Jinny L. Liu

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

BackgroundShark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences.ResultsA semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications.ConclusionWe demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Thermostable Llama Single Domain Antibodies for Detection of Botulinum A Neurotoxin Complex

Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recognition elements. We have constructed a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85 degrees C for 1 h, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long-term storage and in-line monitoring require very rugged yet highly specific recognition elements.

PLGA/hydrogel biopapers as a stackable substrate for printing HUVEC networks via BioLP™

Two major challenges in tissue engineering are mimicking the native cell–cell arrangements of tissues and maintaining viability of three‐dimension (3D) tissues thicker than 300 µm. Cell printing and prevascularization of engineered tissues are promising approaches to meet these challenges. However, the printing technologies used in biofabrication must balance the competing parameters of resolution, speed, and volume, which limit the resolution of thicker 3D structures. We suggest that high‐resolution conformal printing techniques can be used to print 2D patterns of vascular cells onto biopaper substrates which can then be stacked to form a thicker tissue construct. Towards this end we created 1 cm × 1 cm × 300 µm biopapers to be used as the transferable, stackable substrate for cell printing. 3.6% w/v poly‐lactide‐co‐glycolide was dissolved in chloroform and poured into molds filled with NaCl crystals. The salt was removed with DI water and the scaffolds were dried and loaded with a Collagen Type I or Matrigel™. SEM of the biopapers showed extensive porosity and gel loading throughout. Biological laser printing (BioLP™) was used to deposit human umbilical vein endothelial cells (HUVEC) in a simple intersecting pattern to the surface of the biopapers. The cells differentiated and stretched to form networks preserving the printed pattern. In a separate experiment to demonstrate “stackability,” individual biopapers were randomly seeded with HUVECs and cultured for 1 day. The mechanically stable and viable biopapers were then stacked and cultured for 4 days. Three‐dimensional confocal microscopy showed cell infiltration and survival in the compound multilayer constructs. These results demonstrate the feasibility of stackable “biopapers” as a scaffold to build 3D vascularized tissues with a 2D cell‐printing technique. Biotechnol. Bioeng. 2012;109: 262–273.

Development of Antiricin Single Domain Antibodies Toward Detection and Therapeutic Reagents

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricins A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricins biological activity. One of the sdAb (C8) was particularly effective and blocked ricins biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

BackgroundCamelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays.ResultsHere we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL.ConclusionThe anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.

Reconstruction of functional cortical-like tissues from neural stem and progenitor cells.

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

Bacteriophage T4 Nanoparticles as Materials in Sensor Applications: Variables That Influence Their Organization and Assembly on Surfaces

Bacteriophage T4 nanoparticles possess characteristics that make them ideal candidates as materials for sensors, particularly as sensor probes. Their surface can be modified, either through genetic engineering or direct chemical conjugation to display functional moieties such as antibodies or other proteins to recognize a specific target. However, in order for T4 nanoparticles to be utilized as a sensor probe, it is necessary to understand and control the variables that determine their assembly and organization on a surface. The aim of this work is to discuss some of variables that we have identified as influencing the behavior of T4 nanoparticles on surfaces. The effect of pH, ionic strength, substrate characteristics, nanoparticle concentration and charge was addressed qualitatively using atomic force microscopy (AFM).

Immunodiagnostic reagents using llama single domain antibody-alkaline phosphatase fusion proteins.

Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility. AP spontaneously dimerizes in solution, effectively dimerizing the fused sdAbs, imparting avidity in place of the lower affinity monomeric interactions. The sdAb-AP fusion also combines the target recognition domain with a signal transduction domain, commonly used in enzyme-linked immunosorbent assays (ELISAs). The functional affinity of the sdAb-AP fusions, often increased by a factor of 10 over unfused sdAbs, and their utility as tracer reagents in ELISAs was dramatically improved, giving limits of detection of 300 ng/ml or less, whereas parental sdAbs gave no discernible signal at the toxin concentrations tested. The fusion of sdAbs to AP presents a valuable route to facilitate the implementation of sdAb-based immunoreagents rapidly selected from existing naive libraries toward new or emerging threats.

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (KD ) in the range 2.2 × 10−11 to 1.6 × 10−10 M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.