The development of an electrochemical immunosensor utilizing chicken IgY anti-spike antibody for the detection of SARS-CoV-2.

This paper introduces a novel approach for detecting the SARS-CoV-2 recombinant spike protein combining a label free electrochemical impedimetric immunosensor with the use of purified chicken IgY antibodies. The sensor employs three electrodes and is functionalized with an anti-S IgY antibody, ELISA and immunoblot assays confirmed the positive response of chicken immunized with SARS-CoV2 S antigen. The developed immunosensor is effective in detecting SARS-CoV-2 in nasopharyngeal clinical samples from suspected cases. The key advantage of this biosensor is its remarkable sensitivity, and its capability of detecting very low concentrations of the target analyte, with a detection limit of 5.65 pg/mL. This attribute makes it highly suitable for practical point-of-care (POC) applications, particularly in low analyte count clinical scenarios, without requiring amplification. Furthermore, the biosensor has a wide dynamic range of detection, spanning from 11.56 to 740 ng/mL, which makes it applicable for sample analysis in a typical clinical setting.

Review of current in vitro COVID-19 diagnostics methods.

The diagnosis of COVID-19 is considered a significant step in the management of the disease that is causing a major worldwide public health challenge from the time of its emergence in December 2019. Since it has been established that SARS-CoV-2 spreads rapidly, timely detection of the positive cases and isolation of such individuals and their contacts helps in containing viral transmission. In this paper, we review the in vitro technology platforms for testing and diagnosing COVID-19 patients: molecular tests, rapid antigen tests, and serology tests. As part of our review of each category of tests, we discuss the commercialized testing platforms, their analyzing systems, specimen collection protocols, and testing methodologies. Moreover, the efficacy and limitations of each technique are also discussed. The key structural components of the virus are presented to provide an understanding of the scientific principles behind the testing tools.

Freeze-drying of monoclonal antibody-conjugated gold nanorods: Colloidal stability and biological activity.

Maintaining colloidal stability of nanoparticles in suspensions is a major challenge. Therefore, freeze-drying (lyophilization) is recently proposed to preserve colloidal stability of nanoparticles through maintaining them in a solid state. However, freeze-drying would itself induce nanoparticle aggregation unless proper formulation with a careful selection of cryoprotectants is considered. Herein, we evaluate the colloidal stability of gold nanorods (GNRs) conjugated with a rituximab as a model monoclonal antibody upon freeze-drying in the presence of various cryoprotectants (mannitol, trehalose and sucrose). Aggregation-induced optical responses of GNRs were used as a sensitive tool to follow nanoparticle aggregation. In the absence of cryoprotectants, rituximab-conjugated GNRs aggregate irreversibly while evaluated cryoprotectants exhibit a significant protective effect. Maximal colloidal stability of GNRs is observed in the presence of trehalose while mannitol results in best cake formation in terms of shape and integrity. A combination of trehalose and mannitol produces a lyophilized product with satisfactory GNR colloidal stability and cake shape. Moreover, we show that freeze-dried rituximab-conjugated GNRs in presence of proper cryoprotectants maintain typical binding to lymphoma tissues as confirmed via immunohistochemistry assay.

Human periodontal fibroblasts viability stored in Custodiol® , coconut water, and propolis. An ex vivo study.

BACKGROUND/AIMS: Successful replantation of an avulsed tooth depends on the regeneration of periodontal ligament (PDL) attachment which is affected by the transport medium, dry time, and storage time. Various storage media have been studied but the search for the optimum storage medium is still needed to determine the ideal material and storage time to maintain PDL cells. The aim of this study was to determine the ability of Custodiol® , coconut water (CW) from different stages of maturity, and propolis as storage media for avulsed teeth by evaluating the viability of PDL cells for different time intervals. MATERIALS AND METHODS: PDL cultures were subjected to Cutodiol® , immature, half mature, and mature coconut water, and different concentrations of propolis in Dulbecco's Modified Eagles Medium (DMEM). Culture plates with the tested media were incubated for 1, 2, 6, 24, 48, 72, and 168 hour. PDL fibroblast cell viability was assessed by MTT assay. RESULTS: CW showed significantly higher viability of cells than other groups at 6 hour with half mature CW being superior. Propolis at 6.25 mg/mL in DMEM resulted in 138% viable PDL and it was able to preserve PDL cells for up to 168 hour. CONCLUSIONS: Half mature and mature CW are superior storage media if replantation of avulsed teeth is within 6 hour. Propolis in DMEM could be a potential storage media for prolonged storage intervals up to 48 hour.

Cystic echinococcosis in Jordan: A review of causative species, previous studies, serological and radiological diagnosis.

Cystic echinococcosis (CE)/hydatidosis is a zoonotic disease which occur in human and herbivore animals as a result of infection with the larval stage of the taeniid cestode Echinococcus granulosus sensu lato (s. l.). In human, CE is a serious public health concern in many parts of the world including Jordan. The present review will cover CE causative agent: E. granulosus species/genotypes; life cycle of E. granulosus parasite, all published previous studies on CE in Jordan (humans, intermediate hosts, definitive host) as well as its diagnostic methods in human.

Colloidal stability of gold nanorod solution upon exposure to excised human skin: Effect of surface chemistry and protein adsorption.

In this study, we evaluated the colloidal stability of gold nanorods (with positive, negative and neutral surface charge) in solution upon contact with excised human skin. UV-vis absorption, plasmon peak broadening index (PPBI%) and transmission electron microscope analysis were used to follow nanoparticles aggregation in solution. Our results show that positively charged gold nanorods aggregate extensively upon exposure to excised human skin compared to negatively and neutrally charged gold nanorods. Skin-induced aggregation of cationic gold nanorods was linked to the adsorption of proteins released from the dermis layer to the surface of gold nanorods. Protein adsorption significantly screen nanorod's effective surface charge and induce their aggregation. Moreover, we demonstrate that the presence of polyethylene glycol polymer on the surface of cationic gold nanorods minimize this aggregation significantly by providing steric repulsion (non-electrostatic stabilization mechanism). This work highlights the importance of evaluating the colloidal stability of nanoparticles in solution upon contact with skin, which is a "usually overlooked" parameter when studying the nanoparticle-skin interaction.

Blocking of Histamine Release and IgE Binding to FcepsilonRI on Human Basophils by Antibodies Produced in Camels

PURPOSE: The production of camel heavy-chain antihuman IgE (huIgE) that has the potential to block IgE-FcepsilonRI interaction and histamine release by basophils. METHODS: Camels were immunized with a synthetic loop peptide (SLP) designed in a multiple antigen peptide system (MAPS) forming SLP-MAPS immunogen. Camel polyclonal antibodies (PCAs) were produced, purified, characterized using Protein A & G, ELISA, and SDS-PAGE, and tested for their potency to block passive sensitization and histamine release of human basophils using flow cytometry (FCM) and ELISA, respectively. RESULTS: FCM data indicated that camel conventional (IgG1) and heavy chain antibodies (HCAbs; IgG2, and IgG3) had blocking activities of 43.9%, 72%, and 96.6%, respectively. Moreover, both IgG2 and IgG3 achieved remarkable inhibition rates of 93.98% and 97.05% in histamine release, respectively, whereas the IgG1inhibiting activity was 60.05%. CONCLUSIONS: Camel PCAs produced against SLP-MAPS were capable of blocking the IgE-receptor interaction and the release of histamine by basophils with superiority to HCAbs. These findings may pave the way toward the possible use of camel anti-huIgE HCAbs as blocking antibodies in the treatment of IgE-mediated allergy and asthma.

A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins.

Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg Ig G ml⁻¹ resin and a dissociation constant (K(d)) of 4.78 × 10⁻6 M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.

Production of monoclonal antibodies against the 8 kDa subunit of Echinococcus granulosus Antigen B (EgAgB8/2) using DNA immunization.

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.

The induction of T helper type 1 response by cytokine gene transfection protects mice against secondary hydatidosis.

Infection of BALB/c mice with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis (SH) and the associated immune response in immunization and infection trials. This study aimed at testing the efficacy of the cytokine gene expression approach to modulate the immune response and the magnitude of cyst development in mice with secondary hydatidosis. At the time of cyst development (28 days post infection), mice were injected intramuscularly with an expression vector containing murine promoter and carrying the open reading frames of IFN-gamma, IL-12 (Th1 cytokines), or IL-4 (Th2 cytokine). Assessment of cyst load at 22 weeks of infection showed a significant reduction in cyst load in mice injected with IFN-gamma and IL-12 genes at 60% and 47%, respectively. In contrast, the IL-4-gene-injected mice displayed six times higher cyst load in comparison to control-infected mice (injected with empty plasmids). Parasite-specific IgG2a peaked in IL-12-gene-injected mice at week 7 of infection (3 weeks after gene transfection), whereas in IFN-gamma-gene-injected mice IgG2a started to elevate after week 9 and continued to increase steadily until the termination of the experiment (22 weeks post infection). In contrast, in IL-4-gene-transfected mice, the IgG1 elevation started after week 9 and continued steadily thereafter. In conclusion, a significant high protection rate against secondary hydatidosis in BALB/c mice was accompanied with the induction of Th1 response. Moreover, in vivo IL-12 gene expression induced earlier IgG2a in comparison to IFN-gamma.

Humoral and cytokine response during protection of mice against secondary hydatidosis caused by Echinococcus granulosus.

Infection of BALB/c mouse with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis and the associated immune response in immunization and infection trials. The aims of this study were to induce a protective immunity against secondary hydatidosis using conventional vaccination approaches and to analyse the immune responses that accompany this protection. Mice immunized with antigen B (AgB), a component of crude sheep hydatid fluid (CSHF), showed a significant level of protection as indicated by a 98.3% reduction in cyst load. This reduction in cyst development was accompanied by a high concentration of interferon gamma secreted by antigen-stimulated spleen cells, as compared with those secreted by cells of mice immunized with CSHF or protoscoleces homogenate (PSH) antigens. In contrast, interleukin-4 was significantly higher in the supernatants of cells stimulated with CSHF or PSH compared with AgB (191.5, 195.7 and 127.5 pg, respectively). Kinetic analysis of immunoglobulin subclasses showed persistently high levels of IgG1 and IgG2a subclasses in immunized infected animals until 6 months of infection, whereas IgG3 showed a significant decline after 1 month of infection. In infected non-immunized control mice, all IgG subclasses showed a gradual increase after the first month of infection until the experiment termination (8 months after infection).

Canine echinococcosis in northern Jordan: increased prevalence and dominance of sheep/dog strain.

A total of 112 stray and semi-stray dogs (Canis familiaris) from four different geographical areas in northern and middle Jordan were necropsied to evaluate the prevalence and intensity of intestinal helminthiasis. Of these, 33 dogs (29.5%) were infected with Echinococcus granulosus and 61 (54.5%) with other Taenia species. Other cestodes found included Dipylidium caninum in 36 dogs (32.1%), Diplopylidium in 6 dogs (5.4%), Mesocestoides sp. in 3 dogs (2.7%) and Joyuexiella in 1 dog (0.9%). Toxocara nematodes were found in 10 dogs (9.2%) and only 1 dog was positive for acanthocephalans. Among the dogs infected with E. granulosus, 8 dogs (24.2%) had a worm load higher than 1,000 worms. The ratio of infected male to female dogs was 1.9:1.0. Strain analysis of E. granulosus using random primers revealed the dominance of the G1 strain (sheep/dog strain) in the region. Only one dog harbored another E. granulosus strain, which resembled the G4 strain pattern.

Retrospective surgical incidence and case distribution of cystic echinococcosis in Jordan between 1994 and 2000.

A retrospective follow-up study on the surgical incidence of cystic echinococcosis (CE) was carried out in major governmental, military and private hospitals throughout Jordan between 1994 and 2000. A total of 472 cases were recorded over the 7-year period and an overall mean annual surgical incidence (MASI) of 2.3 per 100,000 inhabitants was estimated. The highest number of surgical cases was recorded in hospitals of the middle region of the country. The highest MASI (3.6 per 100,000) was found in the southern region while the lowest (1.4 per 100,000) was in the northern region of the country. Taking into consideration the population size and the origin of surgically confirmed cases of each region, a relative surgical index (RSI) was calculated at which the highest (RSI=3.4) was among cases originated from the southern region and the lowest (RSI=0.7) was among those originated from the middle region of Jordan. The northern region and desert areas (badia) showed comparable RSI at 1.0 and 1.1, respectively. Males younger than 15 years of age showed significantly higher surgical incidence than females of comparable age at a ratio of 1.6:1, whereas the number of female cases of different age groups over 15 years of age was consistently higher than that of males at a ratio of 1.25-2.5:1.0. The liver was the primary site of cyst development in 69.4% of the cases and the lung involvement accounted for 13.3% of the cases. Diagnosis of CE in Jordan relies mostly on imaging methods with serological techniques being rarely used for diagnosis. The frequency of CE recurrence was 27.5% of the cases, which may be attributed to the low use of chemotherapeutic antihelminthics among surgically treated cases.