Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.     

Reliable noninvasive genotyping based on excremental PCR of nuclear DNA purified with a magnetic bead protocol

A new protocol for extraction of DNA from faeces is presented. The protocol involves gentle washing of the surface of the faeces followed by a very simple DNA extraction utilizing the wash supernatant as the source of DNA. Unlike most other protocols, it does not involve the use of proteinase K and/or organic extraction, but is instead based on adsorption of the DNA to magnetic beads. The protocol was tested by microsatellite genotyping across six loci for sheep and reindeer faeces. Comparison with DNA extracted from blood demonstrated that the protocol was very reliable, even when used on material stored for a long time. The protocol was compared with another simple, solid-phase DNA-binding protocol, with the result that the bead-based protocol gave a slightly better amplification success and a lower frequency of allelic drop-outs. Furthermore, our experiments showed that the surface wash prior to DNA extraction is a crucial step, not only for our protocol, but for other solid-phase protocols as well.     

Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray

In this study, we describe a DNA microarray assay by using bead-mediated visible light-assisted signal detection for simultaneous screening of seven clinically important enteric pathogens, including Escherichia coli O157:H7, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella spp., Staphylococcus aureus, Rotavirus, and Norwalk virus (including genogroup I and II). Seven pairs of primers, in which the forward primers were labeled with biotin at the 5′ end, were designed and two sets of multiplex asymmetric PCR system were established to amplify the target genes of the seven pathogens. Twelve type specific oligonucleotides were designed and immobilized onto the aldehyde radical modified glass slide to function as target capture probes. After hybridization and stringency washes, the hybridized biotinylated PCR products were detected by the streptavidin-coated magnetic beads. The final hybridization results were visible to the naked eyes and can be imaged by CCD or digital camera. A total of 86 samples previously identified by conventional microbiological methods and/or PCR method were randomly selected to assess the specificity of this assay by a blind study. A coincidence rate of 100% was obtained. Due to the simplicity and specificity of the magnetic bead based DNA microarray, it is especially appropriate for the diagnosis and monitoring of enteric infectious diseases in the community and seaport.     

DNA glycosylase activity assay based on streptavidin paramagnetic bead substrate capture.

A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5' end with 32P and at the 3' end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.     

A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Efficiency of Simulium sanctipauli as a vector of Onchocerca volvulus in the forest zone of Ghana

The role of Simulium sanctipauli Vajime & Dunbar (Diptera: Simuliidae) as a vector of Onchocerca volvulus (Leuckart) (Spirurida: Onchocercidae) in the forest zone of central Ghana was studied in the Upper Denkyira district, where onchocerciasis is hyperendemic. Simulium sanctipauli was found to be a highly efficient vector, with a mean of 377 infective (L3) larvae in the heads of 1000 parous and 122 in the heads of 1000 biting flies. The overall infection rate of 44% of the parous flies with L1, L2 and L3 stages of O. volvulus (identity confirmed by polymerase chain reaction) demonstrates marked anthropophily. Female flies dispersed over a wide area and can transmit onchocerciasis up to at least 10 km away from their breeding sites. Annual community-directed treatments with ivermectin did not have a noticeable effect on the infection rates and parasitic loads of fly populations, which were as high 2 months after as 3 months before the distribution of ivermectin. This failure can be attributed to poor coverage, with treatment taken by only 24.4% of the population of the six study villages.     

A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection

Background

Onchocerciasis, an infection caused by the filarial nematode Onchocerca volvulus, is a major public health concern. Given the debilitating symptoms associated with onchocerciasis and concerns about recrudescence in areas of previous onchocerciasis control, more efficient tools are needed for diagnosis and monitoring of control measures. We investigated whether luciferase immunoprecipitation systems (LIPS) may be used as a more rapid, specific, and standardized diagnostic assay for Onchocerca volvulus infection.

Methods

Four recombinantly produced Onchocerca volvulus antigens (Ov-FAR-1, Ov-API-1, Ov-MSA-1 and Ov-CPI-1) were tested by LIPS on a large cohort of blinded sera comprised of both uninfected controls and patients with a proven parasitic infection including Onchocerca volvulus (Ov), Wuchereria bancrofti (Wb), Loa loa (Ll), Strongyloides stercoralis (Ss), and with other potentially cross-reactive infections. In addition to testing all four Ov antigens separately, a mixture that tested all four antigens simultaneously was evaluated in the standard 2-hour incubation format as well as in a 15-minute rapid LIPS format.

Findings

Antibody responses to the four different Ov antigens allowed for unequivocal differentiation between Ov-infected and uninfected control sera with 100% sensitivity and 100% specificity. Analysis of the antibody titers to each of these four antigens in individual Ov-infected sera revealed that they were markedly different and did not correlate (r_S = –0.11 to 0.58; P = 0.001 to 0.89) to each other. Compared to Ov-infected sera, patients infected with Wb, Ll, Ss, and other conditions had markedly lower geometric mean antibody titers to each of the Ov 4 antigens (P<0.0002 for each antigen). The simplified method of using a mixture of the 4 Ov antigens simultaneously in the standard format or a quick 15-minute format (QLIPS) showed 100% sensitivity and 100% specificity in distinguishing the Ov-infected sera from the uninfected control sera. Finally, the QLIPS format had the best performance with 100% sensitivity and specificity values of 76%, 84% and 93% for distinguishing Ov from Wb, Ll and Ss-infected sera.

Conclusions

The multi-antigen LIPS assay can be used as a rapid, high throughput, and specific tool to not only to diagnose individual Ov infections but also as a sensitive and potentially point-of-care method for early detection of recrudescent infections in areas under control and for mapping new areas of transmission of Ov infection.     

Fast preparation of fungal DNA for PCR screening

Rapid DNA preparation for the quick screening is highly demanded in diverse research fields. Here, we combined an extraction buffer and heat treatment to generate DNA templates from yeast and filamentous fungal materials for PCR. This method may be widely applicable to diverse fungal species in clinical and basic studies.     

Magnetic bead capture eliminates PCR inhibitors in samples collected from the airborne environment, permitting detection of Pneumocystis carinii DNA

PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S Pneumocystis carinii mitochondrial rRNA gene-based PCR.     

Molting, enzymes and new targets for chemotherapy of Onchocerca volvulus

Parasitic nematodes do not multiply in definitive hosts, but they do molt, grow and mature for a certain period after infection, after which they devote their energies almost entirely to egg production. In this review, Sara Lustigman describes key metabolic enzymes that are essential to the development of the larval stages of Onchocerca volvulus in the host, making them potential therapeutic targets.     

Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands

A magnetic beads based affinity-selection methodology towards the screening of acetylcholine binding protein (AChBP) binders in mixtures and pure compound libraries was developed. The methodology works as follows: after in solution incubation of His-tagged AChBP with potential ligands, and subsequent addition of cobalt (II)-coated paramagnetic beads, the formed bead-AChBP-ligand complexes are fetched out of solution by injection and trapping in LC tubing with an external adjustable magnet. Non binders are then washed to the waste followed by elution of ligands to a SPE cartridge by flushing with denaturing solution. Finally, SPE-LC-MS analysis is performed to identify the ligands. The advantage of the current methodology is the in solution incubation followed by immobilized AChBP ligand trapping and the capability of using the magnetic beads system as mobile/online transportable affinity SPE material. The system was optimized and then successfully demonstrated for the identification of AChBP ligands injected as pure compounds and for the fishing of ligands in mixtures. The results obtained with AChBP as target protein demonstrated reliable discrimination between binders with pK(i) values ranging from at least 6.26 to 8.46 and non-binders.     

DNA extraction method for screening yeast clones by PCR

A simple procedure for isolating yeast DNA suitable for use as a template for PCR amplification is described. SDS treatment alone is sufficient for extraction of chromosomal DNA from yeast cells. Cells of a yeast colony are suspended in a small volume (about 20 microL) of a 0.25% SDS solution, mixed vigorously and centrifuged. The supernatant can be directly used as a template after dilution to give an SDS concentration of less than 0.01% in the final PCR mixture.     

Oligonucleotide Primers for PCR Amplification of Coelomate Introns

Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.     

Cloning, characterization and DNA immunization of an Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH)

In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model.     

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus

Background

Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests.

Methodology/Principal Findings

A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011–2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions.

Conclusions

The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.     

Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

Background

To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length.

Methodology/Principal Findings

We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA_II instrument.

Conclusions/Significance

This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.