A fast and easy one-step purification strategy for plant-made antibodies using Protein A magnetic beads.

A major difficulty to reach commercial- scale production for plant-made antibodies is the complexity and cost of their purification from plant extracts. Here, using Protein A magnetic beads, two monoclonal antibodies are purified in a one-step procedure directly from non-clarified crude plant extracts. This technique provides significant savings in terms of resources, operation time, and equipment.

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during in vitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. OBJECTIVE: We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. METHODS: IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). RESULTS: Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r = 0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n = 15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r = 0.61) and to all blank ImmunoCAPs (r > 0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. CONCLUSIONS: Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

Monoclonal antibody therapy for Junin virus infection.

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Characterization of plants expressing the human ß1,4-galactosyltrasferase gene.

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human ß1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.