An ELISA-based high throughput protein truncation test for inherited breast cancer

Abstract Introduction Breast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis. Methods Segments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format. Results 100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 were achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated. Conclusions Overall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance

Dynamic expression of LIM cofactors in the developing mouse neural tube

The developmental regulation of LIM homeodomain transcription factors (LIM-HD) by the LIM domain-binding cofactors CLIM/Ldb/NLI and RLIM has been demonstrated. Whereas CLIM cofactors are thought to be required for at least some of the in vivo functions of LIM-HD proteins, the ubiquitin ligase RLIM functions as a negative regulator by its ability to target CLIM cofactors for proteasomal degradation. In this report, we have investigated and compared the protein expression of both factors in the developing mouse neural tube. We co-localize both proteins in many tissues and, although widely expressed, we detect high levels of both cofactors in specific neural tube regions, e.g., in the ventral neural tube, where motor neurons reside. The mostly ubiquitous distribution of RLIM- and CLIM-encoding mRNA differs from the more specific expression of both cofactors at the protein level, indicating post-transcriptional regulation. Furthermore, we show that both cofactors not only co-localize with each other but also with Isl and Lhx3 LIM-HD proteins in developing ventral neural tube neurons. Our results demonstrate the dynamic expression of cofactors participating in the regulation of LIM-HD proteins during the development of the neural tube in mice and suggest additional post-transcriptional regulation in the nuclear LIM-HD protein network

Multiplexed VeraCode bead-based serological immunoassay for colorectal cancer

AbstractColorectal cancer (CRC) is the second leading cause of cancer deaths in the US and Western world. Despite increased screening and advances in treatment, the mortality rate (ca. 50,000/year) and high national health-care burden for CRC are likely to remain high unless an effective non-invasive screening test for CRC is instituted for a large segment of the population. Blood-based protein biomarkers hold great promise for early disease diagnosis and personalized medicine; yet robust and reproducible multiplexing platforms and methodologies have lagged behind their genomic counterparts.Here, we report the development of a novel, multiplexed, hybrid immunoassay for CRC that is formatted on barcoded VeraCode™ micro-beads, which have until now only been used for genomic assays. The method combines a sandwich immunoassay format for detection of serum protein biomarkers with an antigen assay for autoantibody detection. The serum protein biomarkers CEA and GDF15 as well as autoantibodies to the p53 tumor associated antigen (TAA) were used to exemplify the method. This multiplex biomarker panel was configured to run on Illumina's holographically barcoded VeraCode™ micro-bead platform, which is capable of measuring hundreds of analytes simultaneously in a single well from small volumes of blood (<50μL) using a 96-well industry standard microtiter plate. This novel use of the VeraCode™ micro-bead platform translates into a potentially low volume, high throughput, multiplexed assay for CRC, for the purposes of biomarker validation, as well as patient screening, diagnostics and prognostics. In an evaluation of a 186 patient sera training set (CRC and normal), we obtained a diagnostic sensitivity of 54% and a specificity of 98%. We anticipate that by expanding and refining the biomarkers in this initial panel, and performing more extensive clinical validations, such an assay could ultimately provide a basis for CRC population screening to complement the more invasive, expensive and low throughput (but highly sensitive and specific) colonoscopy

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

<div><p>Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP^® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP^®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.</p></div

AllerBead with PC-PURE in reference to ImmunoCAP^® on 205 Boston Children’s Hospital serum samples.

<p>AllerBead with PC-PURE in reference to ImmunoCAP^® on 205 Boston Children’s Hospital serum samples.</p

Concentrating patient IgE with PC-PURE: Increased low-end sensitivity for sIgE.

<p>PC-PURE was used to simultaneously purify and concentrate IgE from 46 food allergy samples, followed by analysis on the multiplex AllerBead assay. To achieve the concentrating effect, the input sample volume for the PC-PURE step was 500 μL and the photo-release volume was 100 μL (“5x”), which was then input into the AllerBead assay. This was compared to PC-PURE used to only purify but not concentrate the IgE (“1x”; 100 μL input and photo-release volumes). AllerBead sensitivity (percent of sIgE-positives detected) was assessed in the low-end of the ImmunoCAP^® scale, defined as between 0.35 kIU_A/L and 5 kIU_A/L (sIgE positives determined <i>a priori</i> by ImmunoCAP^® testing).</p

Example ImmunoCAP^®-correlation of AllerBead with and without PC-PURE.

<p>Correlation plot of the multiplex AllerBead assays with and without PC-PURE, compared to the standard, FDA-cleared, non-multiplex ImmunoCAP^® test (for the tree nut cashew) for all 205 Boston Children’s Hospital patients. Pearson’s r and slope of the linear regression lines are provided. Note that AllerBead results were converted to kIU_A/L by heterologous interpolation from a standard curve (5 points; R² of linear regression = 0.99) comprised of purified IgE from the serum of patients with various known amounts of sIgE (based on ImmunoCAP^® testing). Pearson’s r for all foods are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191987#pone.0191987.g004" target="_blank">Fig 4b</a>.</p

Eliminating the matrix effect with photocleavage based affinity purification of biomarkers (PC-PURE): Application to a downstream multiplex in vitro allergy assay (AllerBead).

<p>The biomarker, serum IgE in this case, is pre-purified using photocleavable antibodies (PC-Antibodies) attached to beads (PC-Beads), in an overall process termed PC-PURE. This eliminates interference from sample matrix constituents in the downstream multiplex immunoassay. The multiplex immunoassay shown here is based on Luminex^® coded microspheres which contain the “bait” allergen (antigen) for binding and quantifying sIgE (termed the AllerBead assay). Y-shaped structures are antibodies. The red antibody is the patient’s total IgE and blue is the anti-IgE PC-Antibody used for PC-PURE. The yellow oval is the photocleavable linker that attaches the PC-Antibody to high capacity agarose beads to form the PC-Beads. The processing of the PC-Beads is performed in microtiter filter plates. Following PC-PURE, the photocleaved PC-Antibody can be used also for detection in the AllerBead assay, or, a separate detection antibody (binding a different epitope) can be used, as was the case in this work (not depicted). The blue triangle at Step 5 represents the allergen(s) (antigens) immobilized on the Luminex^® assay surface (the coded microsphere).</p

Performance metrics of AllerBead with and without PC-PURE.

<p>Serum samples from 205 subjects presenting at Boston Children’s Hospital with suspicion of or known food allergy were analyzed by the multiplex AllerBead assay against all eight food allergens under study. AllerBead was performed with and without PC-PURE. Results from the standard, FDA-cleared, non-multiplex ImmunoCAP^® test for all eight foods were used as a reference and to determine true positives and negatives for sIgE. (a) Signal-to-Noise of AllerBead. Signal-to-noise was calculated on a per-food basis as the average AllerBead result for ImmunoCAP^®-positives (≥0.10 kIU_A/L) divided by the average AllerBead result of ImmunoCAP^®-negatives (<0.10 kIU_A/L). (b) Pearson’s r as a metric for ImmunoCAP^®-correlation of the AllerBead assays. (c) Sensitivity of the AllerBead assays. *Sensitivity was defined as the percent of ImmunoCAP^®-positives detected in the range of the maximum measurable by ImmunoCAP^® (100 kIU_A/L) down to the cutoffs for 95% negative predictive value (NPV) for determining clinical allergy. 95% NPV cutoffs ranged from 0.35 kIU_A/L to 5 kIU_A/L depending on the food. 95% NPV cutoffs were based on prior literature reports using ImmunoCAP^® or equivalent assays in comparison to food challenge (see main manuscript for references); if 95% NPV was not reached in those studies, cutoff for best achieved NPV was used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191987#pone.0191987.t001" target="_blank">Table 1</a> for cutoffs and NPVs). Note NPV cutoffs have not been published for all eight foods under study and thus Shrimp and Cashew are omitted. AllerBead sensitivity for peanut is a composite of peanut extract and Ara h 8, and for milk, a composite of milk extract and lactalbumin (Bos d 4).</p