An evaluation of the diagnostic performance characteristics of the Yellow Fever IgM immunochromatographic rapid diagnostic test kit from SD Biosensor in Ghana.

Yellow fever is endemic in Ghana and outbreaks occur periodically. The prodromal signs due to Yellow Fever Virus (YFV) infection are non-specific, making clinical signs unreliable as the sole criteria for diagnosis. Accurate laboratory confirmation of suspected yellow fever cases is therefore vital in surveillance programs. Reporting of ELISA IgM testing results by laboratories can delay due to late arrival of samples from the collection sites as well as limited availability of ELISA kits. In this study, the diagnostic performance characteristics of a rapid immunochromatographic Standard Q Yellow Fever IgM test kit (SD Biosensor) was evaluated for the rapid diagnosis of Yellow Fever infection in Ghana. A panel of 275 sera, comprising 81 confirmed YFV positives and 194 negatives were re-tested in this study using the Standard Q Yellow Fever IgM test kit. Using the CDC/WHO Yellow Fever IgM capture ELISA as a benchmark, the sensitivity, specificity and accuracy of the Standard Q Yellow Fever test kit were 96.3%, 97.9% and 97.5%, respectively. The false positivity rate was 5.1% and there was no cross-reactivity when the Standard Q Yellow Fever test kit was tested against dengue, malaria and hepatitis B and C positive samples. In addition, inter-reader variability and invalid rate were both zero. The results indicate that the diagnostic performance of the Standard Q Yellow Fever IgM test kit on serum or plasma is comparable to the serum IgM detection by ELISA and can be used as a point of care rapid diagnostic test kit for YFV infection in endemic areas.

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

Application of an S-layer protein as a self-aggregating tag for cost-effective separation of recombinant human and yeast D-amino acid oxidases in the aqueous two-phase system.

OBJECTIVE: To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast D-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. RESULTS: In aqueous two-phase (PEG-phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). CONCLUSIONS: The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast D-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast D-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG-phosphate aqueous system.

Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

Optimized Continuous Multicolumn Chromatography Enables Increased Productivities and Cost Savings by Employing More Columns.

The advantages of continuous chromatography with respect to increased capacity are well established. However, the impact of different loading scenarios and total number of columns on the process economics has not been addressed. Here four different continuous multicolumn chromatography (MCC) loading scenarios are evaluated for process performance and economics in the context of a Protein A mAb capture step. To do so, a computational chromatography model is validated experimentally. The model is then used to predict process performance for each of the loading methods. A wide range of feed concentrations and residence times are considered, and the responses of operating binding capacity, specific productivity, and the number of process columns are calculated. Processes that are able to add more columns proved to be up to 65% more productive, especially at feed concentrations above 5 g L-1 . An investigation of the operating costs shows that discrete column sizing and process performance metrics do not always correlate and that the most productive process is not necessarily the most cost effective. However, adding more columns for the non-load steps at higher feed concentrations allows for overall cost savings of up to 32%.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the µCols compared with conventional columns for similar protein yields.

A multi-column plate adapter provides an economical and versatile high-throughput protein purification system.

Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

A Serological Point-of-Care Test for the Detection of IgG Antibodies against Ebola Virus in Human Survivors.

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

Effective purification of recombinant peptide ligands for GPCR research.

Peptide purification from natural sources and chemical synthesis is cumbersome with various shortcomings such as low yield, high cost of production, error prone, and restricted by nature of amino acids. Though recombinant DNA technology had overcome all these setbacks for larger proteins, it is still a challenge to produce peptides that are salt free and without impurities. Our approach discussed in this chapter deals with easy and effective purification of peptides of varying sizes (up to 10kDa), expressed as fusion proteins in bacterial system. This includes cleavage of fusion affinity tag by "PreScission protease" in volatile buffer followed by selective acetonitrile precipitation of high-molecular-weight tag in order to purify peptides in solution. This method can be used to purify peptides in large scale for various biochemical and physiological studies.

Feasibility of using continuous chromatography in downstream processing: Comparison of costs and product quality for a hybrid process vs. a conventional batch process.

The protein A capture step is the main cost-driver in downstream processing, with high attrition costs especially when using protein A resin not until end of resin lifetime. Here we describe a feasibility study, transferring a batch downstream process to a hybrid process, aimed at replacing batch protein A capture chromatography with a continuous capture step, while leaving the polishing steps unchanged to minimize required process adaptations compared to a batch process. 35g of antibody were purified using the hybrid approach, resulting in comparable product quality and step yield compared to the batch process. Productivity for the protein A step could be increased up to 420%, reducing buffer amounts by 30-40% and showing robustness for at least 48h continuous run time. Additionally, to enable its potential application in a clinical trial manufacturing environment cost of goods were compared for the protein A step between hybrid process and batch process, showing a 300% cost reduction, depending on processed volumes and batch cycles.

An integrated experimental and economic evaluation of cell therapy affinity purification technologies.

AIM: To present an integrated techno-economic analysis assessing the feasibility of affinity purification technologies using the manufacture of induced pluripotent stem cell-derived progenitor photoreceptors for retinal dystrophies as a case study. MATERIALS & METHODS: Sort purity, progenitor yield and viable cell recovery were investigated for three cell sorting techniques: fluorescent-activated cell sorting (FACS); magnetic-activated cell sorting (MACS); and a novel technology SpheriTech beads. Experimentally derived metrics were incorporated into an advanced bioprocess economics tool to determine cost of goods per dose for each technology. RESULTS & CONCLUSION: Technical and bioprocess benefits were noted with SpheriTech beads which, unlike FACS and MACS, require no cell labeling. This simplifies the bioprocess, reduces cell loss and leaves target cells label free. The economic tool predicted cost drivers and a critical dose (7 × 107 cells per dose) shifting the most cost-effective technology from FACS to MACS. Process optimization is required for SpheriTech to compete economically.

Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis.

BACKGROUND: Schistosomiasis is a neglected infection affecting millions of people, mostly living in sub-Saharan Africa. Morbidity and mortality due to chronic infection are relevant, although schistosomiasis is often clinically silent. Different diagnostic tests have been implemented in order to improve screening and diagnosis, that traditionally rely on parasitological tests with low sensitivity. Aim of this study was to evaluate the accuracy of different tests for the screening of schistosomiasis in African migrants, in a non endemic setting. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective study was conducted on 373 patients screened at the Centre for Tropical Diseases (CTD) in Negrar, Verona, Italy. Biological samples were tested with: stool/urine microscopy, Circulating Cathodic Antigen (CCA) dipstick test, ELISA, Western blot, immune-chromatographic test (ICT). Test accuracy and predictive values of the immunological tests were assessed primarily on the basis of the results of microscopy (primary reference standard): ICT and WB resulted the test with highest sensitivity (94% and 92%, respectively), with a high NPV (98%). CCA showed the highest specificity (93%), but low sensitivity (48%). The analysis was conducted also using a composite reference standard, CRS (patients classified as infected in case of positive microscopy and/or at least 2 concordant positive immunological tests) and Latent Class Analysis (LCA). The latter two models demonstrated excellent agreement (Cohen's kappa: 0.92) for the classification of the results. In fact, they both confirmed ICT as the test with the highest sensitivity (96%) and NPV (97%), moreover PPV was reasonably good (78% and 72% according to CRS and LCA, respectively). ELISA resulted the most specific immunological test (over 99%). The ICT appears to be a suitable screening test, even when used alone. CONCLUSIONS: The rapid test ICT was the most sensitive test, with the potential of being used as a single screening test for African migrants.

Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.

Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Evaluation of a rapid antigen test for detection of Streptococcus pneumoniae in cerebrospinal fluid.

Detection of Streptococcus pneumoniae antigen in cerebrospinal fluid (CSF) using lateral flow immunochromatography tests (ICTs) is an effective, rapid and low-cost method to diagnose pneumococcal meningitis. This study evaluated the diagnostic accuracy of the Uni-Gold ICT to detect pneumococcal antigen in CSF specimens, compared with gold standard bacteriology and quantitative real-time PCR (qPCR) testing. CSF specimens (n=69) from patients with suspected bacterial meningitis were included in the study. 13/69 (19%) were positive and 56/69 (81%) were negative for pneumococcus by the gold standard tests. The ICT had sensitivity of 85% (55%-98%), specificity of 96% (88%-100%), positive likelihood ratio of 23.7 (6-94) and negative likelihood ratio of 0.16 (0.04-0.57). Overall, a strong correlation between the ICT and qPCR results was seen (κ=0.81). In contrast, CSF microscopy and culture were exceptionally insensitive. The ICT method is sufficiently robust and accurate for use in algorithms to diagnose bacterial meningitis.

A Low-Cost HPV Immunochromatographic Assay to Detect High-Grade Cervical Intraepithelial Neoplasia.

OBJECTIVE: To evaluate the reproducibility and accuracy of the HPV16/18-E6 test. METHODS: The study population was comprised of 448 women with a previously abnormal Pap who were referred to the Barretos Cancer Hospital (Brazil) for diagnosis and treatment. Two cervical samples were collected immediately before colposcopy, one for the hr-HPV-DNA test and cytology and the other for the HPV16/18-E6 test using high-affinity monoclonal antibodies (mAb). Women with a histologic diagnosis of cervical intraepithelial neoplasia grade 2 or 3 were considered to be positive cases. Different strategies using a combination of screening methods (HPV-DNA) and triage tests (cytology and HPV16/18-E6) were also examined and compared. RESULTS: The HPV16/18-E6 test exhibited a lower positivity rate compared with the HPV-DNA test (19.0% vs. 29.3%, p<0.001) and a moderate/high agreement (kappa = 0.68, 95%CI: 0.60-0.75). It also exhibited a significantly lower sensitivity for CIN2+ and CIN3+ detection compared to the HPV-DNA test and a significantly higher specificity. The HPV16/18-E6 test was no different from cytology in terms of sensitivity, but it exhibited a significantly higher specificity in comparison to ASCH+. A triage test after HPV-DNA detection using the HPV16/18-E6 test exhibited a significantly higher specificity compared with a triage test of ASCH+ to CIN2+ (91.8% vs. 87.4%, p = 0.04) and CIN3+ (88.6% vs. 84.0%, p = 0.05). CONCLUSION: The HPV16/18-E6 test exhibited moderate/high agreement with the HPV-DNA test but lower sensitivity and higher specificity for the detection of CIN2+ and CIN3+. In addition, its performance was quite similar to cytology, but because of the structural design addressed for the detection of HPV16/18-E6 protein, the test can miss some CIN2/3+ lesions caused by other high-risk HPV types.

Conformation-sensitive antibody-based point-of-care immunosensor for serum Ca(2+) using two-dimensional sequential binding reactions.

To assess the homeostasis of Ca(2+) metabolism, we have developed a rapid immunosensor for ionic calcium using a membrane chromatographic technique. As calcium-binding protein (CBP) is available for the recognition and undergone conformation change upon Ca(2+) binding, a monoclonal antibody sensitive to the altered structure of CBP has been employed. The sequential binding scheme was mathematically simulated and shown to match with the experimental results. At the initial stage, the rapid analytical system using lateral flow was constructed by immobilizing the antibody on the immuno-strip nitrocellulose membrane and labeling CBP with colloidal gold as a tracer. A major problem with this system in measuring ionic calcium levels was retarded migration of the gold tracer along the immuno-strip. It was conceivable that the divalent cation at a high concentration caused a change in the physical properties of the tracer, resulting in a non-specific interaction with the membrane surface. This problem was circumvented by first eluting a sample containing biotinylated CBP along the immuno-strip and then supplying the gold coupled to streptavidin across the signal generation pad of the strip. The color signal was then generated via biotin-SA linkage and measured using a smartphone-based detector developed in our laboratory. This two-dimensional chromatographic format completed the Ca(2+) analysis within 15min, the analytical performance covered the clinical dynamic range (0.25-2.5mM) and highly correlated with that of the reference system, i-STAT. These results inspired us to eventually investigate a dual-immunoassay system that measures simultaneously ionic calcium and parathyroid hormone, which regulates the ionic calcium level in serum. This will significantly simplify the current diagnostic protocols, which involve separate devices.

Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens.

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 10(4) cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. Graphical abstract Location of binding zones in the developed multiarray on a test strip (MATS) for simultaneous detection of eight pathogens.