摘要:

Tumor Necrosis Factor α (TNFα) is an inflammatory cytokine which exists mainly as a 51kD complex built up of 3 identical, noncovalently-linked polypeptide subunits. We have raised monoclonal antibodies (mAb) against human TNFα (huTNFα). One of these mAb (mAb78, mouse IgGlk) was studied in detail. mAb78 expresses a recurrent idiotype typical of the BALB/c anti-huTNFα antibody response. HuTNFα bound to mAb78 with an affinity constant (Kobs) of 3.2 x lO10M-1. The number of huTNFα-binding sites per mAb78 molecule was ≈0.7. At concentrations higher than the KobsmAb78 neutralized huTNFα at a ≈ 1.3 : 1 molar ratio. mAb78 precipitated huTNFα in a double immunodiffusion assay in agar. Gel-filtration experiments of mAb78-huTNFα mixtures, that had been set up in large antigen excess, detected complexes of 570 kD as the smallest ones formed under these conditions.We propose that these results are accommodated best by a model according to which cyclic complexes built up of 3 mAb78 and 2 huTNFα molecules are the smallest units formed upon interaction of the reagents. In view of this model we discuss how huTNFα and mAb78 can undergo a prec

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.1

年代:1993

数据来源: MAL

摘要:

BrE-3 is an IgG1,κ murine monoclonal antibody that binds to human breast epithelial mucin and that has been shown to be promising for imaging and treatment of breast cancer. We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of BrE-3. VLbelongs to group II and resulted from a Vκ-Jκ1fusion. VHbelongs to group IIIc and arose from a V-D-JH3non-conservative fusion which left little or nothing of the original D minigene. Thus, the third VHCDR contains only 4 amino-acids. We constructed an IgG1,κ human/mouse chimeric antibody (by joining the murine variable domains to human constant domains) and expressed it in SP2/0 myeloma cells. This chimeric monoclonal antibody stains breast carcinoma tissue sections by the ABC immunoperoxidase method. Its affinity for the BrE-3 antigen is 2.68x108M-1, which, considering the experimental error, is indistinguishable from the affinity of the original murine antibody (3.75x108M-1). The VLand VHdomains alone are respectively 73%, and 63% identical to the human VKIIand VHIIIconsensus sequences. If the CDRs are excluded, these numbers become respectively 82% and 80%. Therefore, we expect the reported chimeric BrE-3 to be considerably less immunogenic to humans than the original murine antibody, while retaining the original binding propert

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.15

年代:1993

数据来源: MAL

摘要:

A large battery of anti-CD23 mAb were compared for their epitope specificities and for their abilities to alter both IgE binding to cell-associated CD23 and IgE production in vitro in response to three sets of stimulants. The nine mAb tested can be divided into four families which define four antigenic epitopes (A-D) of CD23. Of these four families, two bind antigenic sites, (A and D) that appear to lie outside the IgE ligand binding site and two bind sites (B and C) that appear to be located within or close to this site, as determined by the abilities of appropriate mAb to alter IgE binding to CD23.The effects that these mAb had on IgE secretion by normal peripheral blood mononuclear cells (PBMNC) varied depending on the stimulant employed to induce IgE production. Interactions with epitope A, which was found to lie outside the ligand binding site and to be made more accessible by binding of mAb to other epitopes, had different effects on IgE production than interactions with the other epitopes. Indeed, mAb binding to this epitope lead to as much as a 10 fold enhancement in IgE biosynthesis induced by IL-4 alone or by IL-4 + hydrocortisone whereas interactions at the other sites resulted in almost complete inhibition of IgE production. In addition, mAb reactive with epitopes B and C had minimal effects on IgE production induced by IL-4 + anti-CD40 mAb whereas interactions at epitope A consistently enhanced IgE production. Finally, no apparent direct correlation was found between the ability of individual anti-CD23 mAb to alter IgE binding to cell-associated CD23 and their ability to modulate IgE production by PBMNC.These studies suggest that IgE binding to cell-associated CD23 does not have a major role in the de novo synthesis of IgE that involves CD23 interactions. In addition, the different effects that binding to epitope A vs B or C have on IgE synthesis suggest that molecular interactions between distinct portions of the CD23 molecule and other cell surface molecules expressed on the same B cell or adjacent communicating cells may lead to divergent cellular effects on IgE production. Finally these studies imply that only epitope A is involved in the generation of an IgE response through the CD40 pathway.

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.25

年代:1993

数据来源: MAL

摘要:

Splenocytes from mice immunized with homogenous, polyclonal, rabbit kinin antibody (BK21) were fused using polyethylene glycol with the mouse myeloma cell line SP2/o. Eleven monoclonal antibodies, whose binding to BK21 could be inhibited by bradykinin, were obtained from 3 fusions. All of these anti-idiotypic antibodies were of the IgGlkisotype, except for one, which was an IgG2ak. An IgMk, auto-anti-idiotypic antibody, reactive with BK21 was obtained from a fusion of SP2/o cells and splenocytes from a mouse immunized with bradykinin conjugated with carbodiimide to keyhole limpet hemocyanin. Bradykinin could completely inhibit the binding of all of the anti-idiotypic antibodies to BK21 in an enzyme-linked immunosorbent assay. This result is consistent with the anti-idiotypic antibodies being reactive with the ligand binding sites of BK21. It was possible to separate the anti-idiotypic antibodies into 2 groups. The first group, 10 of the 12 antibodies tested, was more sensitive to inhibition by bradykinin than the second group and was not readily inhibited by des-Arg9-bradykinin. The second group was about 7 times more sensitive to inhibition by des-Arg9-bradykinin than by bradykinin. Further experiments will be needed to determine whether or not these monoclonal anti-idiotypic antibodies are "internal image" antibodies.

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.45

年代:1993

数据来源: MAL

摘要:

Ten monoclonal anti-idiotypic antibodies (mAB2s) were obtained from fusions of myeloma cells, X63/Ag8.653, and splenocytes from mice immunized with one of two monoclonal kinin antibodies (mAB1s). The interactions of these mAB2s, with four different mAB1s, which have similar kinin binding specificities, was examined. Five of the ten mAB2s cross-reacted with similar affinities, with all four mAB1s. In addition, these five mAB2s were able to inhibit biotinylated-kallidin binding to the mAB1s. This indicated that these mAB2s interact with the mAB1s at, or near, their ligand binding sites. These immunological results are consistent with these five mAB2s being "internal image" β type anti-idiotypic antibodies

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.55

年代:1993

数据来源: MAL

摘要:

Monoclonal antibody BQ16, raised against UM-UC-9, a human bladder cancer cell line, exhibited strong reactivity with most bladder carcinoma tissue samples and cell lines. In normal urothelium, BQ16 stained only the basal surface of urothelial cells at the junction with the lamina propria. BQ16 immunoprecipitated two protein bands of ̃140 and 180 kDa (under non-reducing conditions), while on Western blots, BQ16 identified only the 140 kDa protein indicating that BQ16 binds to one chain of a dimeric protein complex. The dimeric structure, molecular size, and basal orientation of the BQ16 antigen prompted a comparison with the α6β4 integrin identified by monoclonal antibody UM-A9. In most tissues BQ16 and UM-A9 produced identical staining patterns. However, normal lymphocytes and certain bladder cancer cell lines were BQ16 positive but failed to react with UM-A9, indicating that the BQ16 and UM-A9 epitopes can be expressed independently. Pulse-chase immunoprecipitation experiments showed that the α6 subunit was more prominent in early BQ16 precipitates and the β4 subunit was more prominent in early UM-A9 precipitates. Furthermore, preclearing cell extracts with the anti-α6 antibody GoH3 removed all BQ16 reactivity and m UM-A9-negative, BQ16-positive cells, BQ16 precipitated the α6β1 complex. We conclude that BQ16 identifies the α6 integrin subunit and that α6β4 integrin is strongly expressed in most bladd

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.67

年代:1993

数据来源: MAL

摘要:

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated β-subunit (β-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross0reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and β-HCG, or exclusively with the non-associated β-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyreoidea-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated β-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or β-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or β-HCG-producing tumors before and after t

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.81

年代:1993

数据来源: MAL

摘要:

Spleen cells from a Robertsonian mouse immunized with rat α-CGRP were fused with FOXNY cells to induce hybridoma cells. Antibody activities were screened by radioimmunoassay, and hybridomas producing high affinity antibodies were cloned by limiting dilutions. Ascites were produced from the highest affinity clone in pristine-primed Balb/c mice. Ascites fluid contained approximately 20 mg/ml IgG which was of subclass IgG2aas determined by immunodiffusion analysis. The titer of this IgG2aantibody titled #4901, was 1:2,000,000 and the ID50for rat α-CGRP, rat β-CGRP and human α-CGRP were 350, 4000, and 4500 pg/ml respectively. Protein A purified CGRP antibody #4901 (5-10 mg/kg) completely abolished the portal release of somatostatin and the inhibition of gastric acid secretion induced by intravenous infusion of rat αCGRP (15-20 μg/kg/h) in anesthetized rats. The unpurified antibody (25 mg/kg) also prevented the fall in mean arterial blood pressure and the increase in heart rate caused by intravenous injection of rat α-CGRP. Immunohistochemistry showed that CGRP monoclonal antibody stains nerve fibers and endocrine-like cells in the pancreas, and neuronal elements in the gastrointestinal tract. These results show that CGRP monoclonal antibody #4901, which is relatively specific for rat α-CGRP, is useful for in vivo immunoneutralization of CGRP and is also an excellent reagent for immunohistochemical localization of α- and β-CGRP i

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.93

年代:1993

数据来源: MAL

摘要:

Hybridoma cell lines producing monoclonal antibody (Mab) against recombinant human prolactin (rhPrl) were established from fusion between X63-Ag8 myeloma cells and Balb/c mice splenocytes. Four Mabs numbered I to IV were selected by ELISA, purified and characterized. All these Mabs were of the Iglκ isotype and able to recognize oxidized as well as reduced rhPrl. As shown by a competitive inhibition assay, Mab IV did not compete with any of the three others. Moreover, both rhPrl and hPrl extracted from human pituitaries, were recognized equally by this Mab. Properties displayed by Mab IV make it very attractive for the evaluation of prolactin levels by sandwich immunoassays

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.107

年代:1993

数据来源: MAL

摘要:

Human transformed T cell lines were able to induce polyclonal B cell activation and immunoglobulin (Ig) secretion from peripheral blood mononuclear cells, spleen cells and tumor-infiltrating lymphocytes(TIL). Cells from one of the lines tested, MOT, did not require any exogenous stimuli to induce maximal responses and under similar conditions induced higher levels of response than peripheral blood T cells or other T cell lines. MOT-induced B cell activation and Ig secretion required cell contact and factors present in the MOT culture supernatant. MOT cells induced B cell responses from TIL in the three tumors tested (melanoma, ovarian and colon cancer) and HIV-specific immunoglobulin secretion by spleen cells from an HIV+ patient.

ISSN:0272-457X    

DOI:10.1089/hyb.1993.12.115

年代:1993

数据来源: MAL