High affinity ScFvs from a single rabbit immunized with multiple haptens.

We report the generation of single-chain Fv (scFv) fragments with high affinities against four different hapten molecules from a single immunised rabbit. The rabbit was immunised with a mixture of protein conjugates of four different haptens, namely the herbicide mecoprop and derivatives of the herbicides atrazine, simazine, and isoproturon. An scFv phage display library was constructed, and several scFvs with high affinity against each hapten were isolated. For each hapten, a single binder was selected by k(off) ranking and used for affinity determination. The affinities were in sub-nanomolar range and the lowest K(d) value obtained was 6.75 x 10(-10) M. An unusual feature of one of the anti-isoproturon scFvs was its ability to retain binding activity at pH1.7. The utility and potential of using a single animal and immunisation with multiple antigens for the production of multiple, specific, high affinity scFvs by phage display is discussed.

Cytoplasmic expression of a soluble synthetic mammalian metallothionein-alpha domain in Escherichia coli. Enhanced tolerance and accumulation of cadmium.

Bacteria are commonly used for bioremediation of heavy metal pollution and strategies to improve their performance in this respect are desirable. In this study, an Escherichia coli strain was engineered to express a common metallothionein-alpha domain. The metallothionein-alpha domain was over-expressed in the cytoplasm of E. coli as a fusion to the carboxyl terminal of maltose binding protein. The fusion protein was highly soluble in the cytoplasm of E. coli. When grown in the presence of cadmium, cells expressing the metallothionein-alpha fusion protein showed increased viability compared with control cells. Cells expressing the metallothionein-alpha also demonstrated increased accumulation of cadmium.

Selection of phage antibodies to surface epitopes of Phytophthora infestans.

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

Selection of phage-display peptides that bind to cucumber mosaic virus coat protein.

Several discrete peptides that bind specifically to the coat protein of cucumber mosaic virus (CMV) were isolated from a diverse phage library displaying random nonapeptides on the major coat protein VIII. Enrichment was shown by polyclonal phage enzyme linked immunosorbent assay (ELISA) after three rounds of selection. Sequencing of the genes encoding 10 of these peptides revealed an absence of any conserved motifs, although nine of them contained a high proportion of proline residues. Some of the selected peptides were displayed at the N-terminus of thioredoxin and expressed in the cytoplasm of Escherichia coli. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CMV and CMV present in crude leaf extracts from infected plants. By dot blot analysis, a thioredoxin-peptide fusion could readily detect as little as 5 ng of CMV. The peptides did not bind to other plant viruses. These peptides have been shown to be specific and highly sensitive tools in the detection of CMV and, as well as their diagnostic potential, they could form the basis for a novel disease resistance strategy.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector.

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion.

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains of Staphylococcus aureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein delta pIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.

Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment.

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli.

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates.

A microtiter plate-based assay for phosphoenolpyruvate carboxylase.

A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography.

The stimulation of CAM activity in Mesembryanthemum crystallinum in nitrate and phosphate-deficient conditions.

Plants of Mesembryanthemum crystallinum L. were grown from seed in phosphate-sufficient and phosphate-deficient conditions. Separate plants were subjected to nitrate deficiency by removing nitrate from the rooting medium of previously nitrate-sufficient plants. Subsequently, they were also irrigated with 400 mM NaCl to induce CAM. Plants of M. crystallinum with induced CAM that had been grown in complete nutrient medium were deinduced by rehydration. Nitrate and phosphate deficiencies caused enhanced CAM activity prior to salt treatments. After salinisation, phosphate deficiency also caused higher background levels of malate. Prolonged nitrate deficiency reduced CAM activity. Endogenous P1 levels in phosphate-sufficient plants correlated with CAM activity. However in phosphate-deficient plants CAM occurred without a significant rise in P1 content. Levels of endogenous P1 appeared more related to nitrate deficiency than to CAM or salt treatment. Nitrate and phosphate deficiencies and salt treatment could all cause a nitrate deficiency within the leaf, causing high endogenous P1 when available in the rooting medium. It is speculated that a change in nitrogen status could play a role in the initiation of CAM induction in M. crystallinum. A further possibility is that CAM could be induced by reduced water potential due to dehydration and increased ion content in saline conditions, and by an inhibition of growth and concomitant accumulation of solutes in phosphate- and nitrate-deficient conditions.

The work of the WHO Consultative Group on Poliomyelitis Vaccines.

PIP: Through a review of the work on the control of poliomyelitis carried out under the auspices of the World Health Organization (WHO) during the past 20 years, the importance of international collaboration is shown. Because of efforts in planning and coordinating, the production and control of the Sabin strains of the live oral vaccine provide safe, reliable, and potent vaccines. The cooperative efforts have included working not only with national control laboratories but with poliomyelitis vaccine producers in many countries. In the early 1970s, a Consultative Group of WHO became active. Their initial efforts included an extensive epidemiological study in 13 interested countries. Later, the group saw to studying the reliability of the marker tests used in the intratypic differentiation of poliovirus stains of different origins. Additionally, they saw to standardizing tests for the neurovirulence of vaccine lots, including analyzing and recording results, and to ensuring that adequate supplies of vaccine will be available for the next 200 years. After 15 years of continual surveillance of vaccine-associated cases by WHO epidemiologists and clinicians, the findings show the following: Type 1 live poliovirus vaccine is almost never implicated in postvaccination paralysis; type 2 strain occasionally causes of paralysis in contacts of the vaccine, and type 3 strain causes most of the few cases of postvaccine paralysis. The occurrences of the cases from type 2 and 3 strains remains an enigma. Current research of the group suggests an even more effective vaccine may become available in the future.^ieng