Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy

Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.

A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes

Understanding patterns of diversification, genetic exchange, and pesticide resistance in insect species of human health concern is necessary for effective population reduction and management. With the broad availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for large numbers of genetic markers from across the known genome. To this end, the targeting of gene sequences of known function or inheritance can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens that are detrimental to endangered vertebrate species such as bird malaria. Here we describe our development of a targeted gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we examined taxonomic divergence among samples from several members of the complex, as well as distinct populations of the relatively under-studied Culex quinquefasciatus, an urban pantropical species. We also examined the presence of known insecticide-resistance conferring alleles. Broadly, our developed gene-based assay proved effective for examining patterns of taxonomic and geographic clustering within the species complex, as well as for surveying genetic variants that have been associated with insecticide resistance. This assay will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of ubiquitous and increasingly damaging disease vectors.

A New Range of Chitosan Based Nano-antiviral Agents

ABSTRACT The current study involves the synthesis of fourteen analogs of oligochitosan and their screening for antiviral potential against human immunodeficiency virus (HIV), respiratory syncytial virus (RSV) and Coxsackie virus. The synthesized oligochitosan analogs were characterized by nuclear magnetic resonance (NMR) and FTIR techniques. HIV-1 p24 ELISA was performed using HIV-1 p24 antigen capture assay in order to estimate the viral infectivity loss. It was observed that sulfated oligochitosan was devoid of antiviral activity as compared to oligochitosan UN102 analog. The rest of UN102 analogs which include N-thiol (UN105), N-glutaryl (UN106), N-Azido (UN111) and N-phthaloyl (UN114) and N-citric analog (UN117) exhibited antiviral activity against HIV. The UN102 also decreased viral infection caused by RSV. In addition, UN102 was found to bind Coxsackie virus, which causes autoimmune myocarditis. The findings were of great interest to proceed for the development of novel antiviral agents.

Using Y-chromosome capture enrichment to resolve haplogroup H2 shows new evidence for a two-path Neolithic expansion to Western Europe

Uniparentally-inherited markers on mitochondrial DNA (mtDNA) and the non-recombining regions of the Y chromosome (NRY), have been used for the past 30 years to investigate the history of humans from a maternal and paternal perspective. Researchers have preferred mtDNA due to its abundance in the cells, and comparatively high substitution rate. Conversely, the NRY is less susceptible to back mutations and saturation, and is potentially more informative than mtDNA owing to its longer sequence length. However, due to comparatively poor NRY coverage via shotgun sequencing, and the relatively low and biased representation of Y-chromosome variants on capture assays such as the 1240 k, ancient DNA studies often fail to utilize the unique perspective that the NRY can yield. Here we introduce a new DNA enrichment assay, coined YMCA (Y-mappable capture assay), that targets the "mappable" regions of the NRY. We show that compared to low-coverage shotgun sequencing and 1240 k capture, YMCA significantly improves the mean coverage and number of sites covered on the NRY, increasing the number of Y-haplogroup informative SNPs, and allowing for the identification of previously undiscovered variants. To illustrate the power of YMCA, we show that the analysis of ancient Y-chromosome lineages can help to resolve Y-chromosomal haplogroups. As a case study, we focus on H2, a haplogroup associated with a critical event in European human history: the Neolithic transition. By disentangling the evolutionary history of this haplogroup, we further elucidate the two separate paths by which early farmers expanded from Anatolia and the Near East to western Europe.

SARS-CoV-2 IgG detection in human oral fluids

Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Non-invasive sampling methods are advantageous to reduce the need for venepuncture, which may be a barrier to investigations particularly in paediatric populations. Oral Fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. ELISA based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody ELISAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-COV-2 IgG ELISAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG ELISA, intended for use in children. All three assays were assessed using a panel of 1999 paired serum and oral fluids from children and adults participating in national primary school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the UK. The anti NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% CI: 71–79%) specificity of 99% (95% CI: 78–99%) when compared with paired serum antibodies measured using a commercial assay SARS-CoV-2 nucleoprotein IgG assay (Abbott, Chicago, IL, USA). Higher sensitivity was observed in children (80%, 95% CI: 71–88%) compared to adults (67%, CI: 60%-74%). Oral fluid assays using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI (68%-86%) and 53%, 95% CI (43%-64%), respectively).

LncRNA GACAT3 contributes to osteoarthritis progression by suppressing growth and inducing apoptosis of chondrocytes through miR-195/TGF-β/Smad5 axis

IntroductionLong non-coding RNAs (lncRNAs) were identified as an important regulator involved in the pathogenesis of osteoarthritis (OA). We aimed to evaluate whether lncRNA GACAT3 regulate OA progression by miR-195/TGF-β/Smad5 axis.Material and methodsExpression levels in tissue or chondrocytes were detected by RT-qPCR and Western blot. Effects of GACAT3 on cell viability, proliferation, apoptosis were evaluated. Targeted interactions of GACAT3, miR-195 and Smad5 were confirmed by dual luciferase reporter gene assay and biotin-coupled miRNA capture assay. Transfected or non-transfected cells were treated with TGF-β1 to verify role of TGF-β in mechanisms of OA progression.ResultsGACAT3 was overexpressed in OA tissues and associated with OA severity. After GACAT3 overexpression, the ability of cell viability and proliferation as well as proliferation-related genes was inhibited with enhanced level of apoptosis-related genes and Smad5. miR-195-5p was negatively targeted by GACAT3 and reversed effects caused by GACAT3. miR-195-5p negatively targeted Smad5. In the presence of TGF-β1, miR-195-5p mimics inhibited activation of Smad1/5, Smad5 and Smad2 compared with negative control. Immunofluorescence showed that miR-195-5p inhibited Smad1/5 activation and transfer to nucleus. In the presence of TGF-β1, GACAT3 facilitated the activation of TGF-β signaling. In clinical sample analysis, GACAT3 was positively correlated with Smad5 expression in OA patients.ConclusionsWe confirmed the up-regulation of GACAT3 in OA patients, and found that GACAT3 regulated the chondrocytes phenotypes by miR-195/TGF-β/Smad5 axis and in turn contributed to OA progression. Findings indicated that GACAT3 may act as a novel therapeutic target for controlling OA progression.