Human IgG Molecules Research Articles

Acoustofluidics-enhanced biosensing with simultaneously high sensitivity and speed

Simultaneously achieving high sensitivity and detection speed with traditional solid-state biosensors is usually limited since the target molecules must passively diffuse to the sensor surface before they can be detected. Microfluidic techniques have been applied to shorten the diffusion time by continuously moving molecules through the biosensing regions. However, the binding efficiencies of the biomolecules are still limited by the inherent laminar flow inside microscale channels. In this study, focused traveling surface acoustic waves were directed into an acoustic microfluidic chip, which could continuously enrich the target molecules into a constriction zone for immediate detection of the immune reactions, thus significantly improving the detection sensitivity and speed. To demonstrate the enhancement of biosensing, we first developed an acoustic microfluidic chip integrated with a focused interdigital transducer; this transducer had the ability to capture more than 91% of passed microbeads. Subsequently, polystyrene microbeads were pre-captured with human IgG molecules at different concentrations and loaded for detection on the chip. As representative results, ~0.63, 2.62, 11.78, and 19.75 seconds were needed to accumulate significant numbers of microbeads pre-captured with human IgG molecules at concentrations of 100, 10, 1, and 0.1 ng/mL (~0.7 pM), respectively; this process was faster than the other methods at the hour level and more sensitive than the other methods at the nanomolar level. Our results indicated that the proposed method could significantly improve both the sensitivity and speed, revealing the importance of selective enrichment strategies for rapid biosensing of rare molecules.

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System

Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a Kd of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.

Development of a Novel SPR Assay to Study CXCR4-Ligand Interactions.

G protein-coupled receptors (GPCRs) are involved in a plethora of different diseases. Consequently, these proteins are considered as an important class of drug targets. Measuring detailed kinetic information on these types of proteins has been challenging. Surface plasmon resonance (SPR) can provide this information, however, the use of SPR on GPCRs remains a complex issue. Here, we report an SPR assay to investigate the interactions between the full-length chemokine receptor CXCR4 and nanobody-Fc (Nb-Fc) ligands. Nb-Fcs consist of two monovalent VHH domains fused with an Fc domain of a human IgG molecule. The CXCR4 protein used in this assay was produced with a C-terminal 10x-histidine tag and was immobilized on a nitrilotriacetic acid chip. In order to verify the sensitivity and effectiveness of this assay, the results were compared to data obtained from cellular assays as well as from another SPR assay using CXCR4 virus-like particles (VLPs). CXCR4 remained intact and stable for at least 12 h, and the kinetic results correlated well with both the cellular assays and the VLP SPR assay results. Apart from determining the binding kinetics of Nb-Fc with CXCR4, our results contributed to understanding CXCR4 interaction dynamics. In conclusion, this assay provides a viable experimental platform that has high potential to be expanded for studying other molecules as well as other histidine-tagged GPCRs.

BiAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Characterization of the interactions of rabbit neonatal Fc receptor (FcRn) with rabbit and human IgG isotypes.

Despite the increasing importance of rabbit as an animal model in pharmacological studies like investigating placental transfer of therapeutic IgGs, little is known about the molecular interaction of the rabbit neonatal Fc receptor (FcRn) with rabbit and human IgG molecules. We analyzed the interactions of the rabbit and human FcRn with rabbit and human IgG isotypes using surface plasmon resonance assay. Similar to FcRn of other species, rabbit FcRn functions in pH-dependent manner, as it binds IgGs at pH 6.0, but no binding occurs at pH 7.4. We also showed that rabbit FcRn binds rabbit IgG and human IgG1 with nearly identical affinity, whereas it has stronger interactions with the other human IgG isotypes. The similar affinity of rabbit IgG and human IgG1 for rabbit FcRn was confirmed by in vitro FcRn-mediated recycling assay. These data verify that rabbit is an appropriate animal model for analyzing the pharmacokinetics of human therapeutic monoclonal antibodies.

Abstract P6-12-13: Single domain antibody (SBT-100) inhibits growth of human HER2+ and triple negative breast cancers (TNBC) in xenografts by binding STAT3 and P-STAT3

Abstract SBT-100 is a single domain antibody (sdAb), developed by Singh Biotechnology, that binds unphosphorylated signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (P-STAT3). SBT-100 is approximately 13 kD or less than 1/10th the size of a human IgG molecule, and is able to cross the cell membrane to bind intracellular STAT3 and P-STAT3. This in turn inhibits its effects on genes that promote malignant behavior of cancer cells. SBT-100 has a short serum half-life but a long biological half-life. Since certain types of human breast cancers express P-STAT3, we wanted to determine if SBT-100 could inhibit the growth of human breast cancers in vitro and in vivo by studying its effects on MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) cells. BACKGROUND: Many different types of human cancers (solid tumors, leukemias, and lymphomas) are dependent on constitutive expression of (P-STAT3) for their malignant phenotype. Growth factors, tyrosine kinase receptors, cytokines (IL-6, IL-11, IL-12, IL-23), BCR-ABL, and Src are some ways that STAT3 can be activated. In turn P-STAT3 turns on genes such as Cyclin D1 & D3, MMPs, Bcl-xL, Mcl-1, survivin, VEGF, and HIF-1 alpha. Constitutive expression of P-STAT3 has been shown to promote cancer cell proliferation, survival, angiogenesis, immune suppression, and metastasis. Additionally there is increasing evidence suggesting that unphosphorylated STAT3 contributes to malignant phenotype of cancers. STAT3 is also important for the survival of cancer stem cells as well as for some human breast cancers. METHODS: Immunoprecipitation and Western blot analyses were carried out to test whether SBT-100 binds cytoplasmic STAT3 and P-STAT3 in various malignant cell lines (e.g., MDA-MB-231, PANC-1, DU145, and HeLa). MTT assays were done to determine if SBT-100 could suppress the growth of different types of human breast cancers in vitro. Xenograft cancer models using ER+/PR+ (MCF-7), HER2+ (BT474), and TNBC (MDA-MB-231) cancer cells were used to evaluate treatment with SBT-100 1mg/kg/BID (IV and/or IP route). RESULTS: Immunoprecipitation and Western blot studies demonstrated that SBT-100 binds to both STAT3 and P-STAT3 in human cancers cells (MDA-MB-231, PANC-1, DU145, and HeLa). In a three day MTT assay, at least 90% growth suppression was achieved for all three subtypes of human breast cancer, which is highly significant. In the xenograft cancer models, SBT-100 (1mg/kg/BID) treatment for 28 days, yield growth suppression as follows: MDA-MB-231 44.8% (p<0.05) versus its control group and BT474 52% (p<0.07). While the MCF-7 xenograft cancer model showed no suppression. CONCLUSION: Singh Biotechnology's novel sdAb, SBT-100 suppresses growth of TNBC and HER2+ human breast cancers in vivo and suppresses growth of ER+/PR+, HER2+, and TNBC cells in vitro. The most significant anti-cancer effects of SBT-100 is observed against human TNBC. Citation Format: Singh S, Murillo G, Chen D, Singh A, Singh S, Singh A, Mehta R, Parihar A. Single domain antibody (SBT-100) inhibits growth of human HER2+ and triple negative breast cancers (TNBC) in xenografts by binding STAT3 and P-STAT3 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-13.

Molecular Cloning, Expression and Peroxidase Conjugation of Staphylococcus aureus Protein A.

Background Staphylococcal protein A (SPA) is a cell wall component of Staphylococcus aureus that binds to different IgG subclasses of human and several animal species. This bacterial protein can be used as an antibody detector in various immunological assays or as an isolation reagent for the purification of antibody molecules via immuno-chromatography procedures. Objectives Molecular cloning and expression of SPA followed by the purification and conjugation of the recombinant protein to peroxidase enzyme. Material and Methods Encoding DNA fragment of SPA was amplified and inserted into a prokaryotic plasmid vector for the expression of recombinant SPA fused to a maltose binding protein (MBP). The recombinant protein was purified using amylose resin column chromatography and conjugated to horseradish peroxidase (HRP) enzyme. Finally, the reactivity of the recombinant SPA was examined against human IgG molecules in ELISA. Results The results indicated that the recombinant peroxidase-conjugated SPA has a good recognition capacity for human IgG molecules and it was able to produce significant OD values after reacting with human IgG molecules at a concentration up to 0.06 μg.well-1. Conclusions This recombinant protein can be very useful in all research laboratories and may decrease some of the expenses, e.g. those for preparing conjugated anti-antibodies.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

Generation of antigen-specific monoclonal antibodies by in vitro immunization (TECH2P.904)

Abstract Monoclonal antibodies are one of the most important tools in biomedicine but the generation by hybridoma technology is very time consuming and laborious. The adaptation of the process to an in vitro approach is therefore a smart alternative to reduce the disadvantages of hybridoma technology. In this study a protocol for in vitro immunization was developed which enables hematopoietic stem cells to differentiate into naïve dendritic cells. These cells were specifically activated with the antigen of interest and cocultivated with naïve B and T lymphocytes in order to generate antigen specific B lymphocytes. After 4 days the cells were fused with myeloma cells and antigen specific hybridomas were selected. The produced antibody was purified and characterised in comparison to a conventional monoclonal antibody reacting against the same antigen. Further this protocol could be adapted to human cells and the generation of antigen specific hybridoma cells secreting human IgG molecules could be established. The developed protocols allowed a generation of antigen specific hybridoma cells of murine and human origin within 3 weeks. Therefore it seems that the immunization in vitro is useful for avoiding the disadvantages of conventional hybridoma technology.

High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies

Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development.

Teaching the structure of immunoglobulins by molecular visualization and SDS‐PAGE analysis

This laboratory class combines molecular visualization and laboratory experimentation to teach the structure of the immunoglobulins (Ig). In the first part of the class, the three-dimensional structures of the human IgG and IgM molecules available through the RCSB PDB database are visualized using freely available software. In the second part, IgG and IgM are studied using electrophoretic methods. Through SDS-PAGE analysis under reducing conditions, the students determine the number and molecular masses of the polypeptide chains, while through SDS-PAGE under nonreducing conditions, the students assess the oligomerization of these Ig molecules. The aims of this class are to expand upon the knowledge and understanding of the Ig structure that the students have gained from classroom lectures. The combination of this molecular visualization of the Ig molecules and the SDS-PAGE experimentation ensures variety in the teaching techniques, while the implication of the Ig molecules in human disease promotes interest for biomedical students.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Synthesis and characterization of biomimetic nanogels for immunorecognition

Biomimetic nanoparticles are promising materials for biomedical and biotechnological applications. Cationic poly(N-isopropylacrylamide) (PNIPAM) nanogels containing charged amine groups brought by addition of 2-aminoethylmethacrylate hydrochloride (AEMH) or N-(3-aminopropyl) methacrylamide hydrochloride (APMH) as co-monomers were prepared by surfactant-free precipitation polymerization. The influence of the relative amount and mode of addition of the co-monomer on both the size and the amine group density of the nanogel particles was studied. Two nanogels, one prepared using APMH (1%mol/mol NIPAM, in batch) and another with AEMH (2%mol/mol NIPAM, by shot addition) as co-monomers, were selected for the covalent coupling of a Protein L-mimic ligand to free amine groups on the particles. The ability of the synthesized biomimetic nanoparticles for recognizing and binding human IgG (hIgG) molecules was assessed and the selectivity toward immunoglobulin molecules evaluated.

Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

Allergen-Specific IgG Antibodies Purified from Mite-Allergic Patients Sera Block the IgE Recognition ofDermatophagoides pteronyssinusAntigens: AnIn VitroStudy

One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%–51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.

Molecular factors modulating the anti-inflammatory activity of IVIg in ITP

Background Intravenous Immunoglobulin G (IVIg) therapy is widely used to treat autoimmune diseases. A variety of mechanisms have been suggested to be responsible for the antiinflammatory activity of IVIg. Among these, terminal sialic acid residues in the sugar moiety of the IgG Fc-fragment have been shown to be critical for its anti-inflammatory activity in a model of serum transfer arthritis. Moreover, splenic resident cells expressing specific-ICAM3 grabbing nonintegrin-related1 (SIGNR1) were shown to have the capacity to bind antibodies rich in sialic acid residues, suggesting a role of the spleen in this immunomodulatory pathway. Recently, B cells and the sialic acid-binding protein CD22 were suggested to be involved in the IVIgmediated anti-inflammatory process. Material and methods To induce a murine model of thrombocytopenia (ITP), mice were injected with the specific anti-platelet antibody 6A6-IgG2a. Mice were either pretreated with IVIg or PBS two hours prior to platelet depletion. Splenectomized mice and several mouse strains lacking B cells, CD22 or SIGNR1 were used for experiments. Flow cytometric analysis revealed in vivo binding of human IgG-molecules to different cell populations in peripheral blood of IVIg treated mice. Deglycosylation of IVIg preparations was achieved by enzymatic cleavage with neuraminidase or PNGaseF. Results Mice treated with de-glycosylated IVIg lacking either the whole sugar moiety or the terminal sialic acid residue were not protected from ITP. We demonstrated that IVIg activity was still functional in splenectomized mice but could be blocked with a specific anti-SIGNR1 antibody. Despite the capacity of IVIg to recognize sialic acid rich IgG via CD22 on B cells, neither B cells nor CD22 were involved in IVIg dependent suppression in autoantibody induced ITP. Binding efficiency of IVIg to myeloid cells and leukocytes was neither altered in mice lacking B cells or CD22 nor in mice treated with de-syalylated IVIg. Conclusions These results suggest a spleen-independent but sialic acidand SIGNR1-dependent mechanism responsible for IVIg effects in ITP. Moreover, neither B cells nor CD22 are critical for the anti-inflammatory activity of IVIg.

Atomic force microscope based biomolecular force-clamp measurements using a micromachined electrostatic actuator

The authors describe a method for biomolecular force clamp measurements using atomic force microscope (AFM) cantilevers and micromachined membrane-based electrostatic actuators. The actuators comprise of Parylene membranes with embedded side actuation electrodes and are fabricated on a silicon substrate. The devices have a displacement range of 1.8 μm with 200 V actuation voltage, and displacement uncertainty is 0.8 nm, including the noise and drift. The settling time, limited by the particular amplifier is 5 ms, with an inherent range down to 20 μs. A force clamp measurement setup using these actuators in a feedback loop has been used to measure bond life-times between human IgG and anti-human IgG molecules to demonstrate the feasibility of this method for biological experiments. The experimental findings are compared with a molecular pulling experiment and the results are found to be in good agreement.

Silicon photonic crystal nanocavity-coupled waveguides for error-corrected optical biosensing

A photonic crystal (PhC) waveguide based optical biosensor capable of label-free and error-corrected sensing was investigated in this study. The detection principle of the biosensor involved shifts in the resonant mode wavelength of nanocavities coupled to the silicon PhC waveguide due to changes in ambient refractive index. The optical characteristics of the nanocavity structure were predicted by FDTD theoretical methods. The device was fabricated using standard nanolithography and reactive-ion-etching techniques. Experimental results showed that the structure had a refractive index sensitivity of 10 −2 RIU. The biosensing capability of the nanocavity sensor was tested by detecting human IgG molecules. The device sensitivity was found to be 2.3 ± 0.24 × 10 5 nm/M with an achievable lowest detection limit of 1.5 fg for human IgG molecules. Additionally, experimental results demonstrated that the PhC devices were specific in IgG detection and provided concentration-dependent responses consistent with Langmuir behavior. The PhC devices manifest outstanding potential as microscale label-free error-correcting sensors, and may have future utility as ultrasensitive multiplex devices.

Single-Domain Protein A-Engineered Magnetic Nanoparticles: Toward a Universal Strategy to Site-Specific Labeling of Antibodies for Targeted Detection of Tumor Cells

Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T(2) contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor.

Thrombin Binding Predicts the Effects of Sequence Changes in a Human Monoclonal Antiphospholipid Antibody on Its In Vivo Biologic Actions

The mechanisms by which antiphospholipid Abs (aPL) cause thrombosis are not fully understood. It is clear that binding to a number of phospholipid-associated Ags is important but it is difficult to identify which Ag-binding properties are most closely linked to the ability to cause biologic effects such as promotion of thrombosis and activation of endothelial cells. We have previously used an in vitro expression system to produce a panel of human monoclonal IgG molecules between which we engineered small differences in sequence leading to significant well-defined changes in binding properties. In this study, we assess the properties of five of these IgG molecules in assays of biologic function in vitro and in vivo. The i.p. injection of these IgG into mice subjected to a femoral vein pinch stimulus showed that only those IgG that showed strong binding to thrombin promoted in vivo venous thrombosis and leukocyte adherence. However, this finding did not hold true for the effects of these IgG on activation of cultured endothelial cells in vitro, where there was a less clear relationship between binding properties and biologic effects.