Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction.

A heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of the monoclonal antibodies F4, F5, and LT1 directed against mouse polyomavirus large T-antigen. Phage selected by biopanning was cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay. In phage reacting with the F5 antibody the deduced amino acid sequence of the displayed peptides corresponded to a segment of large T-antigen. In phage reacting with the antibodies F4 and LT1, no such similarity was observed. The kinetics of phage particle-monoclonal antibody complex formation and dissociation was analyzed in an optical biosensor instrument. Sensor chips of standard quality were useful for binding analysis of T7 phage in crude lysates of infected Escherichia coli. We synthesized peptides corresponding to selected consensus sequences and showed by biosensor analysis that these peptides (linear NH3-CPNSLTPADPTMDY-COOH and NH3-NSLTPCNNKPSNRC-COOH with an intramolecular S--S bridge) were able to compete with large T-antigen in binding to the corresponding antibodies (LT1 and F4). These synthetic peptides were also used for gentle and specific dissociation of large T-antigen-antibody complexes. The results demonstrate the potential of phage T7 for display of peptides and for rapid analysis of interactions of these peptides with ligands.

Ultrasonic reflection mode imaging of the nonlinear parameter B/A. II: Signal processing.

The nonlinear acoustic interaction between a reflected single-frequency sinusoid and a broadband pump waveform propagating in the opposite direction produces phase changes in the probe proportional to the nonlinear parameter B/A in the spatial region of interaction. The instantaneous phase change along the received probe can be expressed as the convolution of the pump waveform with the spatial distribution of B/A along the propagation path over which the pump and reflected probe interact. In theory, the phase modulated sinusoidal probe can be processed (phase detection and deconvolution) to produce an "A-mode" representation of B/A. If the pump is an intense unipolar impulse and the probe a swept-frequency sinusoid, then the pump interacts with the probe at each point along the propagation path at a unique frequency. Thus the phase modulation that carries information about the spatial distribution of B/A can be extracted from the phase spectrum by a simple Fourier transformation analogous to the space to frequency mapping so basic to magnetic resonance imaging. If the impulsive pump is replaced by another swept-frequency sinusoid, then the phase change in the probe due to B/A at a particular point along the propagation path will be spread out for the duration of the pump along the probe. Passage of the received signal through an appropriate matched filter restores spatial coherence to the phase information in the probe so that it can be processed as if the pump were a broadbanded impulse. This approach suggests a means of approaching the design of effective pump waveforms that can resolve a wide range of spatial frequencies in (B/A)(x).

Effect on polyomavirus T-antigen function of mutations in a conserved leucine-rich segment of the DnaJ domain.

The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen.