A new approach to detection of incomplete antibodies using hydrogel chromatography medium.

In assays for incomplete antibody detection, several washing steps are required to remove unbound globulins which may cause false negatives. Here, we present an improved approach employing hydrogel chromatography medium (HCM) in the detection of incomplete antibodies. After a rapid single-step centrifugation, incomplete antibodies, attached to red blood cells (RBCs), were separated from the reaction mixture using HCM and sedimentation. This method obviates the need for multiple centrifugation steps found in conventional Tube-Coombs tests. The HCM-Coombs tests may have a wide range of applications for incomplete antibody detection.

The US2 protein is involved in the penetration and cell-to-cell spreading of DEV in vitro.

Previous studies have shown that the unique short 2 (US2) protein contributes to the infection of several herpesviruses. However, little is known about whether the US2 protein of duck enteritis virus (DEV) could also contribute to the DEV infection in susceptible cells. This study is aimed at investigating the role of US2 protein in the DEV infection in vitro. The US2 gene was amplified from DEV and cloned into pET32a (+). The expression of recombinant US2 protein was induced in Escherichia coli. After purification, the recombinant US2 protein was immunized into rabbits to generate anti-US2 sera. The immunoreactivity of anti-US2 sera was determined by Western blot and immunofluorescent assays. The US2 protein was detected in the cytoplasm and plasma membrane of DEV-infected duck embryo fibroblast (DEF) cells by immunofluorescent assay using the anti-US2 serum. When DEV infected DEF cells in vitro, pre-treatment with anti-US2 serum did not affect the attachment of virus, but significantly reduced the penetration and cell-to-cell spreading of DEV in DEF cells. Our data suggest that the US2 protein is involved in the penetration and cell-to-cell spreading during the DEV infection of susceptible cells in vitro.

Prevalence of GP. Mur variant phenotype among Malaysian blood donors.

BACKGROUND AND OBJECTIVE: A number of glycophorin variant phenotypes or hybrid glycophorin variants of the MNS blood group system bear multiple immunogenic antigens such as Mia, Mur, and MUT. In the East and Southeast Asian populations, glycoprotein (GP.) Mur is the most common glycophorin variant phenotype expressing those three immunogens. The aim of this study was to detect MNS system glycophorin variant phenotypes (GP. Mur, GP. Hop, GP. Bun, GP. HF, and GP. Hut) among Malaysian blood donors. MATERIALS AND METHODS: In this cross-sectional study, 144 blood donors were selected under stratified random sampling. The deoxyribonucleic acid was extracted from whole blood samples, followed by a polymerase chain reaction assay. Sanger sequencing was used to identify the specific MNS variants and then validated by a serological crossmatch with known anti-Mur and anti-MUT. RESULTS: GP. Mur was identified among Malaysian blood donors with a prevalence of 6.94%, and no other variants of the MNS system were found. CONCLUSION: The present study substantiates that GP. Mur is the main variant of the MNS system glycophorin (B-A-B) hybrid in Malaysian blood donors. GP. Mur-negative red blood cells must therefore be considered in the current transfusion policy in order to prevent alloimmunization and immune-mediated transfusion reactions, particularly in transfusion-dependent patients.

Chicken Egg Yolk Antibodies (IgYs) block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2.

The SARS-CoV-2 virus is still spreading worldwide, and there is an urgent need to effectively prevent and control this pandemic. This study evaluated the potential efficacy of Egg Yolk Antibodies (IgY) as a neutralizing agent against the SARS-CoV-2. We investigated the neutralizing effect of anti-spike-S1 IgYs on the SARS-CoV-2 pseudovirus, as well as its inhibitory effect on the binding of the coronavirus spike protein mutants to human ACE2. Our results show that the anti-Spike-S1 IgYs showed significant neutralizing potency against SARS-CoV-2 pseudovirus, various spike protein mutants, and even SARS-CoV in vitro. It might be a feasible tool for the prevention and control of ongoing COVID-19.

Application of lyophilised human platelets for antibody detection in solid phase red cell adherence assay.

Antibodies against human platelets cause a variety of thrombocytopenic disorders, which lead to potentially fatal haemorrhage. Therefore, their prompt detection is mandatory for successful patient treatment. Solid phase red cell adherence (SPRCA) assay allows for platelet antibody detection widely. However, preparation of fresh platelets with HLA-I and human platelet antigens (HPA)1-5,15 genotyped as target cells is inconvenient and fresh platelets have a short shelf life. In this study, the lyophilised human platelets for antibody detection in SPRCA were prepared. Firstly, platelets were resuspended in lyophilisation buffer and freeze-dried. Then the characteristics of lyophilised platelet were analysed. Rehydrated platelets were recovered with a mean rate of 80.91% ± 2.87%, and still retained spherical morphology. Indirect flow cytometry showed that glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, CD109, and HLA class I were present on the surface of the lyophilised platelets at a comparable level to that of fresh platelets. The consistent results obtained with WHO reference reagents containing anti-HPA-1a, anti-HPA-3a, and anti-HPA-5b, as well as clinical samples from the same donors containing anti-HLA antibodies when reacting with lyophilised versus fresh platelets confirmed good antigenicity preservation of platelets after freeze-drying. Further investigation showed that the lyophilised platelets could be stored at 2-8 °C for up to 14 months and the reconstituted suspension was stable for 48 h. Therefore, lyophilised platelets can be a convenient alternative to fresh platelets to use for anti-platelet antibody detection in SPRCA tests.

Molecular cloning and characterization of CD3ε in Chinese domestic goose (Anser cygnoides).

CD3 is one of the most important cell surface markers of T lymphocytes which play an important role in signal transmission of antigen recognition. In this study, goose CD3ε gene was cloned by touchdown PCR with the template of goose thymus cDNA. The complete open reading frame of goose CD3ε encoded 178 amino acid residues with a 21 signal peptide. Sequence alignments showed that goose CD3ε had an amino acid sequence similarity to duck (80.3%) and chicken (66.3%). The extracellular domain of goose CD3ε was efficiently expressed as fusion protein in Escherichia coli, purified by a Ni-NTA agarose column, and the purified recombinant protein was used to produce anti-GoCD3εex polyclonal antibodies. The characteristics of PAb were identified by Western blot, cellular ELISA, IFA, FCM, and LSCM analysis. These results may be useful for a better understanding of goose CD3ε and have a foundation for the study of T cell mediated immune mechanism in waterfowl.

An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.

Real-time polymerase chain reaction (RT-PCR) enables effective and sensitive screening for infectious risk in the field of blood safety. However, when using RT-PCR to detect bacterial contamination, several intractable points must be considered, one of which is the lack of appropriate quality control. In this study, we developed a simplified RT-PCR assay in which the same primer set and two distinct probes were used to detect both, an internal reference control and the target in a reaction. The copy number of the internal reference control represents the positive detection limit of the assay; therefore, when the threshold-cycle value of the target is less than or equal to that of the internal reference control, the result obtained for the target can be considered to be a true positive. When human gDNA was spiked with Escherichia coli gDNA and the detection limit for the internal reference control was set to five copies, the measured detection limit for E. coli gDNA was two copies. The internal reference control duplex RT-PCR assay showed high efficiency (0.91-1.02), high linearity (R2 > 0.99), and good reproducibility in intra- and inter-assay comparisons. Lastly, when human platelet-rich plasma samples were spiked with E. coli or other bacterial species, all species were detected efficiently, and the results of a two-sample pooled t test showed that the limit of detection for E. coli was 1 cfu/mL. Here, we present a synthetic internal reference control molecule and a new statistical method for improving the reliability of RT-PCR assays when screening for bacterial contamination in blood products.

Cloning and characterization of goose interleukin-17A cDNA.

Interleukin-17 (IL-17 or IL-17A) is a proinflammatory cytokine produced by activated T cells. IL-17A plays important roles in inflammation and host defense. In this study, the cDNA of the goose IL-17A (GoIL-17A) gene was cloned from thymocytes. Recombinant GoIL-17A (rGoIL-17A) was expressed using a baculovirus expression system and then biologically characterized. The complete open reading frame (ORF) of GoIL-17A contains 510 base pairs that encode 169 amino acid residues, including a 29-amino acid signal peptide and a single potential N-linked glycosylation site. This protein has a molecular weight of 18.9kDa. The amino acid sequence showed 95.9%, 84.6%, 45.0% and 38.4% similarity with the corresponding duck, chicken, rat, and human IL-17A sequences, respectively. The six conserved cysteine residues were also observed in GoIL-17A. A recombinant, mature form of GoIL-17A was produced and its biological activities in goose embryonic fibroblasts were investigated. RT-PCR analysis revealed a marked up-regulation of IL-6 and IL-8 mRNA expression in goose embryonic fibroblasts treated with 1-50 µg of rGoIL-17A for 12h. The GoIL-17A gene sequence and the biologically active recombinant protein may be useful for understanding the role of IL-17A in immune regulation.

Recombinant duck enteritis virus expressing the HA gene from goose H5 subtype avian influenza virus.

The duck enteritis virus (DEV) may be a promising candidate viral vector for an aquatic poultry vaccination that can protect against multiple pathogens because it has a very large genome and a narrow host range. Recently, we described two DEV recombinants that contained deletions of the viral US2 or gIgE genes. The hemagglutinin (HA) gene of an H5N1-type highly pathogenic avian influenza virus (HPAIV) of goose origin was inserted into the deletion sites to construct two rDEVs expressing the AIV HA antigen. The resulting rDEV-ΔgIgE-HA or rDEV-ΔUS2-HA recombinant DEV viruses were used to infect duck embryo fibroblasts. Reverse transcription PCR, immunofluorescence and western blot analysis results indicated that rDEV-ΔgIgE-HA and rDEV-ΔUS2-HA were successfully expressed in duck embryo fibroblasts (DEFs). To investigate whether the HA gene could be stably maintained in the recombinant viruses, the viruses were passaged in DEFs 18 times. The HA gene in both recombinants could be detected by PCR amplification. The immunized four-week-old ducks induced specific antibodies against DEV and AIV HA and were protected against challenge infections with DEV AV1221 viruses.