Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

<p>Abstract</p> <p>Background</p> <p>This research was carried out to develop a reliable monoclonal antibody (MoAb)-based sandwich enzyme linked immunosorbent assay (ELISA) for the diagnosis of active <it>Fasciola gigantica </it>infection in both serum and stool for comparative purposes.</p> <p>Methods</p> <p>From a panel of MoAbs raised against <it>F. gigantica </it>excretory/secretory antigens (ES Ags), a pair (12B/11D/3F and 10A/9D/10G) was chosen due to its high reactivity and strict specificity to <it>F. gigantica </it>antigen by indirect ELISA.</p> <p>Results</p> <p>The two MoAbs were of the IgG₁and IgG_2asubclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of <it>F. gigantica </it>infected patients and the OD readings of ELISA in both stool and serum samples (<it>r </it>= 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively).</p> <p>Conclusions</p> <p>These data showed that the use of MoAb-based sandwich ELISA for the detection of <it>F. gigantica </it>coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active <it>F. gigantica </it>infection.</p

Non-Invasive Detection of SARS-CoV-2 Antigen in Saliva versus Nasopharyngeal Swabs Using Nanobodies Conjugated Gold Nanoparticles

The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values &le; 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values &le; 35 and &le;40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples

Antifibrotic effects of gallic acid on hepatic stellate cells: In vitro and in vivo mechanistic study

Few studies reported the antifibrotic effects of gallic acid (GA) despite its known hepatoprotective and antioxidant activities. Accordingly, this study investigated the antifibrotic effects of GA through clarifying its mechanisms on hepatic stellate cells' (HSCs) activation, proliferation and/or apoptosis. In vitro effects of GA on HSC-T6 activation/proliferation, morphology and safety on hepatocytes were assessed. In vivo, hepatic fibrosis was induced via chronic thioacetamide (TAA)-intoxication. TAA-intoxicated rats were treated with silyamrin or GA. At end of experiment, liver functions, hepatic MDA, GSH, PDGF-BB, TGF-β1, TIMP-1 and hydroxyproline were determined. Histological analysis and Sirius red staining of hepatic sections, expressions of alpha-smooth muscle actin (α-SMA), proliferating cellular nuclear antigen (PCNA) and caspase-3 were examined. In vitro, GA resulted in a concentration and time-dependent inhibition in HSCs activation, proliferation (IC50= 45 and 19 μg/mL at 24 and 48 h respectively); restored the quiescent morphology of some activated HSCs plus its safety on hepatocytes. In vivo, GA reduced ALT, AST, MDA, PDGF-BB levels, collagen deposition and fibrosis score (S1 vs S4); increased caspase-3 expression and restored GSH stores, TGF-β1 level, α-SMA and PCNA expressions. In conclusion, GA counteracted the progression of hepatic fibrosis through reduction of HSCs proliferation/activation mutually with their apoptosis induction. Keywords: Gallic acid, Hepatic stellate cells, Hepatocytes, Proliferation/activation, Apoptosi