Covalent functionalization of polyhedral graphitic particles synthesized by arc discharge from graphite.

Carbon materials including carbon nanoparticles, such as nanographite, graphene and graphenic materials, and carbon nanotubes are known to be highly hydrophobic. Oxidation treatments are widely used as the best methods to improve their affinity in a liquid medium or a polymer matrix so that they can be dispersed, handled and processed. Here, we have applied eight different oxidation treatments in order to graft oxygen-containing functional groups at the surface of polyhedral graphitic particles synthesized by arc discharge from graphite, also called astralenes. The used functionalization approaches include both standard chemical attack by strong oxidants and radical functionalization of the sp2 network by direct C[double bond, length as m-dash]C bond opening. Commonly efficient functionalization methods were unsuccessful to functionalize astralenes while radicals generated from arylhydrazine could lead to functionalization of the outer surface of astralenes. The occurrence of functionalization could be shown by TGA coupled with MS and XPS. The reported method represents the first example of functionalization of astralenes. The efficiency of the applied functionalization methods is discussed considering the chemical reactivity of different carbon nanomaterials including graphene and carbon nanotubes.

Accuracy of an automated knowledge base for identifying drug adverse reactions.

INTRODUCTION: Drug safety researchers seek to know the degree of certainty with which a particular drug is associated with an adverse drug reaction. There are different sources of information used in pharmacovigilance to identify, evaluate, and disseminate medical product safety evidence including spontaneous reports, published peer-reviewed literature, and product labels. Automated data processing and classification using these evidence sources can greatly reduce the manual curation currently required to develop reference sets of positive and negative controls (i.e. drugs that cause adverse drug events and those that do not) to be used in drug safety research. METHODS: In this paper we explore a method for automatically aggregating disparate sources of information together into a single repository, developing a predictive model to classify drug-adverse event relationships, and applying those predictions to a real world problem of identifying negative controls for statistical method calibration. RESULTS: Our results showed high predictive accuracy for the models combining all available evidence, with an area under the receiver-operator curve of ⩾0.92 when tested on three manually generated lists of drugs and conditions that are known to either have or not have an association with an adverse drug event. CONCLUSIONS: Results from a pilot implementation of the method suggests that it is feasible to develop a scalable alternative to the time-and-resource-intensive, manual curation exercise previously applied to develop reference sets of positive and negative controls to be used in drug safety research.

[Determinants of psychotropic medication in adults with mild or moderate intellectual disabilities]. / Bedingungsfaktoren psychopharmakologischer Behandlung bei leichter oder mittelgradiger Intelligenzminderung.

BACKGROUND: During the past years the provision of mental healthcare for adults with intellectual disabilities (ID) has repeatedly been criticized; however, the number of relevant studies is still relatively few. OBJECTIVE: The aim of the present study was to identify determinants for utilization of mental healthcare services and prescription of psychotropic medication in adults with mild to moderate ID. MATERIAL AND METHODS: Analyses were based on data from 417 adults with mild to moderate ID, which had been collected within the cross-sectional MEMENTA study in three different regions of Germany. Logistic regression analyses were conducted to identify clinical and sociodemographic variables as predictors of utilization of mental healthcare services (n = 282) and psychotropic medication (n = 351). RESULTS: Utilization of healthcare services and psychotropic medication were both associated with mental disorders and problem behavior. In addition, the likelihood of being treated with psychotropic medication and antipsychotic drugs was higher in adults living in residential homes. CONCLUSION: The findings indicate a lack of adherence to existing guidelines in the treatment of adults with ID living in residential homes.

[Treatment pathways in the care of patients with schizophrenia and depression]. / Behandlungspfade in der Versorgung von Patienten mit Schizophrenie und Depression.

BACKGROUND: In mental healthcare the concept of pathways addresses diverse issues and problem areas, such as heterogeneous health service offers, the regional variability of treatment concepts and clear-cut guidelines on how and where to obtain treatment for a particular mental disorder. The ambiguous aspects of the concept require international and national definitions and consensus which must also cover quality criteria. METHODS: This article gives an overview of currently available evidence for the analysis of clinical pathways and pathways to care in international mental healthcare, covering studies on schizophrenia and depression from 2010 to 2014. RESULTS AND DISCUSSION: The ambiguity of the concept impedes the overview and does not provide unequivocal results. The development, implementation and analyses of guidelines or clear-cut clinical and pathways to care must consider individual, clinical and care system aspects as well as the interplay of these factors. Results suggest that system aspects tend to dominate over clinical factors of schizophrenia and depression. As a consequence, the definition, implementation and evaluation of clinical pathways or pathways to mental healthcare is first and foremost a responsibility of the respective national mental healthcare system and must be understood on that level, before findings are summarized internationally and models of best practice are debated.

2-Chlorodeoxyadenosine (cladribine) induces apoptosis in human monocyte-derived dendritic cells.

2-Chlorodeoxyadenosine (cladribine, CdA) is an immunosuppressive drug that is licensed to treat hairy cell leukaemia, and has been shown recently to have beneficial effects in patients with multiple sclerosis (MS). The therapeutic effects of CdA have been suggested to be mediated partly through its potent toxicity towards lymphocytes. However, the effects of CdA on other immune cells are poorly understood. In the present study, we investigated the effects of CdA on the induction of apoptosis in human monocytes, monocyte-derived immature (ImDC) and mature (mDC) dendritic cells. Treatment of monocytes with CdA strongly induced apoptosis after 24 h, while apoptosis induction in DC was evident after 72 h. Furthermore, CdA treatment strongly induced caspase-3 and caspase-9 in monocytes, whereas activation of caspases was undetected in DC. The mitochondrial membrane potential in DC was reduced significantly after CdA treatment. DNA hypodiploid assessment showed fragmented nuclei in DC after CdA treatment together with activation of p53 protein. These results revealed that CdA induces caspase-independent apoptosis in DC and suggest cell type specific effects of CdA. This mechanism may contribute to the effect of CdA in autoimmune diseases.

cAMP-mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium.

Before addition of cAMP, Dictyostelum amoebae rapidly translocating in buffer are elongate, exhibit expansion zones primarily at the anterior end and filamentous actin (F-actin) localization primarily in the anterior pseudopodia. Intracellular particle movement is primarily in the anterior direction, and the average rate of particle movement is roughly five times the rate of cellular translocation. Within seconds after the addition of 10(-6)M cAMP, there is a dramatic suppression of cellular translocation, an inhibition of pseudopod formation, a freeze in cellular morphology, a dramatic depression in intracellular particle movement, loss of F-actin localization in pseudopodia concomitant with relocalization of F-actin in the general cytoplasmic cortex under the plasma membrane, and a doubling of F-actin content. After 10 s, expansion zones are again visible at the cell perimeter, but they no longer are localized in the original anterior portion of the cell. There is a slight rebound in particle movement after 10 s, but particles with persistent tracks now show no directionality towards the original anterior portion of the cell, as they did before cAMP addition. Finally, in parallel with the resumption of peripheral expansion and the small rebound in particle movement, there is a decrease in total cellular F-actin to the untreated level. The pattern of microtubule organization is unaffected by the addition of cAMP.

Prolonged weightlessness and humoral immunity.

Preflight, inflight, and postflight serum samples obtained from crewmen aboard STS-9 were analyzed for immunoglobulin content. Control studies for circadian rhythm were conducted to further validate the analyses. Quantitation of immunoglobulins G, M, A, D, and E indicated relatively minor fluctuations in the concentration of each class of immunoglobulin during the experiment. Thus, microgravity effects on immunoglobulin levels during a 10-day flight were considered insignificant.

Differential regulation of right and left ventricular beta-adrenergic receptors in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia secondary to an intracardiac right-to-left shunt alters regulation of the myocardial beta-adrenergic receptor/adenylate cyclase system, we produced chronic hypoxemia in nine newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 wk. Eight lambs served as normoxemic controls. beta-receptor density (Bmax) and ligand affinity (KD) were determined with the radio-ligand [125I]iodocyanopindolol and adenylate cyclase activity determined during stimulation with isoproterenol, sodium fluoride (NaF), and forskolin. During chronic hypoxemia, Bmax decreased 45% (hypoxemic, 180.6 +/- 31.5 vs. control, 330.5 +/- 60.1 fmol/mg) in the left ventricle (exposed to hypoxemia alone) but was unchanged in the right ventricle (exposed to hypoxemia and pressure overload). KD was not different from control in either ventricle. Left ventricular isoproterenol-stimulated adenylate cyclase activity was decreased by 39% (30.0 +/- 4.3% increase vs. 44.1 +/- 9.5% increase) whereas right ventricular adenylate cyclase activity was unchanged. Stimulation of adenylate cyclase with NaF or forskolin was not different from control in either ventricle. Circulating epinephrine was increased fourfold whereas circulating and myocardial norepinephrine were unchanged. These data demonstrate a down-regulation of the left ventricular beta-adrenergic receptor/adenylate cyclase system during chronic hypoxemia secondary to an intracardiac right-to-left shunt.

Relative binding properties of fluorescein and 9-hydroxyphenylfluoron (HPF) with murine monoclonal anti-fluorescein antibodies.

Contribution of the fluorescein (F1) carboxyl group to hapten binding by idiotypically related murine monoclonal anti-F1 antibodies 4-4-20, 9-40 and 12-40 was studied by comparing relative liganded active site properties with bound Fl or 9-hydroxyphenylfluoron (HPF). Kinetic studies revealed similar association rate constants between Fl and HPF to 4-4-20 (approximately 1.1 x 10(7) M-1 s-1); however, the 4-4-20 dissociation rate for Fl was approximately 200 times slower, relative to HPF, which resulted in relative intrinsic affinity values of 1.2 x 10(10) and 6.5 x 10(7) M-1, respectively. Mabs 9-40 and 12-40 also displayed a reduced affinity for HPF and affinity constants of 5.5 x 10(5) M-1 and 6.7 x 10(5) M-1 were obtained from a competitive ELISA. Additionally, previous studies revealed that upon binding Fl, Mabs 4-4-20 (92.1%), 9-40 (44.7%) and 12-40 (73.4%) quenched Fl fluorescence. Similar analyses with HPF resulted in 64.4% and 2.0% fluorescence quenching by 4-4-20 and 12-40, respectively; however, 9-40 increased HPF fluorescence by approximately 24%. Steady-state fluorescence polarization experiments revealed that in solution, Fl (P = 0.019) and HPF (P = 0.048) were polarized to different degrees. When bound, however, Fl and HPF expressed similar polarization values (P approximately 0.455), except 9-40 bound HPF which was significantly depolarized (P = 0.428). Fluorescence lifetime experiments revealed Fl to possess two discrete lifetimes: a 3.96 ns component (free Fl) and either a 0.52 ns (4-4-20), 2.23 ns (9-40) or 0.96 ns (12-40) short component that corresponded to bound Fl. HPF, however, when bound by 4-4-20 or 9-40, was best fit by three discrete exponentials: a relatively long 4.0 ns component, a 1.11 ns lifetime (free HPF) and either a 0.52 ns (4-4-20) or 2.23 ns (9-40) component. Finally, HPF bound by Mab 12-40 exhibited a single lorenzian distributed lifetime of 1.36 ns (+/- 0.43 ns). Results are discussed in terms of Mab active site structure and conformational state dynamics.

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein.

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.

Perturbation of antibody bound bifluorescent-ligand probe by polyclonal anti-metatype antibodies interacting with epitopes proximal to the liganded antibody active site.

General localization of metatypic determinants recognized by polyclonal anti-metatype antibodies relative to the antibody active site of the high-affinity anti-fluorescein monoclonal antibody 4-4-20 was achieved through use of a unique bifluorescent-ligand probe. The fluorescent probe possessed intrinsic energy-transfer properties with the fluorescein hapten serving as the energy acceptor. The donor group 5-(2-iodoacetyl) aminoethylaminonaphthalene-1-sulfonic acid (IAEDANS) proved environmentally sensitive both to binding of the FITC-cys-AEDANS ligand and to subsequent anti-metatype antibody interactions involving the antibody variable domains of 4-4-20. Spectral changes in ligand-conjugated AEDANS upon specific reactivity of the antibody with FITC suggested secondary interactions between AEDANS and the topological protein surface adjacent to the 4-4-20 active site. Results indicated that some anti-metatype antibodies (Fab fragments) within the polyclonal population bound to sites immediately surrounding the liganded active site and perturbed the interactions of AEDANS with topological sites. The results are discussed in terms of the types of interactions that may occur between the AEDANS moiety and the 4-4-20 antibody protein surface and subsequent perturbation of those interactions by anti-metatype antibodies.

Interaction of dual-specific autoantibodies with dsDNA and a synthetic dimer peptide simulating the hinge region of IgG2a molecules.

Anti-dsDNA autoantibodies and immune complex formation are major factors in SLE pathogenesis. Understanding stable immune complex formation is critical in deciphering mechanisms of autoimmune pathogenesis. Previous studies identified a subpopulation of murine lupus monoclonal autoantibodies that exhibited dual specificity (anti-DNA and anti-IgG2a hinge) and formed stable immune complexes [J. Mol. Rec. 10(1997)225]. Two monoclonal autoantibodies, BV 17-45 and BV 16-13, were extensively studied because of their dual specificity. To quantitatively assess the role of each specificity in the formation of stable immune complexes, studies were performed to determine binding affinities for various sized dsDNA fragments (21, 43, 84, and 114 bp) and the covalent dimer of a nine amino acid hinge peptide. Results characterizing BV 17-45 showed that the affinity for dsDNA directly correlated with increased dsDNA size. Results with BV 16-13 revealed a generally lower affinity for the various dsDNA fragments. Binding inhibition studies, using a covalently linked dimer of a nine amino acid synthetic hinge peptide as an inhibitor of the antibody-43 bp dsDNA interaction, yielded relative affinities for the anti-hinge activity. Binding affinities for the synthetic hinge specificity were lower than affinities measured for the anti-dsDNA activity. Collectively, the binding and inhibition studies provided insight into the correlation between dual specificity and avid immune complex formation. A model was proposed based on the concept that large dsDNA fragments caused localization of the dual-specific antibodies through the anti-dsDNA activity, thereby facilitating subsequent binding and cross-linkage via the anti-hinge specificity. These synergistic interactions resulted in the formation of avid immune complexes.

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.

Autoimmune VH gene family: PCR-generated murine germline VH10 genes.

A relatively large number of variable region genes (V) contribute, via gene rearrangements with smaller numbers of additional gene elements (D and J), to generate diversity in the immune response. While some VH gene families are thought to contain 100- 1000 members, the VH10 family has only two known functioning members with 99% sequence homology. Both members (monoclonal antibodies) are capable of binding DNA, and since they were derived from inbred mice afflicted with the lupus syndrome they are considered autoimmune antibodies. Relative uniqueness of the VH10 primary nucleotide sequence presents a model system with which to examine unrearranged VH genes and attempt to identify germline genes eventually expressed as autoantibodies. PCR amplified germline sequences of the VH10 family are highly conserved, with few base substitutions evenly distributed between both framework and CDR regions. It was determined that the PCR amplified germline sequences are highly similar to the DNA sequences of the two monoclonal VH10 antibodies, and a non-functional psuedo-germline gene was found that is identical to a non-functional cDNA derived from a hybridoma cell line. These findings indicate that the use of unique CDR DNA sequences for the identification and amplification of specific germline V genes via PCR can yield vital information that may answer fundamental questions about the origins of autoimmune anti-DNA antibodies in afflicted individuals. The nature of the germline gene populations and the possible microheterogeniety of these genes may prove to be important in understanding the role of autoimmune antibodies in normal and diseased individuals.

Effects of secondary forces on the ligand binding properties and variable domain conformations of a monoclonal anti-fluorescyl antibody.

Biochemical interactions occurring external to the antibody active site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4-4-20 active site as a series of thermally averaged substates is proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.

Consequences of avidity in lymphocyte receptor-multivalent antigen binding in affinity maturation.

A mathematical model proposed by Crothers & Metzger (Immunochemistry 9, 341-357, 1972), describing the influence of multiple site interactions on the effective antigen binding property of antibodies was used to assess the possible role of lymphocyte receptor affinity in antibody affinity maturation. The model proposed that observed association constants (KobsS) were primarily dependent on the number of allowed active site-antigen interactions (n), the density and orientation of antigenic sites and the intrinsic association constant (K1) of an univalent antibody active site interacting with an univalent antigen. Because of the inherent uncertainty involved in the estimation of lymphocyte membrane structure and molecular parameters necessary to utilize other mathematical models, the Crothers and Metzger model was applied to the case of surface membrane bound immunoglobulin (smIg) on B-lymphocytes interacting with polyvalent antigens as a simple and direct concept. Consideration of red blood cells, dinitrophenyl16 guinea-pig albumin, and fluorescyl220 keyhole limpet hemocyanin as model antigens revealed that Kobs was many orders of magnitude higher than K1 when only a few interactions were allowed. A dissociation rate of several hours for release of polyvalent antigen from cooperating smIg was calculated for receptors with a K1 as low as 10(3) M-1. Assuming prolonged antigen contact as necessary to activate lymphocytes, cells bearing low-affinity receptors are functionally indistinguishable from those carrying higher-affinity smIg in their ability to retain antigen. The presumed role for antigen-driven selection of lymphocytes bearing increasingly higher-affinity smIg, which results in antibody affinity maturation, must be reexamined if the K1 of smIg does not confer a selective advantage to lymphocytes for retaining antigen.

Specificity of anti-DNA antibodies in SLE--II. Relative contribution of backbone, secondary structure and nucleotide sequence to DNA binding.

Fine specificity of a population of anti-DNA antibodies which bound both ssDNA and dsDNA with apparently equal affinity was studied in two SLE plasma. Sensitivity of DNA binding to increasing sodium chloride concentration indicated that electrostatic interactions occurred between antibody and phosphate moieties of DNA. Secondary nucleic acid structure was important to DNA binding as double-stranded synthetic deoxynucleotide polymers were more effective inhibitors than their substituent single-stranded polymers. Nucleotide bases were also found to play a role in recognition of DNA by these cross-reactive antibodies, as ssDNA binding was sensitive to increasing temperature which caused unstacking of the nucleotide bases. Differing patterns of reactivity with synthetic deoxynucleotide polymers with similar secondary structures but different nucleotide compositions further indicated the importance of nucleotide bases to dsDNA binding by cross-reactive anti-DNA antibodies in SLE plasma.

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: localization and characterization of idiotypic determinants.

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (Ka values ranging from 5 X 10(6) to 10(10) M-1) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (gamma, kappa) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15-20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained gamma 1 heavy (H) chains and 4-6-10 contained both gamma 1 and gamma 2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which defined binding mechanism diversity. The location of determinants recognized by anti-idiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotype by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (approximately 25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the VH- and VL-chain interaction must exist for the expression of these idiotypes.

Monoclonal murine anti-nucleic acid antibody with double-stranded specificity.

The NZB/NZW F1 murine model for the autoimmune disease systemic lupus erythematosus (SLE) has been employed in somatic cell hybridizations to develop hybridoma autoantibodies with double-stranded (ds) DNA specificity. Monoclonal anti-DNA antibodies from one hybridoma cell line were purified and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Results of comparative binding studies with tritiated [3H]-colicin E1 plasmid DNA probes suggested preferential binding for the native DNA conformation relative to single-stranded DNA. [3H]dsDNA binding was inhibited by several ribohomopolymers (poly G, U and I) but not by free nucleotides, indicating that the phosphodiester-ribose backbone may contribute to the binding specificity of the clonotype.

Effects of secondary forces on a high affinity monoclonal IgM anti-fluorescein antibody possessing cryoglobulin and other cross-reactive properties.

The effects of secondary forces on monoclonal IgM anti-fluorescein antibody 18-2-3 reactivity were investigated and the results correlated with similar studies characterizing anti-fluorescein mAbs 4-4-20 and 9-40. mAb 18-2-3 was considered an important model for further elucidation of secondary forces since it possessed ligand binding properties similar to mAb 4-4-20, such as a similar affinity, but due to a very different primary structure it was idiotypically and metatypically distinct. mAb 18-2-3 also possessed cryoglobulin (anti-Ig) and extensive cross-reactive properties (e.g. anti-phenyloxazolone) suggestive of an atypical anti-fluorescein active site. The reactivity of mAb 18-2-3 with model fluorescein-peptides was modulated by secondary forces in a manner that differed from both mAbs 4-4-20 and 9-40. Thus, the effects of secondary forces seemed to vary with each monoclonal antibody even though each of the immunoglobulins studied were specific for the same homologous ligand. Results indicated that secondary forces impacted immune complex stability, variable domain conformation and protein dynamics. Models were postulated to account for secondary effects on the mAb 18-2-3 active site relative to mAbs 4-4-20 and 9-40. Levels of hydration, active site architecture and local amino acid dynamics were among the models cited.