Number Of mAbs Research Articles

Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

A large number of Endotrypanum stocks (representing an heterogeneous population of strains) have been screened against a panel of monoclonal antibodies (MAbs) derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12) or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5) reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved) or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent), but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains crossreacted with a number of MAbs that were produced against members of the L. major-L. tropica complex.

Structural characterization of immunodominant regions of streptokinase recognized by murine monoclonal antibodies.

In order to determine the nature of the antigenic and functional determinants of streptokinase (SK), we produced monoclonal antibodies (MAbs) by immunizing A/J mice with native SK protein. By virtue of their differential binding to a large panel of recombinant SK truncated proteins, and their effect on the formation of functional SK-plasminogen activator complex (SKPAC), these MAbs were found to recognize 6 unique and minimally overlapping epitopes on the SK protein. The fine epitope specificity of the anti-SK MAbs derived from A/J mice was compared with that of MAbs derived from BALB/c mice. A number of MAbs from both inbred strains of mice were directed against the same sequences of SK (1-13, 1-253, 120-352) suggesting that these regions of the molecule contain peptide sequences that are immunodominant. Two of the "immunodominant" sequences of SK protein appeared to be important for SK function, since the formation of SKPAC could be inhibited by MAbs against these sequences.

Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain

A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.

Monoclonal antibodies against human breast tumor-associated antigens: characterization of B21 antigen.

Monoclonal antibodies (MAbs) show promise in the early detection and monitoring of cancer and may have therapeutic applications as well. The purpose of this study was to characterize MAb B21, a novel murine-derived antibody that has strong reactivity with MCF-7 and T47D cell lines from human breast cancer. A number of MAbs that react with breast cancer cell lines were obtained from cultured mouse spleen cells, and one, MAb B21, was selected for detailed analysis. MAb B21 was characterized by immunocytochemical, immunofluorescence, immunoprecipitation, and Western blotting procedures. We found a strong reactivity of MAb B21 with cultured breast cancer cells and cells from human breast tumors, although some reactivity was seen sporadically in non-breast or normal tissue. Negligible reactivity was detected in a series of non-breast cell lines and with normal breast epithelial cell line MCF-10A. However, when MCF-10A cells were permeabilized, allowing the antibody to penetrate within the cells, reaction became apparent. MCF-10A cells, when transfected with the oncogene c-Ha-ras (MCF-10T), gave a positive immunostaining similar to that observed with MCF-7 and T47D cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of L-[35S]methionine-labeled MCF-7 and T47D cell extracts showed distinct immunoprecipitated components, with molecular weight values ranging from 150,000 to 20,000 with the addition of MAb B21. Western blot assays using MAb B21 of SDS-PAGE fractionated/electroblotted proteins from breast cancer cell lines and MCF-10A cells showed specific reaction with a 95,000 component. Our results indicate that B21 antigen is expressed in neoplastic cells of epithelial origin, mainly breast cancer, and to a minor extent in other cell lines. In addition, MAb B21 recognizes an antigen that is differentially localized during cell transformation. Our future studies will address the full characterization of MAb B21 and explore its capacity as a tool for therapeutic manipulation.

The feasibility of radioimmunotherapy of head and neck cancer.

Since the introduction of the hybridoma technology by Kohler and Milstein (Nature 1975, 256, 495-497), tremendous effort has been put in the realisation of Ehrlich's concept of the magic bullet, which was proposed as early as the beginning of the century. The first clinical studies for radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) with radiolabelled antibodies were undertaken in the early 1980s. Since then, RIS has been performed on thousands of patients with various types of malignancies, like colon carcinoma, lung carcinoma, breast carcinoma, neuroblastoma, T-cell lymphoma and ovarian carcinoma. In addition, a substantial number of therapy trials with radiolabelled antibodies have been performed. The developments for head and neck squamous cell carcinoma (HNSCC) have only recently been able to catch up with these events to some extent. One of the main reasons for this slow progress has been the lack of monoclonal antibodies (Mab) with specificity for HNSCC. Although there are as yet no real tumour specific antigens known for HNSCC, which also holds true for the majority of malignancies arising from other tissues, we now have the availability of a number of Mab with high specificity for HNSCC and with a very restricted reaction pattern with normal tissues. Labelled with 131I, these Mab have been shown to be highly capable to localise in HNSCC xenografts in nude mice. Based on these promising data, patient studies with one of these Mab, designated Mab E48, labelled with 99mTc, were started to evaluate the feasibility of RIS in patients with head and neck cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

6.22 An investigation of temporary workshop clusters reacting with cells of the mononuclear phagocytic system

Monoclonal antibodies (mAbs) within ten temporary workshop clusters (TC3, TC8, TC9, TC11, TC16, TC17, TC19, TC20, TC21 and TC31) which from preliminary evidence appeared to recognize molecules expressed by cells of the mononuclear phagocytic system were studied by immunohistology and flow cytometry. A number of mAbs were identified that were potentially useful for immunohistological studies of the mononuclear phagocytic system. The results confirmed the specificity of a number of CD11a, CD11b, CD11c and CD44 mAbs. The findings also indicated that for several TCs the mAbs could not be regarded as recognizing the same molecule.

Structure-function analyses of diphtheria toxin by use of monoclonal antibodies

A large panel of hybridomas, secreting monoclonal antibodies (MAbs) specific for diphtheria toxin (DT) and prepared by immunization with either intact DT or its A or B fragment (DTA or DTB), have been isolated and characterized. The 213 MAbs were initially screened for reactivity to DT by enzyme-linked immunosorbent assay analyses and then were classified for their reactivity with DT, DTB, or DTA by solid-phase Western blot (immunoblot) analyses; 129 DTB-specific, 51 DTA-specific, and 33 non-fragment-assignable MAbs were obtained. Of the DTB MAbs, 118 recognize epitopes between residues 194 and 453, 10 recognize epitopes between residues 454 and 481, and 1 recognizes an epitope present in denatured toxin but not present in native DT located within the carboxyl-terminal receptor-binding region of DT (residues 482 to 535). Those MAbs that were the most protective in a cytotoxicity assay recognized native toxin in solution and inhibited binding of radiolabeled toxin to Vero cells to the greatest extent. A number of MAbs were able to detect epitopes that became more or less accessible when the toxin was preincubated at acidic (endosomal-mimicking) pH, suggesting that the epitopes they recognize may be important in the low-pH-induced insertion and/or translocation of DT across the endosomal membrane.

The next human leukocyte differentiation antigen workshop

The Workshop on Human Leukocyte Differentiation Antigens organizes international exchanges of monoclonal antibodies (mAbs), cooperative research on cell surface molecules, and the CD nomenclature. Plans have just been laid for the fifth workshop, and 1 have been asked by the organizing committee* to summarize these for members of the scientific community so that they may understand how this important scientific undertaking works, how to participate, and how new features such as sections on adhesion structures, cytokine receptors and endothelial cells broaden the scope of the workshop. This broadening of scope reflects an increasing emphasis in the workshops on the function of cell surface molecules. The workshop is divided into sections:: to increase efficiency and spread out the burden among the organizers of handling and distributing the large number of mAbs that is studied (over 1 100 in the last workshop) 1. The sections will be forums where antibodies that are of interest to people working on a specific cell type or specific class of surface molecules are gathered. The antibodies do not have to be specific for a given cell lineage, but must react with a given cell lineage in the case of sections that focus on a specific cell type. Each section will sponsor a main panel containing un-

Evidence that the M antigen ofHistoplasma capsulatumvar.capsulatumis a catalase which exhibits cross-reactivity with other dimorphic fungi

A panel of monoclonal antibodies (Mabs) was raised against histoplasmin, the antigen derived from the mycelial phase of Histoplasma capsulatum var. capsulatum which contains the diagnostically useful H and M antigens. A number of Mabs were obtained which recognized a 70-75 kD component of an antigenic preparation of H. capsulatum var. capsulatum by Western blotting. When reacted with histoplasmin by Western blotting the Mabs recognized a similar 70-75 kD band, together with a series of higher molecular mass bands at approximately 130, 190 and 230 kD, a pattern which correlates strongly with both the published relative molecular mass (Mr) of the M antigen and the known subunit structure of the enzyme catalase. These Mabs were also shown to recognize a commercial preparation of Aspergillus niger catalase by ELISA. Other dimorphic fungi were also reactive with these Mabs by Western blotting, indicating the presence of common epitopes on the catalase molecules of these species.

Multiple regions of HLA-DR beta 1 chains determine polymorphic epitopes recognized by monoclonal antibodies.

To investigate the locations of antibody binding epitopes on HLA class II molecules, four DR4/7 beta 1 hybrid cDNA were constructed by exchanging the DNA encoding the NH2-terminal portions (amino acids 1 to 40) or the COOH-terminal portions (amino acids 41 to 94) of the first domains of DR4 beta 1- and DR7 beta 1-chains, in association with DNA encoding either the DR4 beta 1 or DR7 beta 1 second domains. Transfectants expressing a DR alpha cDNA and a wild-type DR4 beta 1 or DR7 beta 1 cDNA or one of four hybrid DR4/7 beta 1 cDNA were produced, and the binding to the transfectants of anticlass II mAb, which detect polymorphic epitopes on either DR4 or DR7 molecules, was analyzed. Four different patterns of mAb binding to the transfectants were observed, indicating that multiple regions of DR beta 1-chains play the predominant roles in the contributions of these chains to polymorphic epitopes recognized by mAb on intact molecules. The relevant regions of these chains and the number of mAb that recognize the associated polymorphic epitopes are: 1) the COOH-terminal portion of the first domain of DR4 beta 1; a DR4-specific mAb, 2) the NH2-terminal portion of the first domain of DR7 beta 1; two mAb, including a DR7-specific mAb, 3) the NH2-terminal portion of the first domain of DR4 beta 1; seven mAb, and 4) the second domain of DR4 beta 1; one mAb.

The majority of human CD3 epitopes are conferred by the epsilon chain

Transgenic mouse T cells expressing the human CD3 epsilon chain bind the majority (29/36) of monoclonal antibodies (mAbs) specific for human CD3. A proportion of these mAbs are also able to recognize isolated CD3 epsilon in a soluble, recombinant form. Thus, CD3 epsilon can confer most CD3 epitopes on the TCR--CD3 complex, but many determinants may require assembly of the complex for their formation. A number of mAbs did not recognize epsilon-transgenic T cells and probably need other CD3 subunits for binding. CD3-specific mAbs from each of the three groups defined here, as well as mAbs directed against the TCR alpha beta heterodimer, are all able to activate T cells. Therefore mAb attachment at several different sites on the TCR--CD3 complex can give rise to activation signals. This suggests that the cross-linking function of mitogenic antibodies may be their most significant property, rather than the perturbation of a particular 'functional epitope'.

Effect of estrogen on the expression of a cell-surface antigen associated with rat anterior pituitary somatotrophs

The long-term in vivo effect of diethylstilbestrol (DES) on the expression of a cell-surface antigen associated with the anterior pituitary somatotroph was studied in two strains of female rats using double immunofluorescence techniques. Mab WHC-1, a recently generated and characterized monoclonal antibody, was used to detect the antigen associated with somatotrophs, whereas rabbit anti-rat prolactin (rPRL) and anti-human growth hormone (hGH) antisera were used to identify mammotrophs and somatotrophs, respectively. In F344 rats, Mab WHC-1-positive cells increased from 13.8 ± 0.5% of total pituitary cells in normal anterior pituitaries to 34.2 ± 4.0% in DES-induced pituitary tumors. The number of mammotrophs also increased significantly from 58.0 ± 3.2% in controls to 75.9 ± 2.2% in tumors. On the other hand, somatotrophs decreased significantly in number following ovariectomy (OVX) and DES implantation (19.7 ± 0.5% vs. 6.1 ± 1.2%). Based on double immunofluorescence, the percentage of Mab WHC-1-positive cells, which were somatotrophs, decreased from 85.5 ± 2.7% in normal controls to 6.7 ± 1.5% in DES-induced tumors. On the other hand, the percentage of Mab WHC-1-positive cells which were mammotrophs increased significantly from 14.0 ± 1.4% to 86.1 ± 1.8% following OVX and DES implantation. A similar change was found in the number of somatotrophs and mammotrophs following the same treatment in Sprague-Dawley (SD) rats which did not develop pituitary tumors. In contrast to F344 rats, the number of Mab WHC-1-positive cells in SD rats decreased significantly from 32.4 ± 2.8% in sham-operated controls to 19.3 ± 2.9% in OVX + DES-implanted rats. Significant strain differences were found in the relative population of Mab WHC-1-positive cells, somatotrophs and mammotrophs. The results indicate a shift in the expression of Mab WHC-1-defined cell-surface antigens from the majority of somatotrophs to an increasing number of mammotrophs following long-term treatment of estrogen.

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF 1 (BALB/c × A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG 1), recognized an epitope within residues 30–51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7–35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, an IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.

Monoclonal antibodies specific to the alpha-subunit of the mast cell's Fc epsilon R block IgE binding and trigger histamine release.

In an attempt to block the interactions between IgE and its receptor on mast cells (Fc epsilon R), we have established anti-Fc epsilon R monoclonal antibodies (mAb) by fusion of myeloma cells with mouse splenocytes immunized with irradiated rat basophilic leukemia (RBL) cells. Two anti-Fc epsilon R mAb were obtained (denoted 4.7 and 5.14) that could specifically bind to RBL and mast cells. This binding could be inhibited by IgE. The mAb and their F(ab')2 fragments inhibited 125I-IgE binding to RBL cell and triggered cell degranulation. The Fab' fragments, on the other hand, could only inhibit IgE binding but did not stimulate cell degranulation. Furthermore, these monovalent fragments inhibited RBL and mast cell degranulation induced by IgE-antigen complexes both in vitro and in vivo in the passive cutaneous anaphylaxis reaction. The number of mAb 4.7 and 5.14 molecules bound per RBL cells was similar to that of IgE; nevertheless, mAb 4.7 and 5.14 recognized different epitopes on the IgE receptor. Immunoprecipitation and immunoblotting analysis demonstrated that the mAb reacted with the alpha-subunit of the Fc epsilon R. Our findings establish the anti-Fc epsilon R mAb as a useful reagent for the isolation and characterization of the Fc epsilon R's alpha-subunit and the monomeric (Fab') for blocking the IgE-Fc epsilon R interactions.