Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.
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The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo. (+info)
Selection of antibodies for intracellular function using a two-hybrid in vivo system.
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Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences. (+info)
Murine embryos as a direct target for some human autoantibodies in vitro.
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The involvement of one or another autoantibody in reproductive failure have long been thought to be through post-implantation thrombosis and/or peri-implantation trophoblast dysfunction and/or maternal hormonal imbalance. It can be postulated that the embryo may be a direct target for some autoantibodies prior to implantation. Mouse embryos have been labelled and cultured with affinity purified immunoglobulin (IgG) and IgA from positive for antiphospholipid antibody sera, as well as IgG from positive for antinuclear antibody sera and positive for antithyroid antibody sera. Intact IgG and IgA from healthy individuals were used as controls. All embryos cultured with purified antiphospholipid IgG or IgA, and anti-nuclear IgG exhibited strong immunofluorescence. No difference in fluorescent intensity was observed whether antiphospholipid or anti-nuclear antibodies were used, but the pattern of antibody distribution seemed to be different. Antiphospholipid IgG was more dominant on the zona pellucida, while antiphospholipid IgA and antinuclear IgG had predominant distribution on the embryonic cells. None of the embryos cultured with antithyroid IgG or with control immunoglobulins showed strong immunofluorescence. Embryos cultured with purified antiphospholipid and antinuclear immunoglobulins experienced significant growth impairment or death compared to those cultured with antithyroid or control immunoglobulins. (+info)
Cloning and characterization of a novel activating Ly49 closely related to Ly49A.
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The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily. (+info)
Thermodynamic analyses reveal role of water release in epitope recognition by a monoclonal antibody against the human guanylyl cyclase C receptor.
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The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site. (+info)
Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines.
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We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type. (+info)
Counter-electrophoresis as a possible method for typing ECHO and Coxsackie B viruses.
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A method for typing ECHO and Coxsackie B viruses using counter-electrophoresis has been developed using stock strains of 15 different ECHO types and 5 Coxsackie B viruses. Work is continuing to see whether the method is a practical one to use for typing of strains from patients infected with ECHO or Coxsackie B viruses. (+info)
Response to influenza vaccine in adjuvant 65-4.
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A comparison was made of the antibody response and subjective reactions to zonally-purified influenza vaccine in aqueous suspension and in peanut oil adjuvant 65-4. Both preparations contained 700 CCA units of A/Aichi/2/68, and 300 CCA units of B/Mass/1/71. Subjective reactions were recorded by asking the volunteers to complete a record daily for 5 days. Pain at the injection site was recorded by 64 per cent of the recipients of the oil adjuvant vaccine compared with 35 per cent of the aqueous recipients, but local redness was more frequent after aqueous vaccine. Systemic symptoms was recorded a little more frequently after aqueous than oil adjuvant vaccine. When measured 71/2 weeks after a single dose of vaccine, the HAI geometric mean antibody titre (G.M.T) to the A/Hong Kong/1/68 antigen (antigenically similar to the A/Aichi/2/68 antigen in the vaccine) increased 2-7 fold after aqueous and 16-4 fold after adjuvant vaccine. Sixty-two weeks after vaccination the antibody titres remained higher in those given adjuvant vaccine. The G.M.T. to B/Mass/1/71 increased 1-9 fold 71/2 weeks after aqueous vaccine and 3-7 fold after adjuvant vaccine. The antibody response to both influenza A and B antigens was broader in the recipients of adjuvant vaccine. The G.M.T. to A/England/42/72 increased 2-8-fold after aqueous and 13-fold after adjuvant vaccine; and to B/England/847/73 it increased 1-3-fold after aqueous and 1-9-fold after adjuvant vaccine. (+info)