Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Engineered discoidin domain from factor VIII binds αvβ3 integrin with antibody-like affinity

Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to αvβ3 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 Å resolution. We have christened this discoidin domain alternative scaffold the “discobody.”

Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein

More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design.

Model systems for the study of drug hypersensitivity

I. It was not possible to produce anti-tetracycline antibody in laboratory animals by any of the methods tried. Tetracycline protein conjugates were prepared and characterized. It was shown that previous reports of the detection of anti-tetracycline antibody by in vitro-methods were in error. Tetracycline precipitates non-specifically with serum proteins. The anaphylactic reaction reported was the result of misinterpretation, since the observations were inconsistent with the known mechanism of anaphylaxis and the supposed antibody would not sensitize guinea pig skin. The hemagglutination reaction was not reproducible and was extremely sensitive to minute amounts of microbial contamination. Both free tetracyclines and the conjugates were found to be poor antigens.

II. Anti-aspiryl antibodies were produced in rabbits using 3 protein carriers. The method of inhibition of precipitation was used to determine the specificity of the antibody produced. ε-Aminocaproate was found to be the most effective inhibitor of the haptens tested, indicating that the combining hapten of the protein is ε-aspiryl-lysyl. Free aspirin and salicylates were poor inhibitors and did not combine with the antibody to a significant extent. The ortho group was found to participate in the binding to antibody. The average binding constants were measured.

Normal rabbit serum was acetylated by aspirin under in vitro conditions, which are similar to physiological conditions. The extent of acetylation was determined by immunochemical tests. The acetylated serum proteins were shown to be potent antigens in rabbits. It was also shown that aspiryl proteins were partially acetylated. The relation of these results to human aspirin intolerance is discussed.

III. Aspirin did not induce contact sensitivity in guinea pigs when they were immunized by techniques that induce sensitivity with other reactive compounds. The acetylation mechanism is not relevant to this type of hypersensitivity, since sensitivity is not produced by potent acetylating agents like acetyl chloride and acetic anhydride. Aspiryl chloride, a totally artificial system, is a good sensitizer. Its specificity was examined.

IV. Protein conjugates were prepared with p-aminosalicylic acid and various carriers using azo, carbodiimide and mixed anhydride coupling. These antigens were injected into rabbits and guinea pigs and no anti-hapten IgG or IgM response was obtained. Delayed hypersensitivity was produced in guinea pigs by immunization with the conjugates, and its specificity was determined. Guinea pigs were not sensitized by either injections or topical application of p-amino-salicylic acid or p-aminosalicylate.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

Immunodetection of planktonic algae

Interest in the identification and characterisation of cyanobacteria and dinoflagellates in aquatic environments is increasing rapidly due to the perceived roles of these organisms in primary production and nuisance aspects in terms of water treatment and public health. Techniques for the identification and quantification of these organisms currently are limited, and the application of molecular approaches provides fundamental taxonomic information and techniques of practical value. Antigenic properties of algal cells may be useful taxonomic markers. Immunodetection techniques utilise the specificity of the antibody/antigen association as a probe for recognising and distinguishing between microorganisms according to their cell- surface chemistry. Immunofluorescent detection of unicellular cyanobacteria and dinoflagellates has been studied with success in marine and freshwater ecosystems and a range of techniques and results are presented and discussed. The most recent advances in the study of planktonic algae have come with the application of continuous flow cytometric methods (CFC). Flow cytometry makes use of the autofluorescence properties of the algal cells, which alone can be used to demonstrate their presence and permit their quantification in natural water samples. When used in conjunction with immunolabelling techniques, the potential of CFC analysis is broadened to study the serological/strain composition of plankters in natural populations. Changes in algal strains represented within and between waters over periods of time are reported and discussed, along with the ecological issues thus raised.

Direct detection of Yersinia pestis from the infected animal specimens by a fiber optic biosensor

The FOB-3, anew type fiber optic biosensor, is designed to rapidly detect a variety of biological agents or analytes with better stability, sensitivity and specificity. In order to detect Y. Pestis, a sandwich immunoassay was developed by using the purified antibody against antigen FI immobilized on polystyrene probes as the capture antibody and the monoclonal antibody-Cy5 conjugate as the detector. After a series of optimization for the stability, sensitivity and specificity of the FOB-3, 50-1000 ng/ml of antigen FI and 6 x 10(1)-6 x 10(7) CFU/ml Y. pestis could be detected constantly in about 20 min, and Y pestis could be detected specifically from Y. pseudotuberculosis, Y. enterocolitica, B. anthracis and E. coli. Then, 39 blind samples, including 27 tissues of mice infected with Y pestis and 12 tissues of healthy mice as negative control, were detected with the FOB-3. 92.6% infected tissues were identified from the tissues of healthy mice and the tissues containing more than 100 CFU/ml bacteria could be detected by the biosensor. The results demonstrated the feasibility of the FOB-3 as an effective method to detect Y. pestis rapidly and directly from the infected animal specimens with the advantage of portability, simple-operation as well as high sensitivity and specificity. (c) 2006 Elsevier B.V. All rights reserved.

Novel Papillomaviruses in Free-Ranging Iberian Bats: No Virus-Host Co-evolution, No Strict Host Specificity, and Hints for Recombination

Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus. Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus-host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species (E. serotinus and E. isabellinus) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Specificity of reflex adaptation for task-relevant variability.

The motor system responds to perturbations with reflexes, such as the vestibulo-ocular reflex or stretch reflex, whose gains adapt in response to novel and fixed changes in the environment, such as magnifying spectacles or standing on a tilting platform. Here we demonstrate a reflex response to shifts in the hand's visual location during reaching, which occurs before the onset of voluntary reaction time, and investigate how its magnitude depends on statistical properties of the environment. We examine the change in reflex response to two different distributions of visuomotor discrepancies, both of which have zero mean and equal variance across trials. Critically one distribution is task relevant and the other task irrelevant. The task-relevant discrepancies are maintained to the end of the movement, whereas the task-irrelevant discrepancies are transient such that no discrepancy exists at the end of the movement. The reflex magnitude was assessed using identical probe trials under both distributions. We find opposite directions of adaptation of the reflex response under these two distributions, with increased reflex magnitudes for task-relevant variability and decreased reflex magnitudes for task-irrelevant variability. This demonstrates modulation of reflex magnitudes in the absence of a fixed change in the environment, and shows that reflexes are sensitive to the statistics of tasks with modulation depending on whether the variability is task relevant or task irrelevant.

Interfacial immobilization of monoclonal antibody and detection of human prostate-specific antigen.

Antibody orientation and its antigen binding efficiency at interface are of particular interest in many immunoassays and biosensor applications. In this paper, spectroscopic ellipsometry (SE), neutron reflection (NR), and dual polarization interferometry (DPI) have been used to investigate interfacial assembly of the antibody [mouse monoclonal anti-human prostate-specific antigen (anti-hPSA)] at the silicon oxide/water interface and subsequent antigen binding. It was found that the mass density of antibody adsorbed at the interface increased with solution concentration and adsorption time while the antigen binding efficiency showed a steady decline with increasing antibody amount at the interface over the concentration range studied. The amount of antigen bound to the interfacial immobilized antibody reached a maximum when the surface-adsorbed amount of antibody was around 1.5 mg/m(2). This phenomenon is well interpreted by the interfacial structural packing or crowding. NR revealed that the Y-shaped antibody laid flat on the interface at low surface mass density with a thickness around 40 Å, equivalent to the short axial length of the antibody molecule. The loose packing of the antibody within this range resulted in better antigen binding efficiency, while the subsequent increase of surface-adsorbed amount led to the crowding or overlapping of antibody fragments, hence reducing the antigen binding due to the steric hindrance. In situ studies of antigen binding by both NR and DPI demonstrated that the antigen inserted into the antibody layer rather than forming an additional layer on the top. Stability assaying revealed that the antibody immobilized at the silica surface remained stable and active over the monitoring period of 4 months. These results are useful in forming a general understanding of antibody interfacial behavior and particularly relevant to the control of their activity and stability in biosensor development.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons receiv

SECRETORY MONOCLONAL IGA ANTIBODY TO HUMAN SPERM PRODUCED BY GASTROINTESTINAL IMMUNIZATION INHIBITS HUMAN SPERM ACTIVITY AND MOUSE INVITRO FERTILIZATION

BALB/c mice were immunized intragastrically with human sperm. Cells from the Peyer's patches and spleens of the immunized mice were for the preparation of hybridomas secreting antisperm monoclonal IgA (mcIgA). The specific ratio of IgA-secreting cells in Peyer's patches was much higher than that in spleen. The binding site on human sperm of 9 of 19 mcIgA was in the post-acrosomal region using an immunofluorescent assay. Two of eight selected mcIgA caused strong human sperm agglutination and three of them produced significant inhibition of mouse in vitro fertilization. No mcIgA tested caused obvious human sperm immobilization or inhibited mouse in vivo fertilization. In vitro assembly of selected mcIgA in ascites with mouse secretory component (SC) caused no significant changes in effects on sperm function and in vitro fertilization. By use of Western blotting, dimer or higher polymers were demonstrated in all selected mcIgAs and corresponding protein antigens in 6 of 8 selected mcIgAs. These results suggest that human sperm function may be inhibited and fertilization rate reduced by specific secretory IgA to human sperm and that secretory immunity to protein antigens of human sperm could be induced by intragastrointestinal immunization.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.