Production of monoclonal antibodies against fullerene C60 and development of a fullerene enzyme immunoassay.

The aim of the present study was to produce monoclonal anti-fullerene C(60) antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C(60) both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C(60) carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein-conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C(60) a specially selected water-organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C(60) in solution was 2 µg L(-1). Fullerene C(60) was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C(60) from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C(60) in different tissues.

Binding of antigen by immunocytes. II. Effect of specific Ig on antigen binding by MOPC 315 cells.

The effect of specific immunoglobulin (Ig) on specific binding of antigen to cells has been studied in a model system consisting of nurine myeloma cells (MOPC 315), MOPC 315 serum, and DNP conjugates. MOPC 315 serum, which has IgA specific for DNP, specifically inhibited the binding of DNP conjugates to these cells. Using this model it was found that cells have a marked advantage over free specific Ig in binding multivalent antigen molecules and retaining them in a bound state. Cells were able to specifically bind multivalent antigen in the presence of a large excess of free specific Igm the kinetics of antigen binding to cells was slow, and prolongation of time of incubation increased the amount of specific binding. Both antihapten and anticarrier Ig augmented nonspecific binding of multivalent but not of univalent hapten to control cells. Furthermore, antihapten Ig at low concentration increased antigen binding to specific cells.

Intramolecular heterogeneity of ligand binding by two IgM antibodies derived from murine hybridomas.

The ligand binding properties of two murine anti-DNP IgM hybridoma antibodies were analyzed. These IgM antibodies, designated NP3-17 C1-20 and SP2/0 I-64 C1-12, each displayed an average of only five high affinity binding sites for the DNP moiety. Reductive 7S subunits of both of these proteins each adsorbed to and were hapten eluted from DNP affinity columns. These subunits, when examined by equilibrium dialysis, each contained an average of one high affinity binding site. Structural analysis of these molecules indicated each to be homogeneous. Results obtained from trypsin hydrolysis of each molecule indicated that differences in conformation of the binding sites may account for the observed binding heterogeneity.

Immunogenicity of biotinylated hapten-avidin complexes.

The efficacy of avidin as a carrier for the generation of anti-hapten antibodies was assessed in mice by immunization with complexes of avidin and synthetic peptides containing biotin and an epsilon-dinitrophenyl (DNP) lysine residue. The synthetic haptens were constructed with 0, 1 or 2 6-aminocaproyl groups as spacers between the biotin and DNP-lysine moieties. Complexes without a spacer did not induce anti-DNP antibody responses, while those with two spacers induced stronger responses than those with only one spacer. However, the anti-DNP responses to avidin-biotinylated hapten complexes were considerably weaker than responses to a conventional hapten-protein conjugate (DNP-ovalbumin), and, like "T-independent" antigens, failed to induce significant immunological memory. The distribution of isotypes in the anti-DNP antibodies produced to avidin-biotin-6-aminocaproyl-epsilon-DNP-lysine-alanine and DNP-ovalbumin was similar, but the former antigen induced significantly lower levels of antibody in (CBA/N X BALB/c) F1 male mice with the xid defect than in phenotypically normal female littermates, and also induced significant responses in nu/nu mice, in contrast to DNP-ovalbumin. These findings suggest that there is at least a "T-independent" or "T-efficient" component in the response to avidin-biotin complexes, perhaps due to the tetrameric structure of the molecule. Estimates of the depth of the receptor site for biotin were obtained by using the complexes to competitively inhibit the binding of anti-DNP antibody to plates coated with DNP-protein. The findings were consonant with the data on immunogenicity (capacity to induce anti-DNP antibody responses) and suggested that the receptor site has a depth of 16-26 A.

The affinity and spectrum of cross reactivity of antibody production in senescent mice: the IgM response.

Experiments were performed to determine whether the IgM component of the primary humoral immune response of senescent mice differs from that of young-adult mice in its affinity, specificity or heterogeneity. For this purpose, mice were immunized with the T-dependent antigen, TNP-KLH, or the T-independent antigen, TNP-LPS. Affinity and specificity of the anti-TNP response were studied at the cellular level by the plaque inhibition technique using the cross-reacting haptens TNP-epsilon-aminocaproic acid )TNP-EACA),DNP-EACA and ONP-EACA. It was found that when the immunogen was TNP-KLH, the peak IgM response was delayed in senescent animals by several days as compared to the response in young adults. However, the number of PFC on the day of peak response was comparable. No age related delay in the peak of response was detected when the antigen was TNP-LPS although the magnitude of response was reduced in old age. The average affinity of the plaque forming cell responses to both immunogens is comparable in young and old mice.

Stimulation and inhibition of anti-hapten responses in guinea pigs immunized with hybrid liposomes.

Guinea pigs were immunized with liposomal model membranes containing phosphatidylethanolamine (PE) or glycerophosphorylethanolamine (GPE) derivatives in which the amino function was substituted with either dinitrophenylaminocaproyl (Dnp-Cap) or mono(p-azobenzenearsonic acid)tyrosyl (ABA-Tyr) residues. Previous studies have demonstrated that hapten-specific antibodies are elicited by DNP-Cap-PE or ABA-Tyr-PE sensitized liposomes and that cell-mediated immunity is induced by ABA-Tyr-PE (but not Dnp-Cap-PE) sensitized liposomes. These liposomes differ from conventional immunogens in which haptens are covalently attached to immunogenic carriers. This investigation describes two new aspects of liposomal immunogenicity in animals immunized with hybrid liposomes containing both Dnp-Cap-PE and ABA-Tyr-PE. (1) Stimulation of the anti-Dnp response by incorporation of increasing amounts of ABA-Tyr-PE; (2) inhibition of anti-ABA antibody formation by incorporation of increasing amounts of DNnp-Cap-PE. The two phenomena are dependent on the presence of each determinant in the same lipid bilayer. Thus, entrapment of the water-soluble deacylated derivative of ABA-Tyr-PE (i.e., ABA-Tyr-GPE) in a aqueous compartments of Dnp-Cap-PE sensitized liposomes does not enhance anti-Dnp antibody production. Similarly, entrapment of the non-amphipathic derivative of DNP-Cap-PE (i.e., Dnp-Cap-GPE) within ABA-Tyr-PE sensitized liposomes does not suppress anti-ABA antibody formation. Furthermore, mixtures of Dnp-Cap-PE sensitized liposomes and ABA-Tyr-PE sensitized liposomes neither stimulated nor inhibited the anti-hapten responses. These results indicate that preparation of hybrid liposomes with different N-substituted PE derivatives provides an extremely convenient method for controlling hapten and/or immunologic carrier determinant density.

A comparison of flexible and constrained haptens in eliciting antibody catalysts for paraoxon hydrolysis.

A new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of 20 that catalyzed paraoxon hydrolysis. The current hapten design differs from that previously described in that the molecule is inherently more flexible than its constrained predecessor. One of the seven antibody catalysts, mAb 1H9, showed the highest activity and was selected for detailed study. At pH = 8.77, the catalytic hydrolysis of paraoxon by mAb 1H9 followed Michaelis Menten kinetics affording a k(cat) = 3.73 x 10(-4) min(-1) and a Km = 1.12 mM with a rate acceleration k(cat)/k(uncat) = 56. The hapten was found to be a competitive inhibitor of antibody-catalyzed paraoxon hydrolysis with a Ki = 0.54 mM. A comparison of both the number and proficiency of antibody catalysts obtained when utilizing a flexible versus constrained hapten indicates that, for paraoxon hydrolysis, constrained haptens elicit superior catalysts, suggesting that further development should begin with the use of constrained haptens in producing more proficient antibody catalysts for paraoxon hydrolysis.

Interaction of purified precipitating and non-precipitating (coprecipitating) antibodies with hapten and with haptenated protein. Evidence of an asymmetric antibody molecule.

The interaction of monovalent hapten dinitrophenyl epsilon-amino caproic acid (DNP-EACA) with purified IgG1 sheep anti-DNP precipitating and non-precipitating antibodies, and their F(ab')2, F(ab') and Fab fragments, was studied by fluorescence quenching and by a radioimmunoassay. The Scatchard plots of whole non-precipitating antibody and its F(ab')2 fragment showed a bi-modal curve that could be interpreted as due to the existence of two populations of sites with very different affinity for the ligand, each population representing 50% of the total number of sites. The F(ab) fragments of the non-precipitating antibody could be fractionated by immunoadsorption into two populations of high and low affinity whose association constants differed by more than 2 logs. The study of the interaction of whole antibodies with DNP-bovine serum albumin (BSA) demonstrated that each molecule of precipitating antibody can combine with two molecules of antigen but non-precipitating antibody cannot combine with more than one molecule of antigen. It is concluded that the molecule of non-precipitating antibody is asymmetric and has a site of high affinity and another of low affinity. As a consequence of this structure the non-precipitating antibody behaves functionally as univalent and is unable to form precipitates with the multivalent antigen and to activate effector mechanisms.

Phospholipase A2 stimulation during cell secretion in rat basophilic leukemia cells.

The bridging of IgE receptors on rat basophilic leukemia cells (RBL-2H3) results in a number of biochemical events that accompany histamine secretion. Prominent among these is the release of arachidonic acid from cellular phospholipids, which could be due to the activation of phospholipase enzymes. In the present experiments we studied the intracellular activation of phospholipase A2 (PLA2) during histamine release. RBL-2H3 cells were stimulated through the IgE receptor, and the homogenates were prepared and tested for phospholipase A2 activity on 1-stearoyl-2-[14C]arachidonyl-sn-3-phosphatidylcholine. The amount of activity in the homogenates was dependent on the concentration of secretagogue used to activate the cells. Under optimal conditions there was a 1.86 +/- 0.12-fold (mean +/- SEM, N = 44) increase in the activity found in homogenates of stimulated cells. Activity was present in homogenates prepared 30 sec after cell activation, was optimal between 5 and 10 min, and decreased later. In time course experiments the PLA2 activation preceded histamine release. The activation of the enzyme in the cell occurred in the presence of 10 microM EGTA in the extracellular medium, which completely inhibited release of arachidonic acid and histamine. However, the activity of the enzyme required Ca2+. The PLA2 activity in the homogenates and the extent of cell stimulation for histamine release were maximal at the same concentration of antigen, and both were blocked by the addition of a monovalent hapten. The enzyme in the homogenates was capable of cleaving arachidonic acid from different phospholipids. The production of lysophospholipids could play a critical role in histamine release from cells. These results demonstrate the activation of PLA2 enzyme in cellular homogenates during the secretory process.

Use of structural alterations in the synthesis of halothane metabolite antigens to mimic halothane-induced immunogen.

Four hapten-carrier conjugates were synthesized to evaluate any potential antigenic similarities between these synthetic compounds and the immunogens induced in vivo by the anesthetic, halothane and, thus, be used eventually as a more sensitive probe to detect the presence of these halothane-induced antibodies in halothane-exposed individuals. In this study, antibodies from five halothane hepatitis patients were used to evaluate these antigenic alterations since the specificity of these antibodies would most accurately reflect the antigenic structure of halothane-induced immunogens. Quantitation of antibody binding to these synthetic proteins was determined in an enzyme linked immunosorbent assay and immunoblot techniques. Trifluoroacetylated rabbit serum albumin was 5 times more reactive with these antibodies and thus more antigenic than the homologous acetylated moiety confirming the importance of the trifluoromethyl moiety as an epitope in the immunogen in vivo. Insertion of a spacer arm, aminocaproic acid, between the hapten and carrier moieties and an epitope density of 40% acetylation also increased antigenicity. Through these structural alterations produced in vitro, antigenic compounds have been produced which may resemble more closely the immunogen elicited in vivo and which may ultimately serve as more sensitive probes for halothane-induced antibodies from exposed individuals.