced MUC1 Abs can effect tumor cell killing. MUC1 Ab-dependent cell-mediated tumor cell killing may occur in vivo and the ADCC assay can be applied to monitor MUC1 vaccination trials.

Amino Acid Sequence↗
PubMed2001

T cell infiltration and MHC I and II expression in the presence of tumor antigens: An immunohistochemical study in patients with serous epithelial ovarian cancer.

OBJECTIVES: Ovarian cancer is a frequent cause of death among women with gynaecologic malignancies despite the introduction of combination chemotherapy. There is therefore a need for new therapeutic strategies for patients with ovarian cancer, such as cellular immunotherapy. In this immunohistochemical study we analysed the expression of three tumor antigens, p53, HER-2/neu and MUC-1 in relation to the expression of major histocompatibility complex (MHC) class I and II on tumor cells, and we searched for the presence of (activated) immune effector cells at the tumor site. STUDY DESIGN: The study was carried out retrospectively in tumor tissue from 29 patients with serous ovarian cancer. Material used had been formalin fixed and paraffin embedded. Material had been obtained from 15 patients at staging laparotomy and from 14 patients during second look or intervention laparotomy. RESULTS: A positive staining for p53 was found in 19/29 (66%) of the tumors, with a high positivity in 13/29 (45%). HER-2/neu and MUC-1 staining was positive in 8/29 (28%) and 21/28 (75%), respectively. Downregulation of MHC class I on tumor cells was found in a minority of the patients, beta-2-microglobin (beta2m) was expressed on tumor cells in all patients. High staining for CD45RO correlated with a high positive staining for granzyme-B (R=0.40, P=0.04) and TIA-1 (R=0.39, P=0.04). A statistically significant better survival in the group with lower stage of disease was found. CONCLUSIONS: As only three out of 29 patients were negative for the tumor antigens p53, HER-2/neu and MUC-1, immunotherapy aiming at all three could serve almost all patients with ovarian cancer. We found that granzyme-B, TIA-1 and CD45RO+ T cells are present in the tumor biopsies, increasing this number by immunotherapy may be beneficial. The immune escape mechanism by MHC class I and beta2m downregulation seems to be of minor importance. Our data support the view that immunotherapy may offer new possibilities with high specificity in ovarian cancer.

Adult↗
PubMed2001

MUC1 (EMA) is preferentially expressed by ALK positive anaplastic large cell lymphoma, in the normally glycosylated or only partly hypoglycosylated form.

AIMS: To investigate whether MUC1 mucin, a high molecular weight transmembrane glycoprotein, also known as epithelial membrane antigen (EMA), differs in its expression and degree of glycosylation between anaplastic large cell lymphoma (ALCL) and classic Hodgkin's disease (HD), and whether MUC1 immunostaining can be used to differentiate between CD30 positive large cell lymphomas. METHODS/RESULTS: Using five different monoclonal antibodies (E29/anti-EMA, DF3, 139H2, VU-4H5, and SM3) that distinguish between various MUC1 glycoforms, high MUC1 expression (50-95% of tumour cells positive) was found in 13 of 17 anaplastic lymphoma kinase (ALK) positive systemic nodal ALCLs, and in one of 20 cases of classic HD. Scattered or focal staining (< 25% of tumour cells) was seen in two additional ALK positive systemic ALCLs, two additional classic HD cases, and in three of 20 cases of ALK negative systemic nodal ALCL. Primary cutaneous ALCL showed no staining with the anti-MUC1 antibodies. Antibodies detecting hypoglycosylated MUC1 were found to be absent in all lymphomas (SM3) or present in only six of 15 ALK positive ALCLs (VU-4H5). CONCLUSIONS: MUC1 is preferentially expressed by a subtype of systemic nodal ALCL, characterised by ALK expression, but is found in only a few cases of classic HD and ALK negative ALCL. Therefore, although MUC1 could be used in a panel of markers for CD30 positive lymphomas, it is probably not a valuable tool to differentiate between ALK negative CD30 positive large cell lymphomas. Finally, the degree of MUC1 glycosylation in lymphomas is relatively high, compared with the aberrant hypoglycosylation found in adenocarcinomas.

Anaplastic Lymphoma Kinase↗
PubMed2000

Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma.

Soluble MUC1 (sMUC1) levels are elevated in many MUC1(+) cancers. We and others have shown that MUC1 is expressed on multiple myeloma (MM) plasma cells and B cells. In this study, we measured sMUC1 levels in bone marrow (BM) plasma from 71 MM patients and 21 healthy donors (HDs), and in peripheral blood (PB) plasma from 42 MM patients and 13 HDs using an immunoassay that detects the CA27.29 epitope of MUC1. sMUC1 levels were found to be significantly greater (mean 31.76 U/mL, range 5.69 to 142.48 U/mL) in MM patient BM plasma versus HD BM plasma (mean 9.68 U/mL, range 0.65 to 39.83 U/mL) (P <. 001). Importantly, BM plasma sMUC1 levels were related to tumor burden because sMUC1 levels were significantly higher for MM patients with active disease (34.62 U/mL, range 5.69 to 142.48 U/mL) versus MM patients with minimal residual disease (16.16 U/mL, range 5.7 to 56.68 U/mL) (P =.0026). sMUC1 levels were also elevated in the PB plasma of MM patients (32.79 U/mL, range 4.15 to 148.84 U/mL) versus HDs (18.47 U/mL, range 8.84 to 42.49) (P =.0052). Lastly, circulating immunglobulin M (IgM) and IgG antibodies to MUC1 were measured in 114 MM patients and 31 HDs, because natural antibodies to MUC1 have been detected in patients with other MUC1-bearing malignancies. These studies demonstrated lower levels of circulating IgM (P <.001) and IgG (P =.078) antibodies to MUC1 in MM patients compared with HDs. Our data therefore show that in MM patients, sMUC1 levels are elevated and correlate with disease burden, whereas anti-MUC1 antibody levels are decreased.

Autoantibodies↗
PubMed2000

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Animals↗
PubMed2000

Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and n-acetylgalactosamine (GalNAc) peptides.

Antibodies (Abs) to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. We compared the specificity of natural and induced MUC1 Abs with the objective of defining an effective MUC1 vaccine for active immunotherapy of adenocarcinoma patients. Serum samples, selected out of a screened population of 492 subjects for their high levels of IgG and/or IgM MUC1 Abs, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients immunized with MUC1 peptides coupled to keyhole limpet hemocyanin (KLH) and mixed with QS-21. The samples were tested with enzyme-linked immunoassays for reactivity with (1) overlapping hepta- and 20-mer peptides spanning the MUC1 tandem repeat sequence; (2) two modified 60-mer peptides with substitutions in the PDTR (PDTA) or in the STAPPA (STAAAA) sequence of each tandem repeat; and (3) four 60-mer glycopeptides with each 1, 2, 3 and 5 mol N-acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined, indicating that Abs directed to more than one region of the MUC1 peptide core can coexist in one and the same subject. The most frequent minimal epitopic sequence of natural MUC1 IgG and IgM Abs was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG Abs predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 Abs from breast cancer patients reacted more strongly with the N-acetylgalactosamine (GalNAc) peptides than with the naked 60-mer peptide, while reactivity with the GalNAc-peptides was significantly reduced (2-tailed p < 0.0001) in the MUC1 IgG and IgM Abs induced by MUC1 peptide vaccination. Whereas in cancer patients glycans appear to participate in epitope conformation, the epitope(s) recognized by MUC1 Abs induced by peptide vaccination are already masked by minimal glycosylation. Therefore, our results indicate that a MUC1 glycopeptide would be a better vaccine than a naked peptide.

Acetylgalactosamine↗
PubMed2000

Functional topography of discrete domains of human CD38.

In addition to its catalytic activities as ecto-NAD+ glycohydrolase (NADase), CD38 displays the ability to transduce signals of biological relevance. Indeed, ligation of CD38 on peripheral blood mononuclear cells (PBMC) by agonistic monoclonal antibodies (mAbs) is followed by the transcription and secretion of a vast array of regulatory cytokines. The present work addresses the issue of whether the signals leading to calcium (Ca2+) mobilization, lymphocyte proliferation and release of cytokines is dependent on the epitopes recognized by the individual mAbs. Competition binding analysis identifies two families of mAbs, namely IB4, IB6 and AT2 on one side and OKT10, SUN-4B7 and AT1 on the other. Each mAb family binds epitopes that are completely or partially common. However, the functional activities of the CD38 molecule can not be simply attributed to the epitopes engaged: for instance, IB4 and OKT10 mAbs, which bind different epitopes, perform as agonistic mAbs in inducing PBMC proliferation and interferon (IFN)-gamma secretion. SUN-4B7 yields intermediate effects, whereas IB6, AT1 and AT2 mAbs are totally ineffective. The effects mediated by IB4 and OKT10 mAbs are apparent in 80% of the healthy individuals studied, whereas the effects of SUN-4B7 mAb operate only in 25% of the donors. Interleukin (IL)-6 secretion was observed in all individuals analyzed, irrespective of the epitopes triggered and of mAbs used to ligate the CD38 molecule. In addition, IB4 is the only mAb able to induce significant intracellular Ca2+ fluxes.

ADP-ribosyl Cyclase↗
PubMed2000

Survival in early breast cancer patients is favorably influenced by a natural humoral immune response to polymorphic epithelial mucin.

PURPOSE: Polymorphic epithelial mucin (PEM or MUC1) is being studied as a vaccine substrate for the immunotherapy of patients with adenocarcinoma. The present study analyzes the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relates the presence of these antibodies in pretreatment serum to outcome of disease. MATERIALS AND METHODS: We measured immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to MUC1 with an enzyme-linked immunoassay (PEM.CIg), which uses a MUC1 triple-tandem repeat peptide conjugated to bovine serum albumin, in pretreatment serum samples obtained from 154 breast cancer patients (52 with stage I disease and 102 with stage II) and 302 controls. The median disease-specific survival time of breast cancer patients was 74 months (range, 15 to 118 months). A positive test result was defined as MUC1 IgG or IgM antibody levels equal to or greater than the corresponding rounded-up median results obtained in the total breast cancer population. RESULTS: A positive test result for both MUC1 IgG and IgM antibodies in pretreatment serum was associated with a significant benefit in disease-specific survival in stage I and II (P =.0116) breast cancer patients. Positive IgG and IgM MUC1 antibody levels had significant additional prognostic value to stage (P =.0437) in multivariate analysis. Disease-free survival probability did not differ significantly. However, stage II patients who tested positive for MUC1 IgG and IgM antibody and who relapsed had predominantly local recurrences or contralateral disease, as opposed to recurrences at distant sites in the patients with a negative humoral response (P =.026). CONCLUSION: Early breast cancer patients with a natural humoral response to MUC1 have a higher probability of freedom from distant failure and a better disease-specific survival. MUC1 antibodies may control hematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1-expressing tumor cells. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides in an adjuvant setting may favorably influence the outcome of disease.

Adolescent↗
PubMed1999

Aberrant expression of MUC1 mucin in ductal hyperplasia and ductal carcinoma In situ of the breast.

MUC1 mucin is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of normal glandular epithelia. In many human adenocarcinomas, this protein is up-regulated and/or underglycosylated, and its expression changes from apical to the entire cell membrane. It is thought that entire cell membrane expression of MUC1 reduces cell-cell and cell-extracellular matrix interactions and therefore may facilitate invasive growth and development of metastases. In this study, we determined immunohistochemically the expression of normal and underglycosylated MUC1 in normal breast tissue (n = 8) and in a spectrum of breast lesions, including usual ductal hyperplasia (n = 23), atypical ductal hyperplasia (n = 7), and ductal carcinoma in situ (DCIS) (n = 22). We used 4 monoclonal antibodies; 115D8 is directed to a glycopeptide, the other 3 to the peptide core of the molecule, of which 139H2 is not affected by the degree of glycosylation of MUC1, whereas SM3 and VU-4-H5 stain only underglycosylated forms. All cases showed apical positivity for 115D8 and 139H2. Entire cell membrane expression of fully (normal) glycosylated MUC1 was mainly found in DCIS lesions. Apical staining of SM3 was found in 38% of normal cases and 60% of the ductal lesions with no difference between the different subgroups. Apical staining of VU-4-H5 was found more often in DCIS (27%) than in normal tissue or ductal hyperplasia (3%). Membrane expression of underglycosylated MUC1 was found only in poorly differentiated DCIS. In conclusion, aberrant expression of MUC1, i.e., on the entire cell membrane and/or underglycosylated forms, can be found in ductal hyperplasia with atypia and especially in DCIS of the breast. This finding implies that these lesions with aberrant expression are at higher risk for developing subsequent invasive breast carcinoma.

Antibodies, Monoclonal↗
PubMed1999

Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads.

The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3 weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens.

Adult↗
PubMed1999

Cellular and humoral immune responses to MUC1 mucin and tandem-repeat peptides in ovarian cancer patients and controls.

The objective of this study was to demonstrate the presence of proliferative T cell responses to human polymorphic epithelial mucin (MUC1) and its tandem-repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and to correlate these cellular responses to a humoral response to MUC1. PBMC were obtained from 6 healthy women, from 13 women in the third trimester of pregnancy and from 21 ovarian cancer patients. Only 1 of the 6 healthy women showed a weak primary proliferative response (stimulation index, SI <2) to a 20-mer MUC1 tandem-repeat peptide in the presence of interleukin-2 (IL-2). In PBMC from 5/13 pregnant women (38%) a weak response could be induced by the 20-mer and/or 60-mer tandem-repeat peptides (SI < or =3.0) and in PBMC from 8/15 ovarian cancer patients (53%) 20-mer and/or 60-mer MUC1 tandem-repeat peptides induced primary responses (SI < or =5.4). MUC1 mucin purified from a breast tumor cell line and/or from urine of a healthy donor had a relatively strong stimulating effect (SI < or =19) on PBMC from 4 of 16 ovarian cancer patients (25%). In contrast, in PBMC of 9 ovarian cancer patients stimulated by the addition of a Candida albicans extract, MUC1 mucin strongly inhibited proliferation. This inhibition could partially be abrogated by the addition of IL-2. MUC1 (CA 15.3 assay) and free circulating MUC1 IgG and IgM antibodies (PEM.CIg assay) were determined in the plasma of all subjects. The MUC1 and the free circulating MUC1 IgG antibody plasma levels were significantly higher in the ovarian cancer patients than in the healthy women. Although no significant correlations were found between MUC1 mucin, MUC1 Ab plasma levels and the individual proliferative responses to the MUC1 antigens, an association may exist between them, since all three are significantly higher in the ovarian cancer patients than in the healthy women.

Amino Acid Sequence↗
PubMed1999

Quantification of MUC1 in breast cancer patients. A method comparison study.

OBJECTIVE: To compare the performance of four serum assays for the quantification of MUC1 in breast cancer patients. STUDY DESIGN: A total of 282 serum samples were evaluated with two automated (Boehringer Mannheim Enzymun-Test CA 15-3 and Chiron ACS BR) and two manual assays (Centocor CA 15-3 radioimmunoassay [RIA] and Biomira Truquant BR RIA). Sera were obtained from healthy controls (n=50), patients with benign (n=25) and malignant breast disease (n=77) and patients with other malignancies (n=69). In addition, sera from pregnant women (n=56) and patients with liver cirrhosis (n=5) were included. RESULTS: Intraassay coefficients of variation (C.V.s) were highest for the manual Centocor CA 15-3 assay (7.4% for values below 50 kU/l and 8.1% for values above 180 kU/l). Interassay C.Vs were highest for the manual Truquant BR assay (11.7% for the lower concentration values and 18.6% for the higher concentration values). False positive rates ranged between 0% for the Centocor CA 15-3 RIA and 14% for the ACS BR assay (cut-off: 30 kU/l). In monitoring breast cancer patients all four assays show similar patterns, although absolute MUC1 values found may differ up to 50%. CONCLUSION: For monitoring purposes all assays perform equally well, however, automated assays show lower inter- and intraassay variability, especially in the higher value range. Therefore we recommend the use of the same, automated, assay for quantification of MUC1 during the follow-up of breast cancer patients.

Biomarkers, Tumor↗
PubMed1999

Influence of chemotherapy on the expression of p53, HER-2/neu and proliferation markers in ovarian cancer.

OBJECTIVE: Mutated p53 and HER-2/neu play a role in the etiology of ovarian cancer. It is important to know whether the expression of these proteins is affected by platinum-containing chemotherapy. STUDY DESIGN: Together with the cell proliferation markers Ki-67 and PCNA, the expression of p53 and HER-2/neu was assessed before and after chemotherapy. Paraffin-embedded tumor sections from 20 patients with ovarian cancer and four patients with benign disorders of the ovaries (controls) were analyzed. The expression of p53 was determined by the antibodies DO-1 and BP53-12. In addition to HER-2/neu and PCNA specific antibodies, MIB-1 was used to detect Ki-67. RESULTS: The expression of all markers was higher in ovarian cancer patients than in non-malignant controls. MIB-1 showed a significant increase of expression after chemotherapy (P=0.002). HER-2/neu, p53 and PCNA also showed a clear increase after treatment, but this was not statistically significant. HER-2/neu is of prognostic relevance with respect to the response to chemotherapy (P=0.005) and survival (P=0.0002). CONCLUSION: The different markers tested all increase after chemotherapy, but the differences are not statistically significant. Low HER-2/neu expression correlates with good outcome at second look.

Adult↗
PubMed1999

Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen.

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Antibodies, Monoclonal↗
PubMed1998

Epitope specificity of 30 monoclonal antibodies against cytokeratin antigens: the ISOBM TD5-1 Workshop.

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins. i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology. All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270-400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340-365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270-429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322-342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311-335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346-367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356-370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients' blood for tumor marker evaluation.

Amino Acid Sequence↗
PubMed1998

An enzyme-linked immunosorbent assay for the measurement of circulating antibodies to polymorphic epithelial mucin (MUC1).

INTRODUCTION: About one-third of breast and ovarian carcinoma patients have circulating antibodies reactive with polymorphic epithelial mucin (MUC1), either free or bound to immune complexes. While the presence of these immune complexes has prognostic significance in breast cancer patients, the significance of free MUC1 antibodies is less clear. The objective of this study was to develop a reliable assay for the accurate determination of circulating free antibodies to MUC1. MATERIAL AND METHODS: We developed an enzyme-linked immunosorbent assay (ELISA) (PEM.CIg) employing a 60 mer peptide (a triple tandem repeat sequence of the MUC1 peptide core) conjugated to bovine serum albumin and peroxidase-labeled antihuman immunoglobulin G or M antibodies. The assay was standardized and its analytical performance evaluated. A total of 492 serum samples were obtained from 40 healthy men, from 201 healthy women (including 55 women without a history of pregnancy and 45 pregnant women), and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients. MUC1 serum levels were determined with commercial CA 15-3 tests. RESULTS: Circulating antibodies to MUC1 are present both in healthy subjects and in breast cancer patients. The within- and between-assay coefficients of variation were, respectively, 2 and 12% for the IgG determinations and 1.2 and 3% for the IgM determinations. Correlation coefficients for serially diluted serum samples ranged from 0.9998 to 0.9920 for IgG and from 0.9996 to 0.9818 for IgM determinations. The reactivity of serum samples was partially blocked by the addition of various MUC1 peptides and by MUC1 mucin. The inhibiting effect of modified 60 mer peptides suggests the presence of antibodies directed to more than one epitope. CONCLUSIONS: The PEM. CIg assay is a reliable ELISA for measuring free MUC1 antibodies in serum. We are in the process of relating the results obtained in the breast cancer group to disease outcome to evaluate its prognostic significance. In addition, the assay may become a useful tool for vaccine therapy monitoring.

Autoantibodies↗
PubMed1998

Epitope mapping of human alpha-fetoprotein.

Thirty monoclonal antibodies (MoAbs) to human alpha-fetoprotein (AFP) were compared with one another by two methods: Immunoaffinity electrochromatography or additive ELISA. The first method permitted to analyse the epitopes of native AFP in solution [Abelev et al., Immunol Lett 1994;40:133-138] while the other approach also detects the epitopes of conformationally modified (partly denatured) AFP fixed on the plastic [Yazova et al., Immunol Lett 1990;25:325-330]. Competitive analysis of all MoAbs revealed 10 epitopes, 9 expressed on native AFP and 1 only on the partly denatured molecule. The cross-reactions between separate MoAbs allowed to include them into 6 distinct epitope clusters, or immunodominant groups with the characteristic patterns of reactivity. The obtained epitope map of AFP is necessary for the construction of AFP detection kits as well as for the identification of its antigenic and functional subfractions.

Antibodies, Monoclonal↗