Biomedical subjects

B T Messmer

Publications and source records attributed to B T Messmer.

4 recordsLinked to original sources

PubMed2001

Specific blocking to improve biopanning in biological samples such as serum and hybridoma supernatants.

Screening phage-displayed peptide libraries (biopanning) is an important technique for acquiring peptide ligands and for mapping peptide epitopes recognized by antibodies (Ab). In biological samples, other materials, not only contaminants but also natural constituents, often interfere with biopanning. Capture methods use anchoring Abs that reduce the need for purification of the intended panning target from a crude sample. This capture method is analogous to sandwich ELISA. However, when the target molecule concentration is low in the initial mixture, the panning of a captured target often yields epitopes that bind to the capture antibody rather than the target of interest. We have developed a methodology that utilizes specific blocking reagents of the capture Ab to extend the sensitivity and applicability of the capture approach to phage panning. A flowchart is presented to enable the worker to begin panning with the simplest approach and then to employ sandwich capture and specific blocking reagents as necessary.

Animals↗

PubMed2000

Sequential determination of ligands binding to discrete components in heterogeneous mixtures by iterative panning and blocking (IPAB).

Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.

Amino Acid Motifs↗

PubMed2000

C1q-binding peptides share sequence similarity with C4 and induce complement activation.

Two peptide motifs that bind to C1q have been identified from phage displayed libraries. A first panning cycle recovered phage that displayed a [N/S]PFxL motif. A synthetic peptide with that motif blocked those phage from binding to C1q. A second panning cycle was conducted with the [N/S]PFxL motif peptide present, leading to recovery of phage displaying a different motif, SHY. The two motifs are specific for C1q and are competed by DNA and the cognate synthetic peptide but not by immunoglobulins. Phage displayed peptide sequences containing the [N/S]PFxL have significant sequence similarity to a region of complement component C4, suggesting a possible site of interaction between C4, or one of its processed forms, and C1q. The SHY motif peptide induces C4 consumption in a hemolytic assay, suggesting that it activates C1 independent of immune complexes. This peptide may activate C1 by a mechanism similar to the beta-amyloid peptides found in Alzheimer's disease.

Animals↗

PubMed1999

Two human neonatal IgM antibodies encoded by different variable-region genes bind the same linear peptide: evidence for a stereotyped repertoire of epitope recognition.

Two monoclonal IgM Abs have been produced from lymphocytes isolated from two human umbilical cord bloods. These mAbs recognize a conformational epitope present in a CNBr digestion fraction of lactoferrin. Linear epitopes recognized by each mAb were selected from several phage display peptide libraries. In each case, phages displaying a peptide with a motif defined by [WF],G,[EQS],N were recovered. Phages displaying that motif bound equally well to either mAb but did not bind to control IgM. A peptide bearing this motif competed with the phage-displayed peptides for binding to either mAb. The same peptide also competes with a component of the CNBr digestion fraction of lactoferrin for Ab binding in ELISA. The Abs use different families of VH, JH, and VK gene cassettes but use the same JK cassette. All segments are virtually identical to their germline gene counterparts. This work provides further evidence that certain innate specificities are stereotyped among individuals.

Antibodies, Monoclonal↗