Seshi R. Sompuram

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.

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.

Recent efforts toward the development of peptide secondary structure mimetics at Molecumetics Ltd. for the discoveries of new drug candidates utilizing combinatorial chemistry with solid phase synthesis are described

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen escape variants, and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review her e our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Abstract The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.

A bidirectional phage display vector for the selection and mass transfer of polyclonal antibody libraries

An approach to the creation of antigen-specific polyclonal libraries of intact antibodies is presented. A polyclonal library of Fab antibody fragments would be expressed using a phage display vector, and selected for reactivity with an antigen or group of antigens. For conversion into a sublibrary of intact polyclonal antibodies, the selected heavy (H) and light (L) chain variable (V) region gene combinations would be transferred in mass, as linked pairs, to a eukaryotic expression vector which provides immunoglobulin (Ig) constant (C) region genes. To enable this selection and transfer, a bidirectional phage display vector was generated, in which the V region gene pairs are linked head to head in opposite transcriptional orientations. The functionality of this vector was demonstrated by the selection, transfer and expression of linked V region gene pairs derived from an A/J mouse that had been immunized with p-azophenylarsonate (Ars)-coupled keyhole limpet hemocyanin (KLH). As expected, the expressed IgG2b anti-Ars antibodies with selected V region gene pairs were shown to have V region sequences and Ars-binding characteristics similar to those of anti-Ars hybridoma antibodies. The technology presented here has potential for many diagnostic and therapeutic applications. These include the generation of polyclonal antibody libraries against multiple epitopes on infectious agents or cancer cells, and of polyclonal libraries encoding chimeric molecules composed of antibody V regions and T cell receptor C regions.

Bioinformatic Requirements for Protein Database Searching Using Predicted Epitopes from Disease-associated Antibodies

We describe a new approach to identify proteins involved in disease pathogenesis. The technology, Epitope-Mediated Antigen Prediction (E-MAP), leverages the specificity of patients’ immune responses to disease-relevant targets and requires no prior knowledge about the protein. E-MAP links pathologic antibodies of unknown specificity, isolated from patient sera, to their cognate antigens in the protein database. The E-MAP process first involves reconstruction of a predicted epitope using a peptide combinatorial library. We then search the protein database for closely matching amino acid sequences. Previously published attempts to identify unknown antibody targets in this manner have largely been unsuccessful for two reasons: 1) short predicted epitopes yield too many irrelevant matches from a database search and 2) the epitopes may not accurately represent the native antigen with sufficient fidelity. Using an in silico model, we demonstrate the critical threshold requirements for epitope length and epitope fidelity. We find that epitopes generally need to have at least seven amino acids, with an overall accuracy of >70% to the native protein, in order to correctly identify the protein in a nonredundant protein database search. We then confirmed these findings experimentally, using the predicted epitopes for four monoclonal antibodies. Since many predicted epitopes often fail to achieve the seven amino acid threshold, we demonstrate the efficacy of paired epitope searches. This is the first systematic analysis of the computational framework to make this approach viable, coupled with experimental validation.

Experimental Validation Of Peptide Immunohistochemistry Controls

Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance among a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with coefficients of variation of 4% to 8%. Moreover, the peptide controls were stable at ≤4°C for at least 7 months, the longest time duration we tested. A national study of 109 participating clinical laboratories demonstrated a good correlation between a laboratorys ability to properly stain formalin-fixed peptide controls to their ability in properly staining a 3+ HER-2 formalin-fixed tissue section mounted on the same slide (r=0.87). Therefore, peptide IHC controls accurately reflect the analytical component of an IHC stain, including antigen retrieval. Besides its use in proficiency survey testing, we also demonstrate the feasibility of applying peptide IHC controls for verifying intralaboratory IHC staining consistency, using Levy-Jennings charting.

A Novel Microscope Slide Adhesive for Poorly Adherent Tissue Sections

Abstract We describe a protected isocyanate (PI) microscope slide-coating technology that significantly enhances tissue section adhesion. This technology is particularly useful for problematic tissue sections that detach during staining. The glass slide coating is unique in that it involves a highly reactive covalent coupling to cellular proteins. Namely, we coat the slides with a modified isocyanate group capable of binding to amine, hydroxyl, and carboxylic functionalities on cells and tissue sections. This contrasts to presently available slide coatings, which primarily provide a weak charge interaction with tissue proteins. We compared the tissue adhesiveness of our isocyanate-coated slides to commercially available aminosilane (“plus”), poly-L-lysine, and polysineTM slides. Serial tissue sections were mounted on PI-coated slides versus one of the other existing coatings and treated in an otherwise-identical manner for staining. In instances where tissue adhesion was problematic, PI-coated slides produced better tissue retention almost every time. The difference was sometimes dramatic and of significant diagnostic value. We believe that this technology can be useful for capturingt issue sections that otherwise will not adhere well to glass slides. (The J Histotechnol 26:117, 2003) Submitted November 4, 2002; Accepted with revisions April 7, 2003

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.

B-cell epitopes in GroEL of Francisella tularensis.

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.