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Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests. [Display omitted] [ABSTRACT FROM AUTHOR]

In 2022, the outbreak of the monkeypox virus occurred in many non-endemic countries, and the World Health Organization (WHO) assessed that this outbreak was "atypical". The establishment of a rapid and effective assay that can be used for the early diagnosis of monkeypox virus infection is crucial for outbreak prevention and control. In this study, the monkeypox virus A29 protein and the homologous vaccinia virus A27 protein and cowpox virus 162 protein were expressed in Escherichia coli BL21 for screening. We synthesized the monkeypox virus A2917–49 peptide as the immunogen and obtained 25 monoclonal antibodies (mAbs) against the A29 protein using mouse hybridoma techniques. Then an immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity and lowest limit of detection: 50 pg mL−1 for purified A29 and specificity tests showed that the strips did not cross-react with other orthopox viruses (vaccinia virus or cowpox virus) as well as common respiratory pathogens (SARS-CoV-2, influenza A and influenza B). Therefore, this method can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. [ABSTRACT FROM AUTHOR]

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml–100 ng/ml) were needed to form the antigen–antibody complexes during 20–30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses. [ABSTRACT FROM AUTHOR]

[Display omitted] • A µPAD was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide. • TO evaluate the applicability of the proposed sensor, nasopharyngeal swabs samples in COVID-19 patient samples collected. • The µPES displayed the attributes of low cost , reliability, selectivity, and high sensitivity for low-level detection of the target. In the offered study, the proposed sensor combined the innovative disposable microfluidic paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. An electrochemical microfluidic paper-based analytical device (µPAD) was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide (NiFe LDH). Its electrocatalytic activity asthe platform of proposed the analytical device enhanced the signal output via redox-recycling of the species the ferri/ferrocyanide [Fe(CN) 6 ]3-/4- in solution and is applied to µPAD response signal. Layered NiFe double hydroxide (LDH) nanosheets with a suitable redox potential were prepared by incorporating Fe into the Ni-based LDH. With the help of Fe, the charge-transfer kinetics for the reduction of Ni3+ to Ni2+ was improved and the formation of unwanted Ni components with higher oxidation state was suppressed. The incorporated Fe as the electron transfer mediator enhanced the process of Ni(OH) 2 /NiOOH redox cycle. Under the optimum conditions, the dependency of impedance signal and SARS-CoV-2 concentration showed a linear region from 10 fg/mL-5 ng/mL with a detection limit of 0.23 fg/mL. This Paper-based immunosensor device also showed long stability and good reproducibility, which can be used for the quantitative assay of the SARS-CoV-2. The results of nasal samples analysis revealed the applicability of the resulting biosensor. [ABSTRACT FROM AUTHOR]

In this paper, we developed a simple and disposable electrochemical paper-based immunosensor (e-PI) based on manual screen printing method via patterned sticker label film on paper. The proposed electrochemical sensors provide the opportunity for economical singleuse analysis of biological samples. The e-PI was constructed with hydrophobic glossy paper layers on a wax paper substrate to define three-electrode system. A carbon ink was used for construction of e-PI which composed of the graphite powder and cellulose acetate. The stability of the e-PI was evaluated using electrochemical methods. Carbohydrate antigen 15-3 (CA15-3) antibodies to the target CA15-3 antigen were immobilized on gold-modified graphite electrodes on the e-PI. Electrochemical properties of e-PI were investigated by cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. The fabricated e-PI offers a vast linear range from 0.5 to 200 U mL-1 with a low limit of detection 0.15 U mL-1. Furthermore, the fabricated e-PI has acceptable stability, accuracy and high sensitivity. [ABSTRACT FROM AUTHOR]

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The objective of the present work was to standardize and evaluate indirect plate and dot- enzyme linked immunosorbent assay (ELISA) using purified Paramphistomum epiclitum homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen (PeSAg) in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of the molecular weight ranging from 14 -100 kDa. Two step ethanolic precipitation of supernatant of in-vitro culture of the fluke yielded P. epiclitum excretory-secretory antigen (PeESAg) of molecular weight 28 kDa. The animals (Goats=123; Sheep=91) were broadly kept into post-mortem and faecal examined groups. At many occasion the PeSAg found to cross reacts with other helminths parasites thus minimizing the specificity of the tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The noted prevalence rate after combining the results of post-mortem examination and PeESAg based ELISA (plate and paper/ dot) was 30.08% (37/123) in goats and 28.57% (26/91) in sheep. While using PeESAg, the calculated overall sensitivity% was 92.86 (goats)/ 100 (sheep) in both plate and dot- ELISA, specificity% was 91.58 (goats)/ 91.55 (sheep) in plate ELISA while 88.42 (goats)/ 92.96 (sheep) in dot-ELISA, positive predictive value% was 76.47 (goats)/ 76.92 (sheep) in plate ELISA while 70.27 (goats)/ 80 (sheep) in dot-ELISA and negative predictive value% was 97.75 (goats)/ 100 (sheep) in plate ELISA while 97.67 (goats)/ 100 (sheep) in dot- ELISA, these values were optimum for the field sera sample so the tests and PeESAg can be recommended for the detection P. epiclitum infection in the small ruminants. [ABSTRACT FROM AUTHOR]

A paper-based electrochemical immunosensor was developed for the detection of cancer antigen 125 (CA125) by screen-printing technique. The reduced graphene oxide/thionine/gold nanoparticles (rGO/Thi/AuNPs) nanocomposites were compounded and coated onto the working electrode of immunosensor for CA125 antibody (anti-CA125) immobilization and detection signal amplification. The detection principle was based on the fact that the immunocomplex formed by specify binding of CA125 antibody and antigen could reduce the current response of thionine, which was proportional to the corresponding concentration of CA125 antigen. The immunoassay results showed that the linear range of CA125 was from 0.1 U mL−1 to 200 U mL−1 with the limit of detection (LOD) of 0.01 U mL−1 at signal to noise of 3. Quality control serum samples measured by our proposed immunosensor showed acceptable agreement with traditional ELISA method with the relative error less than 8.05%. The immunosensor exhibited good electrochemical performance with high reproducibility, reliability, stability and accuracy. The proposed immunosensor could be used for the determination of CA125 and had the potential for point-of-care testing (POCT) of other tumor marker. • A novel electrochemical immunosensor based on rGO/Thi/AuNPs nanocomposites was developed for the determination of CA125. • The rGO/Thi/AuNPs nanocomposites were synthesized to improve the sensitivity of immunosensor. • The immunosensor showed acceptable agreement with traditional ELISA method in clinical diagnosis. • It could be used for point-of-care testing of tumor markers and had the potential for disease screening in remote region. [ABSTRACT FROM AUTHOR]

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN) 6 ]3-/4- redox solution. The current change of [Fe(CN) 6 ]3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis. [Display omitted] • The electrochemical paper-based device integrated with a label-free immunoassay was developed for detecting SARS-CoV-2. • A novel plant-based mAb CR3022 offers a remarkable simplicity and cost-effectiveness in terms of Ab production. • CNC containing the abundant COOH functional groups has the potential to be used as the immobilizing material. • The proposed immunosensor was successfully applied to detect SARS-CoV-2 from nasopharyngeal swabs and saliva samples. [ABSTRACT FROM AUTHOR]