Cheryl L. Baird

Surface chemistries for antibody microarrays.

Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

Immobilization strategies for single-chain antibody microarrays.

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust‐affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single‐chain antibodies (scFv) are affinity reagents that have greater potential for high‐throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope‐tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate‐specific antigen (PSA) are highly specific when tested against 21 different IgG‐based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R2 = 0.95) to a commercial 96‐well ELISA when tested using human serum samples. In addition, we find that TRX‐scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.

Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR

We evaluated digital PCR (dPCR) to directly enumerate plasmid and chromosome copies in three strains of Bacillus anthracis. Copy number estimates based on conventional quantitative PCR (qPCR) highlighted the variability of using qPCR to measure copy number whereas estimates based on direct sequencing are comparable to dPCR.

Engineering an ultra-stable affinity reagent based on Top7

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.

Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray® system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores.

Reducing heterophilic antibody interference in immunoassays using single-chain antibodies

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.

Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

These protocols describe a yeast surface display–based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single‐chain antibody fragments (scFvs) is created from antigen‐binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic‐activated cell sorting (MACS) and fluorescence‐activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen. Curr. Protocol. Cytom. 45:4.7.1‐4.7.30.

Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast‐display libraries. Yeast‐display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non‐immune libraries is the conversion of highly active yeast‐displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast‐display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast‐displayed and ‐secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast‐displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast‐displayed scFv that bind non‐overlapping or non‐competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast‐displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast‐displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast‐display libraries. Biotechnol. Bioeng. 2010;105: 973–981.

Molecular basis of the structural stability of a Top7-based scaffold at extreme pH and temperature conditions

The development of stable biomolecular scaffolds that can tolerate environmental extremes has considerable potential for industrial and defense-related applications. However, most natural proteins are not sufficiently stable to withstand non-physiological conditions. We have recently engineered the de novo designed Top7 protein to specifically recognize the glycoprotein CD4 by insertion of an eight-residue loop. The engineered variant exhibited remarkable stability under chemical and thermal denaturation conditions. In the present study, far-UV CD spectroscopy and explicit-solvent MD simulations are used to investigate the structural stability of Top7 and the engineered variant under extreme conditions of temperature and pH. Circular dichroism measurements suggest that the engineered variant Top7(CB1), like Top7, retains its structure at high temperatures. Changes in CD spectra suggest that there are minor structural rearrangements between neutral and acidic environments for both proteins but that these do not make the proteins less stable at high temperatures. The anti-parallel beta-sheet is well conserved within the timescale simulated whereas there is a decrease of helical content when low pH and high-temperature conditions are combined. Concerted alanine mutations along the alpha-helices of the engineered Top7 variant did not revert this trend when at pH 2 and 400K. The structural resilience of the anti-parallel beta-sheet suggests that the protein scaffold can accommodate varying sequences. The robustness of the Top7 scaffold under extreme conditions of pH and temperature and its amenability to production in inexpensive bacterial expression systems reveal great potential for novel biotechnological applications.

Controlled Activation of Protein Rotational Dynamics using Smart Hydrogel Tethering

Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications that take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a poly(ethylene glycol) (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with (13)C and (15)N, permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. The protein dynamics is suppressed upon initial formation of hydrogels, with a concomitant increase in protein stability. Relaxation of the hydrogel matrix following transient heating results in enhanced protein dynamics and resolution of substrate-induced large-amplitude domain rearrangements.