Twist on protein microarrays: layering wax-patterned nitrocellulose to create customizable and separable arrays of multiplexed affinity columns.

We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 µL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

Enzyme-linked immunosorbent assay for p16(INK4a) - a new triage test for the detection of cervical intraepithelial neoplasia?

OBJECTIVE: To evaluate enzyme-linked immunosorbent assay (ELISA) for cyclin-dependent kinase inhibitor 2A protein (p16(INK4a) ) on self-collected cervicovaginal lavage samples as an additional triage test to identify women with high-grade cervical intraepithelial neoplasia (CIN). DESIGN: Retrospective feasibility, sensitivity and specificity study. SETTING: University Medical School, Germany. SAMPLE: One hundred and fifty-two patients from the colposcopy clinic were included. METHODS: All women used a cervico-vaginal lavage device (Delphi Screener) for self-sampling and had gynecological examinations with Pap smears, cervical smears in ThinPrep PreservCyt solution and Cervatec medium for human papillomavirus (HPV) testing (Qiagen Hybrid Capture 2) and colposcopic examinations with biopsies if abnormalities were detected (72 women; 51%). All cytological samples were examined by p16(INK4a) ELISA. MAIN OUTCOME MEASURES: Sensitivity and specificity of p16(INK4a) ELISA for high-grade CIN. RESULTS: Complete data were available for 140 women. Among these, 62 women (46%) presented with an atypical Pap smear and 65 (46.4%) were high-risk HPV positive in the reference smear sample. Seventeen women (12%) had CIN 3+. Twenty-seven (19%) physician-collected samples were p16(INK4a) ELISA positive. In contrast, p16(INK4a) ELISA turned out to be positive in only one (1%) vaginal lavage sample. CONCLUSIONS: Our study shows that self-sampling with cervicovaginal lavage followed by p16(INK4a) ELISA is not suitable for the detection of high-grade CIN.

Exploring the sheep (Ovis aries) immunoglobulin repertoire by next generation sequencing.

Next-generation sequencing (NGS) has revolutionized the way we determine the antibody repertoires encoded by B cells in the blood or lymphoid organs and transformed our understanding of adaptive immune responses in many species. Sheep (Ovis aries) have been widely used as a host for therapeutic antibody production since the early 1980s, however, little is known about their immune repertoires or immunological processes affecting the antibody generation. The objective of this study was to employ NGS for a comprehensive analysis of immunoglobulin heavy and light chain repertoires in four healthy sheep. We obtained > 90 % complete antibody sequences and nearly 130,000, 48,000 and 218,000 unique CDR3 reads for the heavy chain (IGH), kappa chain (IGK), and lambda chain (IGL) loci, respectively. Consistent with other species, we observed biased usage of germline variable (V), diversity (D) and joining (J) genes in the heavy and kappa loci, but not in the lambda loci. Moreover, the enormous diversity of CDR3 sequences was observed through sequence clustering and convergent recombination. These data will build a foundation for future studies investigating immune repertoires in health and disease as well as contribute to further refinement of ovine-derived therapeutic antibody drugs.

Evaluation of a new multiplex real-time polymerase chain reaction assay for the detection of human papillomavirus infections in a referral population.

OBJECTIVES: Human papillomavirus (HPV) testing is an important part of cervical cancer screening and management of women with atypical screening results. This study was conducted to evaluate the analytical and clinical performance of the Abbott RealTime High-Risk HPV assay (RealTime) in a referral population, in comparison to the Qiagen Hybrid Capture 2 High-Risk HPV DNA Test (hc2). METHODS: RealTime is a new polymerase chain reaction assay that detects 14 high-risk HPV genotypes with simultaneous differentiation between HPV 16 and HPV 18. Five hundred forty-five routine cervical smear samples (ThinPrep) from women who were referred to 2 German colposcopy clinics were included in the study. All samples were tested with both assays for the detection of high-risk HPV DNA. Specimens with repeatedly discordant results were genotyped by Linear Array (Roche) and in-house polymerase chain reaction assays. RESULTS: Both assays showed excellent overall agreement (92.8%; κ = 0.86) on 545 samples. Analytical sensitivity of RealTime was comparable to that of hc2 (97.6% vs 95.1%, P = 0.189), whereas RealTime demonstrated significantly higher analytical specificity compared with hc2 (100% vs 93.1%, P < 0.0001). RealTime showed no cross-reactivity with untargeted HPV genotypes in this study. The clinical performance of the assays was evaluated based on histology results available from 319 women (90 nonpathological, 73 cervical intraepithelial neoplasia [CIN] 1, 75 CIN 2, 74 CIN 3, and 7 invasive cancers). High-risk HPV detection rates observed in women with CIN 1, CIN 2+, and CIN 3+ diagnosis, respectively, were comparable for both assays: 47.9%, 92.3%, and 97.5% (RealTime) and 47.9%, 92.3%, and 93.8% (hc2). Detection of HPV 16/18 with RealTime was highly correlated with severity of dysplasia: less than CIN 2, 30.5%; CIN 2+, 59.0%; CIN 3+, 71.6%. CONCLUSIONS: These results support the use of RealTime for routine detection of HPV infections in a referral population.

Vesicles for Signal Amplification in a Biosensor for the Detection of Low Antigen Concentrations.

The sensitivity of biosensors is often not sufficient to detect diagnostically relevant biomarker concentrations. In this paper we have utilized a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to detect dissipative losses induced by the attachment of intact vesicles. We modified a sandwich assay by coupling the secondary antibodies to vesicles. This resulted in an increase of detection sensitivity, achieving a diagnostically relevant detection limit of 5 ng/ml or 30 pM antigens. In addition, we could combine the individual assay steps to decrease the total time to result in about 30 minutes.

Improving FoRe: A New Inlet Design for Filtering Samples through Individual Microarray Spots.

In this publication we present an improvement to our previously introduced vertical flow microarray, the FoRe array, which capitalizes on the fusion of immunofiltration and densely packed micron test sites. Filtering samples through individual microarray spots allows us to rapidly analyze dilute samples with high-throughput and high signal-to-noise. Unlike other flowthrough microarrays, in the FoRe design samples are injected into micron channels and sequentially exposed to different targets. This arrangement makes it possible to increase the sensitivity of the microarray by simply increasing the sample volume or to rapidly reconcentrate samples after preprocessing steps dilute the analyte. Here we present a new inlet system which allows us to increase the analyzed sample volume without compromising the micron spot size and dense layout. We combined this with a model assay to demonstrate that the device is sensitive to the amount of antigen, and as a result, sample volume directly correlates to sensitivity. We introduced a simple technique for analysis of blood, which previously clogged the nanometer-sized pores, requiring only microliter volumes expected from an infant heel prick. A drop of blood is mixed with buffer to separate the plasma before reconcentrating the sample on the microarray spot. We demonstrated the success of this procedure by spiking TNF-α into blood and achieved a limit of detection of 18 pM. Compared to traditional protein microarrays, the FoRe array is still inexpensive, customizable, and simple to use, and thanks to these improvements has a broad range of applications from small animal studies to environmental monitoring.

Microarrays made easy: biofunctionalized hydrogel channels for rapid protein microarray production.

We present a simple, inexpensive, and sensitive technique for producing multiple copies of a hydrogel-based protein microarray. An agarose block containing 25 biofunctionalized channels is sliced perpendicularly to produce many identical biochips. Each microarray consists of 500 µm spots, which contain protein-coated microparticles physically trapped in porous SeaPrep agarose. Proteins diffuse readily through SeaPrep agarose, while the larger microparticles are immobilized in the hydrogel matrix. Without major assay optimization, the limit of detection is 12 pM for a sandwich assay detecting human IgG. These highly flexible, multiplexed arrays can be produced rapidly without any special instrumentation and are compatible with standard fluorescence-based read-out.

On-chip surface-based detection with nanohole arrays.

A microfluidic device with integrated surface plasmon resonance (SPR) chemical and biological sensors based on arrays of nanoholes in gold films is demonstrated. Widespread use of SPR for surface analysis in laboratories has not translated to microfluidic analytical chip platforms, in part due to challenges associated with scaling down the optics and the surface area required for common reflection mode operation. The resonant enhancement of light transmission through subwavelength apertures in a metallic film suggests the use of nanohole arrays as miniaturized SPR-based sensing elements. The device presented here takes advantage of the unique properties of nanohole arrays: surface-based sensitivity; transmission mode operation; a relatively small footprint; and repeatability. Proof-of-concept measurements performed on-chip indicated a response to small changes in refractive index at the array surfaces. A sensitivity of 333 nm per refractive index unit was demonstrated with the integrated device. The device was also applied to detect spatial microfluidic concentration gradients and to monitor a biochemical affinity process involving the biotin-streptavidin system. Results indicate the efficacy of nanohole arrays as surface plasmon-based sensing elements in a microfluidic platform, adding unique surface-sensitive diagnostic capabilities to the existing suite of microfluidic-based analytical tools.