Dendronized ions

In dieser Arbeit wurde die Frage untersucht, inwieweit die divergente Synthese von starren dendritischen Gerüsten genutzt werden kann, um die Eigenschaften von Salzen und Ionen zu verändern und gezielt zu steuern. Motiviert wurde diese Fragestellung durch das Ziel, größere und dadurch schwächer koordinierende Anionen als die bereits in der Literatur beschriebenen Anionen zu erzeugen. In der Tat konnten durch das divergente Wachstum starrer Polyphenylen-Dendronen sterisch sehr anspruchsvolle Borat-Anionen von bislang unerreichter Größe im Nanometerbereich erzeugt werden. Durch die Größe ihrer hydrophoben Hülle und die damit einhergehende sterische Abschirmung der zentralen Ladung weisen starr dendronisierte Ionen eine deutlich verminderte Koordination zu Gegenionen auf. Die Koordinationkraft ließ sich sowohl durch eine stärkere Verzweigung der dendritischen Hülle als auch durch die Perfluorierung der Ionenoberfläche weiter herabsetzen. Schließlich konnten durch den Einbau lichtschaltbarer Funktionen in das starre Dendrimergerüst sogar Anionen mit schaltbarer, veränderlicher Größe und Koordinationskraft hergestellt werden. Darüber hinaus wurde gezeigt, wie sich Gegenionen in Salzen dendronisierter Ionen austauschen lassen, und es wurde eine neue Klasse von Aryl-Triazol-Dendrimeren entwickelt.

Synthese und Evaluierung makrozyklischer 68 Ga-MPI-Tracer auf Pyridaben-Basis

Herz-Kreislauf-Erkrankungen zählen weltweit zu den Hauptursachen, die zu frühzeitigem Tod führen. Pathophysiologisch liegt eine Gefäßwandverdickung durch Ablagerung arteriosklerotischer Plaques (Arteriosklerose) vor. Die molekulare Bildgebung mit den nuklearmedizinischen Verfahren SPECT und PET zielt darauf ab, minderperfundierte Myokardareale zu visualisieren, um den Krankheitsverlauf durch frühzeitige Therapie abschwächen zu können. Routinemäßig eingesetzt werden die SPECT-Perfusionstracer [99mTc]Sestamibi und [99mTc]Tetrofosmin. Zum Goldstandard für die Quantifizierung der Myokardperfusion werden allerdings die PET-Tracer [13N]NH3 und [15O]H2O, da eine absolute Bestimmung des Blutflusses in mL/min/g sowohl in der Ruhe als auch bei Belastung möglich ist. 2007 wurde [18F]Flurpiridaz als neuer Myokardtracer vorgestellt, dessen Bindung an den MC I sowohl in Ratten, Hasen, Primaten als auch in ersten klinischen Humanstudien eine selektive Myokardaufnahme zeigte. Um eine Verfügbarkeit des Radionuklids über einen Radionuklidgenerator gewährleisten zu können, sollten makrozyklische 68Ga-Myokard-Perfusionstracer auf Pyridaben-Basis synthetisiert und evaluiert werden. Die neue Tracer-Klasse setzte sich aus dem makrozyklischen Chelator, einem Linker und dem Insektizid Pyridaben als Targeting-Vektor zusammen. Struktur-Affinitätsbeziehungen konnten auf Grund von Variation des Linkers (Länge und Polarität), der Komplexladung (neutral und einfach positiv geladen), des Chelators (DOTA, NODAGA, DO2A) sowie durch einen Multivalenzansatz (Monomer und Dimer) aufgestellt werden. Insgesamt wurden 16 neue Verbindungen synthetisiert. Ihre 68Ga-Markierung wurde hinsichtlich pH-Wert, Temperatur, Vorläufermenge und Reaktionszeit optimiert. Die DOTA/NODAGA-Pyridaben-Derivate ließen sich mit niedrigen Substanzmengen (6 - 25 nmol) in 0,1 M HEPES-Puffer (pH 3,4) bei 95°C innerhalb 15 min mit Ausbeuten > 95 % markieren. Für die DO2A-basierenden Verbindungen bedurfte es einer mikrowellengestützen Markierung (300 W, 1 min, 150°C), um vergleichbare Ausbeuten zu erzielen. Die in vitro-Stabilitätstests aller Verbindungen erfolgten in EtOH, NaCl und humanem Serum. Es konnten keine Instabilitäten innerhalb 80 min bei 37°C festgestellt werden. Unter Verwendung der „shake flask“-Methode wurden die Lipophilien (log D = -1,90 – 1,91) anhand des Verteilungs-quotienten in Octanol/PBS-Puffer ermittelt. Die kalten Referenzsubstanzen wurden mit GaCl3 hergestellt und zur Bestimmung der IC50-Werte (34,1 µM – 1 µM) in vitro auf ihre Affinität zum MC I getestet. In vivo-Evaluierungen erfolgten mit den zwei potentesten Verbindungen [68Ga]VN160.MZ und [68Ga]VN167.MZ durch µ-PET-Aufnahmen (n=3) in gesunden Ratten über 60 min. Um die Organverteilung ermitteln zu können, wurden ex vivo-Biodistributionsstudien (n=3) vorgenommen. Sowohl die µ-PET-Untersuchungen als auch die Biodistributionsstudien zeigten, dass es bei [68Ga]VN167.MZ zwar zu einer Herzaufnahme kam, die jedoch eher perfusionsabhängig ist. Eine Retention des Tracers im Myokard konnte in geringem Umfang festgestellt werden.

Synthese von selbstaggregierenden Glycopeptidkonjugaten mit (per)fluorierten Membranankern

Für eine effektive Erkennung tumorassoziierter Kohlenhydratantigene durch das Immun-system in der Krebs¬immuntherapie ist eine multivalente Präsentation der Haptene notwendig. In der vorliegenden Arbeit wurde ein neuer Zugang zu einer solch räumlichen Konzentration der Haptene untersucht, indem MUC1-Antigene mit perfluorierten Alkylketten funktionalisiert und in einer geeigneten Lipidmatrix entmischt wurden. Perfluoralkyl-Amphiphile zeichnen sich durch eine hohe Entmischungstendenz in Alkyllipiden aus und bewirken dadurch eine Anreicherung der Erkennungsstrukturen (Haptene) in Analogie zu den natürlichen raft-Domänen auf der Zelloberfläche.rnDazu wurden zunächst verschiedene Membranankersysteme mit unterschiedlichem Fluorierungsgrad entwickelt. Beispielsweise konnte ausgehend von einem zentralen Glycerin-fragment ein Membrananker mit zwei Perfluoralkylketten hergestellt werden. Letztere wurden mittels radikalischer Perfluoralkylierung eingeführt, wobei der Fluorgehalt der Verbindung über die Kettenlänge gesteuert wurde. Daneben konnte ein weiteres Ankersystem, basierend auf der Aminosäure Lysin, synthetisiert werden, dass einen bequemen Einbau der Perfluoralkylketten durch Peptidkupplungen von entsprechenden perfluorierten Aminen bzw. perfluorierten Carbonsäuren erlaubte. In diesem Fall wurde der Fluorgehalt durch die Einführung von Alkyl- bzw. Perfluoralkylketten verändert.rnBeide Systeme konnten für erste Untersuchungen ihres Phasenverhaltens mit polaren Kopf-gruppen ausgestattet werden, wobei neben einem hydrophilen, nicht-immunogenen Triethylenglycolspacer vor allem ein TN-Antigen tragendes Dipeptid zum Einsatz kam. In Gegenwart des Matrixlipids DODAMA konnten in Langmuir-Blodgett-Untersuchungen mit diesen Verbindungen eine Entmischung und die Ausbildung mikroseparierter Bereiche nachgewiesen werden. Auch war es möglich, durch Anbindung eines Fluoreszenzfarbstoffes zu zeigen, dass solche amphiphilen Membrananker auf perfluorierten Oberflächen effektiv und dauerhaft immobilisiert werden können. Damit eröffnet diese Verbindungsklasse interessante Anwendungsmöglichkeiten in der Entwicklung von diagnostischen Microarray-Formaten.rnUm eine Anbindung der fluorierten Membrananker an den N-Terminus eines an fester Phase aufgebauten mucinanalogen Glycopeptids als antigene Einheit zu ermöglichen, wurde ein entsprechendes Ankersystem auf Basis von Glutaminsäure entwickelt. Dabei wurden an diese Verbindung neben dem TN-Antigen noch weitere komplexe tumorassoziierte Kohlenhydrat-antigene des Mucintyps angebunden, wobei der Aufbau der resultierenden amphiphilen Glycolipopeptide vollständig an der festen Phase gelang. Insgesamt konnten so mithilfe des teilfluorierten Lysinankers und des zweifach perfluorierten Glutaminsäureankers erste amphiphile Glycopeptid-Konjugate hergestellt werden, deren antigene Kopfgruppe aus einer 20 Aminosäuren umfassenden Wiederholungseinheit des Mucins MUC1 mit TN-, T- bzw. STN-Antigen-Seitenkette besteht. Derartige Verbindungen stellen reizvolle Bausteine für die Tumordiagnostik und für die Entwicklung von stabilen liposomalen Tumorvakzinen dar, da die verwendeten Perfluoralkylanker die Antigenpräsentation nicht wesentlich beeinflussen und die Bindung des Antikörpers nicht behindern. rn

High-resolution electrophoretic simulations: performance characteristics of one-dimensional simulators

Three comprehensive one-dimensional simulators were used on the same PC to simulate the dynamics of different electrophoretic configurations, including two migrating hybrid boundaries, an isotachophoretic boundary and the zone electrophoretic separation of ten monovalent anions. Two simulators, SIMUL5 and GENTRANS, use a uniform grid, while SPRESSO uses a dynamic adaptive grid. The simulators differ in the way components are handled. SIMUL5 and SPRESSO feature one equation for all components, whereas GENTRANS is based on the use of separate modules for the different types of monovalent components, a module for multivalent components and a module for proteins. The code for multivalent components is executed more slowly compared to those for monovalent components. Furthermore, with SIMUL5, the computational time interval becomes smaller when it is operated with a reduced calculation space that features moving borders, whereas GENTRANS offers the possibility of using data smoothing (removal of negative concentrations), which can avoid numerical oscillations and speed up a simulation. SPRESSO with its adaptive grid could be employed to simulate the same configurations with smaller numbers of grid points and thus is faster in certain but not all cases. The data reveal that simulations featuring a large number of monovalent components distributed such that a high mesh is required throughout a large proportion of the column are fastest executed with GENTRANS.

Syntheses and structures of molybdenum and tungsten complexes capable of epoxidaton and copper coordination polymers and dendrimers

We are interested in the syntheses of new complexes and in their characterization by single crystal X-ray diffraction techniques. Once we understand the structures, studies aimed at understanding uses of these complexes in the field of catalytic epoxidation using complexes soluble in water and syntheses of thin films (not assessed) were conducted. The syntheses, characterization and catalytic properties of a series of mononuclear, dinuclear and tetranuclear molybdenum and tungsten oxo complexes are described. The syntheses and structural characterization of two copper coordination polymers with 3,5-dihydroxylbenzoate ligand, and five paddlewheel shaped copper dendrimers coordinated with Fréchet-type dendrons are also detailed. The background of this dissertation is outlined in Chapter 1. Chapter 2 describes the syntheses, and characterization of two new mononuclear molybdenum(VI) and tungsten(VI) oxo complexes, MoO2Cl2(OPPh2CH2OH)2, and WO2Cl2(OPPh2CH2OH)2, bearing hydrophilic phosphine oxide ligand. The catalytic properties of these complexes for the epoxidation of cis-cyclooctene were also studied. Two new dinuclear molybdenum(VI) and tungsten(VI) oxo complexes Mo2O4Cl2[(HOCH2)PhPOO]2, and (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, bearing organophosphinate ligand are described in Chapter 3 and 4. Chapter 4 and 5 describes the syntheses and characterization of tetranuclear molybdenum(V) oxo complexes bearing various organophosphinate ligands. The catalytic abilities of these complexes for the epoxidation of cis-cyclooctene in the presence of hydrogen peroxide as oxidant were explored as well. Various spectroscopic methods, such as IR, UV-vis, and NMR are used to characterize the nature of these complexes. Crystal structures of compounds MoO2Cl2(OPPh2CH2OH)2, WO2Cl2(OPPh2CH2OH)2, Mo2O4Cl2[(HOCH2)PhPOO]2, (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, and Mo4(µ3-O)4(µ-O2PR2)4O4 (R=Ph, Me, ClCH2, o-C6H4(CH2)2) are also presented. The syntheses, and structural characterization of three copper(II) coordination polymers bearing 3,5-dihydroxybenzoate ligand are described in Chapter 6. Two copper(II) coordination polymers, [Cu2(3,5-dhb)2(pyridine)4]n, and [Cu2(3,5-dhb)4]n were afforded based on different amount of pyridine used in the reaction. The structures of these complexes are further built into 2D or 3D networks via inter or intra hydrogen bonds. The syntheses and structural characterization of the zinc(II) monomer, Zn(3,5-dhb)2(pyridine)2 is also described in this Chapter. Chapter 7 describes the syntheses, and characterization of five dendronized dicopper complexes bearing different generations of Fréchet-type dendrons. The structures of 3,5- bis(benzoyloxl)benzoic acid, 3,5-(PhCOO)2PhCOOH (G1), Cu2(3,5-dhb)4(THF)2, Cu2(G1)4(pyridine)2, and Cu2(G1)4(CH3OH)2 were characterized unambiguously by single X-ray diffraction. In addition, all compounds were characterized by FT-IR, UV-vis spectroscopy and elemental analyses.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

Prevalence of antileptospiral serum antibodies in dogs in Ireland.

A total of 474 serum samples from client owned Irish dogs were tested for the presence of antibodies to serovars Canicola, Icterohaemorrhagiae, Bratislava, Autumnalis, Pomona, Altodouro, Grippotyphosa, Mozdok, Hardjobovis and Ballum. Six per cent of dogs presented to veterinary practitioners for problems unrelated to leptospirosis showed evidence of prior exposure to leptospiral serovars belonging to the serogropus Ballum, Australis, Pomona and Sejroe. One unvaccinated dog suspected to have leptospirosis showed seroconversion to serogroup Icterohaemorrhagiae. Based on these results the authors conclude that canine exposure to serogroup Ballum should be monitored because dogs may serve as sentinels for this serovar in the environment. Vaccination with multivalent vaccines containing serovar Bratislava in addition to serogroups Icterohaemorrhagiae and Canicola is advisable.

Liquid-Crystalline Dendrimers Designed by Click Chemistry

Liquid-crystalline dendrimers have been prepared from second-generation Percec-type poly(benzyl ether) dendrons or second-generation poly(aryl ester) dendrons carrying cyanobiphenyl mesogens. The Janus dendrimer, which combines the two types of dendromesogens, has also been synthesized. Those compounds have been prepared under copper-catalyzed azide–alkyne cycloaddition conditions. The mesomorphic properties have been studied by thermal analysis (POM, DSC) and small-angle X-ray scattering. Smectic A, nematic, and columnar phases have been observed depending on the dendritic building blocks. The click reaction has proven to be a powerful and elegant synthetic tool for the design of complex dendritic liquid-crystalline architectures.

Superactivation of an immune response triggered by oligomerized T cell epitopes

The peptides bound to class II major histocompatibility complex (MHC) molecules extend out both ends of the peptide binding groove. This structural feature provided the opportunity to design multivalent polypeptide chains that cross-link class II MHC molecules through multiple, repetitive MHC binding sites. By using recombinant techniques, polypeptide oligomers were constructed that consist of up to 32 copies of an HLA-DR1-restricted T cell epitope. The epitope HA306–318, derived from influenza virus hemagglutinin, was connected by 12- to 36-aa long spacer sequences. These oligomers were found to cross-link soluble HLA-DR1 molecules efficiently and, upon binding to the MHC molecules of a monocyte line, to trigger signal transduction indicated by the enhanced expression of some cell surface molecules. A particularly strong effect was evident in the T cell response. A hemagglutinin-specific T cell clone recognized these antigens at concentrations up to three to four orders of magnitude lower than that of the peptide or the hemagglutinin protein. Both signal transduction in the monocyte and the proliferative response of the T cell were affected greatly by the length of the oligomer (i.e., the number of repetitive units) and the distance of the epitopes within the oligomer (spacing). Thus, the formation of defined clusters of T cell receptor/MHC/peptide antigen complexes appears to be crucial for triggering the immune response and can be used to enhance the antigenicity of a peptide antigen by oligomerizing the epitope.

Influence of follicular dendritic cells on decay of HIV during antiretroviral therapy

Drug treatment of HIV type 1 (HIV-1) infection leads to a rapid initial decay of plasma virus followed by a slower second phase of decay. To investigate the role of HIV-1 retained on follicular dendritic cells (FDCs) in this process, we have developed and analyzed a mathematical model for HIV-1 dynamics in lymphoid tissue (LT) that includes FDCs. Analysis of clinical data using this model indicates that decay of HIV-1 during therapy may be influenced by release of FDC-associated virus. The biphasic character of viral decay can be explained by reversible multivalent binding of HIV-1 to receptors on FDCs, indicating that the second phase of decay is not necessarily caused by long-lived or latently infected cells. Furthermore, viral clearance and death of short-lived productively infected cells may be faster than previously estimated. The model, with reasonable parameter values, is consistent with kinetic measurements of viral RNA in plasma, viral RNA on FDCs, productively infected cells in LT, and CD4+ T cells in LT during therapy.

Virus neutralization by germ-line vs. hypermutated antibodies

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Cκ) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in VH increased by 300-fold the binding strength of monovalent scFv-Cκ. The multivalent binding avidity of germ-line scFv-Cκ was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Cκ did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show “affinity maturation” effects of up to 1000-fold.

Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.

Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.