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Seshi R Sompuram

Publications and source records attributed to Seshi R Sompuram.

10 recordsLinked to original sources
PubMed2006

A high throughput combinatorial library technique for identifying formalin-sensitive epitopes.

We present a technique for identifying the amino acids responsible for a loss of immunoreactivity in response to treating an antigen with a chemical modifier. This is of particular interest for the chemical formaldehyde, the cross-linking agent in formalin. Formalin is a commonly used fixative to preserve the cellular architecture of cells and tissues and to prevent degradation from proteases and nucleases. Formalin is also routinely used in the preparation of vaccines, to inactivate both toxins and microbes. Formalin fixation attenuates infectivity and pathogenicity by cross-linking while often preserving antigenicity. However, some epitopes are irreversibly modified by formalin while others are not. An understanding of how formalin affects epitope immunoreactivity may be useful in vaccine development or in the development of diagnostic antibody reagents for formalin-fixed tissues. In this report, we describe a method for systematically identifying formalin-sensitive and formalin-insensitive epitopes in a high throughput fashion, for any particular antibody. The data from this effort underscore the importance of certain amino acids, notably lysine, in affecting antibody immunoreactivity after formalin fixation. The method can be generally applicable in exploring the sensitivity of protein epitopes to an agent or condition of interest.

Amino Acids↗
PubMed2006

Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes.

It is not clearly understood why some monoclonal antibodies bind to their antigens informalin-fixed, paraffin-embedded tissue sections but others do not. To address this question, we analyzed the protein epitopes of 9 monoclonal antibodies that are immunoreactive after formalin fixation and antigen retrieval. We identified the antibody contact sites by using phage display and synthesized corresponding peptides derived from the GenBank database sequence that contain the predicted antibody binding sites. Our data indicate that all 9 antibodies bind to linear epitopes, ie, composed of contiguous amino acids. In addition, the amino acids proline, tyrosine, glutamine, and leucine are highly represented in these antibody contact sites. The epitopes tend to be mildly to moderately hydrophilic. These findings are the first detailed studies of antibody epitopes associated with antigen retrieval and suggest that antibodies must recognize linear sequences to bind after formalin fixation and antigen retrieval.

Amino Acid Sequence↗
PubMed2006

A molecular model of antigen retrieval using a peptide array.

Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.

Animals↗
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↗
PubMed2004

Recent trends and advances in immunodiagnostics of solid tumors.

The development of new cancer immunodiagnostic tests measuring soluble markers can be divided along the lines of single analyte measurement versus multiplex analysis. In the measurement of single analytes, newly proposed test analytes still struggle with the same issues as their predecessors; namely, can the measurement of a single biomarker be sufficiently sensitive and specific for screening the general population? Probably the best example of this challenge is in the area of bladder cancer detection, where several newly identified markers are being clinically evaluated in multicenter trials. In order to surmount this hurdle, multiplex analysis has become an increasingly important research focus. By combining the statistical power of measuring many cancer-associated analytes, it is hoped that highly specific diagnostic tests can be developed that are suitable for screening the general population. Some of the most impressive data for multiplex cancer biomarker detection derive from a non-immunologic technique - mass spectroscopy. Multiplex analysis has also recently been applied to the measurement of serum antibodies to tumor-associated antigens. Recent data link the development of antibodies to tumor-associated antigens with the presence of solid tumors. This strategy is a departure from the more traditional assay format of measuring the antigens themselves, and is another promising emerging area of investigation for the early detection of solid tumors. Solid tumor analysis by quantitative immunohistochemical staining is another rapidly growing area of cancer immunodiagnosis. This field has become especially important in the context of pharmacodiagnostics - the coupling of cancer therapy to the outcome of a test measurement from a patient biopsy. Standardization and assay reproducibility appear to be the most significant challenges in this context. In summary, developments over the past several years give reason for excitement and optimism about the potential for cancer immunodiagnostics to meaningfully impact cancer patient survival. In this review we take a fresh look at the field of cancer immunodiagnostics, to identify these recent and emerging trends that may impact on clinical practice over the next few years.

Animals↗
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

Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones.

A combinatorial Fab phage display library was generated from the antibody variable region genes of each of 2 BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries were shown to be diverse by nucleotide sequencing and diagnostic restriction enzyme digestion (fingerprinting) of individual members. The two libraries were combined and selected for binding to a suspension of formaldehyde-fixed human colorectal cancer cells in two successive rounds of selection and phage amplification by infection of bacteria. Analysis of the selected libraries as well as individual library clones by ELISA, showed binding to the cancer cell lines in both formaldehyde-fixed and native forms. Fifty five percent and 94% of library clones were positive for colorectal cancer cell binding after the first and second rounds of selection, respectively. Fingerprinting of individual clones showed the first round selected library to be very diverse and the second round selected library to be of more limited diversity. After absorption with normal human cell types, these anti-cancer selected libraries could be used to develop therapeutic and/or diagnostic agents.

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↗