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M R Suresh

Publications and source records attributed to M R Suresh.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates.

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP-IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5-20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.

Alkaline Phosphatase↗
PubMed1998

A new method to generate quadromas by electrofusion and FACS sorting.

Bispecific monoclonal antibodies (bsMAbs) are unique molecules incorporating two different paratopes in a single antibody molecule. BsMAbs can be useful in different areas of research as well in clinical applications. Traditionally, bsMAbs are produced by hybrid-hybridomas that are generated by the fusion of two pre-established hybridomas. The development of such hybrid-hybridomas can be difficult and time-consuming. Here, we introduce a new technique to generate such hybrids, electro-FACS-fusion. In this procedure, before the electrofusion, one of the hybridomas is labeled with fluorescein isothiocyanate (FITC) and the other with tetramethylrhodamine isothiocyanate (TRITC). The mixture of cells is then electrofused, and cells exhibiting dual fluorescence are selected by fluorescence activated cell sorting (FACS). The fused cells are directly plated in microplates for clonal growth. Using this technique, we produced three new hybrid-hybridomas secreting bsMAb that could be used for the next generation of immunoassays.

Animals↗
PubMed1998

Summary report on the ISOBM TD-6 workshop: analysis of 20 monoclonal antibodies against Sialyl Lewisa and related antigens. Montreux, Switzerland, September 19-24, 1997.

The ISOBM TD-6 Workshop is the first international workshop on monoclonal antibodies against the Sialyl Lewisa (SLea) antigen. Eight research groups participated in a blind study to characterize the epitope binding, relative affinity and performance in immunoradiometric assays, of a panel of 20 monoclonal antibodies. The antibodies were tested against a diverse panel of neoglycoconjugates, purified antigens and human serum pools from gastrointestinal malignancies. Epitope specificities were determined for the majority of antibodies in the panel. Cross-reactivity with related saccharide structures was noted in several antibodies. Overall, the results of the TD-6 Workshop show further development of SLea immunoassays may yield yet more specific assays for the detection and management of gastrointestinal and other malignancies.

Antibodies, Monoclonal↗
PubMed1998

Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin. San Diego, Calif., November 17-23, 1996.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.

Amino Acid Sequence↗
PubMed1998

Analysis of the role of type 1 core O-glycans in the binding of anti-MUC1 antibodies by cytofluorometry and synthetic peptide/glycopeptide binding inhibition studies.

A panel of 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop were analysed in two systems. These systems were designed to screen for peptide type 1 core O-glycan-related reactivities. Using synthetic MUC1 mucin-related peptides and glycopeptides, the panel of MAbs were tested for relative binding affinities to type 1 core O-glycan-substituted MUC1 structures. These studies utilized a competitive binding format with a native human adenocarcinoma-derived mucin as a solid phase. This system allows for analysis of the type 1 core glycoform subspecificity of each MAb. The second approach taken in parallel, utilized MCF-7 (BrCa) and OVCAR (OVCa) cell lines which were grown in the presence or absence of phenyl-N-acetylgalactosaminide (p-gal), a blocker of mucin O-linked glycosylation. These cells were analysed by FACS to examine the role these same glycan substitutions play with regard to either the diagnostic or therapeutic application of these MAbs. By FACS analysis there was a consistent increased 'epitope exposure' for peptide-specific MAbs binding in the presence of p-gal. In addition, a single MAb (TD-4 #150) is interpreted to react with a type 1 core O-glycan, probably with Tn, TF or STn specificity.

Amino Acid Sequence↗
PubMed1997

Novel bispecific immunoprobe for rapid and sensitive detection of prostate-specific antigen.

Prostate-specific antigen (PSA) is one of the most useful tumor markers for the screening and follow-up of prostate cancer. Bispecific monoclonal antibodies (bsMAbs) are unique immunoprobes that incorporate two different binding sites in the same antibody molecule. This antibody designing can bring important advantages in the development of new immunoassays. We have developed a new hybrid hybridoma that secretes bsMAb anti-PSA x anti-horseradish peroxidase. This bsMAb has shown rapid kinetics and an excellent detection limit in a sandwich single-step assay with a total incubation time of 15 min and a 5-min substrate development. This assay in a manual format has a detection limit of 0.028 microgram/L. Comparison with the Hybritech Tandem-E PSA assay yielded a regression equation with slope = 0.433 [95% confidence interval (CI) = 0.415-0.451], intercept = 0.88 (CI = 0.45-1.31), and Sy/x = 1.83 micrograms/L (r = 0.98). This new immunoprobe can be used to develop a new generation of assays for clinical laboratories and can be adapted to screening devices for physicians' offices and even home diagnostics.

Ammonium Sulfate↗
PubMed1996

An antibody-lectin sandwich assay for the determination of CA125 antigen in ovarian cancer patients.

A two-step forward sandwich assay was developed for the determination of the ovarian tumour associated glycoconjugate antigen CA125 with anti-CA125 Monoclonal antibody B27.1 on the solid phase and 125I-labelled wheat germ lectin as tracer in the solution phase. This Mab-lectin heterosandwich assay was optimized and the clinical utility was evaluated in sera from healthy volunteers and ovarian cancer patients. A correlation was established between Mab-lectin assay and the dual monoclonal antibody sandwich assay, TRUQUANTOV2 RIA, that uses the same MAb B27.1 on the solid phase and a second 125I-labelled B43.13 MAb in the solution phase. A potentially improved clinical utility is suggested for the Mab-lectin assay. The unique format seems to identify novel isoforms of CA125 with different carbohydrate side chains that would otherwise be undetectable in the MAb-MAb sandwich assay wherein the paratopes are likely directed to protein determinants.

Antibodies↗
PubMed1996

Specificity and affinity of 26 monoclonal antibodies against the CA 125 antigen: first report from the ISOBM TD-1 workshop. International Society for Oncodevelopmental Biology and Medicine.

The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines. Immunometric assays using all possible antibody combinations were used to study the topography of antibody binding sites on the antigen. We conclude that the CA 125 antigen carries only two major antigenic domains, which classifies the antibodies as OC125-like (group A) or M11-like (group B). One antibody, OV 197, showed binding specificity related to some of the OC125-like antibodies, but was classified into a separate group C. The OC125-like group of antibodies has four subgroups with different binding specificities. These are A1 = OC 125 and K 95, A2 = K 93, A3 = B43.13, and A4 = ZS 33, B27.1 and CCD 247. Binding of nonlabelled OC 125 or K 95 to CA 125 caused a marked increase in binding of labelled OV 197 to the complex. This conformational change was not observed with any other antibody combinations. Antibody B43.13 could form immunometric assay combinations particularly with antibodies of subgroup A4, indicating that the B43.13 epitope is in the periphery of the binding area of OC125-like antibodies. The M11-like group of antibodies is more homogenous with strong cross-inhibition between most antibodies. Only one antibody, ZR 38, would form an immunoassay combination with other M11-like antibodies and thus represents a distinct subgroup. The main group of M11-like antibodies are M 11, ZR 45, MA602-6, K 91, OV 185, K 101, K 90, K 96, K 97, K 102, CCD 242, 145-9, and 130-22. Antibody OV 197 binds to a domain designated C and is unique, as stated above. Antibody pairs from any two of the three groups may be used in immunometric assays. Three antibodies were not studied by complete cross-inhibition due to low affinity (OV 198 and K 100) or lack of material (MA602-1). OV 198 and K 100 are most likely OC125-like and MA602-1 is M11-like. Antibody affinity was estimated with labelled antigen in solution or with antigen absorbed on microtiter wells. Western blot analysis showed staining both in the stacking gel and corresponding to a molecule of 200 kDa. There was a marked difference between the antibodies in their ability to bind to CA 125 immobilized on a membrane. Strongest binding was observed with the M11-like antibodies, particularly M 11, K 96, K 97, MA602-6, 145-9. Antibodies belonging to the subgroup A4 were the only OC 125-like antibodies which reacted well with CA 125 in Western analysis. Digestion of CA 125 with proteolytic enzymes showed it to be particularly sensitive to trypsin cleavage. However, no low molecular weight fragments with preserved immunoreactivity were found.

Animals↗
PubMed1996

Immunohistochemical characterization of 22 monoclonal antibodies against the CA125 antigen: 2nd report from the ISOBM TD-1 Workshop.

We evaluated the immunohistological (IH) characteristics of 22 different antibodies that were submitted for study in the frame of the TD-1 ISOBM Workshop on monoclonal antibodies against CA125. Information on relative affinities and epitope similarities was obtained from a parallel immunochemical study. Antibodies were tested at concentrations of 10 and 1 micrograms/ml on frozen and paraffin sections. Paraffin sections were stained according to the streptavidin-biotin complex protocol, and frozen sections according to a two-step immunoperoxidase technique. Aminoethylcarbazole served as the chromogen. The tissues were from normal proliferative endometrium (formalin-fixed paraffin-embedded) material and clear-cell adenocarcinoma of the ovary (formalin-fixed paraffin-embedded and frozen material). Sections were scored for staining in epithelial cells, basal, apical and diffuse cytoplasmic and in stromal components. Intensity was graded as 1, 2 or 3 for epithelial cells and as -1, -2 or -3 for stroma. The cumulative scores for each antibody expressed the discriminative properties of specific epithelial staining against background. M11 and M11-like antibodies, as well as OC125 and OC125-like antibodies, in general showed good staining results. Although there was a trend for high-affinity antibodies to show higher scores, there was no clear relationship between affinity and staining result. For nine antibodies (ZR45, MA602-1, K102, K94, K90, OV185, K97, K96, OV198), the reactions in paraffin and frozen sections were of similar intensity. Most of these were of low affinity with one exception: antibody ZR45, a rat monoclonal antibody (MAb) which had a high relative affinity. For eight antibodies (M11, K101, MA602-6, ZS33, B27.1, B43.13, K93, OC125), a loss of specific staining was observed in frozen sections. All but two of these antibodies (MA602-6 and OC125) were of high relative affinity. With four antibodies (K91, ZR38, K95, K100), the reverse situation was observed. One (K100) was of low affinity, two (K95 and K91) of high affinity and the fourth (ZR38) was a rat MAb of high affinity. Mainly due to the increased cytoplasmic staining in carcinoma, the reactivity in paraffin sections was less extensive in normal endometrium compared to ovarian carcinoma for the majority of antibodies, irrespective of their affinity or epitope group. The IH characterization of these antibodies may be of help in selecting antibodies with specific properties for further comparative studies. The reactivity of normal endometrium with all useful antibodies makes it a good candidate for standard external IH tissue control.

Adenocarcinoma, Clear Cell↗
PubMed1996

Epitope mapping of prostate-specific antigen with monoclonal antibodies.

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Amino Acid Sequence↗
PubMed1995

A rapid non-selective method to generate quadromas by microelectrofusion.

A simple non-selective methodology was developed and standardized to generate desired hybrid-hybridoma or quadroma secreting bifunctional antibodies. This novel protocol is based on microelectrofusion on a meander chamber using a few hundred cells of each of the two parental hybridomas with no laborious drug selection procedures. Seeding approximately 10 cells per well in a 96-well microtitre plate after fusion in 200 microliters standard medium containing 20% FBS and 10% Origen growth factor generated positive quadromas secreting bispecific antibodies with good stability after the second reclone. Compared to the conventional PEG fusion and other methods this simple protocol is both rapid and economical. Generally, conventional methods to make quadromas and triomas require the introduction of drug selection markers into one or both of the parental cells, a procedure that could take 3-6 months. Utilizing the non-selective microelectrofusion method described here, we have generated several quadromas in a very short time. Further, such a protocol could also be potentially adopted to generate human hybridomas with few B cells isolated from peripheral blood lymphocytes enriched by antigen specific panning or affinity microelectrofusions.

Animals↗
PubMed1995

Passive immunization with an anti-substance P antibody prevents substance P- and neurokinin A-induced bronchospasm in anesthetized guinea-pigs.

In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs.

Amino Acid Sequence↗
PubMed1994

Activating anti-idiotypic human anti-mouse antibodies for immunotherapy of ovarian carcinoma.

Human anti-mouse antibodies (HAMA) are observed frequently after immunoscintigraphy with monoclonal antibodies (MoAb) directed against CA-125. As the authors have shown previously, HAMA can cause false-positive CA-125 values in routine CA-125 immunoradiometric assay (IRMA) tumor-marker assays (in one case, up to 900 days after immunoscintigraphy). In 32 patients, the authors found a HAMA frequency of 34% (11/32: 3/7 after the first administration, 6/13 after the second, and 2/2 after the third). Ten patients developed extremely high CA-125 levels after undergoing the CIS IRMA assay (up to 80,000 U/ml) in parallel to a significant HAMA increase. The use of different assays, or HAMA removal before in vitro testing, can solve this problem. After a new CA-125 assay containing antibodies that recognize different epitopes on the CA-125 antigen (Biomira Tru-Quant OV) was applied, only mildly increased assay results or normal levels were measured. Most of HAMA-positive patients demonstrated a predominantly anti-idiotypic response, determined with two different HAMA assays. Seven patients with anti-idiotypic HAMA responses after OC-125 immunoscintigraphy remained free of tumor or had stable disease (2-42 or more months), contrary to their poor prognoses that had been made based on the underlying stages of their tumors. All of these patients are currently doing well (Karnofsky Index > 70%) and show no significant tumor progression. In light of their extremely poor prognoses (5-year survival rates of 3-5% in recurrent International Federation of Gynecology and Obstetrics III/IV stages), without further chemotherapy, these courses are extremely unusual. Preliminary in vitro experiments lead to the postulation that anti-idiotypic HAMA may trigger an antitumor effect either by suppressing the growth of CA-125-expressing cancer cells directly, or by activating the patient's immune response via induction of Ab3. Similar results are observed after immunoscintigraphy with a technetium-99m-labeled anti-CA-125 monoclonal antibody (B43.13), which the authors now also use for immunotherapy of ovarian cancer patients by repeated injections, hoping that induction of anti-idiotypic HAMA will be beneficial for prolonged survival of patients with ovarian carcinoma.

Animals↗
PubMed1991

Immunoassays for cancer-associated carbohydrate antigens.

The advent of hybridoma technology and the availability of virtually unlimited amounts of specific high affinity monoclonal antibodies to desired antigens have ushered in a new revolution in immunoassay methodology. An ever increasing number of monoclonal antibody-based immunoassays are replacing those employing polyclonal antisera. A host of hitherto unknown tumor-associated antigens have also been identified due to the development of unique monoclonal antibodies. The exquisite specificities and affinities of monoclonals has led to the development of sensitive and reproducible immunoassays for various analytes, especially tumor-associated antigens. Most tumor-associated antigens are glycoconjugates and among these many are blood group antigens and their derivatives, found both as glycoproteins and glycolipids. The development and clinical applications of new immunoassays for cancer-associated carbohydrate antigens in the last decade has had a major positive impact on the management of cancer patients.

Antibodies↗
PubMed1990

Analysis of human tumor associated Thomsen-Friedenreich antigen.

The Thomsen-Friedenrich (TF) antigen is a precursor structure of MN blood group antigens and is also expressed by about 90% of human carcinomas. The immunodominant group of TF antigen [beta-galactosyl(1-3)-alpha-N-acetylglactosamine] is present in cryptic form in normal RBC and is revealed by neuraminidase treatment. A murine monoclonal antibody (Mab 49H.8) developed against neuraminidase treated human RBC was reactive against a variety of human tumors. We have characterized the human tumor associated TF antigen detected by this antibody from a human transitional bladder carcinoma cell line (647V), a human colon adenocarcinoma cell line (LS174T), and a pleural effusion fluid of a breast adenocarcinoma patient (PE 89). A heterologous sandwich radioimmunoassay for TF antigen was developed using Mab 49H.8 as the catcher and 125I-peanut agglutinin as the probe. Detergent extracts of 647V and LS174T cells, media conditioned by culturing these cells, and PE 89 were shown to contain the antigen by this assay. The specificity of the antigen capture by Mab 49H.8 in this assay was demonstrated by its selective inhibition by nitrophenyl-beta-D-galactoside, phenyl-beta-D-galactoside, and a TF hapten. Preliminary studies on TF antigen in serum samples using this assay showed that about 53.7% of the carcinoma samples contained an antigen concentration greater than 200 units/ml whereas for 90.9% of the normal samples the antigen concentration was below 200 units/ml. These studies demonstrated that the TF antigen is shed by the tumor cells both in vitro and in vivo. The TF antigen was sensitive to treatment with alkali (0.1 M NaOH for 5 h at 37 degrees C) and periodate (10 mM sodium periodate for 1 h at room temperature), was resistant to acidic pH (50 mM acetate buffer, pH 4.5, for 5 h at 37 degrees C), and could be extracted with perchloric acid (0.6 M for 1 h at 4 degrees C). The antigen was shown to be a high molecular weight glycoprotein (Mr greater than 1,000,000) by gel filtration chromatography. The density of the antigen was estimated to be about 1.35 g/ml by cesium chloride density gradient centrifugation. The antigen could be isolated from conditioned media by a combination of affinity chromatography and gel filtration with an overall purification of about 61,432-fold and a final recovery of 53.2%.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1989

Importance of pH in defining the specificity and clinical utility of monoclonal antibodies--an evaluation of two anti-CA 19-9 monoclonal antibodies.

The effect of pH on the binding characteristics of monoclonal antibodies against tumor associated antigens has been studied, using anti-CA 19-9 monoclonal antibodies as an example. The 1116-NS-19-9 monoclonal antibody is used in several commercially available two-step sandwich IRMAs for the measurement of the CA 19-9 antigen in cancer patient sera (Centocor CA 19-9 RIA, Malvern, PA, U.S.A., or similar kits by Centocor licensees). The B25.10 monoclonal antibody is in use in the Truquant GITM RIA (Biomira Inc., Edmonton, Alberta, Canada), a competitive inhibition assay measuring the CA 19-9 antigen. We determined the Ka value of the B25.10 and the 1116-NS-19-9 antibodies at pH 4.5 and 7.3 on a solid phase coated with CA-19-9 bearing mucin. The 1116-NS-19-9 antibody had a Ka value of 4.1 x 10(8) at pH 4.5 and approximately 0.3 x 10(8) at pH 7.3. The Ka value of B25.10 was in the range of 0.7-4.1 x 10(9) at either pH, but somewhat lower at pH 4.5. At pH 4.5 (the pH optimum for binding of 1116-NS-19-9 to the CA 19-9 antigen Del Villano, B.C., 1984]) the binding of 125I-labeled B25.10 to the solid phase was comparably inhibited by 1116-NS-19-9 and B25.10. However, at neutral pH (pH 7.2) the apparent affinity of the 1116-NS-19-9 antibody for the CA 19-9 antigen is significantly diminished [Del Villano, B.C., 1984] while that of B25.10 is slightly higher. Consequently, the ability of the 1116-NS-19-9 to inhibit the binding of the B25.10 antibody to the CA 19-9 solid phase is greatly reduced in the physiological pH range. The consequences of these findings for in vivo applications are discussed.

Antibodies, Monoclonal↗