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Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes 1 -bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies 2,3 . Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to β2 microglobulin and RIFINs through their apical domains 4,5 . By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (β/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the β zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Colloidal gold-antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl 4 , is reduced with dilute solutions of sodium citrate, as described here. This will cause the gold to form small aggregates that will associate with proteins. Gold particles of specific sizes can be isolated and differentiated microscopically, allowing these particles to be used for multiple-label experiments. Colloidal gold-labeled antibodies are widely used in electron microscopy (EM), and can be used for light microscopy but require additional steps (silver enhancement)., (© 2020 Cold Spring Harbor Laboratory Press.)

The present study aims to identify specific staining methods for acoustic histology. We compared attenuation-of-sound (AOS) images from scanning acoustic microscopy (SAM) with light microscopy (LM) images. Ethanol-fixed tissue or cytology samples and formalin-fixed surgical or autopsy specimens were examined. Nuclei, collagen, elastic fibers and polysaccharides and various antigens, including cell surface, cytoplasmic, nuclear and stromal substances, were observed. Samples with various fixation methods were used. Hematoxylin staining had significantly higher AOS values in accordance with staining duration. Specific staining for collagen, elastic fibers and polysaccharides increased the AOS values of the specific substance. Using diaminobenzidine tetrahydrochloride in NiCl 2 solution as a substrate for horseradish peroxidase increased the AOS values to those suitable for acoustic immunostaining. Collagenase digestion after collagen staining decreased AOS values, reflecting collagen density and distribution. Staining with specific dyes or acoustic immunostaining enabled the histologic localization of specific substances by SAM, similar to LM., (Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4 BS ) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1-3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design., Competing Interests: AB, CW, MG, BF, JS, MJ, IG, GS, PK, AM, FD, LS, MP, DV No competing interests declared

For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

Taking advantage of imprinting technology, artificial antibody-microbial imprinted Ag-TiO(2) materials are fabricated for microbial inactivation using a facile and green method. Due to the induced shape and size recognition elements, the artificial antibodies specifically recognize and kill target microbes under visible-light irradiation with minimal toxic side effects toward mammalian cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Alzheimer's disease is a slowly progressive dementia that is characterized by Aβ amyloid aggregation, neurofibrillary tangles, and neuronal death. The major clinical symptoms are memory disturbance, aphasia, visuospatial disturbance, disturbance of executive function, and behavioral changes. Diagnosis is defined by the presence of dementia, the exclusion of non-Alzheimer type dementia, progressive atrophy of the hippocampus and temporoparietal cortex, decreased blood flow and glucose metabolism in the posterior cingulate gyrus and precuneus, decreased levels of Aβ42, and increased levels of tau in the cerebrospinal fluid accompanied by positive amyloid proton emission tomography. The development of disease modifying drugs is desired.

We report the straightforward oriented covalent attachment of antibodies (Abs) on the surface of carboxylated multiwalled carbon nanotube-polystyrene (MWCNT-PS) materials. The combination of this composite material, applied as a robust electrochemical transducer platform, and its covalent functionalization with Abs in a controlled way by means of a two-step process, could contribute to the development of highly sensitive immunosensor devices. Using the simple and versatile carbodiimide chemistry, Abs were attached to the carboxylic groups of the MWCNT-PS composite surfaces via their superficial amine groups. By taking into account the Ab isoelectric point and the net charge of the composite surface, we engineered an immobilization process to achieve the oriented binding of the Ab molecules by favoring an ionic pre-adsorption step before covalent binding occurred. Thus, the antigen binding capacity of the attached Abs was enhanced by up to 10 times with respect to the capacity estimated for a random spatial distribution of these molecules. The proposed strategy would also serve as a model for the efficient biofunctionalization of other carboxylated carbon-based polymer composite materials with potential applications in the biosensor field., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this paper we demonstrate the possibility of modifying porous silicon (PSi) particles with surface chemistry and recognition molecules (antibodies) such that these devices could potentially be used for single-cell identification or sensing. This is achieved by modifying PSi Rugate filters via hydrosilylation with surface chemistry that serves firstly, to protect the silicon surfaces from oxidation; secondly, renders the surfaces resistant to nonspecific adsorption of proteins and cells and thirdly, allows further functionality to be added such as the coupling of antibodies. The surface chemistry remained unchanged after sonication of the PSi to form PSi microparticles. The ability to monitor the spectroscopic properties of microparticles, and shifts in the optical signature due to changes in the refractive index of the material within the pore space, is demonstrated. The particles are shown to remain stable in physiological buffers and human blood for longer than one week. Finally, the modification of the PSi particles with functional antibodies is achieved.

In this paper, a cell separation technique has been explored using antibody-functionalized Ni nanowires. An antibody (anti-CD31) against mouse endothelial cells (MS1) was conjugated to the Ni nanowire surface through self-assembled monolayers (SAMs) and chemical covalent reactions. The measured cytotoxicity was negligible on the CD-31 antibody-functionalized nanowires by the tetrazolium salt (MTT) assay. The use of functionalized nanowires for magnetically separating MS1 cells revealed that the cell separation yield was closely related to cell concentration and the nanowire/cell ratio. Cell separation yield using functionalized Ni nanowires was compared with that using commercial magnetic beads. Considering the volume difference of the material used between the beads and nanowires, antibody-functionalized nanowires showed an obvious advantage in cell separation. Further study on the effect of Ni nanowires on MS1 cells for extended culture confirmed that cell morphology remained comparable to control cells with a lower proliferation rate. This work demonstrates that antibody-functionalized Ni nanowires provide an effective means to separate target cells.

Simultaneously Manufactured Nano-In-Micro (SIMANIM) particles for the pulmonary delivery of antibodies have been prepared by the spray-drying of a double-emulsion containing human IgG (as a model antibody), lactose, poly(lactide-co-glycolide) (PLGA) and dipalmitoylphosphatidylcholine (DPPC). The one-step drying process involved producing microparticles of a diameter suitable for inhalation that upon contact with aqueous media, partially dissolved to form nanoparticles, approximately 10-fold smaller than their original diameter. Continuous release of the model antibody was observed for 35 days in pH 2.5 release media, and released antibody was shown to be stable and active by gel electrophoresis, field-flow fractionation and enzyme linked immunosorbent assay. Adding 1% L-leucine to the emulsion formulation, and blending 'SIMANIM' particles with 1% magnesium stearate, achieved a fine particle fraction of approximately 60%, when aerosolised from a simple, capsule-based, dry powder inhaler device. 'SIMANIM' particles could be beneficial for the delivery of antibodies targeted against inhaled pathogens or other extracellular antigens, as well as having potential applications in the delivery of a wide range of other biopharmaceuticals and certain small-molecule drugs., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

X-ray and neutron-scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are useful when either a large protein cannot be crystallized, in which case scattering yields a solution structure, or a crystal structure has been determined and requires validation in solution. These solution structures are determined by the application of constrained modelling methods based on known subunit structures. First, an appropriate starting model is generated. Next, its conformation is randomized to generate thousands of models for trial-and-error fits. Comparison with the experimental data identifies a small family of best-fit models. Finally, their significance for biological function is assessed. We illustrate this in application to structure determinations for secretory immunoglobulin A, the most prevalent antibody in the human body and a first line of defence in mucosal immunity. We also discuss the applications to the large multi-domain proteins of the complement system, most notably its major regulator factor H, which is important in age-related macular degeneration and renal diseases. We discuss the importance of complementary data from analytical ultracentrifugation, and structural studies of protein-protein complexes. We conclude that constrained scattering modelling makes useful contributions to our understanding of antibody and complement structure and function.

Unlabelled: We describe a web server for the automatic prediction of immunoglobulin variable domains based on the canonical structure model. The server is user-friendly and flexible. It allows the user to select the templates for the frameworks and the loops using different strategies. The final output is a full-fledged 3D model of the variable domains of the target immunoglobulin., Availability: The server is openly accessible to academic users at the address: http://arianna.bio.uniroma1.it/pigs. It does not require registration and there is no limit to the number of sequences that can be submitted.

Protein misfolding and aggregation are a common thread in numerous diseases including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, diabetes, and prion-related diseases. Elucidation of the role played by the various protein forms in these diseases requires reagents that can target specific protein forms. Here we present a method to isolate antibodies that bind to a specific protein form. We combined the imaging and nanomanipulation capabilities of atomic force microscopy (AFM) with the protein diversity of phage display antibody libraries to develop a technology that allows us to recover a single antibody molecule that is bound to a single protein molecular target. The target protein-antibody complex is first imaged by AFM, the AFM tip is then manipulated by nanolithography over the target antibody to recover the associated phage, and the antibody gene is recovered from the single phage particle by polymerase chain reaction.

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

Complex DNA nanostructures have been developed as structural components for the construction of nanoscale objects. Recent advances have enabled self-assembly of organized DNA nanolattices and their use in patterning functional bio-macromolecules and other nanomaterials. Adapter molecules that bind specifically to both DNA lattices and nanomaterials would be useful components in a molecular construction kit for patterned nanodevices. Herein we describe the selection from phage display libraries of single-chain antibodies (scFv) for binding to a specific DNA aptamer and their development as adapter molecules for nanoscale construction. We demonstrate the decoration of various DNA tile structures with aptamers and show binding of the selected single-chain antibody as well as the self-assembly of mixed DNA-protein biomolecular lattices.

Antibody-antigen binding events at a monolayer protein concentration have been demonstrated on nanostructured adaptive silver films (ASFs) using surface-enhanced Raman scattering (SERS) and luminescence-based assays. It is shown that proteins stabilize and restructure the ASF to increase the SERS signal while preserving antigen-binding activity. Evidence for antibody-antigen binding on the ASF substrates is the distinct SERS spectral changes of the surface-bound antibody or antigen without special tags. The activity of the surface-bound proteins and their practical application are validated by independent immunochemical assays. Results are presented to demonstrate that these surfaces can be extended to protein arrays with detection applications distinct from current SERS, fluorescence, or luminescence methods.

A systematic evaluation of the effects of antibody immobilization strategy on the binding efficiency and selectivity (e.g., ability to distinguish between specific and nonspecific interactions) of immunosurfaces prepared with F(ab') antibody fragments of rabbit Immunoglobulin G (IgG) is described. F(ab') was attached to gold surfaces either (1) directly via the formation of a gold-thiolate bond or (2) indirectly through a series of a bifunctional linkers containing an alkane chain or ethylene glycol spacer. Immobilization of F(ab') via the sulfhydryl reactive group located opposite the antigen binding site ensured optimum orientation of the antigen binding site. X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) were used to confirm surface modification with the bifunctional linkers and antibody immobilization, respectively. Binding efficiency assays performed with SPR indicated that increasing the length of the linker increased the antigen binding efficiency. Atomic force microscopy (AFM) adhesion force measurements indicated that AFM probes functionalized with directly immobilized F(ab') more effectively discriminated between specific and nonspecific surface-bound proteins than probes modified indirectly via linker-immobilized F(ab'). In addition, a greater number of antibody-antigen binding events were observed with directly immobilized F(ab')-functionalized probes.

Tissue factor (TF) is a membrane-anchored protein that initiates the extrinsic cascade of blood coagulation. TF forms a complex with serine protease Factor VIIa, and then activates Factor X zymogen to Factor Xa, leading to the blood coagulation. Humanized anti-TF antibody hATR-5 strongly inhibits TF-initiated blood coagulation, and is of potential use for various thrombotic diseases. The Fab fragment of antibody hATR-5 is obtained for crystallization. The crystal structure of the complex of the Fab with extracellular domains of human TF was determined with the molecular replacement method, and refined to an R factor of 0.196 at 2.1 A resolution. All the complementarity-determining regions (CDRs) of the Fab are involved in interaction with the C-terminal-side extracellular domain of TF through 19 hydrogen bonds. The interface between the Fab and TF molecules contains 15 water molecules, and yields buried surface areas as wide as 2000 A2. The TF surface in the interface is possibly involved in the activation of Factor X, by forming a transient ternary complex of Factor X-TF-Factor VIIa. Electrostatic interactions are predominantly observed between the heavy-chain CDRs and TF. These hydrogen-bonding and electrostatic interactions together with the wide buried areas contribute to the high affinity of the antibody toward TF, leading to the effective inhibition of the TF-initiated blood coagulation.

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.

The aim of this work is to detect immune complexes without any kind of labelling of each of the immunological species, with a view to create a very sensitive biosensor. This is achieved by using the atomic force microscopy. We have proceeded by imaging the antibody (anti-rabbit IgG) or anti-rabbit IgG moieties adsorbed onto mica surface, before and after incubation of two kinds of antigens: a specific (rabbit IgG) and a non-specific one (sheep IgG). The analysis using the height histograms reveals many interesting features. We propose a general framework for interpreting these analysis, which enables the discrimination between specific and non-specific complexes.

A chimeric human-simian IgG, antigen specific for CD4, when exposed to 0.5 M SO(=)(4) containing 0.4% polyethylene glycol or Jeffamine, self-assembles into discreet, roughly spherical particles 23 nm in diameter. Increasing SO(=)(4) to 1.55 M induces the IgG particles to crystallize in either a hexagonal or a monoclinic form. From X-ray diffraction, the former crystal is of space group P622, with one IgG particle in the unit cell; thus the particle itself must have 622 point group symmetry. Both crystal forms have been imaged using atomic force microscopy. Detailed features of the duodecamer were evident, including the symmetry and a large solvent channel along the sixfold axis. The particles in some ways resemble the hexameric IgG aggregates believed to activate compliment upon antigen binding and, therefore, may have physiological relevance. Investigation of seven other IgGs of diverse origins and subclasses indicates that many, if not most, IgGs form similar particles. To our knowledge, this is the first observation of the assembly of IgG into high symmetry aggregates in the absence of antigen or their crystallization., (Copyright 2000 Academic Press.)

A force microscope operated with an amplitude modulation feedback (usually known as tapping-mode atomic force microscope) has two tip-sample interaction regimes, attractive and repulsive. We have studied the performance of those regimes to imaging single antibody molecules. The attractive interaction regime allows determination of the basic morphologies of the antibodies on the support. More importantly, this regime is able to resolve the characteristic Y-shaped domain structure of antibodies and the hinge region between domains. Imaging in the repulsive interaction regime is associated with the irreversible deformation of the molecules. This causes a significant loss in resolution and contrast. Two major physical differences distinguish the repulsive interaction regime from the attractive interaction regime: the existence of tip-sample contact and the strength of the forces involved.

Extended studies were performed to prepare substrates for scanning force microscopy of biological samples with a surface roughness below 1 nm rms over an area of 500 x 500 nm2. The substrate smoothness and the lack of tip obscuring material are indispensable in order to visualize detailed structures of membrane surfaces, particularly when scanning the lamellipodia of mechanically sensitive goldfish glial cells where peripheral lamellipodia are only 20-30 nm in thickness. Appropriate substrates are poly(vinyl phenyl ketone) or furan polymers with corrugations of 0.3 and 0.15 nm rms, respectively, to which the growth-promoting protein laminin adsorbs directly with an acceptably increased roughness. Cells show normal growth behavior on these substrates and stick to the substrates in a stable fashion during several scans. Thus details of the membrane's surface may be resolved and are not obscured by the substrate texture. The uncoated furan polymer substrates are suitable for immobilization of membrane preparations such as purified membrane fragments containing bacteriorhodopsin or Na, K-ATPase and protein preparations such as antibodies., (Copyright 1999 Academic Press.)

Hypothesis: The etiological hypothesis is that there might be factors triggering an immunological chain reaction that eventually leads to tympanosclerosis formation., Background: Tympanosclerosis is a condition leading to a calcification process in the middle ear and, occasionally, also to the lining of the inner ear. This sometimes leads to hearing loss due to fixation of the middle ear ossicles. In severe cases. deafness may occur as a result of the inner ear impairment. Surgery is the treatment offered, often with poor long-term results, and, alternatively, prescription of hearing aids. Some patients develop tympanosclerosis after mild inflammatory otitis media processes whereas some heal without tympanosclerosis after more aggressive infections. This difference may be due to individual variations in the inflammatory response. The biological mechanism of calcification in tympanosclerosis is probably similar to that occurring in other calcifying tissues due to diseases., Methods: The present investigation was performed to develop methods for immunohistochemical analyses of this delicate tissue consisting of both hard bone and the very thin tympanic membrane. Sprague-Dawley rats were inoculated with a suspension of Streptococcus pneumoniae, type 3, into the middle ear and sacrificed after 1 week up to 6 months. A new technique was elaborated where the whole specimen was prefixed briefly and then en bloc incubated with the primary antibodies and after that decalcified in edetic acid (EDTA). Primary antibodies against macrophages were used for the immunohistochemical staining., Results: Acute otitis media was successfully induced in the rats and myringosclerosis was seen in 30% of the animals, often localized close to the bony frame where macrophages could also be detected., Conclusions: Acute otitis media and myringosclerosis were introduced in the animals. Conventional immunological techniques were tested on this delicate tissue. A new method for immunohistochemical staining was elaborated in which specimens were stained en bloc before decalcification and sectioning were performed. Expression of macrophages was demonstrated in the tympanic membrane.

We have analysed antigen-contacting residues and combining site shape in the antibody Fv and Fab crystal structures now available from the Protein Data Bank. Antigen-contacting propensities are presented for each antibody residue, allowing a new definition for the complementarity determining regions (CDRs) to be proposed based on observed antigen contacts. Contacts are more common at CDR residues which are located centrally within the combining site; some less central CDR residues are only contacted by large antigens. Non-contacting residues within the CDRs coincide with residues identified by Chothia and co-workers as important in defining "canonical" conformations. An objective means of classifying protein surfaces by gross topography has been developed and applied to the antibody combining site surfaces. The surfaces have been clustered into four topographic classes: concave and moderately concave (mostly hapten binders), ridged (mostly peptide binders) and planar (mostly protein binders). We have determined the topographic classes for ten pairs of complexed and uncomplexed antibody-antigen crystal structures; four change topographic class on complexation. The results will be of use in antibody engineering, antigen docking and in clinical immunology. To demonstrate one application, we show how the data can be used to locate the antigen binding pocket on antibody structures.

A range of partial denaturation antibody pre-treatments that affect immunoassay performance have been evaluated. Monoclonal anti-ferritin antibody was either partially denatured by heat, urea or pH pre-treatment or left untreated and then passively adsorbed to polystyrene microtiter wells. The adsorption characteristics and functionality of the different surfaces produced have been evaluated by dynamic contact angle (DCA) analysis and scanning tunnelling microscopy (STM) imaging respectively. The DCA data show that the effect of partial denaturation is to change the wetting characteristics of the antibody surfaces, while, in addition, STM imaging reveals marked effects seen in the aggregation properties of the denatured antibodies.

The most sensitive analytical techniques available today for detecting immuno assay complexes are radio or enzyme immuno analytical techniques, by which quantities of 10(7)-10(8) analyte molecules can be detected. With the introduction of scanning force microscopy, a new method for detecting biological processes became available. Here, we examine the feasibility of using scanning force microscopy as a biosensitive tool. We demonstrate that single or multiple rabbit anti-human serum albumin molecules form complexes with preadsorbed single human serum albumin molecules on mica. However, no interaction is observed between human immunoglobulin G molecules and preadsorbed single albumin molecules; only separate antigens and antibodies are observed at random positions on the mica. This shows the ability of scanning force microscopy to act as a biosensor for detection of immunocomplexes, and to act as a very powerful tool to study molecule-surface interactions in general.

Objective: Anti-Purkinje cell antibodies have been found specifically in patients with paraneoplastic cerebellar degeneration. We investigated the ultrastructural localization of antigen-antibody reaction products by means of immune electron microscopy, using an antibody identical to anti-Yo antibody, which occurs most frequently and which immunostains the cytoplasm of Purkinje cells., Design: The antibody used here (anti-Us) was obtained from a patient with paraneoplastic cerebellar degeneration. Based on results indicating 94% homology between the amino acid sequences of the antigens recognized by anti-Us and anti-Yo antibodies, these two antigens are considered to be the same. After immunostaining of the specimen from the rat cerebellum by the avidin-biotin peroxidase complex method, ultrathin sections were examined with an electron microscope without counterstaining., Results: Immunoreaction products were found to be localized on membrane-bound ribosomes and free ribosomes. There were no reaction products in the endoplasmic reticulum lumen, smooth endoplasmic reticulum, Golgi complex, mitochondria, or nucleus., Conclusion: Although it has been noted that there is a leucine-zipper motif in the amino acid sequence of the antigenic protein that can bind to DNA, our findings indicate that this protein exists on the ribosome and may regulate protein synthesis rather than gene expression in the nucleus.

Antibodies specific for xylose-containing plant complex N-linked glycans are used for indirect immunolocalization of xylosyltransferase in sycamore cells. The use of high pressure freezing and freeze substitution for sample preparation resulted in very good morphological preservation of the different Golgi cisternae. Xylosyltransferase shows a diffuse distribution all over the Golgi stacks and xylosylation appears to be an early processing event that is initiated in the cis Golgi compartment.