Production of antibody to tetanus toxoid by continuous human lymphoblastoid cell lines.

Peripheral lymphocytes from human volunteers boosted with tetanus toxoid were cultured after in vitro infection with Epstein-Barr virus. Forty-four continuous lymphoblastoid lines were established which continued to secrete human gamma globulin; seven of these secreted antibody to tetanus toxoid. Subcultures derived from limiting dilution experiments continued to secrete the antibody. Some of these antibody-secreting cells have been in continuous culture for more than 6 months.

CA 19-9 and CA 125 levels in the sera of normal blood donors in relation to smoking history.

CA 19-9 and CA 125 serum levels were evaluated among smoking and nonsmoking healthy blood donors. Smoking did not elevate mean levels of either CA 19-9 or CA 125 in the sera of 496 of these blood donors from Philadelphia, PA. Mean CA 19-9 levels were slightly higher among females than among males. Among smokers there was a trend toward slightly increasing CA 19-9 serum levels with increased age, which was significant among the male donors. Trends toward slightly decreased mean serum levels of CA 125 among smokers were of borderline significance. Serum CA 19-9 and CA 125 levels in none of these donor subpopulations was elevated compared to levels reported by others for gastrointestinal or ovarian carcinoma patients, respectively. Therefore, smoking status should not interfere with use of either the CA 19-9 or CA 125 assays for diagnostic or monitoring applications.

Characterization of the CA 125 antigen associated with human epithelial ovarian carcinomas.

The murine monoclonal antibody OC125 reacts with an antigenic determinant (CA 125) found on a high-molecular-weight glycoprotein complex present in the serum of greater than 80% of women with epithelial ovarian cancer. The antigen expressing CA 125 (CA 125 antigen) isolated from the sera of ovarian carcinoma patients was shown by gel electrophoresis, molecular size exclusion chromatography, and buoyant density ultracentrifugation to have similar immunological and physical characteristics to antigen isolated from an ovarian cancer cell line (OVCA 433) and human milk. A composite sodium dodecyl sulfate: polyacrylamide:1.0% agarose gel resolved the CA 125 activity from the three sources of antigen into disperse bands of similar electrophoretic mobilities with apparent masses of 200,000 to 1 million daltons. The buoyant densities of the CA 125 antigen complexes from human serum, OVCA 433 cells, and human milk were in the range of 1.36 to 1.46 g/ml. Isolation of CA 125 antigen of higher purity from OVCA 433 supernatant was achieved by a series of steps including OC125 immunoaffinity chromatography. Subsequent resolution of this purified CA 125 antigen complex by sodium dodecyl sulfate:polyacrylamide gel electrophoresis gave rise to a band at approximately 200,000 daltons. Treatment of the CA 125 antigen from OVCA 433 cells with 10 mM periodic acid resulted in no loss of activity. Reduction and alkylation in 6 M guanidine-HCl or treatment at 100 degrees C for 20 min resulted in complete loss of activity. Exoglycosidase treatments did not result in loss of activity, whereas protease digestion eradicated all activity. These data strongly suggest that the CA 125 antigenic determinant is composed of, at least in part, conformationally dependent peptide.

Purification and composition of a novel gastrointestinal tumor-associated glycoprotein expressing sialylated lacto-N-fucopentaose II (CA 19-9).

Monoclonal antibody 1116NS 19-9 (Mab 19-9) exhibits selective reactivity with human gastrointestinal carcinomas and recognizes a carbohydrate determinant (CA 19-9) defined as a sialylated lacto-N-fucopentaose II. A scheme was devised for the purification of a human gastrointestinal tumor-associated glycoprotein antigen expressing CA 19-9 from colorectal carcinoma cell line SW1116 culture media in high yield. The key steps in the purification were immunoaffinity column chromatography with Mab 19-9 followed by reduction and alkylation of the specifically bound proteins in the presence of 6 M guanidine hydrochloride and a second Mab 19-9 immunoaffinity fractionation. The purified CA 19-9 containing glycoprotein ran as a single band on sodium dodecyl sulfate-polyacrylamide gradient gels with an apparent molecular mass of 210 kilodaltons. In the absence of detergents, this purified glycoprotein apparently reassociated to form aggregates of 600-2000 kilodaltons molecular mass as determined by size-exclusion chromatography. Amino acid analysis of CA 19-9 containing glycoprotein revealed that serine, threonine, and proline together accounted for greater than 35% of the amino acid residues, consistent with a mucin-like structure for the protein. Carbohydrate compositional analysis, however, was in contrast to a typical mucin with a fucose:mannose:galactose:N-acetylgalactosamine: N-acetylglucosamine:N-acetylneuraminic acid molar ratio of 4:1:12:2.5:5:5. The presence of both N-acetylgalactosamine and mannose suggested that both O- and N-linked oligosaccharides may exist on CA 19-9 containing glycoprotein. Protein and carbohydrate analyses indicated that this novel tumor-associated glycoprotein was 85% carbohydrate by weight. This purification procedure may be applicable to the isolation of other epithelial tumor-associated antigens.

Chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing.

The MOv18 (gamma 1, kappa) and MOv19 (gamma 2a, kappa) murine monoclonal antibodies (MAbs) recognize different epitopes on the human folate binding receptor which is overexpressed on 90% of nonmucinous epithelial ovarian tumors. A chimeric murine-human (human gamma 1, kappa) version of both antibodies was constructed and expressed. The genes encoding the murine heavy and light chain variable regions of the MOv18 and MOv19 MAbs were cloned from the parental hybridomas, fused with genes encoding the human heavy (gamma 1) and light (kappa) chain constant regions, respectively, and expressed in the SP2/0 murine myeloma cell line. Using human peripheral blood mononuclear cells as effector cells and conditions that provide for maximum lysis (effector target = 50:1, saturating antibody concentration), the murine MOv18 MAb (IgG1) mediated variable levels of specific cytolysis of the target ovarian cancer cell line IGROV1. In contrast, the chimeric MOv18 MAb mediated higher and more consistent lysis even at a 10-100-fold lower antibody concentration. The murine MOv19 MAb (IgG2a) mediated specific lysis of IGROV1 cells, and the chimeric version of this antibody mediated an amount of lysis at least equal to that mediated by its murine counterpart. A comparison of the ED50 values obtained for the murine MOv19 and chimeric MOv19 antibodies indicates that the chimeric MOv19 MAb was 3 to 10 times more potent than the murine MOv19 antibody. In addition, the ED50 values obtained for the chimeric MOv18 and chimeric MOv19 MAbs were similar, indicating that these MAbs are equally potent. The level of maximal lysis obtained was dependent on the number of target molecules/cell; the same high level of lysis mediated by cMOv18, MOv19, and cMOv19 was observed with both IGROV1 and OvCA432 target cells. However, only low levels of lysis were obtained when the SW626 cell line, which expresses 1 x 10(4) folate binding protein sites/cell, was used as a target. An equimolar mixture of the chimeric MOv18 and MOv19 MAbs was no more effective in the mediation of lysis than an equivalent amount of either chimeric MAb alone. These data suggest that the folate binding receptor is expressed on IGROV1 cells at a density sufficient to provide for optimal levels of antibody-mediated lysis using a single chimeric antibody directed at the folate binding receptor.

Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas.

CA 125 is an antigenic determinant expressed by greater than 80% of nonmucinous epithelial ovarian carcinomas. An immunoradiometric assay has been developed using a murine monoclonal antibody (OC125) to quantitate CA 125 in human serum. This immunoradiometric assay was optimized for specificity, sensitivity, and performance characteristics. Using a simultaneous immunoradiometric assay, the mean CA 125 concentration in 56 sera from healthy individuals was 11.2 +/- 5.4 (S.D.) units/ml, with 9.7 +/- 3.2 units/ml for 30 males and 13.1 +/- 6.8 units/ml for 26 females. A reference value of 35 units/ml included all 56 normals and excluded 86 of 105 (82%) ovarian carcinoma patients. This reference value also excluded 9 of 142 patients (6%) with benign diseases, but if the upper limit of normal was set at 65 units/ml, only 3 of 142 (2%) patients with benign diseases had elevated serum CA 125 levels, whereas 77 of 105 (73%) ovarian carcinoma patient sera remained positive. The ability of researchers, with this assay, to discriminate between CA 125 values in sera of patients with ovarian carcinoma and those of healthy individuals and patients with benign disease suggests that the assay deserves continued evaluation for monitoring and early diagnosis of ovarian cancer.

Monoclonal antibodies to carcinoembryonic antigen produced by somatic cell fusion.

Hybridoma cell lines secreting monoclonal antibodies to carcinoembryonic antigen (CEA) were generated by fusing mouse immune lymphocytes with the mouse myeloma variant cell line, NS-1. Antibody secreted by one cloned hybrid cell line could bind only a select portion of the CEA bound by the commercially available goat anti-CEA antiserum used in clinical assays. Radiolabeled CEA could be purified on a monoclonal antibody affinity column. Incorporation of this purified radiolabeled CEA in a double-antibody solid-phase assay with goat anti-CEA antiserum led to an approximately 2.5-fold increase in sensitivity of the assay. Genetically stable hybrid clones may be sources of virtually unlimited quantities of such antibodies which may have potential utility in improving the cancer specificity of clinical assays.

Human T-lymphocytes targeted against an established human ovarian carcinoma with a bispecific F(ab')2 antibody prolong host survival in a murine xenograft model.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.

Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis.

In the past year, gp38, a glycosyl-phosphatidylinositol linked membrane protein that is overexpressed in some malignant tissues, has been shown to be the folate receptor. Using immunohistochemical techniques with the monoclonal antibody MOv19 against gp38, we evaluated the cellular localization of folate receptors in normal human tissues, which are potential target sites for drugs that utilize this uptake mechanism. The choroid plexus was intensely positive with staining limited to the epithelium, which in some foci had a distinct bilaminar pattern limited to the luminal and basal surfaces. The epithelium of the fallopian tube, uterus, and epididymis was highly immunoreactive. The acinar cells of the breast, submandibular salivary, and bronchial glands also showed intense staining as did the trophoblastic cells of the placenta. In the kidney reactivity was localized to the proximal tubules. Lung alveolar lining including type I and II pneumocytes stained intensely. Limited but focal reactivity was noted in the vas deferens, ovary, thyroid, and pancreas. This study in conjunction with previous work showing marked overexpression of folate receptor in some malignant cells suggests that the folate receptor may be an important target for diagnostic or therapeutic exploitation and indicates sites of potential drug toxicity.

Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues.

In some epithelial cells studied in vitro a membrane-bound folate receptor initiates the process for cell accumulation of 5-methyltetrahydrofolic acid. This receptor was found to be GP38, an overexpressed, glycosyl-phosphatidylinositol anchored glycoprotein, recognized by two monoclonal antibodies, designated MOv18 and MOv19. Using immunoblotting with MOv19, radioimmunoassay with MOv18 and 19, Northern blot analysis, and radioligand binding when possible, we describe the limited expression of the folate receptor in a large number of normal tissues from four autopsies. The immunoblot technique detected as little as 40 pg (approximately 1 fmol) of receptor protein. Choroid plexus consistently had the largest amount of folate receptor. Other tissues containing substantial amounts of receptor included lung, thyroid, and kidney. The liver, intestines, muscle, cerebellum, cerebrum, and spinal cord were immunologically nonreactive. Folate receptor gene expression determined by Northern blot analysis confirmed these observations. We also show that several malignant cell lines express significantly more receptor than normal epithelial cells or fibroblasts. Specifically, malignant cells bound greater than or equal to 20 pmol [3H]folate/10(6) cells, while normal epithelial cells and fibroblasts bound less than or equal to 1 pmol radioligand/10(6) cells. We also demonstrate that 4 of 6 brain tumors overexpress the folate receptor. These studies reveal the limited normal tissue distribution of the folate receptor, a cell surface protein which may be a useful immunological or pharmacological target for the development of selective cancer therapy.

Tumorigenicity in athymic mice of the human colon carcinoma cell line SW1116 expressing the tumor-associated antigenic determinant CA 19-9.

The human colorectal carcinoma cell line SW1116 under optimal growth conditions synthesized and shed antigens bearing the monoclonal antibody-defined carbohydrate determinant CA 19-9. Antigen expressing CA 19-9 in cell culture supernatant was quantitated by an immunoradiometric assay for CA 19-9. Injection of SW1116 cells s.c. into athymic BALB/c mice resulted in the growth of moderately differentiated tumors possessing a distinct morphological resemblance to a typical adenocarcinoma of the colon. Intervals to tumor appearance were dependent on inoculum dose, but 95% of mice at both 5 X 10(6) and 10(7) cells/mouse developed tumors within 14 to 21 days. CA 19-9 antigen was detected in the sera of all nude mice with SW1116 tumors, and antigen concentration correlated (r = 0.77) with tumor volume throughout the 9-week study. The half-life of this antigen in serum following tumor excision from nude mice was 6.5 +/- 1.5 (S.D.) hr. Carcinoembryonic antigen was also detected in serum from mice bearing SW1116 tumors by an immunoradiometric assay for carcinoembryonic antigen, but its concentration correlated (r = 0.86) with tumor volume for only the first 4 weeks of tumor growth. Significant levels of endogenous immunoglobulin G1 and immunoglobulin G3 antibodies to CA 19-9 antigen were found in the serum of nude mice with SW1116 tumors by radioimmunodiffusion, but no apparent relationship between antibody titer and tumor growth or CA 19-9 antigen level in serum was evident. This tumor model may be useful in devising radioimmunodetection and immunotherapeutic strategies for primary and metastatic human colon carcinomas.

Coexpression of different antigenic markers on moieties that bear CA 125 determinants.

CA 125 has been extensively evaluated as a serum marker for monitoring patients with epithelial ovarian carcinoma. Recently, consideration has been given to the use of CA 125 as one component in a strategy for early detection of this disease. A number of benign conditions can, however, increase CA 125 in serum, limiting the utility of a single antigen determination for identifying ovarian cancer patients. Coexpression of different epitopes on the high molecular weight complexes that express CA 125 determinants might provide a more specific test for malignant disease, provided that adequate sensitivity were maintained. To determine how frequently determinants are coexpressed, macromolecular moieties containing CA 125 determinants have been isolated from ascites fluid of ovarian cancer patients by immunoaffinity chromatography. CA 125+ moieties have been probed on Western transfers with several murine monoclonal antibodies that recognize distinct tumor-associated epitopes. Marked heterogeneity was observed between patients with regard to antigenic determinants that could be coexpressed with CA 125. A fraction of ascites fluids from different ovarian cancer patients contained moieties which bound to OC 125 on a solid phase immmunoadsorbent and which also bound 125I-labeled monoclonal antibodies NS 19-9, B72.3, DF3, or the novel murine monoclonal antibody OC 3632 in a double determinant immunoradiometric assay. Serum samples were evaluated from patients with ovarian cancer and from apparently healthy individuals. Coexpression of TAG 72 and CA 125 was observed most frequently. When the double determinant assay for coexpression of TAG 72 and CA 125 was compared to assays for the individual antigens, the assay for coexpression was substantially less sensitive than those for the individual markers.

Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.

Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.

New chimeric anti-pancarcinoma monoclonal antibody with superior cytotoxicity-mediating potency.

The monoclonal antibodies (MAbs) 323/A3 and 17-1A both recognize a 40-kDa carcinoma-associated epithelial glycoprotein (EGP40). MAb 17-1A has been used in many therapeutic trials as an immunotherapeutic agent to combat advanced colorectal cancer, and about 5-10% overall responses have been observed. It has been shown that MAb 323/A3 has a higher affinity than 17-1A, which might be an advantageous feature for a therapeutic agent. In our immunohistological studies different reaction patterns of these two MAbs were observed, suggesting that MAb 323/A3 reacts more intensely with carcinoma cells than MAb 17-1A. This also suggests that MAb 323/A3 might be a more effective immunotherapeutic tool. Because chimerization may reduce the immunogenicity of the murine MAb 323/A3 and increase the interaction with human effector mechanisms, we developed a chimeric form of murine MAb 323/A3. MAb 323/A3 heavy and light chain variable genes were cloned and grafted onto human C gamma 1 and C kappa domains, respectively. A chimeric antibody-producing cell line was established by transfection of the chimeric constructs into a nonproducing myeloma cell. The chimeric and murine 323/A3 MAbs were evaluated for efficacy of inducing complement-mediated cytotoxicity (CMC) and mediating antibody-dependent cellular cytotoxicity against LS 180 cells derived from human colon carcinoma. Both forms were found to mediate similar levels of CMC in the presence of human complement; however, higher levels of lysis of target cells were observed with human peripheral blood lymphocytes when the chimeric 323/A3 was used. Chimeric 323/A3 mediated higher maximal cytotoxicity than chimeric 17-1A in both CMC and antibody-dependent cellular cytotoxicity assays and was equally active as chimeric 17-1A at 100- to 1000-fold lower concentrations. The superior reactivity of chimeric 323/A3 with EGP40 on carcinoma cells and its higher cytotoxicity-mediating capacity, compared to chimeric 17-1A, are important characteristics, which support further clinical studies with chimeric MAb 323/A3 in immunotherapy of carcinomas.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Comparison of circulating CA15-3 and carcinoembryonic antigen levels in patients with breast cancer.

An immunoradiometric assay (IRMA) has been used to determine circulating levels of the breast cancer-associated antigen, CA15-3. Of 1,050 normal control subjects, serum from 99 (9.4%) had CA15-3 antigen levels greater than 22 U/mL, while that from 58 (5.5%) and 14 (1.3%) had levels greater than 25 U/mL and 30 U/mL, respectively. In contrast, 115 of 158 patients (73%) with metastatic breast cancer had CA15-3 levels greater than 22 U/mL. Thirteen of 26 patients (50%) with only local metastases, 27 of 34 (79%) of those with only bone metastases, and 20 of 24 (83%) with hepatic metastases had CA15-3 levels greater than 22 U/mL. Furthermore, nine of 31 patients (29%) with primary breast cancer had CA15-3 levels greater than 22 U/mL. CA15-3 and carcinoembryonic antigen (CEA) levels were compared for the same patient population. Significantly more patients with metastatic breast cancer had elevated CA15-3 levels than had elevated CEA levels (P less than .001). Furthermore, the CA15-3 IRMA was more sensitive than the CEA assay in patients with only bone metastases, as well as those with only local metastases. Significantly more patients with primary carcinoma of the breast also had elevated CA15-3 than had elevated CEA levels (P less than .02). CA15-3 levels were greater than 22 U/mL in patients with nonmalignant conditions, including five of 25 patients (20%) with benign breast diseases, and 23 of 52 patients (44%) with benign liver diseases. Furthermore, CA15-3 levels were also greater than 22 U/mL in 24 of 54 patients (44%) with gastrointestinal (GI) malignancies, 12 of 17 patients (71%) with bronchogenic carcinoma, and 29 of 44 patients (66%) with epithelial ovarian carcinoma. Serial CA15-3 levels correlated with clinical disease course. Nineteen of 21 patients (91%) with tumor progression had at least a 25% increase in CA15-3 levels. Conversely, seven of nine patients (78%) with tumor regression had at least a 50% decrease in CA15-3 levels. Among 27 patients with stable disease, 16 (59%) had levels that did not vary by more than +/- 25% of the original CA15-3 levels. These results indicate that the CA15-3 antigen is a sensitive marker for the evaluation and monitoring of patients with breast cancer.

Serial levels of CA 19-9 and CEA in colonic cancer.

The use of serial carbohydrate antigen (CA) 19-9 assays was assessed by comparison with serial carcino-embryonic antigen (CEA) levels on the plasmas of 53 patients with colorectal carcinoma. The patients had all undergone resection for their primary tumors and in six instances subsequent resections for hepatic metastases. Initial CA 19-9 levels were greater than or equal to 37 U/mL in 22 of the 53 patients (41%) and in 68% of the patients with metastatic disease. Similar trends of serial CA 19-9 and CEA levels were found in 79% of the 53 patients. One patient with initially normal CEA levels had elevated CA 19-9 levels from the start. In ten of the 53 patients (19%), serial CA 19-9 levels remained low despite tumor recurrence or progression, and despite increasing CEA levels above 5 ng/mL. The increasing serial CEA trends predicted recurrence in 88% and increasing CA 19-9 trends in 50% of cases, which was increased to 70% by including trends of CA 19-9 levels below 37 U/mL. Following hepatic lobectomy, both serial CEA and CA 19-9 levels decreased rapidly. Used alone, serial CA 19-9 levels did not appear to be as sensitive as standard CEA in this retrospective study of selected patients.

Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay.

In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.

Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1.

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent Mr of 36 kDa after SDS-PAGE, whereas the membrane form appeared as a single band with an apparent Mr of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N-glycanase-treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross-reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor-product relationship between them.

Monoclonal antibodies directed against human myoglobin: characterization and application in a bideterminant radioimmunoassay.

Monoclonal hybridoma cell lines secreting antibodies directed against human myoglobin were selected. Two of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for human myoglobin that uniquely recognizes two different epitopes on the same molecule.