Search

Immune function shows intraspecific variation, with diet being a crucial factor that could explain such variation. Using the isotopic signature δ15N and C:N ratio, we evaluated the relationship between immune state (hemolysis and hemagglutination score) and natural variation in resource use in individuals of the species Diuca diuca. We found that some components of the immune state, specifically the hemagglutination score, correlated positively and significantly with the incorporation of animal material (high levels of δ15N), while the hemolysis score correlated positively with the C:N ratio. In this sense, we conclude that individuals of Diuca Finches that fed with a variety of prey exhibit a better immune state. [ABSTRACT FROM AUTHOR]

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays. [ABSTRACT FROM AUTHOR]

Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples. [ABSTRACT FROM AUTHOR]

Brucellosis is a zoonotic disease that causes enormous losses in livestock production worldwide and has a significant public health impact. None of the brucellosis-free countries is currently able to guarantee their ability to prevent the introduction of the pathogen due to the increase in tourism and the expansion of migration. The timely identification of infected animals is an effective means of preventing brucellosis and minimizing the epidemiological risk. The tube agglutination test, Rose Bengal plate test, complement fixation test, and enzyme-linked immunosorbent assay, which are routinely used to identify seropositive productive animals, have limitations and results that do not always correlate. The indirect hemagglutination assay (IHA) stands out among non-traditional methods because it is affordable, has a simple protocol, and is more reliable than classical serological tests, especially in cases of questionable and/or false-negative results. The diagnostic value of the IHA has long been studied by laboratories in several countries, but mostly by post-soviet research teams; therefore, the results continue to be published in Russian-language journals, ensuring that the local scientific community can access the results. In addition, the efficacy of this test for the diagnosis of brucellosis and other infectious diseases has not yet been reviewed. The purpose of this review was to summarize the results of studies on the development and use of IHA for the diagnosis of brucellosis and to determine the prospects for further improvement. [ABSTRACT FROM AUTHOR]

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target a, 2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures. IMPORTANCE The H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for humantype receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is a common resistant bacterium, whose resistance has expanded to commonly used antibiotics. It is crucial to create novel treatments to tackle bacterial resistance. Trans-resveratrol and curcumin are naturally occurring phenolic compounds, whose effects on MRSA virulence are the subject of this investigation. Sub-MICs of trans-resveratrol and curcumin were tested on the virulence factors of 50 MRSA clinical isolates (CIs), including biofilm, hemolysin, hemagglutination, protease, and lecithinase. The distribution of the virulence factors of the CIs was as follows: hemolysin: 98%, hemagglutination: 70%, protease: 62%, biofilm: 56%, and lecithinase: 52%. The sub-MIC that could reduce the effect of the tested virulence factors by 50% or more (IC50) was observed in the strains that showed susceptibility to the individual administration of trans-resveratrol at 50 µg/mL and curcumin at 20 µg/mL. Hemagglutination and hemolysin activity were inhibited by at least 50% in the majority of CIs (57–94%). Meanwhile, the IC50 for protease and biofilm was observed in 6.5–17.8% of the CIs. A few of the CIs were susceptible to lecithinase inhibition, but all showed a full inhibition. This research supports the possibility of the use of these compounds to reduce the bacterial virulence that can reduce antibiotic utilization, and eventually, they can become a potential alternative treatment in combating bacterial resistance. [ABSTRACT FROM AUTHOR]

The present study describes the case of a 52-year-old male patient who presented with subacute onset dysarthria and oral-facial-lingual dyskinesia, with normal blood glucose and acanthocyte levels, and no history of drug use. The patient tested negative for autoimmune encephalitis-related antibodies and paraneoplastic-related antibodies. The level of cerebrospinal fluid (CSF) protein was slightly elevated, and the Treponema pallidum hemagglutination assay and rapid plasma reagin test were positive in both serum and CSF samples. After 1 month of treatment with doxycycline, the patient's oral-facial-lingual dyskinesia was significantly improved, suggesting the diagnosis of neurosyphilis. [ABSTRACT FROM AUTHOR]

Abstract—As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus. [ABSTRACT FROM AUTHOR]

Background: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. Results: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111–124 and 154–162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111–124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154–162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111–124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. Conclusions: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111–124 and 154–162 could inhibit the adhesion of E.coli to the chicken trachea. [ABSTRACT FROM AUTHOR]

Haemorrhagic septicaemia (HS) is a highly contagious and fatal disease of cattle and buffaloes and causes major economic losses to farmers. Though indirect hemagglutination (IHA) test has been used to detect a specific antibody against P. multocida, it has low specificity for sero-diagnosis of HS. Therefore, development of a rapid, highly sensitive and specific serological test is a prerequisite for detection of antibodies against HS. In this context, we explored an in-house ELISA method using recombinant antigens for detection of antibodies against P. multocida in bovines. nanH gene from P. multocida B:2 strain P52 was cloned and the recombinant mature protein with a C- and N-terminal truncation was produced as a fusion protein (~63 kDa) in Escherichia coli. The immunogenic potential of purified rNanH-Tr was assessed by the Western blot method using specific anti-rNanH-Tr antibody responses in sera collected from immunized rabbits. An indirect-ELISA based on rNanH-Tr was developed and optimized. Furthermore, the rNanH-Tr ELISA was applied to screen bovine serum samples (n=250). The receiver operating characteristic curve analysis for the detection of anti P. multocida specific antibodies indicated a diagnostic sensitivity of 86.2 (CI 73.26-96.80%) and specificity of 80.0 (63.06-91.56%). No cross reactivity was noted with antibodies against other bovine diseases (e.g., foot-and-mouth disease and brucellosis). Screening of random bovine serum samples showed a 22% sero-positivity for anti P. multocida specific antibodies. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is an acute, highly contagious, and economically significant avian disease worldwide. Vaccination is the most effective measure for controlling ND. In recent years, vaccines matched with the prevalent strains of genotype VII have been developed and are now commercially available. These vaccines can provide full protection for chickens against clinical disease and mortality after challenges with genotype VII viruses and significantly decrease virus shedding compared to conventional vaccines belonging to genotypes I and II. Vaccinated hens can transfer antibodies to their offspring through the egg yolk. Maternally derived antibodies can provide passive protection against diseases but can also interfere with vaccination efficacy early in life. This study was conducted on chicks hatched from hens vaccinated with a commercial genotype VII NDV-matched vaccine to investigate the correlation between hemagglutination inhibition (HI) antibody levels in chicks and hens and the decaying pattern of maternally derived HI antibodies, and to evaluate the protective efficacy of different levels of maternally derived HI antibodies against challenge with a virulent NDV strain of genotype VII based on survivability and virus shedding. The HI antibody titers in chicks at hatching were about 1.3 log2 lower than those in hens, indicating an antibody transfer rate of approximately 41.52%. The estimated half-life of these antibodies was about 3.2 days. The protective efficacy of maternally derived HI antibodies was positively correlated with the titer. These antibodies could effectively protect chicks against mortality when the titer was 7 log2 or higher, but they were unable to prevent virus shedding or infection even at a high titer of 11 log2. The obtained results will greatly assist producers in determining the immune status of chicks and formulating appropriate vaccination schedules against ND. [ABSTRACT FROM AUTHOR]

Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3–4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo , complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection. [ABSTRACT FROM AUTHOR]

The disease is linked to an underlying BM-based clonal B-cell proliferation.[[3]] Cases associated with overt lymphoma, in particular lymphoplasmacytic lymphoma (LPL), or infections are excluded from the pCAD category and are considered secondary cold agglutinin syndromes. PCAD diagnosis needs appropriate correlation of clinical and laboratory findings with bone marrow histology and molecular data. When clinical and laboratory findings are suggestive of pCAD and BM histology shows a low-grade clonal B-cell infiltrate, the mutational analysis of I MYD88 i status is mandatory to exclude an LPL. [Extracted from the article]

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of β-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225–I412 according to the numbering of amino acid residues in HlyII). A soluble form of HlyIILCTD was obtained using the SlyD chaperone protein. HlyIILCTD was first shown to be capable of agglutinating rabbit erythrocytes. Monoclonal antibodies against HlyIILCTD were obtained by hybridoma technology. We also proposed a mode of rabbit erythrocyte agglutination by HlyIILCTD and selected three anti-HlyIILCTD monoclonal antibodies that inhibited the agglutination. [ABSTRACT FROM AUTHOR]

BACKGROUND: In pretransfusion blood typing, pretreatments such as centrifugation and suspension of red blood cells (RBCs) and mixing them with sufficient amounts of reagents are required, but these steps are time-consuming and costly. OBJECTIVE: Aiming to develop a new blood typing method that requires no dilution and only a small amount of reagent, we attempted to determine blood type using syllectometry, an easy-to-use and rapid optical method for measuring the RBC aggregation that occurs when blood flow is abruptly stopped in a flow channel. METHODS: Samples of whole blood from 20 healthy participants were mixed with antibody reagents for blood typing at mixing ratios of 2.5% to 10% and measured with a syllectometry device. RESULTS: Amplitude (AMP), one of the aggregation parameters, showed significant differences between agglutination and non-agglutination samples at mixing ratios from 2.5% to 10%. Although there were significant individual differences in aggregation parameters, calculation of AMP relative to that of blood before reagent mixing reduced the individual differences and enabled determination of blood type in all participants. CONCLUSIONS: This new method enables blood typing with a small amount of reagent, without the time-consuming and labor-intensive pretreatments such as centrifugation and suspension of RBCs. [ABSTRACT FROM AUTHOR]

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen. [ABSTRACT FROM AUTHOR]

Glycerol is seen in biological systems as an intermediate in lipid metabolism. In recent years, glycerol has been reported to act as a chemical chaperone to correct the conformation of proteins. Here, we investigate the role of glycerol in galectin-7 (Gal-7). The thermal shift and CD assays showed that the thermal stability of Gal-7 increased with glycerol concentration but with little secondary structure changes induced by glycerol. In addition, glycerol can inhibit Gal-7-mediated erythrocyte agglutination. We also solved the crystal structures of human Gal-7 in complex with glycerol in two different conditions. Glycerol binds at the carbohydrate-recognition binding sites of Gal-7, which indicates glycerol as a small ligand for Gal-7. Surprisingly, glycerol can bind a new pocket near the N-terminus of Gal-7, which can greatly reduce the flexibility and improve the stability of this region. Moreover, overexpression of Gal-7 decreased the intracellular triglyceride levels and increased mRNA expression of aquaporin-3 (AQP-3) when HeLa cells were incubated with glycerol. These findings indicate that Gal-7 might regulate glycerol metabolism. Overall, our results on human Gal-7 raise the perspective to systematically explore this so far unrecognized phenomenon for Gal-7 in glycerol metabolism. [ABSTRACT FROM AUTHOR]

We explored the potential for a biphasic pattern in waning of antibody titers after influenza vaccination. We collected blood samples in a randomized controlled trial of influenza vaccination in children and tested them with hemagglutination inhibition assays for influenza A(H3N2) and influenza B/Victoria lineage. Using piecewise log-linear mixed-effects models, we found evidence for a faster initial waning of antibody titers for the first 1-2 years after vaccination and then slower longer-term declines. Children with higher postvaccination titers had faster antibody decay. [ABSTRACT FROM AUTHOR]

Currently, duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is widely spread and becomes endemic in duck populations in Asia, causing significant economic losses in the duck producing industry. To early detection and control of DTMUV, the well‐validated diagnostic tests for efficient detection of DTMUV infection in ducks are needed. In this study, we validated and compared hemagglutination inhibition (HI) and indirect immunofluorescence (IFA) tests for identifying antibodies against DTMUV in duck serum samples. Our results demonstrated that HI and IFA tests can both be used to detect antibodies against DTMUV in duck serum samples with high sensitivity (100%), specificity (>87%) and overall agreement with the gold standard serum neutralization (SN) test (>90%). Additionally, DTMUV‐specific antibody titres determined by HI and IFA tests correlated well with the neutralizing antibody titres obtained by SN test. No cross‐reactivity against common duck viruses and other flaviviruses was observed in both tests. It is interesting to note that HI test had higher diagnostic specificity and exhibited a stronger positive correlation with SN test than IFA test. Evaluating the performance of HI and IFA tests with experimental and field serum samples revealed that both tests showed comparable performance with SN test in terms of antibody kinetic and detection rate. Collectively, these findings support the use of both tests, particularly HI test, as the alternative to SN test for measuring the antibody responses against DTMUV in ducks. These tests could be the suitable choices for DTMUV diagnosis, epidemiological study and vaccine efficacy evaluation. [ABSTRACT FROM AUTHOR]

To improve cross-protection of influenza vaccination, we tested conjugation of conserved M2e epitopes to the surface of inactivated influenza virus (iPR8-M2e*). Treatment of virus with chemical cross-linker led to diminished hemagglutination activity and failure to induce hemagglutination inhibiting antibodies. Conjugated iPR8-M2e* vaccine was less protective against homologous and heterosubtypic viruses, despite the induction of virus-specific binding IgG antibodies. In alternative approaches to enhance cross-protection, we developed a genetically linked chimeric protein (M2e-B stalk) vaccine with M2e of influenza A and hemagglutinin (HA) stalk of influenza B virus. Vaccination of mice with inactivated influenza A virus supplemented with M2e-B stalk effectively induced hemagglutination inhibiting antibodies, humoral and cellular M2e immune responses, and enhanced heterosubtypic protection. This study demonstrates the importance of HA functional integrity in influenza vaccine efficacy and that supplementation of influenza vaccines with M2e-B stalk protein could be a feasible strategy of improving cross-protection against influenza viruses. • Chemical modification of inactivated influenza virus can lead to a loss of hemagglutination activity. • HA functional integrity in influenza vaccines is critical for inducing protective antibodies and homo and hetero protection. • Supplementing M2e immunity to influenza vaccination provides a feasible strategy for enhancing cross protection. [ABSTRACT FROM AUTHOR]

Novel high-performance biosensing devices, based on a microporous cellulose matrix, have been of great interest due to their high sensitivity, low cost, and simple operation. Herein, we report on the design and testing of portable paper-based immunostrips (IMS) for in-field blood typing in emergencies requiring blood transfusion. Cellulose fibrils of a paper membrane were functionalized with antibodies via supramolecular interactions. The formation of hydrogen bonds between IgM pentamer and cellulose fibers was corroborated using quantum mechanical calculations with a model cellulose chain and a representative amino acid sequence. In the proposed immunostrips, paper with a pore size of 3 µm dia. was used to enable functionalization of its channels with antibody molecules while blocking the red blood cells (RBC) from channel entering. Under the optimized test conditions, all blood types of AB0 and Rh system could be determined by naked eye examination, requiring only a small blood sample (3.5 µL). The durability of IgM immunostrips against storing has been tested. A new method of statistical evaluation of digitized blood agglutination images, compatible with a clinical five-level system, has been proposed. Critical parameters of the agglutination process have been established to enable future development of automatic blood typing with machine vision and digital data processing. [ABSTRACT FROM AUTHOR]

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-Di GS , Nb-Z77-Tri GS , Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-Di GS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-Di GS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 μg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection. • A nanobody strain against the AIV H7 subtype was screened using a yeast two-hybrid assay. • The yeast expression conditions for the recombinant nanobody were optimized. • The oligomers showed greater neutralizing activities than did the monomeric nanobody. [ABSTRACT FROM AUTHOR]

Agglutinins/lectins are conventionally defined as proteins/ glycoproteins of non-immune origin with a remarkable ability to specifically and reversibly interact with carbohydrate ligands. Lectins from different sources may essentially exhibit common biological activities. This study was therefore undertaken to survey naturally occurring agglutinins in two species of marine crabs by hemagglutination assay using mammalian erythrocytes. Hemagglutination assay results showed that the hemolymph of the marine crab Grapsus albolineatus showed the highest HA titer with rat erythrocytes. HA titer of the crab Leptodius sanguineus varied from 0 to 32 with all the tested erythrocytes. Among the various tissues of Grapsus albolineatus analyzed for the presence of agglutinins, hemagglutination activity was observed in the hemolymph and hepatopancreas with rat erythrocytes. HA was determined by both male and female crabs of Grapsus albolineatus. HA activity increased with an increase in animal size. Biochemical factors like water, protein and calcium content of the hemolymph did not have any influence on the HA titer. [ABSTRACT FROM AUTHOR]

In Japan, inactivated influenza vaccines are used. We measured titers of antibodies to vaccine strains of three influenza types—influenza A (H1N1), influenza A (H3N2), and influenza B/Victoria—from the 2017/2018 to 2021/2022 seasons, but not for influenza A (H3N2) from the 2018/2019 season, using a single set of serum samples from 34 healthy volunteers, and assessed the consistency in antibody positivity between seasons. The antibody titers in the 2017/2018 season were used as a reference. The influenza A (H1N1) antibody titer in 2019/2020 did not differ significantly from that in the 2017/2018 season, but the titers varied in the two subsequent seasons. The influenza A (H3N2) antibody titers toward the 2019/2020, 2020/2021, and 2021/2022 seasonal viruses differed significantly from that in the 2017/2018 season. The influenza B/Victoria antibody titer toward the 2019/2020 seasonal antigen differed from that in the 2017/2018 season, and the antibody positivity was inconsistent between seasons; however, the antibody titer in the 2020/2021 season did not differ significantly from those in the prior two seasons, and the antibody positivity was consistent between seasons. Antibody titers and their consistency can be used to evaluate cross-immunity of antibodies. [ABSTRACT FROM AUTHOR]

Russell's viper being one of the most venomous snake species in Asia causes significant amount of snake bites and deaths due to neurotoxicity, coagulopathy, haemorrhage, cardiotoxicity, myotoxicity, hypotension, oedema, tissue damage and convulsion. Treatment with monoclonal antibodies, anti-snake venom is the only medically approved treatment available for snake envenomation and has many limitations and side effects such as anaphylactic shock, skin rashes, nausea to vomiting and fever, chills, hypotension, seizures, serum sickness, pruritus, urticaria, arthralgia, lymphadenopathy, encephalopathy etc. Recently, nanoscience and nanotechnology are playing an important role in medical field and has proven to be quite beneficial. In present study, chemically synthesized silver nanoparticles (40±3 nm) are used to inhibit Russell's viper venom activity. The silver nanoparticles and albumin conjugated with curcumin was prepared and characterized using spectroscopic techniques. Venom inhibitory potency of the prepared conjugate was analysed using acidimetry, protease assay and whole blood clotting test. The ultravioletvisible spectra showed enhancement in absorbance of venom with significant hyperchromic shift and thus confirmed modification of venom by silver nanoparticles. This also led to the enhanced interaction with bovine serum albumin-curcumin conjugates. Fluorescence data also showed strong interaction between the conjugates and venom. Interestingly the activity of Russell's viper venom was reduced to 75 %-96 %. The possible mechanism of action of bovine serum albumin-curcumin complex on venom treated with silver nanoparticles is explained at molecular level and biocompatibility of the same was also analysed. Blood agglutination by bovine serum albumin-curcumin complex showed its in vitro biocompatibility. [ABSTRACT FROM AUTHOR]

In the current study, the roots of Quercus agrifolia Nee were subjected to phytochemical analysis to determine the presence of various compound groups. To explore the effect of Quercus agrifolia Nee roots, we evaluated four solvent extracts, namely, crude ethanolic extract (CEE), n-hexane, chloroform (CHCl3), and ethyl acetate (EtOAc), for their antibacterial, antifungal, and hemagglutination activities. Agar well diffusion and agar tube dilution methods were used to determine the antibacterial and antifungal effects, respectively, and hemagglutination activity was measured against human erythrocyte blood groups. The qualitative phytochemical analysis of the solvent extracts of Quercus agrifolia Nee roots showed the presence of different classes of compounds, i.e., flavonoids, coumarins, terpenoids, etc. The CEE and various fractions were tested against Escherichia coli, Listeria monocytogenes, Alcaligenes faecalis, and Pseudomonas aeruginosa, with CEE exhibiting the widest zone of inhibition against A. faecalis (30 mm) and moderate activities against P. aeruginosa (18 mm%), L. monocytogenes (18 mm%), and E. coli (20 mm%). The n-hexane fraction and the remaining fractions had moderate to good activity against all the four bacterial strains. The antifungal ability of plant roots was also tested against four separate fungal strains, i.e., Alternaria solani, Aspergillus niger, Triticum harzianum, and Fusarium oxysporium, and the n-hexane extract showed a higher activity than the CEE. During the investigation by hemagglutination assay, some solvent extracts exhibited a low amount of lectin complex in the selected plant. The current study revealed that the roots of Quercus agrifolia Nee has an ideal medicinal viability and contains a diverse range of phytochemical classes. This research is the first systematic study of Quercus agrifolia Nee roots, demonstrating its versatility in a variety of biological activities. [ABSTRACT FROM AUTHOR]

Proteasomes are major non‐lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p‐proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc‐proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune‐subunits) and intermediate (containing some immune‐subunits) forms of 20S are observed. Moreover, we show that galectin‐3, which displays a highly disordered N‐terminal region, is efficiently cleaved by purified cc‐proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin‐3 is unable to induce red blood cells agglutination when preincubated with cc‐proteasome. These results highlight potential novel physio‐ and pathologic functions for the extracellular proteasome. [ABSTRACT FROM AUTHOR]

This study demonstrated the terminated sialo‐sugar chains (Neu5Acα2,6Gal and Neu5Acα2,3Gal)‐mediated specificity enhancement of influenza virus and chicken red blood cell (RBC) by hemagglutination assay. These glycan chains were immobilized on the gold nanoparticle (GNP) to withhold the higher numbers. With the preliminary optimization, a clear button formation with 0.5% RBC was visualized. On the other hand, intact B/Tokio/53/99 with 750 nM hemagglutinin (HA) displayed a nice hemagglutination. The interference on the specificity of RBC and influenza virus was observed by anti‐influenza aptamer at the concentration 31 nM; however, there is no hemagglutination prevention was noticed in the presence of complementary aptamer sequences. Spiking GNP‐conjugated Neu5Acα2,6Gal or Neu5Acα2,3Gal or a mixture of these two to the reaction promoted the hemagglutination to 63‐folds higher with 12 nM virus, whereas under the same condition the heat‐inactivated viruses were lost the hemagglutination. Neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens at 0.0025 neuraminidase units are able to abolish the hemagglutination. Other enzymes, Glycopeptidase F (Elizabethkingia meningoseptica) and Endoglycosidase H (Streptomyces plicatus) did not show the changes with agglutination. Obviously, sialyl‐Gal‐terminated glycan‐conjugated GNP amendment has enhanced the specificity of erythrocyte–influenza virus and able to be controlled by aptamer or neuraminidases. [ABSTRACT FROM AUTHOR]

Influenza still represents a problematic disease, involving millions of people every year and causing hundreds of thousands of deaths. Only a few drugs are clinically available. The search for an effective weapon is still ongoing. In this scenario, we recently identified new drug-like compounds with antiviral activity toward two A/H1N1 Influenza virus strains, which were demonstrated to interfere with the processes mediated by hemagglutinin (HA). In the present work, the compound's ability to act against the A/H3N2 viral strain has been evaluated in hemagglutination inhibition (HI) assays. Two of the five tested compounds were also active toward the A/H3N2 Influenza virus. To validate the scaffold activity, analogue compounds of two broad-spectrum molecules were selected and purchased for HI testing on both A/H1N1 and A/H3N2 Influenza viruses. Forty-three compounds were tested, and four proved to be active toward all three viral strains. A computational study has been carried out to depict the HA binding process of the most interesting compounds. [ABSTRACT FROM AUTHOR]

Olea ferruginea has been used to treat skin ailments, as well as kidney and ocular problems for a long time. The current study was designed with the aim of investigating and scientifically validating its widespread use. Chloroform, n-hexane, and ethyl acetate were used to assess the antibacterial, hemagglutination, and insecticidal properties of O. ferruginea roots. Escherichia coli, Micrococcus luteus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were among the bacterial strains selected for assessing antibacterial activity. The results showed that ethyl-acetate (EtOAc) extract (56%) and chloroform (CHCl3) extract (56%) showed the widest zone of inhibition against K. pneumonia, while n-hexane extract (13.4%) had the lowest zone of inhibition against P. aeruginosa. All three extracts remained inactive against M. luteus. During hemagglutination activity, the CHCl3 and EtOAc extracts, when used at different concentrations, only agglutinated the AB-ve and O+ve blood groups, respectively, while the n-hexane extract strongly agglutinated the A-ve and B-ve blood groups at different concentrations. The plant extracts were also checked for insecticidal activity against Rhizopertha dominica. The results also revealed the high mortality rate of CHCl3 extract (70%) against R. dominica as compared to other extracts. The aforesaid activities suggest that the roots of O. ferruginea have excellent medicinal viability and contain a wide variety of agglutinins and lectins, as shown in this study. [ABSTRACT FROM AUTHOR]

Rabies vaccine preparations are quantitatively assayed for potency using the in-vivo challenge National Institute of Health (NIH), the main test that consumes a high number of animals, takes a long time, and has wide variability. The Rapid focus fluorescent inhibition (RFFIT) and the passive hemagglutination (PHA) tests, the two serologically based tests, were also used for such purpose. In this study, we aimed to evaluate and correlate the potency of the NIH, RFFIT, and PHA tests according to the World Health Organization (WHO) validity criteria, aiming to validate the use of RFFIT or PHA test as a substitute to the NIH test for determining the potency of commercially available Rabies vaccine preparations. The results showed that, the three tests can be successfully used; however, a higher correlation between RFFIT and NIH than PHA and NIH was recorded (Pearson correlation = 1). The potency of rabies vaccine preparations using NIH, RFFIT, and PHA were 3.73, 3.51, and 4.50, respectively. NIH is the main test for the determination of vaccine potency carried out by conducting 25 experiments and consuming about 5,000 mice compared to 1,200 mice used with RFFIT and 1,000 mice used with PHA test. Taken together, we concluded that (i) in some tested preparations, both RFFIT and PHA tests gave comparable results, and they can be used interchangeably; (ii) RFFIT could successfully replace NIH test, but not PHA; (iii) RFFIT and PHA tests are faster, more accurate, more economic, and more sensitive than NIH; nevertheless, PHA needs further investigations; and (iv) both RFFIT and NIH tests complement and reinforce each other as they provide a comprehensive picture of the product potency. [ABSTRACT FROM AUTHOR]

Background Data from a randomized controlled efficacy trial of an inactivated quadrivalent influenza vaccine in children 6–35 months of age were used to determine whether hemagglutination inhibition (HI) antibody titer against A/H1N1 and A/H3N2 is a statistical correlate of protection (CoP) for the risk of reverse-transcription polymerase chain reaction (RT-PCR)–confirmed influenza associated with the corresponding strain. Methods The Prentice criteria were used to statistically validate strain-specific HI antibody titer as a CoP. The probability of protection was identified using the Dunning model corresponding to a prespecified probability of protection at an individual level. The group-level protective threshold was identified using the Siber approach, leading to unbiased predicted vaccine efficacy (VE). A case-cohort subsample was used for this exploratory analysis. Results Prentice criteria confirmed that HI titer is a statistical CoP for RT-PCR–confirmed influenza. The Dunning model predicted a probability of protection of 49.7% against A/H1N1 influenza and 54.7% against A/H3N2 influenza at an HI antibody titer of 1:40 for the corresponding strain. Higher titers of 1:320 were associated with >80% probability of protection. The Siber method predicted VE of 61.0% at a threshold of 1:80 for A/H1N1 and 46.6% at 1:113 for A/H3N2. Conclusions The study validated HI antibody titer as a statistical CoP, by demonstrating that HI titer is correlated with clinical protection against RT-PCR–confirmed influenza associated with the corresponding influenza strain and is predictive of VE in children 6–35 months of age. Clinical Trials Registration NCT01439360. [ABSTRACT FROM AUTHOR]

Objective. To develop a novel sensitive and accurate assay suitable for high-volume testing of the total complement activity in the serum for clinical laboratories. Methods. The total complement activity (TCA) to be measured was quantified by detecting the number of fragments produced by erythrocyte lysis and the erythrocyte fragmentation index (EFI), indicating TCA. EFI = M × M 2 / M 1 + M 2 , where M is the number of erythrocyte fragments (removed from the background), M 1 is the number of unagglutinated red cells, M 2 is the number of agglutinated red cell groups, and M 2 / M 1 + M 2 is the agglutination coefficient indicating the degree of erythrocyte agglutination. Mild changes in hemolysin and erythrocyte concentrations were made to optimize the testing conditions. The same serum samples were tested for 10 consecutive days to determine the stability of the experimental results. Serum EFI was detected in both nephrotic syndrome patients and healthy subjects. Results. There was a linear relationship between hemolysin and erythrocyte agglutination (r = 0.999 , P < 0.001). A good linear relationship existed between EFI and TCA (r = 0.991 , P < 0.001). The results were not affected by slight fluctuations in the concentrations of hemolysin or erythrocytes. The interbatch CV = 8.6 % of the test results showed good stability. There was a significant difference in the EFI between nephrotic syndrome patients and healthy individuals, P < 0.001 , and EFI was reduced in nephrotic syndrome patients compared to healthy individuals. Conclusion. The flow cytometry-based assay for TCA was sensitive and accurate and had potential value for clinical application. [ABSTRACT FROM AUTHOR]

Diagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as 'NanoSpot.ai', is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens. [ABSTRACT FROM AUTHOR]

Influenza remains one of the most contagious infectious diseases. Approximately, 25 to 50 million people suffer from influenza-like illness in the United States annually, leading to almost 1 million hospitalizations. Globally, the World Health Organization (WHO) estimates 250,000 to 500,000 mortalities associated with secondary respiratory complications due to influenza virus infection every year. Currently, seasonal vaccination represents the best countermeasure to prevent influenza virus spread and transmission in the general population. However, presently licensed influenza vaccines are about 60% effective on average, and their effectiveness varies from season to season and among age groups, as well as between different influenza subtypes within a single season. The hemagglutination inhibition (HAI) assay represents the gold standard method for measuring the functional antibody response elicited following standard-of-care vaccination, along with evaluating the efficacy of under-development influenza vaccines in both animal models and clinical trial settings. However, using the classical HAI approach, it is not possible to dissect the complexities of variable epitope recognition within a polyclonal antibody response. In this paper, we describe a straightforward competitive HAI-based method using a combination of influenza virus and recombinant hemagglutinin (HA) proteins to dissect the HAI functional activity of HA-specific antibody populations in a single assay format. IMPORTANCE The hemagglutination inhibition (HAI) assay is a well-established and reproducible method that quantifies functional antibody activity against influenza viruses and, in particular, the capability of an antibody formulation to inhibit the binding of hemagglutinin (HA) to sialic acid. However, the HAI assay does not provide full insights on the breadth and epitope recognition of the antibody formulation, especially in the context of polyclonal sera, where multiple antibody specificities contribute to the overall observed functional activity. In this report we introduce the use of Y98F point-mutated recombinant HA (HADSA) proteins, which lack sialic acid binding activity, in the context of the HAI assay as a means to absorb out certain HA-directed (i.e., strain-specific or cross-reactive) antibody populations. This modification to the classical HAI assay, referred to as the competitive HAI assay, represents a new tool to dissect the magnitude and breadth of polyclonal antibodies elicited through vaccination or natural infection. [ABSTRACT FROM AUTHOR]

Cross-matching of the giant tortoise of San Cristóbal (Chelonoidis chathamensis) was employed to determine if erythrocyte agglutination occurs in individuals with no transfusion history after exposure to plasma from a conspecific individual. The main islands in the Galápagos archipelago are inhabited by unique species of tortoises. On the largest island, Isabela, there are multiple species isolated by volcanic formations creating unique separate ecosystems. With plans to begin the reintroduction of C. chathamensis into the wild, as well as the recent rediscovery of the Fernandina giant tortoise (Chelonoidis phantasticus), appropriate veterinary care is paramount, and having every medical contingency, especially vehicular/tortoise interaction, under consideration is important. Cross-matching the San Cristóbal giant tortoise was a first step in exploring the compatibility of blood transfusions between conspecifics. Blood was drawn from the jugular vein of 20 juvenile tortoises with the use of a heparinized syringe and each sample was coded. The blood was centrifuged, the erythrocytes washed (five times), and then the erythrocyte pellet and plasma were used for the major cross-match procedure. The erythrocytes represented the "donor" tortoise and the plasma represented the "recipient" tortoise. Each tortoise was cross-matched against itself as an auto control and then 1–5 times against another randomly assigned individual at two incubation temperatures, 23°C (73.4°F) and 34°C (93.2°F). No agglutination was found in any of the trials. As predicted, fibrin and thrombocyte clumps occurred in all trials. Additional research that includes different species of tortoises is the next important step to enhance our knowledge of antigen compatibilities. [ABSTRACT FROM AUTHOR]

Split-virus vaccine serves as a major countermeasure against influenza virus, but its effectiveness and protective action are not complete. We previously demonstrated the effect of Benifuuki, a green tea cultivar in Japan, on enhancing the split-virus vaccine–elicited immune response. However, little is known about the detail mechanisms. Here, we show that EGCG3"Me intake significantly potentiated the vaccine-elicited hemagglutination inhibition titer increase. Flow cytometry analysis revealed the increased Toll-like receptor 5 (TLR5) expression after EGCG3"Me treatment in lamina propria dendritic cells (LPDCs) and macrophages, which play crucial roles in the humoral immune system. TLR5 expression correlated with the level of interleukin-6 (IL-6)/C–C chemokine type receptor 5, which are important mediators of the humoral immunity. Taken together, In vivo and ex vivo studies showed that EGCG3"Me potentiated the split-virus vaccine–elicited immune response accompanied with the upregulation of TLR5 in intestine and splenocyte macrophages. [ABSTRACT FROM AUTHOR]

Purpose: Virus-like particles (VLPs) are being developed as a promising vaccine platform and therapeutic delivery. Various strategies for effectively constructing VLPs have been studied, but relatively few studies have been done on various factors affecting storage. In this study, we investigated the antigenic changes of VLPs in an acidic or basic pH environment using influenza VLPs as an experimental model. Materials and Methods: Influenza VLPs containing hemagglutination and M1 proteins were generated and their antigenicity and protective immunity in vitro and in vivo were evaluated after exposure to acidic (pH 4 and 5) or basic (pH 9 and 10) pH buffers. Results: VLP exposed to basic pH showed similar levels of antigenicity to those stored in neutral pH, while antigenicity of VLP exposed to acidic pH was found to be significantly reduced compared to those expose neutral or basic pH. All groups of mice responded effectively to low concentrations of virus infections; however, VLP vaccine groups exposed to acid pH were found not to induce sufficient protective immune responses when a high concentration of influenza virus infection. Conclusion: In order for VLP to be used as a more powerful vaccine platform, it should be developed in a strategic way to respond well to external changes such as acidic pH conditions. [ABSTRACT FROM AUTHOR]

The hemagglutination inhibition (HI) test has long been used as a standard measure of antibody response for inactivated influenza vaccines. However, the HI test has limitations, such as insensitivity when using some H3N2 virus strains and failure to detect neutralizing antibodies that target regions distant from the receptor binding site. We therefore examined a hemagglutinin pseudovirus neutralization (PVN) test as a possible supplement or alternative to the HI test. We evaluated the association of HI or PVN titres with protection against influenza infection in mice based on morbidity (where the illness was defined as 25% body weight loss). We assessed this relationship using dose–response models incorporating HI or PVN titres as a variable. The morbidity was correlated with the pre‐exposure titres, and such a correlation was well described by a modified dose–response model. The mathematical modelling suggests that PVN titres consistently show a stronger association with in vivo protection as compared to HI titres in mice. Given our findings, the PVN test warrants further investigation as a tool for evaluating antibody responses to influenza vaccines containing hemagglutinin. The resulting models may also be useful for analyzing human clinical data to identify potentially protective antibody titres against influenza illness. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is one of the most important pathogens of zoonotic importance globally, causing various diseases in humans and animals. With the increase of drug resistance and the emergence of multidrug-resistant strains, therapeutic effects of antibiotics against A. hydrophila are limited. Hence, novel agents and strategies are urgently needed to overcome these difficulties. Wavy pili (W-pili), one of the major adhesion molecules, play a crucial role in adhesion and hemagglutination in the pathogenesis of A. hydrophila and regarded as a potential target for therapy. In this study, cinnamaldehyde, a natural compound isolated from cinnamon, could inhibit the production of W-pili and its biological function at the sub-inhibitory concentration (sub-MIC). To determine the active mechanism of cinnamaldehyde, transmission electron microscopy, adhesion inhibition, hemagglutination, electrophoresis, semi-quantitative RT-PCR, biofilm assay, and fish immersion challenge were performed. The results showed that cinnamaldehyde can reduce the production of W-pili and the formation of biofilm without affecting the growth of A. hydrophila. Besides, cinnamaldehyde interfered with the synthesis of W-pili by downregulating the expression of the genes related to pili protein, thereby reducing the adhesion and hemagglutination ability of A. hydrophila and its adhesive ability to the gills and intestines of channel catfish. Therefore, cinnamaldehyde could be employed as a promising candidate for preventing and controlling A. hydrophila infection in aquaculture, especially in antibiotic-resistant strains. [ABSTRACT FROM AUTHOR]

Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, an accidental laboratory-mediated infection is concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:215. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HIFPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6–CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6–CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6–CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies. • Novel, cheap, and fast methods are needed to rapidly detect SARS-CoV-2 antibodies. • Fusion proteins can be designed to cover red blood cells with SARS-CoV-2 antigen. • Fusion proteins trigger red blood cell agglutination with COVID-19 patient serum. • Agglutination takes 1 h, is visible, requires one reagent, and needs no device. • Mammalian fusion proteins function better than bacterial in hemagglutination assays. [ABSTRACT FROM AUTHOR]

The influenza D virus (IDV) was discovered less than ten years ago. Increased interest in this virus is due to its nature (RNA virus with high mutation rate), its worldwide circulation in livestock species, its probable role in bovine respiratory disease and its zoonotic potential. Until currently, the establishment of positivity cut‐off of the hemagglutination inhibition (HI) assay was not formalized in field conditions for the detection of antibodies directed against IDV in cattle (i.e. the proposed reservoir). In this study, the positivity cut‐off of the HI assays was formally established (titre = 10) using a receiver operating characteristic (ROC) curve. This information was used to estimate the sensitivity (68.04 to 73.20%) and the specificity (94.17 to 96.12%) of two different HI assays (HI1 and HI2, with two different IDV antigens) relatively to virus micro‐neutralization test (VNT) as reference test. Based on the above characteristics, the true prevalence of IDV was then estimated in Morocco using a stochastic approach. Irrespective of the HI assays used, the estimation of the true prevalence was statistically equivalent (between 48.44% and 48.73%). In addition, the Spearman rank correlation between HI titres and VNT titres was statistically good (0.76 and 0.81 for HA1 and HA2, respectively). The positive (0.82 and 0.79 for HA1 and HA2, respectively) and the negative (0.86 and 0.85 for HA1 and HA2, respectively) agreement indices between results of HI assays and VNT were good and similar. This study allowed for a formal establishment of a positivity cut‐off in HI assays for the detection of antibodies directed against IDV. This information is of prime importance to estimate the diagnostic sensitivity and specificity of the test relatively to the VNT (i.e. the reference test). Using these characteristics, the true prevalence of IDV should be determined in a country. [ABSTRACT FROM AUTHOR]

Background: Cold agglutinin disease can cause the agglutination of red blood cells and hemolytic anemia due to cold temperature. Herein, we report a case of progressive hemolytic anemia due to cold agglutinin disease during fluid resuscitation and in the absence of exposure to cold.Case Presentation: A 71-year-old Japanese man was admitted to the emergency department with signs of hypotension and disturbed consciousness. He was diagnosed with diabetic ketoacidosis, and treatment with fluid resuscitation and insulin infusion was initiated. Laboratory test results obtained the following day indicated hemolytic anemia. On day 5 after admission, red blood cell agglutination was detected, and the patient was diagnosed with cold agglutinin disease.Conclusions: Cold agglutinin disease should be considered in the differential diagnosis of progressive hemolytic anemia during fluid resuscitation, even if the solution is at room temperature. [ABSTRACT FROM AUTHOR]

Background: A systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-binding antibodies against co-circulating H7 influenza strains. Method: A panel of eight H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000–2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013. Results: VLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain. Conclusion: This study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations. [ABSTRACT FROM AUTHOR]

This study aimed to examine the immunity level against rubella in pregnant women of different birth cohorts. In total, 512 pregnant women who visited a primary clinic between May 2019 and March 2020 were enrolled. Information in terms of the patients' hemagglutination inhibition (HI) titers, birthdate, obstetrical history, and vaccination history were collected. Participants were divided into three generational groups according to the vaccination policy in Japan. Publicly funded vaccination was administered twice as part of a routine program in group A (n = 11), once as part of a routine program and once in a catch-up program in group B (n = 181), and once in group C (n = 320). All groups had some women with negative rubella HI antibody titers (7.6% of all the women, 18.2% of group A, 9.4% of group B, and 6.3% of group C) and those with rubella HI antibody titers of ≤1:16 (45.1% of all women, 90.9% of group A, 56.4% of group B, and 37.2% of group C). Rubella HI antibody titers differed between the groups; group C had higher titers than that in group B. In groups B and C, the proportions of women with rubella HI antibody titers of ≤1:16 were not statistically different between primipara and multipara. Our study showed that an increase in immunity to rubella, a vaccine-preventable disease, is nevertheless required among childbearing women to prevent rubella and congenital rubella syndrome. [ABSTRACT FROM AUTHOR]

Lectins are proteins or glycoproteins that bind specifically and reversibly to the carbohydrate or glycoconjugates. A new lectin is purified from the rhizome of Xanthosoma violaceum Schott. by successive steps of ammonium sulfate fractionation and affinity chromatography with asialofetuin as ligand. The purified lectin was found to be a homotetramer of approximately 49 kDa with a subunit molecular weight of 12 kDa linked by non-covalent bonds. Characterization of the lectin shows that the hemagglutination activity is inhibited by asialofetuin and d -galacturonic acid. Hemagglutination activity is shown only in rabbit RBC but not in the human RBC of all blood groups. It is a metal ion-independent glycoprotein of 1.87% carbohydrate content, stable upto 40 °C and pH from 5.5 to 9. The lectin shows its optimum hemagglutination activity at 0 °C–40 °C and pH 6 to 8.5. From LC-MS/MS analysis it is confirmed that the purified lectin was not purified and characterized earlier. • Purification of a lectin from the rhizome of Xanthosoma violaceum Schott. • Ammonium sulfate fractionation and affinity chromatography techniques were used to purify the lectin. • Native and sub molecular weight were determined using SDS PAGE and gel filtration chromatography • LC MS/MS confirms the purified protein was lectin belonging to mannose binding lectin. • Chemical nature of the lectin were determined. [ABSTRACT FROM AUTHOR]

An efficient one-pot synthesis of a new series of mannosyl triazoles has been achieved through CuAAC reaction where the alkyl chain spacer between the phthalimide moiety and the triazole ring in the aglycone backbone is varied from one methylene to six methylene units. The target compounds were evaluated in terms of their inhibitory potency against FimH using hemagglutination inhibition (HAI) assay. It was found that the length of four methylene units was the optimum for the fitting/binding of the compound to FimH as exemplified by compound 11 (HAI = 1.9 μM), which was approximately 200 times more potent than the reference ligand 1 (HAI = 385 μM). The successful implementation of one-pot protocol with building blocks 1 - 7 and the architecture of ligand 11 will be the subject of our future work for developing more potent FimH inhibitors. A facile single pot strategy was employed to synthesize a group of six mannosyl conjugates through Cu(I)-catalyzed click reaction. These conjugates were screened for their inhibitory potency against FimH. Among them, the conjugate with n = 4 exhibited the highest inhibitory activity with an HAI titer of 1.9 μM. [Display omitted] • One pot synthesis of new six mannosyl triazoles through CuAAC click reaction. • They were screened for their inhibitory potency against FimH. • Biological evaluation revealed the effect of alkyl chain spacer length on binding affinity. • The length of four methylene units was the optimum for the fitting/binding of the compound to FimH. [ABSTRACT FROM AUTHOR]

Turkey red blood cells (tRBCs) are an essential reagent used in the laboratory diagnosis of influenza viruses. Fresh tRBCs when stored at 4 °C have a shelf life of less than a week. Previous studies have shown the utility of glutaraldehyde-fixed tRBCs, with an increased shelf life, for use in hemagglutination (HA) assays. In the present study, we report their functionality after storage for 18 months, at -80 °C. Three influenza A subtypes, namely, H3N2, H1N1 and H5N1, were used in the study. Hemagglutination assay was performed using freshly prepared 0.5 % tRBCs suspension and stored 1 % glutaraldehyde-fixed tRBCs. There was no significant difference in the HA titers obtained using fresh and stored tRBCs. The validation of the HA assay was carried out, to determine the specificity, linearity, precision, accuracy, and robustness of the assay. All of the titers were within the acceptable range, indicating the validity of the HA assay using stored tRBCs. Hemagglutination inhibition assay was also performed to compare the antibody titers obtained using stored and fresh tRBCs. The stored RBCs also gave equivalent antibody titers, as compared to the fresh tRBCs. Thus, the present study demonstrates the utility of glutaraldehyde-fixed tRBCs after one and a half years of storage. • Turkey red blood cells (tRBC) are used for HA and HAI assays of influenza viruses. • Use of Glutaraldehyde-fixed tRBCs in these assays has been demonstrated previously. • We demonstrate their applicability after 18 months' storage at −80 °C. • There was no significant difference in HA and HAI titers using fresh and stored tRBCs. • This is the first report of the use of stored tRBCs after prolonged storage. [ABSTRACT FROM AUTHOR]