Hydrophobin-Protein A Fusion Protein Produced in Plants Efficiently Purified an Anti-West Nile Virus Monoclonal Antibody from Plant Extracts via Aqueous Two-Phase Separation.

The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the ßH-ßI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Development of a microarray detection method for galectin cancer proteins based on ligand binding.

In this article, we describe the development of a novel detection method for the visualization of ligand-binding proteins. Current proteomic tools, such as the enzyme-linked immunosorbent assay (ELISA), are based on protein abundance rather than protein activity and can result in conflicting data. To address this issue, we developed an assay in which ligand binding is detected using a microarray approach with immobilized antibodies on a porous aluminum oxide matrix. The galectin family of proteins was used as a model system to evaluate the performance of this approach. Galectins selectively bind galactosides and are linked to cancer progression. Our assay employed antibodies directed against different galectins. The antibodies were immobilized on the microarray surface by use of protein A/G. In our example, galectin-1 and galectin-9 were then detected in cell lysates. Lysates were exposed to the anti-galectin surface, followed by washing and quantification with a general fluorescent galectin ligand. The optimal galectin ligand allowed detection of nanogram amounts of galectin using only 1 µg of antibody. Galectin-1 was visualized in HeLa and tumor cell lysates, indicating the potential of the method for a clinical setting.

Posttranscriptional regulation of gene expression in epithelial cells by polyamines.

In addition to regulating gene transcription, polyamines also potently modulate gene expression posttranscriptionally. Posttranscriptional gene regulation, which includes processes such as mRNA transport, turnover, and translation, involves specific mRNA sequences (cis-element) that interact with transacting factors such as RNA-binding proteins (RBPs) and microRNAs. U- or AU-rich elements (ARE) are the best characterized cis-acting sequences located in the 3'-untranslated regions of many labile mRNAs. Several RBPs, including AUF1, BRF1, TTP, and KSRP, promote ARE-mRNA decay through the recruitment of the ARE-bearing mRNA to sites of mRNA degradation, whereas RBPs such as HuR, HuB, HuC, and HuD stabilize target mRNAs and stimulate their translation. HuR is one of the best-studied RBPs and has emerged as a key regulator of posttranscriptional control of gene expression and its activity is tightly regulated by cellular polyamines. Ribonucleoprotein immunoprecipitation assays and biotin pull-down assays are two major methods used extensively in experiments investigating the roles and mechanisms of cellular polyamines in the posttranscriptional regulation and are described in detail in this chapter.

Modulation of Interleukin-8 and staphylococcal flora by Avène hydrotherapy in patients suffering from chronic inflammatory dermatoses.

BACKGROUND: A number of studies argue in favour of an important role of microbial colonization, in particular of Staphylococcus aureus, in triggering atopic dermatitis (AD) flare-up and psoriasis, in particular through the superantigenic properties of toxins generated by S. aureus. OBJECTIVES: The aim of this study was to assess the efficacy of a 3-week Avène hydrotherapy on the skin surface of patients suffering from psoriasis or atopic dermatitis. METHODS: Skin samples were taken from healthy subjects or atopic (n = 18) or psoriatic patients (n = 39) undergoing hydrotherapy at Avène at the beginning (D0) and the end of treatment (D18). The severity of the dermatosis was evaluated according to SCORing Atopic Dermatitis (SCORAD) or Psoriasis Area Severity Index (PASI) scores at D0 and D18. Marker of inflammation interleukin 8 (IL-8), S. aureus colonization (protein A) and enterotoxins were assessed in skin samples using RT-PCR. RESULTS: At D0, significant differences were observed between healthy subjects and atopic or psoriatic patients in all the parameters evaluated (IL-8, protein A). At the end of the hydrotherapy, a significant decrease in SCORAD was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin D in patients with atopic dermatitis. Similarly, a significant decrease in PASI was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin N in patients with psoriasis. CONCLUSIONS: This study demonstrates the positive effects of Avène hydrotherapy on the skin of patients suffering from chronic dermatosis, with decreased inflammation and reduced colonization by S. aureus.

Mercury exposure in protein A immunoadsorption.

BACKGROUND: Immunoadsorption is increasingly used to treat antibody-mediated autoimmune diseases. To prevent microbial growth during storage, reusable protein A-Sepharose gel columns are primed with ethyl mercury thiosalicylate (thiomersal, 0.1% solution) and rinsed with phosphate buffer before use. In this study, we tested the hypothesis of systemic mercury exposure in protein A immunoadsorption. METHODS: Whole blood mercury levels were measured by atomic absorption spectroscopy before and after protein A immunoadsorption (11 patients, 26 treatments), anti-IgG immunoadsorption (eight patients, 13 treatments) and LDL apheresis (DALI and Therasorb systems; nine patients, 14 treatments). RESULTS: Patients treated with protein A immunoadsorption had significantly elevated baseline mercury levels compared with the other groups, which were not different from healthy controls. Following protein A immunoadsorption, mercury levels increased from 5.9+/-1.4 microg/l (mean+/-SEM, normal, <5 microg/l) to 32.3+/-5.7 microg/l, P<0.001). In one intensively treated patient, acute neurological toxicity developed at a mercury level of 107 microg/l. Symptoms abated slowly and did not recur after switching to a thiomersal-free system and chelation therapy. No mercury release to patients occurred in anti-IgG immunoadsorption or LDL apheresis treatments. CONCLUSION: This preliminary report suggests that protein A immunoadsorption columns primed with thiomersal during storage may cause a sustained increase of systemic mercury concentrations, which exceed current safety recommendations in a proportion of patients. Considering the potential for mercury-induced toxicity, every effort should be undertaken to reduce systemic mercury exposure, either by adding chelators to the rinsing solution or ideally by replacement of thiomersal.

Identification of a dog IgD-like molecule by a monoclonal antibody.

IgD has not been identified in dogs. We produced a monoclonal antibody (mAb) designated 9B during the production of hybridomas to dog IgE. Using Western blot analysis under non-reducing conditions, the mAb (9B) recognized a predominant protein band of 185 kDa which was also recognized by anti-dog IgG F(ab')2, suggesting that this 185 kDa protein is an immunoglobulin (Ig) containing light chains. Under reducing conditions, the mAb (9B) recognized only one protein band of 55 kDa which presented a distinct molecular weight (MW) and immunoreactivity from the dog tau, mu, alpha, and epsilon chains. The 55 kDa band did not react with anti-dog IgE, IgM, IgA, and IgG, but did react with the mAb (9B). The MW was 75 kDa for the epsilon chain, 77.5 kDa for the mu chain, 58 kDa for the alpha chain, and 52 kDa for the tau chain. Further, by immunofluorescent staining, this Ig recognized by the mAb (9B) was found on the surface of dog lymphocytes. Studies of this dog Ig with the mAb revealed that this Ig bound to protein A and protein G-Sepharose, and that its enzyme-linked immunosorbent assay (ELISA) activity as measured by the mAb (9B) did not change after heating at 56 degrees C for 2 h. Ragweed-specific IgG, IgE, and this newly defined Ig significantly increased when dogs were immunized with ragweed extract. These data suggest that this Ig is a previously unrecognized IgD-like molecule in dogs.

Soluble complex formation of bovine immunoglobulin G2 with staphylococcal protein A studied by gel filtration chromatography.

The binding property of bovine IgG2 to staphylococcal Protein A was investigated by the methods of gel filtration chromatography and affinity chromatography. High performance gel filtration chromatography was carried out using TSK gel G3000SW and G2000SW columns, and immobilized Protein A column was used for affinity chromatography. Although bovine IgG2 did not form any precipitin lines with Protein A by double diffusion method on agar gel, IgG2 could bind to immobilized Protein A column. Moreover, by gel filtration chromatography, peaks of the complex between bovine IgG2 and Protein A were observed in addition to the IgG2 monomer peak. Thus, it is concluded that bovine IgG2 interacts with staphylococcal Protein A and forms "soluble complexes". Carbethoxylated IgG2 lost its affinity to Protein A indicating that histidyl residues in IgG2 is essential for the binding to Protein A.

Selective binding of colloidal gold-protein conjugates to epidermal phosphorus-rich keratohyaline granules and cornified cells.

Colloidal gold solutions conjugated with staphylococcal protein A (SpA) are widely used in high-resolution immunocytochemical studies to visualize antibodies bound at antigenic sites. Here we report that colloidal gold solutions conjugated with SpA, bovine serum albumin (BSA), or gelatin bind selectively to structures in glutaraldehyde-fixed, plastic-embedded epidermis of rabbit, mouse, and human. Two types of keratohyaline granules are present in epidermis, a phosphorus-rich (PR) and a sulphur-rich (SR) type. The PR keratohyaline granules were strongly labeled with gold particles, whereas SR keratohyaline granules or other structures in the living cells of epidermis were unlabeled. The PR keratohyaline granules are assumed to be precursors of the matrix protein of cornified cells, and intense gold labeling occurred over the lower layer of cornified cells (i.e., stratum lucidum). More superficial cornified cells were weakly labeled or unlabeled. The gold labeling pattern was identical whether SpA, BSA, or gelatin was used to stabilize the colloidal gold solution. The mechanism of binding of protein-conjugated gold to PR keratohyaline granules and matrix protein of cornified cells is not clear. It is speculated that the charged gold particles are not completely coated by the stabilizing protein, allowing for an electrostatic interaction with charged proteins in sections of cells.

Measurement of 'blocking antibodies' to pollen antigens by radiolabelled protein A from Staphylococcus aureus.

Radiolabelled protein A from Staphylococcus aureus (125I-SpA) was utilized to measure the relative concentrations of the so-called 'blocking antibodies' (BA) in sera from patients subjected to two different forms of desensitization therapy. In addition, sera from rabbits immunized actively with pollen antigens were assayed. Antigens coupled to CNBr-activated papear discs were used as solid phase, and BA bound to the solid-phase antigens were detected by 125I-SpA. The results obtained with this technique correlated excellently with those of a double-antibody technique for IgG, indicating the 125I-SpA assay to be suitable for the measurement of the antigen-specific IgG. The BA concentration in sera from patients subjected to conventional injection therapy increased significantly during the treatment period, whereas no increase could be detected in sera from patients subjected to sublingual treatment. The increases in BA concentrations of rabbit antisera to pollen antigens could be readily traced by this technique.

The interactions of porcine and ovine, serum and colostral immunoglobulins with staphylococcal Protein A.

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produce by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG1, IgG2, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG1 from most ewes possessed a low affinity for Protein A whereas ovine IgG2 generally possessed a high affinity; 100% of the IgG2 in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.