Modified streptavidin-biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤ 35, and Ct ≤ 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

Gold conjugated nanobodies in a signal-enhanced lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Despite the transfer of COVID-19 from the pandemic to control, we are still in a state of uncertainty about long-term success. Therefore, there is a great need for rapid and sensitive diagnostics to sustain the control status. After several optimization trials, we developed lateral flow test (LFT) strips for rapid detection of SARS-CoV-2 spike 1 (S1) antigen in saliva samples. For signal enhancement of our developed strips, we applied dual gold conjugates. Gold-labeled anti-S1 nanobodies (Nbs) were employed as S1 detector conjugate, while gold-labeled angiotensin-converting enzyme 2 (ACE2) was used as S1 capturing conjugate. In a parallel strip design, we used an anti-S1 monoclonal antibody (mAb) as an antigen detector instead of anti-S1 Nbs. Saliva samples were collected from 320 symptomatic subjects (180 RT-PCR confirmed positive cases and 140 confirmed negative cases) and were tested with the developed strips. In early detection for positive samples with cycle threshold (Ct ≤ 30), Nbs-based LFT strips showed higher sensitivity (97.14%) and specificity (98.57%) than mAb-based strips which gave 90.04% sensitivity and 97.86% specificity. Moreover, the limit of detection (LoD) for virus particles was lower for Nbs-based LFT (0.4 × 104 copies/ml) than for the mAb-based test (1.6 × 104 copies/ml). Our results are in favor of the use of dual gold Nbs and ACE2 conjugates in LFT strips. These signal-enhanced strips offer a sensitive diagnostic tool for rapid screening of SARS-CoV-2 S1 antigen in the easily collected saliva samples.

Lab-scale production of anti-Schistosoma monoclonal antibodies by hybridoma culturing in serum free medium: Enhancement of growth, cryopreservation survival rate, and diagnostic accuracy.

This study was performed to evaluate the effect of two commercially available serum-free culture media; serum free medium (SFM) and chemically defined medium (CDM), on the growth rate, antibody productivity and post adaptation cryopreservation and revival reactivity of hybridoma cells compared to the conventional serum based medium (SBM). In addition, the diagnostic efficacy of MAbs secreted in each culture medium was evaluated by testing their performance in sandwich ELISA for antigen detection. Anti- Schistosoma mansoni soluble egg antigen hybridoma cell line (7A/8F) secreting previously characterized IgG Kappa mAbs, were retrieved and propagated in each of the three aforementioned media. Growth rate and viability were assessed post culturing in each media. The data collected from this study indicated that MAbs secreted from hybridoma cells cultured in SFM were the most abundant, easiest to purify, and the most effective in antigen detection by sandwich ELISA, in comparison to those produced in the other two media. Moreover, combination of fresh and conditioned medium with DMSO 7.5% was the most promising formulation for the cryopreservation of hybridoma cells cultivated in serum independent media (SFM or CDM).

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 ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤ 35 and ≤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.

Cloning of human cord blood-mesenchymal stem cells for isolation of enriched cell population of higher proliferation and differentiation potential.

Heterogeneity of Mesenchymal stem cells (MSCs) imposes limitations for their in vitro expansion and accounts for the lack of reproducibility in some clinical studies. So, this study was designed to isolate and enrich clones of multipotent and self-renewing MSCs from cord blood (CB). Enriched clones with higher proliferation and differentiation potential provide regenerative cells suitable for various clinical demands. MSCA and MSCB original (progenitor) cells were isolated from CB samples, and single cells were cloned by limiting dilution method, in mouse embryonic fibroblast conditioned media. Original MSCs and their single-cell derived clones were characterized by identifying their proliferation rate, immunophenotyping of surface antigens, expression of pluripotency and proliferation genes (Oct4, Sox2, Nanog, KLF4, c-Myc, and PDGFRA), and differentiation potential into multiple lineages (osteogenic, adipogenic, and chondrogenic). Some single-cell clones of MSCA showed a higher proliferation rate and greater differentiation potential than their original cells. However, original MSCB cells were of greater proliferation and differentiation potential than their derived single-cell clones, except for one clone which had comparable results. Cloning of MSCs was attainable when cultured in mouse embryonic fibroblast conditioned media. Single clones with higher proliferation and differentiation potential than their original progenitor cells were obtained by cloning of poorly functioning MSCs progenitor cells, enabling the selection of more therapeutically efficacious MSCs with better performance in clinical applications. Moreover, this study draws attention to the importance of CD105 as a possible MSCs biomarker associated with the multilineage commitment of MSCs.

Induction of Hepatic Regeneration in an Experimental Model Using Hepatocyte-Differentiated Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. MSCs were generated from umbilical cord blood. Hepatogenic differentiation was induced on two-dimensional (2D) and three-dimensional (3D) culture system and characterized by morphology, scanning electron microscopy, immunocytochemistry, and gene expression. Albumin and α-1 antitrypsin (AAT) in culture supernatants were measured. Differentiated cells were administered intravenous into a murine model of carbon tetra induced liver cirrhosis. After 12 weeks of injection, liver pathology was examined. The hepatogenic differentiated MSCs stained positively for albumin, alpha fetoprotein, HepPar1, cytokeratin 7 and 18, and OV6 with more mature cells, hexagonal in shape with central nuclei forming large sheets in groups in 3D culture system. AAT secretion and indocyanine green uptake were significantly increased in 3D system. In experimental model, MSC-3D treated group exhibited maximal restoration of liver architecture with absent septal fibrosis and marked improvement of alanine transaminase (ALT) and aspartate transaminase (AST), and mild increase in albumin. Both 3D and 2D culture system are effective in functional hepatogenic differentiation from MSCs and serve as a vehicle in liver tissue engineering. In vivo hepatogenic differentiation is more effective on 3D scaffold, with better functional recovery.

Liver Macrophage Depletion Ameliorates The Effect of Mesenchymal Stem Cell Transplantation in a Murine Model of Injured Liver.

Mesenchymal stem cells (MSCs) therapy show different levels of effectiveness in the context of different types of liver damage, suggesting that the microenvironment of the injured liver is a key determinant for effective stem cell therapy. The objective was to assess the modulatory effect of hepatic stem cell niche components on the transplanted MSCs during liver injury induced by carbon tetrachloride (CCl4). Superparamagnetic iron oxide (SPIO)-labeled human MSCs were injected intravenously into mice treated with CCl4 and subjected to hepatic macrophage-depletion. Liver tissues were collected at different intervals post transplantation for subsequent histopathological, morphometric, immunohistochemical, gene expression and ultrastructural studies. The homing of the transplanted MSCs was evidenced by tracing them within the niche by iron staining and immunohistochemical studies. MSCs differentiated into hepatocyte-like cells and intimal smooth muscle cells as evidenced by their expression of human albumin and α-smooth muscle actin with a concomitant increase in the level of mouse hepatocyte growth factor. A post transplantation reduction in the liver fibro-inflammatory reaction was found and was promoted by liver macrophages depletion. Thus, it could be concluded from the present study that prior manipulation of the microenvironment is required to improve the outcome of the transplanted cells.

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-ß1, 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-ß1 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.

Potentials of Differentiated Human Cord Blood-Derived Unrestricted Somatic Stem Cells in Treatment of Liver Cirrhosis.

OBJECTIVES: Liver transplantation is the well-known treatment for chronic liver diseases; however, postoperative complications and lack of donors continue to be limitations with this treatment. Investigating new modalities for treatment of chronic liver illness is a must. In the present study, we aimed to clarify the effects of an in vitro hepatocyte-differentiated human unrestricted somatic stem cell transplant as a new cell-based therapy in an experimental model of chronic liver failure. MATERIALS AND METHODS: Human umbilical cord blood-derived unrestricted somatic stem cells were isolated, cultured, propagated, and characterized. Cells were directed to differentiate into hepatocyte-like cells. An animal model of carbon tetrachloride cirrhotic liver failure was prepared, and the human in vitro differentiated unrestricted somatic stem cells were transplanted into the experimental model. Animals that did not receive transplant served as the pathologic control group. Animals were euthanized 12 weeks after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the pathologic control group, the transplant group showed improvements in levels of alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin. Histopathologic examination of the transplant group also showed improvements in hydropic degeneration and fibrosis. CONCLUSIONS: The use of unrestricted somatic stem cells, isolated and propagated from cord blood and then differentiated into hepatocyte-like cells, improved both fibrosis and normal function of cirrhotic livers. These cells could be considered as a line of cell-based therapy in cases of chronic liver disease.

Cord blood-derived mesenchymal stem cells with hepatogenic differentiation potential ameliorate chronic liver affection in experimental models.

BACKGROUND: The liver is one of the major target organs for which cell-based therapies are very promising. The limitations of various cellular therapies, including bone marrow (BM)-derived mesenchymal stem cells (MSCs), urges the exploration of stem cell sources more suitable for transplantation. Human umbilical cord blood (HUCB) can overcome these drawbacks with a favorable reparative outcome. OBJECTIVES: The aim of this study was to evaluate the therapeutic potential of MSCs in 2 groups of chronic liver injury experimental models. MATERIAL AND METHODS: Propagation and characterization of MSCs isolated from cord blood (CB) samples were performed and differentiation into osteogenic, adipogenic and hepatogenic lineages was induced. The 1st experimental model group (80 mice) included a negative control, a pathological control and 60 mice infected with Schistosoma mansoni (S. mansoni) and transplanted with MSCs. The 2nd experimental model group (30 hamsters) included 10 healthy hamsters serving as a negative control and 20 hamsters injected with repeated doses of carbon tetrachloride (CCl4) to induce liver fibrosis; 10 of them were treated with an intrahepatic (IH) injection of 3 × 106 MSCs and the other 10 were untreated pathological controls. Mice and hamsters were sacrificed 12 weeks post-transplantation and their liver sections were stained immunohistochemically for the detection of human hepatocyte-like cells. Moreover, the sections were examined for the levels of fibrosis. RESULTS: In both models, the transplantation of CB-derived MSCs (CB-MSCs) resulted in the engraftment of the fibrotic livers with newly formed hepatocytes, as evidenced by positive immunohistochemistry staining with human Hepatocyte Paraffin 1 (Hep Par 1), alpha-fenoprotein (AFP), cytokeratin 18 (CK18), cytokeratin 7 (CK7), and OV6 monoclonal antibody. The transplanted liver sections showed markedly reduced hepatic fibrosis with a significantly lower fibrotic index, as well as significantly improved liver functions compared to the pathological control (p < 0.001). CONCLUSIONS: This data provides hope that human CB-MSCs can be utilized as multipotent stem cells with unlimited potentiality in regenerative medicine and supports the concept of cellular therapy for the cure of hepatic fibrosis.

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

OBJECTIVES: Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. MATERIALS AND METHODS: Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. CONCLUSIONS: Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

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.

BACKGROUND: This research was carried out to develop a reliable monoclonal antibody (MoAb)-based sandwich enzyme linked immunosorbent assay (ELISA) for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. METHODS: From a panel of MoAbs raised against F. gigantica 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 F. gigantica antigen by indirect ELISA. RESULTS: The two MoAbs were of the IgG1 and IgG(2a) subclasses, 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 F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively). CONCLUSIONS: These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

Mediastinal ganglioneuroblastoma-secreting vasoactive intestinal peptide causing secretory diarrhoea.

In this case report we describe a case of mediastinal ganglioneuroblastoma-secreting vasoactive intestinal peptide (VIP), causing secretory diarrhoea in an 18-month-old child. An 18-month-old girl presented with a 2-month history of diarrhoea, abdominal distension and weight loss. Investigations revealed secretory diarrhoea with hypokalaemia, hyponatraemia and hypochloraemia and metabolic acidosis. Her stool output was 2.5-3.lday(-1) with increased stool sodium. VIP levels were strikingly high with normal glucagon and gastrin levels. X-ray of the chest revealed a well-defined mass in the right upper zone with tracheal shift, which was confirmed with computed tomography (CT) of the chest. The mass was resected and the patient became asymptomatic. This case shows that secretory diarrhoea caused by VIP and produced by ganglioneuroblastoma indicates a favourable prognosis, provided it is resectable.