Binding Of Murine Monoclonal Antibodies Research Articles

Capto MMC mixed-mode chromatography of murine and rabbit antibodies

Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0.

Love wave immunosensor for antibody recognition using an innovative semicarbazide surface functionalization

A new novel and easy functionalization route of a Love wave acoustic sensor based on the α-oxo-semicarbazone bond is described. The interest is firstly to observe in real-time the immobilization of the peptide on the semicarbazide surface of the transducer and secondly to monitor the specific binding of antibodies. Site-specific immobilization of antigenic-peptides as well as binding of murine monoclonal antibodies has been shown by gravimetric measurements. A tetramethylrhodamine-labeled goat antibody directed against murine antibodies was used to further characterize the biomolecular interactions by fluorescence microscopy and surface analysis (by AFM). Our data show that the gravimetric monitoring developed from the prepared Love wave immunosensor is a promising alternative route to characterize chemical and biomolecular events.

Binding of murine monoclonal antibodies to human MHC class I or II antigens increases binding sites for either antibody.

Membrane redistribution of major histocompatibility complex (MHC) class I and II antigens on human B lymphocytes and monocytes was studied quantitatively by fluorescence flow cytometry using monoclonal antibodies (mAb) w6.32 (class I-specific) and 2MC3 (class II-specific). It was shown that the surface density of class I and II antigens to which corresponding mAb had been bound decreased rapidly upon incubation at 37 degrees C. However, the total amount of class I antigens remained practically unchanged, while that of class II antigens increased substantially. This augmentation of class II antigens could be elicited not only by the corresponding class II-specific mAb but, similarly, also by non-corresponding class I-specific mAb. New expression of class I antigens only compensated for removed antigens. The speed of disappearance was comparable for antigens of either class. We conclude that the more pronounced expression of new class II antigens is not due to a greater surface mobility, but to a faster recycling and/or larger intracellular deposits of the antigens. This uneven redistribution of class I and II antigens is likely to reflect differences of function known to be dependent on MHC products.

Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein.

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

Enhanced binding of murine monoclonal antibodies to lipopolysaccharide structures of Enterobacteriaceae after treatment with antibiotics

Four strains of E. coli, two encapsulated and two unencapsulated, were grown in the presence or absence of the β-lactam antibiotics aztreonam, ceftazidime and ceftriaxone, the fluoroquinolone ciprofloxacin, and the aminoglycoside netilmicin. Treatment of the unencapsulated strains with β-lactams and ciprofloxacin resulted in enhanced binding of murine Mabs (all:IgM) directed to rough lipopolysaccharide structures in ELISA. However, binding of these Mabs to antibiotic treated E. coli 07K1 (bearing a thin capsule) was only slightly increased, and was unchanged in E. coli 08K49 (surrounded by a thick capsule). Treatment of bacteria with netilmicin did not result in differences in binding of these monoclonal antibodies. These results show that the inner parts of the lipopolysaccharide become more accessible to antibodies when, especially unencapsulated, bacteria are grown in the presence of certain antibiotics.

Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin

Murine monoclonal IgG 1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.

Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon.

We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.

Topographic and functional assay of antigenic determinants of human prolactin with monoclonal antibodies

Six distinct antigenic determinants were identified on human prolactin (hPRL) by competition assays with murine monoclonal antibodies (MABs). The affinity of binding and the cross-reactivity of the antibodies with two non-primate prolactins was also determined. Binding of 125I-hPRL to MAB-coated microtitre plates in the presence of a second MAB resulted in either inhibition or enhancement of antigen binding to the plate. These results were interpreted in terms of conformational changes to epitopes, induced allosterically by the binding of a second MAB to the antigen. The topographic relationship of epitopes to the biologically active regions on the hormone was examined on the basis of the neutralizing potency of MABs in the proliferation of the prolactin-dependent cell line NB2. The NB2 growth-inhibitory activity was restricted to three distinct epitopes (NE02/6, 1208 and NE03) but absent from three other MABs tested (QB01, WC01/3 and 1200). On the basis of the competition and functional studies, an elemental scheme of the topographic localization of epitopes is presented. The experimental approach employed may contribute to studies on the allocation of biologically active sites on protein hormones.

Quantitative analysis of cell surface HLA structures by means of monoclonal antibodies

Quantitative data on the binding of murine monoclonal antibodies to whole human lymphoblastoid lines and peripheral blood lymphocytes (PBL) are reported. Antibodies reacting with β 2m or a common part of the HLA heavy chains and nonpolymorphic determination of the DR dimer were used. The equilibrium constant (K) of the reaction and the total number of antigenic determinants was graphically estimated. For the above-mentioned antibodies. K ranged between 5 × 10 8 and 4 × 10 9 I lmole at 0°C and progressively decreased with the increasing temperature. T cells expressed less HLA and β 2m determinants that the B cells. The number of determinants per surface unit is higher on the B cell from PBL than on E.B. virus-transformed cell lines and is generally very low, suggesting that the complement-dependent cytotoxic activity is a phenomenon depending on membrane fluidity. A portion of β 2mseems not to be bound to the HLA heavy chains on B cells as well as on T line surface, as already shown for Molt 4 line.