Antibody-labelled gold nanoparticles synthesized by laser ablation to detect SARS-CoV-2 antigen spike.

Background and purpose: Rapid detection test via lateral flow immunoassay (LFIA) is employed as an alternate method to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Gold nanoparticles (AuNPs), a vital component of LFIA, can be synthesized by laser ablation technique. This intense laser radiation may result in monodisperse gold nanoclusters, which are impurity-free and demonstrate innovative biocompatible surface chemistry. In this current research, laser-ablated AuNPs are produced and coupled with an anti-spike SARS-CoV-2 monoclonal antibody (mAb) generated in our prior study. Experimental approach: The AuNPs from 30,000 shots of laser ablation exhibited a robust red color with a maximum absorbance peak at 520 nm. The performance of AuNPs-mAb conjugates as a signal reporter was then evaluated in half-stick LFIA. Key results: The size distribution of AuNPs shows a relatively monodisperse and unimodal distribution with average particle diameters of 44.77 nm and a surface potential of -38.5 mV. The purified anti-spike mAb SARS-CoV-2 yielded two protein bands, representing the mAb heavy chain at 55 kDa and its light chain at 25 kDa. The immobilization of anti-spike mAb onto the surface of AuNPs revealed that 25 g/ml of mAb at phosphate buffer pH 9 was required to stabilize the AuNPs. The functional test of this conjugate was performed using dipstick LFIA, and the result shows that the AuNPs-mAb conjugates could successfully detect commercial spike antigen of SARS-CoV-2 at 10 ng level. Conclusion: In this study, laser-ablated AuNPs were functionalized with anti-spike mAb SARS-CoV-2 and successfully used as a signal reporter in half-stick LFIA for detecting antigen spike SARS-CoV-2.

Point-of-care dengue detection: polydopamine-modified electrode for rapid NS1 protein testing for clinical samples.

Early diagnosis of dengue infection by detecting the dengue virus non-structural protein 1 (DENV-NS1) is important to the patients to initiate speedy treatment. Enzyme-linked immunosorbent assay (ELISA)-based NS1 detection and RT-PCR are time-consuming and too complex to be employed in remote areas of dengue-endemic countries. Meanwhile, those of NS1 rapid test by lateral flow assay suffer from low detection limit. Electrochemical-based biosensors using screen-printed gold electrodes (SPGEs) have become a reliable detection method to convey both ELISA's high sensitivity and rapid test portability. In this research, we developed an electrochemical biosensor for DENV-NS1 detection by employing polydopamine (PDA)-modified SPGE. The electrodeposition of PDA on the surface of SPGE serves as a bioconjugation avenue for anti-NS1 antibody through a simple and low-cost immobilization procedure. The biosensor performance was evaluated to detect DENV-NS1 protein in PBS and human serum through a differential pulse voltammetric (DPV) technique. The developed sensing platform displayed a low limit of detection (LOD) of 1.63 pg mL-1 and a wide linear range of 10 pg mL-1 to 1 ng mL-1 (R2 ∼ 0.969). The sensing platform also detected DEV-NS1 from four different serotypes in the clinical samples collected from dengue patients in India and Indonesia, with acceptable sensitivity, specificity, and accuracy values of 90.00%, 80.95%, and 87.65%, respectively. This result showcased the facile and versatile method of PDA coating onto the surface of screen-printed gold electrodes for a miniaturized point-of-care (PoC) detection device.

Epizootiology, Clinical Signs, and Phylogenetic Analysis of Fowl Adenovirus in Chicken Farms in Indonesia from 2018 to 2019.

Fowl adenovirus (FAdV) infection is an emerging problem in the world poultry industry, especially in broilers, as the causal agent of inclusion body hepatitis or hepatitis-hydropericardium syndrome. From December 2017 to January 2019, we recorded 116 cases of suspected hepatitis-hydropericardium syndrome in chicken farms throughout Indonesia. Necropsy was done on each farm site with three to five freshly dead birds per farm. Tissue samples were collected in virus transport medium and frozen at -20 C. The virus was cultivated in 9-day-old fertilized specific-pathogenic-free chicken eggs. FAdV was detected using polymerase chain reaction with a published primer set. The polymerase chain reaction products were sequenced and subjected to a BLAST search. The phylogeny was inferred using the neighbor-joining method and tested using the bootstrap test. FadV-D and -E are present in Indonesia and confirmed in 40 of 116 suspected cases. The affected chicken ages were 27.27 ± 8.94 days. Most affected farms were raising broiler chickens. The only typical clinical sign was unusual daily mortality of >1%, while the three most frequent pathologic lesions were swelling and hemorrhage of kidney and liver, as well as hydropericardium. To reduce economic loss, a vaccine should be developed immediately.

Epizootiología, signos clínicos y análisis filogenético del adenovirus de pollos en granjas avícolas en Indonesia entre los años 2018 a 2019. La infección por adenovirus de aves (FAdV) es un problema emergente en la industria avícola mundial, especialmente en pollos de engorde, como agente causal de la hepatitis por cuerpos de inclusión y del síndrome de hepatitis-hidropericardio. Desde diciembre del año 2017 hasta enero de 2019, se registraron 116 casos sospechosos de síndrome de hepatitis-hidropericardio en granjas avícolas en toda Indonesia. Se realizaron necropsias en los sitios de las granjas con tres a cinco aves recién muertas por granja. Se recogieron muestras de tejido en medio de transporte viral y se congelaron a -20 C. El virus se cultivó en huevos embrionados de aves libres de patógenos específicos de 9 días de edad. Se detectaron adenovirus del pollo usando una reacción en cadena de la polimerasa con un conjunto de iniciadores previamente publicados. Los productos de reacción en cadena de la polimerasa se secuenciaron y se sometieron a una búsqueda mediante la herramienta básica de búsqueda de alineación local (BLAST). La filogenia se infirió usando el método Neighbor-Joining y se evaluó mediante la prueba bootstrap. Se determinó la presencia de adenovirus del pollo D y E en Indonesia y se confirmó su presencia en 40 de 116 casos sospechosos. Las edades de los pollos afectados fueron de 27.27 ± 8.94 días. Las granjas más afectadas fueron de pollos de engorde. El único signo clínico típico fue una mortalidad diaria inusual mayor al 1%, mientras que las tres lesiones patológicas más frecuentes fueron inflamación y hemorragia de riñón e hígado, así como hidropericardio. Para reducir la pérdida económica, se debe desarrollar una vacuna de inmediato.