Biomedical subjects

Steven A Bogen

Publications and source records attributed to Steven A Bogen.

4 recordsLinked to original sources

PubMed2004

A water-stable protected isocyanate glass array substrate.

We describe the performance of a new glass attachment chemistry for arrays that is particularly well suited to attachment of small molecules, such as peptides. The attachment chemistry is a protected isocyanate (PI) group. Isocyanate groups are well suited to serving as a glass coating for arrays, in that they are highly reactive with many different types of biological compounds. However, they are generally so reactive as to be unstable. The new feature of the PI slide coating is its stability. It can withstand immersion in water without loss of reactivity and has at least a 1-year shelf life. The high reactivity of the PI group results in a rapid coupling reaction (< 15min) and is particularly useful for attaching small molecules, such as peptides. Since isocyanates bind to both amines (forming a urea linkage) and hydroxyl groups (forming a carbamate bond), we tested the ability of the PI coating to bind to a wide variety of compounds. We found that the PI slide coating can directly attach to peptides, proteins, carbohydrates, lipooligosaccharides, and DNA. The sensitivity of detection for these compounds is comparable to that of other previously published array substrates.

Animals↗

PubMed2004

A molecular mechanism of formalin fixation and antigen retrieval.

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.

Amino Acid Sequence↗

PubMed2002

A novel quality control slide for quantitative immunohistochemistry testing.

We introduce a novel quality control technology that may improve intra- and interlaboratory immunohistochemistry (IHC) standardization. The technology involves the creation of standardized antibody targets that are attached to the same slides as the patient sample. After IHC staining, the targets turn the same color as the stained cells or tissue elements. Unlike current clinical practice, our proposed targets are neither cells nor tissue sections. To create reproducible standards that are available in unlimited supply, we use short constrained peptides as antibody targets. These peptides are attached directly to the glass slide. We show that these peptides simulate the portion of the native antigen to which the antibody binds. They are useful in detecting subtle changes in IHC staining efficacy. Moreover, the peptides do not degrade after deparaffinization or antigen retrieval treatments. This technology may be valuable in creating nationally standardized controls to quantify IHC analytical variability.

Animals↗

PubMed2002

Synthetic peptides identified from phage-displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets.

BACKGROUND: Quantitative immunohistochemical (IHC) assays currently lack optimal reference quality-control material for cellular protein targets. To address this problem, we identified peptides that mimic the site on the native analyte to which the primary (monoclonal) antibody binds and used them as surrogate peptide controls. METHODS: We identified peptide candidates from a combinatorial peptide phage-display library that mimic the epitope for the 1D5 estrogen receptor (ER) monoclonal antibody (mAb). The peptide inserts of the phage clones were sequenced. Several phage-encoded peptides were then synthesized and analyzed for affinity and specificity. RESULTS: We identified phage clones that specifically bound to the ER 1D5 mAb. The binding was specific, in that the phage clones did not bind to two other isotype-matched mAbs. Their ability to bind the ER 1D5 mAb was related to the presence of a consensus sequence. Binding analysis revealed a K(d) of 8.3 x 10(-8) mol/L. The peptide was not recognized by any of 15 other mAbs commonly used for clinical IHC testing. Moreover, the peptide was able to inhibit the binding of ER 1D5 mAb to native ER, indicating that the peptide bound to ER 1D5 mAb at or close to the antigen-binding site. CONCLUSIONS: Surrogate peptide controls behave like the native analyte in terms of affinity and specificity. This technology may be especially useful when the native analyte is in short supply.

Amino Acid Sequence↗