摘要:

The coupling of haptens to carrier proteins is required for the generation of antihapten antibody response. To date, different polypeptides (such as gelatin, albumin, and keyhole limpet hemocyanin) have been used to increase the immune response against haptens. Different methods of conjugation have also been used to increase the specificity of the antibody response. Our results, obtained through an analysis of the antibody response at both the polyclonal and the monoclonal levels, show that carrier proteins specifically modulate the antibody titer, the specificity of the response, the fusion efficiency, and the number of specific clones. Moreover, the affinity constants of both serum and randomly selected hybridomas directed against different haptens have been found to be exclusively modulated by carrier proteins. In addition, the results show that the carrier protein should be selected considering its possible physicochemical interaction with haptens. Data obtained suggested that any carrier is suitable for hydrophilic haptens, while the choice of the carrier for hydrophobic haptens is critical in obtaining a specific immune response. The use of specific carriers allowed the production of highly specific antibodies: even IgMs, obtained by using these carriers, were able to specifically react with hydrophobic haptens; the case of some IgM monoclonal antibodies specifically reacting with protoporphyrin IX is reported.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.1

年代:1996

数据来源: MAL

摘要:

The CD45 protein tyrosine phosphatase is expressed in different isoforms that result from alternative splicing of three exons (A, B, and C) encoding regions near the N-terminus of the extracellular part of the molecule. We describe here a novel epitope on the N-terminal end of CD45 that is recognized by the MAb BL-TSub/2. Crossblocking studies showed that BL-TSub/2 and UCHL1 (CD45RO) binding sites are partially overlapping. However, in marked contrast to the CD45RO epitope, protease treatment of cells strongly diminished BL-TSub/2 binding. Similar to the UCHL1 epitope, the BL-TSub/2 binding site involves carbohydrate moieties, since neuraminidase treatment abrogated the reactivity of the MAb. Markedly, preincubation of cells with both CD45 common and CD45RA MAb induced a pronounced increase of BL-TSub/2 binding. This latter finding suggests that crosslinking of the CD45 molecule leads to conformational changes that could influence association of the molecule with putative ligands.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.11

年代:1996

数据来源: MAL

摘要:

Monoclonal antibodies (MAb) were made to two different superpotent guanidino sweet tasting ligands,N-(p-cyanophenyl)-N′-(idiphenylmethyl)-guanidineacetic acid andN-(p-cyanophenyl)-N′-(cyclooctyl -guanidineacetic acid. In the present study we examined the binding specificity of three MAb clones (denoted as NC10.7, NC10.13, and NC37.3). The isotypes of these MAb were determined to be IgG1κ for NC10.7 and NC37.3, while NC10.13 was IgG2b, κ. The dissociation constants for the MAb were 19 nM (NC10.7), 28 nM (NC10.13), and 16 nM (NC37.3). The binding specificity of each MAb was characterized by a competitive inhibition radioimmunoassay using related sweetener analogues. Antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste li

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.17

年代:1996

数据来源: MAL

摘要:

A series of heavy chain switch variants has been isolated from a new B cell-specific monoclonal antibody belonging in the CD20 cluster. The antibodies NKI-B20/1, NKI-B20/2b, and NKI-B20/2a (of isotype IgG, IgG2b, and IgG2a, respectively) have been used to study the influence of isotype and of the target antigen on the capacity to mediate cytotoxicity with a number of effector mechanisms. Unlike many mouse MAbs, NKI-B20/2b and NKI-B20/2a were cytolytic with human complement on human target cells that did not express the complement regulatory factor HRF20. All 3 isotypes of NKI-B20 mediated antibody-dependent cell-mediated cytotoxicity (ADCC) with rIL-2-activated NK cells from mouse spleen. Here the antigen density seemed the most important factor in determining the level of cell kill. With mouse peritoneal macrophages as effector cells again all 3 isotypes of NKI-B20 mediated cytotoxicity. For the IgG1and IgG2bvariants of NKI-B20 this is at variance to what has been reported for MAbs of other specificities. Despite the high activity with murine effector cells none of the NKI-B20 MAbs mediated ADCC with human peripheral blood NK cells, with or without stimulation with rIL-2, due to the lack of interaction of the murine MAbs with the human Fc receptor. The CD20 antigen appears to be a good target antigen for various forms of cytotoxicity, to which its relatively high antigenic density, its resistance to antibody-induced modulation, and its unusual structure all contribute.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.23

年代:1996

数据来源: MAL

摘要:

HIS50 MAb recognizes a GPI-linked molecule on the surface of rat cells committed to the B lineage but is absent from cells of other hematopoietic lineages. Eukaryotic expression cloning of the cDNA has revealed that HIS50 recognizes a rat homologue of human CD24 and murine HSA. CD24/HSA exists in multiple glycoforms and plays an important role in hematopoietic and neural cell development. HIS50 is significant in that it is the only, presently available, anti-rat CD24/HSA MAb. Furthermore it is the only reported MAb with specificity restricted to B cell forms of CD24. Here we report Western blot analysis of HIS50 antigen (ag). In rat bone marrow HIS50 MAb recognized MW species in the range of 35-70 kDa. These species were shown by immunomagnetic cell sorting to be all derived from the HIS50+cell subset. In other tissues with varying HIS50 levels as judged by immunostaining, apparent levels of HIS50 ag in the 35-70 kDa range varied accordingly. Thus, Western blot data corresponded to the immunostaining data of HIS50 ag, and the size distribution of B-restricted forms of rat CD24 appeared as wide as that reported for the more extensively expressed forms of human and murine CD24. N-Deglycosylation of cell lysates from lymphoid organs reduced the signal in the 35-70 kDa range, without appearance of lower MW HIS50-reactive species. Thus, unlike certain epitopes on human and murine CD24, HIS50 epitopes appeared (partially) N-linked carbohydrate dependent. The data reported here provide a basis for the further use of HIS50 MAb in studying the role of the highly heterogeneous CD24 molecules in cell development.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.33

年代:1996

数据来源: MAL

摘要:

Cell adhesion is a complex biophysical process that plays a central role in immunophysiology. Because of the complex force—energy relationships involved, insights into the mechanism of cell adhesion largely depend on comparative measurements. In this report, we describe a comparative approach to the measurement of cell adhesion under conditions of flow. The assay system perfuses fluorescently labeled lymphocytes over a cell monolayer in commercially available multiwell culture plates. The fluorescently labeled cells demonstrate a reproducible flow pattern within the well. Videomicroscopic recordings of cell movement have demonstrated rolling behavior over a wide range of cell velocities. This technique permits the measurement of cell adhesion over a variety of flow velocities, time courses, and treatment conditions. The ability to vary treatment conditions and provide multiple parallel conditions suggests the utility of this approach in the development of monoclonal antibodies (MAb) recognizing cell adhesion molecule

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.43

年代:1996

数据来源: MAL

摘要:

Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung. To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E.coliand purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen. Hybridomas producing high-affinity antibodies were subcloned by limiting dilution. Antibodies from tissue culture fluid from an IgG1clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized. The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lystates of cell lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization. MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung. TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas. These findings demonstrate that anti-TTF-1 MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.49

年代:1996

数据来源: MAL

摘要:

The TATA box-binding protein (TBP) is a key transcription factor required for transcription by all three eukaryotic RNA polymerases. It consists of a conserved carboxy-terminal DNA binding domain and a highly divergent amino terminal domain. TBP and different sets of TBP-associated factors (TAFs) constitute at least four multisubunit complexes referred to as SL1, TFIID, TFIIIB, and SNAPC. SL1, TFIID, and TFIIIB are required for transcription by RNA polymerases I, II, and III, respectively, while the SNAP complex is involved in transcription of the small nuclear RNA (snRNA) genes by RNA polymerases II and III. TBP also associates with a number of basal transcription factors such as TFIIA and TFIIB, and with several regulatory factors such as VP16, E1A, and p53. Here we describe the characterization of a panel of monoclonal antibodies (MAbs) directed against the amino-terminal domain of human TBP. These MAbs recognize different TBP epitopes, some of which have been precisely defined. Different MAbs recognize different TBP-containing complexes and several of them crossreact with TBP from other species. These antibodies can be used to purify TBP-containing complexes in a functional form and should be useful to identify new protein-protein interactions involving TBP.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.55

年代:1996

数据来源: MAL

摘要:

A monoclonal antibody designated B1.9.E-2 was produced and characterized to facilitate study of the immunizing antigen—a serum glycoprotein of 120 kDa (sgp 120) of unknown function. The antibody, produced in mice, reacts with 1-2 epitopes located in the N-terminal region of the sgp 120 molecule. The affinity of the reaction, as determined by Scatchard analysis, is Ka= 1.13 × 10101/M and is of a hydrophobic nature. The monoclonal antibody can be purified to a high degree by a modified caprylic acid method and by protein G FPLC chromatography column. B1.9.E-2 does not trigger the complement cascade, as determined by C3 RIA and immune complex solubilization assays. Both affinity purified (C4b Sepharose) and chromatographically isolated (using jacqualine agarose) sgp 120 were recognized by B1.9.E-2. The monoclonal antibody can be utilized for affinity purification of sgp 120, for detection of intact or cleaved sgp 120 in biological samples of healthy and ill individuals, and for the number of functional and neutralization assays aimed at resolving the physiologic role of sgp 1

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.69

年代:1996

数据来源: MAL

摘要:

The recently discovered human class theta glutathioneS-transferase Tl-1 (GSTT1-1) is responsible for the GSH-dependent detoxification of naturally occurring monohalomethanes. The detoxifying role of GSTT1-1 has not been investigated in cancer susceptibility and the polymorphism of the protein is unknown in different populations. The purpose of our work was to produce a panel of mouse monoclonal antibodies (MAbs) that could bind to different regions of the GSTT1-1 protein and would help us select suitable MAbs for Western blot analyses and immunohistochemistry, and develop an ELISA assay for detection of GSTT1-1 in whole blood. Six highly specific MAbs were generated against GSTT1-1. Out of six MAbs, one was able to recognize only the native form of the enzyme and possesses two binding sites on the dimeric GSTT1-1 molecule. The other five MAbs bind to both native and denatured GSTT1-1 enzyme in direct and antigen capture ELISA or Western blot. The antibodies recognize at least four different epitopes on the GSTT1-1 molecule. Using MAbs 4G1 and 2D8, a sensitive ELISA assay for determination of GSTT1-1 in whole blood was developed.

ISSN:0272-457X    

DOI:10.1089/hyb.1996.15.77

年代:1996

数据来源: MAL