Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries.

Selection of antibodies from large repertoire phage display libraries has become a common technique for isolation of specific antibodies to antigens. Many of these libraries are shown to contain antibodies specific to haptens, but only when these haptens are derivatised or conjugated to an immobilising molecule, such as bovine serum albumin (BSA). There has been little demonstration of the suitability of naive recombinant antibody libraries for isolating antibodies that bind low molecular weight haptens in the absence of a carrier molecule and few have addressed the problems associated with selecting antibodies that only recognize the combination of hapten and the carrier molecule. We have panned two-phage antibody libraries against AflatoxinB1-BSA and screened single-chain antibody fragments for binding to AflatoxinB1-BSA and Aflatoxin-B1. Many of the antibodies isolated specifically bound AflatoxinB1-BSA, but not soluble Aflatoxin-B1 or BSA. Modification of the protocol led to isolation of single-chain fragment variable antibody domain (scFv) antibodies that specifically bound soluble Aflatoxin-B1 with an affinity of 6x10(-9) M.

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate.

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.

Photoimmunodetection: a nonradioactive labeling and detection method for DNA.

We describe a simple detection system for DNA based on antibody detection of UV-induced photoproducts. It includes a convenient and inexpensive labeling procedure, which is completed in 5-10 min. The only equipment required is a UV source such as an ordinary transilluminator or a DNA crosslinker. Using a monoclonal antibody specific for thymine dimers, coupled to horseradish peroxidase, we are able to detect subpicogram amounts of UV-irradiated DNA directly, and approximately 10 pg homologues DNA indirectly by hybridization with an irradiated probe.

The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

Bacteriophage P2 and P4 morphogenesis: assembly precedes proteolytic processing of the capsid proteins.

Several of the structural proteins of phage P2 and its satellite P4 undergo proteolytic processing during development of mature phage particles. Here, we report that uncleaved shell protein, gpN, is present in immature capsids of both P2 and P4, showing that assembly precedes processing. This excludes the possibility that processing of gpN is involved in capsid size determination. We also find that N*, the fully processed version of gpN, produced from a plasmid, can assemble into both P2- and P4-sized particles, implying that the amino-terminal end of gpN is not required for assembly initiation nor for the formation of a T = 4 shell. As may be expected for a scaffolding protein, we find that gpO coexists with gpN in immature P2, as well as P4, capsids. This result supports the conclusion that gpO is required for both phages and strongly suggests that the O derivative, h7 (found in mature capsids), results from proteolytic cleavage after gpN/gpO coassembly.

Bacteriophage P2 and P4 assembly: alternative scaffolding proteins regulate capsid size.

The capsid protein of bacteriophage P2, encoded by the N gene, can assemble into icosahedral capsids of two possible sizes, with diameters of 60 and 45 nm, respectively. Only the larger capsid is used by P2 itself, but the smaller one is exploited by the satellite phage P4. We have analyzed the assembly products of gpN expressed in vivo from a plasmid, i.e., in the absence of any other phage proteins, and find that gpN alone forms closed shells of both sizes, although with poor efficiency. Coexpressing gpN with gpO, the putative P2 scaffolding protein, increases the efficiency of large particle formation. In contrast, introducing the sid gene by P4 infection stimulates the assembly of small particles. Our results suggest that gpO and gpSid act competitively with respect to capsid size determination. Furthermore, we demonstrate that gpN alone undergoes the normal proteolytic maturation steps, implying that gpN processing is either autocatalytic or mediated by a host enzyme.

Bacteriophage P2 and P4 morphogenesis: identification and characterization of the portal protein.

The portal structure has been implicated in several aspects of the bacteriophage life cycle, including capsid assembly initiation and DNA packaging. Here we present evidence that P2 gene Q codes for the P2 and P4 portal protein. First, microsequencing shows that capsid protein h6 is derived from gpQ, most probably by proteolytic cleavage. Second, antibodies against gpQ bind to the portal structure in disrupted P2 phage virions, as observed by electron microscopy. Third, gpQ partially purified from an overexpressing plasmid assembles into portal-like structures. We also show by microsequencing that capsid protein h7 is encoded by the P2 scaffold gene, O, and is probably derived from gpO by proteolytic cleavage. Previous work has demonstrated processing of the major capsid protein. Thus, all essential capsid proteins of P2 and P4 are proteolytically cleaved during the morphogenetic process.