A Reliable Indirect ELISA Protocol for Detection of Human Antibodies Directed to SARS-CoV-2 NP Protein.

A few months ago, the availability of a reliable and cost-effective testing capacity for COVID-19 was a concern for many countries. With the emergence and circulation of new SARS-CoV-2 variants, another layer of challenge can be added for COVID-19 testing at both molecular and serological levels. This is particularly important for the available tests principally designed to target the S gene/protein where multiple mutations have been reported. Herein, the SARS-CoV-2 NP recombinant protein was utilized to develop a simple and reliable COVID-19 NP human IgG ELISA. The optimized protocol was validated against a micro-neutralization (MN) assay, in-house S-based ELISA, and commercial chemiluminescence immunoassay (CLIA). The developed assay provides 100% sensitivity, 98.9% specificity, 98.9% agreement, and high overall accuracy with an area under curve equal to 0.9998 ± 0.0002 with a 95% confidence interval of 0.99 to 1.00. The optical density values of positive samples significantly correlated with their corresponding MN titers. The assay specifically detects IgG antibodies to the SARS-CoV-2 NP protein and does not cross-detect IgG to the viral S protein. Moreover, it does not cross-react with antibodies related to other coronaviruses (e.g., the Middle East respiratory syndrome coronavirus or human coronavirus HKU1). The availability of this reliable COVID-19 NP IgG ELISA protocol is highly valuable for its diagnostic and epidemiological applications.

Immunogenicity of Candidate MERS-CoV DNA Vaccines Based on the Spike Protein.

MERS-coronavirus is a novel zoonotic pathogen which spread rapidly to >25 countries since 2012. Its apparent endemicity and the wide spread of its reservoir host (dromedary camels) in the Arabian Peninsula highlight the ongoing public health threat of this virus. Therefore, development of effective prophylactic vaccine needs to be urgently explored given that there are no approved prophylactics or therapeutics for humans or animals to date. Different vaccine candidates have been investigated but serious safety concerns remain over protein or full-length spike (S) protein-based vaccines. Here, we investigated the immunogenicity of naked DNA vaccines expressing different fragments of MERS-CoV S protein in mice. We found that plasmids expressing full-length (pS) or S1-subunit (pS1) could induce significant levels of S1-specific antibodies (Abs) but with distinct IgG isotype patterns. Specifically, pS1 immunization elicited a balanced Th1/Th2 response and generally higher levels of all IgG isotypes compared to pS vaccination. Interestingly, only mice immunized with pS1 demonstrated significant S1-specific cellular immune response. Importantly, both constructs induced cross-neutralizing Abs against multiple strains of human and camel origins. These results indicate that vaccines expressing S1-subunit of the MERS-CoV S protein could represent a potential vaccine candidate without the possible safety concerns associated with full-length protein-based vaccines.

Association of tomato leaf curl Sudan virus with leaf curl disease of tomato in Jeddah, Saudi Arabia.

Tomato is an important vegetable crop and its production is adversely affected by leaf curl disease caused by begomovirus. Leaf curl disease is a serious concern for tomato crops caused by begomovirus in Jeddah, Kingdom of Saudi Arabia. Tomato leaf curl disease has been shown to be mainly caused either by tomato leaf curl Sudan virus or tomato yellow leaf curl virus as well as tomato leaf curl Oman virus. Many tomato plants infected with monopartite begomoviruses were also found to harbor a symptom enhancing betasatellites. Here we report the association of tomato leaf curl Sudan virus causing leaf curl disease of tomato in Jeddah, Kingdom of Saudi Arabia. The complete genome sequence analysis showed highest (99.9 %) identity with tomato leaf curl Sudan virus causing leaf curl disease in Arabian Peninsula. In phylogenetic relationships analysis, the identified virus formed closest cluster with tomato leaf curl Sudan virus. In recombination analysis study, the major parent was identified as tomato leaf curl Sudan virus. Findings of this study strongly supports the associated virus is a variant of tomato leaf curl Sudan virus causing disease in Sudan, Yemen and Arabian Peninsula. The betasatellites sequence analysis showed highest identity (99.8 %) with tomato leaf curl betasatellites-Amaranthus-Jeddah. The phylogenetic analysis result based on betasatellites formed closed cluster with tomato yellow leaf curl Oman betasatellites. The importance of these findings and occurrence of begomovirus in new geographic regions causing leaf curl disease of tomato in Jeddah, Kingdom of Saudi Arabia are discussed.

Evaluation of propolis, honey, and royal jelly in amelioration of peripheral blood leukocytes and lung inflammation in mouse conalbumin-induced asthma model.

Bee products have been used since ancient times to treat many diseases, including respiratory ailments. The present study aimed to examine the modulatory effect of honey, royal jelly, and propolis extract on peripheral blood leukocytes and lung inflammation in a mouse conalbumin-induced asthma model. The mice in group I were not sensitised or treated; they were kept as controls. The mice in group II were sensitised and challenged with conalbumin. Twenty-four hours after the first challenge with antigen, the mice in group III received 0.5 mg/kg of dexamethasone intraperitoneally per day for 18 consecutive days and kept as positive controls. The mice in groups IV, V, and VI received 650, 1000, and 30 mg/kg of honey, royal jelly, and propolis (aqueous and ethanolic extract), respectively, once per day for 18 consecutive days. Blood was collected from all of the mice for white blood cell differentiation, and the lungs were removed for histopathological studies. The groups treated with propolis extract exhibited considerable ameliorative effects against asthma, which might be explained by the flavonoids and phenolics found in propolis, which might have antioxidative effects. Otherwise, the sensitised and honey- or royal jelly-treated groups exhibited an increased incidence of asthma cascade events due to increased inflammatory cells. These results might be due to the immunostimulatory and vasodilatory effects of royal jelly and honey, which are antagonistic to bronchial asthma cases. Histopathological examination revealed that the sensitised treated propolis extract groups had significant decreases in inflammatory scores compared with other treatments and the sensitised untreated group. These results confirmed the previous data of peripheral blood cells.

Serum P53 Abs in HCC patients with viral hepatitis - type C.

BACKGROUND/AIMS: P53 gene mutations have a higher malignant potential and often leads to the production of p53 Abs. This study was conducted to evaluate the clinical implications of p53Abs in HCV-related HCC and its diagnostic capacity as a new biomarker in HCC. METHODOLOGY: 83 patients with HCV-chronic liver disease (25 with LC and 58 with HCC) were enrolled in this study. Ten healthy individuals (HI) served as control group. The studied group was subjected to clinical examination, imaging radiology, laboratory investigation and liver biopsy. Serum p53 Abs was assessed by (ELISA). RESULTS: Serum p53 Abs in HCC (0.5567±0.227) was significantly elevated (p<0.0001) than LC (0.252±0.0099) and HI (0.214±0.068) (p=0.001). Serum P53 Abs was significantly (p=0.01) increased with the progression of child score but there was no significant difference with regard to age, sex, tumor size or serum liver profile. However, serum p53 Abs showed no significant positive correlation with AFP in HCV-related HCC (r=0.09, p value= 0.6) but serum p53 Abs in combination with AFP showed higher diagnostic sensitivity (82.2%) of HCC than either alone. CONCLUSIONS: P53 Abs could be regarded as a specific biomarker for cancer process and its use in combination with AFP may increase the diagnostic sensitivity of HCC.

Prognostic significance of three hepatitis markers (p53 antibodies, vascular endothelial growth factors and alpha fetoprotein) in patients with hepatocellular carcinoma.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world. Of patients with HCC, the diagnostic capacity of Alpha Fetoprotein (AFP) depends on its elevation in the serum. Concentration of AFP greater than the upper reference limit indicate the presence of HCC, but values below this level are less useful because they may also occur in chronic liver disease. To improve the sensitivity of HCC detection by AFP, this work was conducted to study serum expression of p53 Antibodies (p53 Abs) and Vascular Endothelial Growth Factors (VEGF) as a biomarkers in combination with AFP in patients with HCC. METHODOLOGY: The study included 67 patients with HCC (58 males and 9 females with a mean age of 53.7 years) and 27 patients with liver cirrhosis (23 males and 4 females with a mean age of 42 years). Ten healthy volunteers served as control group. Sera of all cases were examined for p53 Abs and VEGF by Enzyme linked immunosorbent assay (ELISA) and correlate its levels with serum AFP expression. RESULTS: Serum level of p53 Abs was detected in HCC patients (0.54 +/- 23) with a significant elevation (p < 0.0001) than liver cirrhosis (0.26 +/- 0.1) and healthy individuals (0.21 = 0.068). The higher percentage of p53 Abs (73.07%) was detected in HCC patients than in liver cirrhosis (7.4%) (p < 0.0001). Serum expression of VEGF was significantly elevated (p < 0.0001) in HCC patients and in cirrhotic patients than healthy individuals (0.52 +/- 0.25, 0.55 +/- 0.25 vs 0.17 +/- 0.034) while there was no significant difference in VEGF between HCC and cirrhotic patients (p > 0.05). There was no association between either p53 Abs or VEGF and AFP concentrations. However, a greater incidence of VEGF and accumulation of p53 Abs expression was detected in positive cases for AFP where VEGF was detected in 85.3% and p53 Abs was detected in 83.3% of positive cases for AFP. Also, p53 Abs positive patients showed a significant high serum level of VEGF; so both can be used in association for screening of patients with HCC. CONCLUSION: It could be concluded that p53 Abs can be considered as an additional tumor marker to increase the diagnostic potential of AFP in HCC patients and VEGF may offer a novel diagnostic value for HCC.

Efficacy of passive immunization with IgY antibodies to a 58-kDa H. pylori antigen on severe gastritis in BALB/c mouse model.

Consecutive triple doses of 1 x 10(8) CFU/mL of a pathogenic H. pylori strain isolated from stomach of Egyptian patients with severe gastritis were used to establish infection in BALB/c mice model. White Leghorn hens were immunized with H. pylori whole cell lysate (HpLysate) antigen and with a highly reactive 58-kDa H. pylori (Hp58) antigen. Two months later, IgY antibodies (IgY-HpLysate & IgY-Hp58) were purified from egg yolk and its efficacy was evaluated in the adopted model. Microbiological culture and immunohistochemical staining revealed that H. pylori infection was inhibited 1 week after oral passive immunization in 70% of infected BALB/c mice with a significant decrease (p < 0.05) in the degrees of gastritis. In conclusion, we have adapted BALB/c mice model for human H. pylori pathogenic strain and oral passive immunization with specific IgY antibodies to the 58-kDa antigen inhibited active H. pylori infection and decreased gastritis.

In vitro evaluation of bovine turbinate cells and Sarcocystis falcatula proliferation in response to serum-free medium and other media supplements.

A serum-free medium (SFM) was evaluated for the growth of bovine turbinate (BT) cells used for the production of Sarcocyvstis falcatula merozoites. Serum free cultures used to propagate S. falcatula were compared to cultures maintained in media supplemented with fetal calf serum (FCS) or horse serum (HS). Serum free cultures were more.effective and very promisin, than the others in supporting the proliferation of S. falcatula merozoites. However, the serum free cultures were unable to adequately support BT cell proliferation compared to the serum-supplemented cultures. No significant differences were seen between cultures supplemented with HS or FCS used for the production of S. falcatula merozoites or BT cells. The rate of BT cell proliferation in response to SFM and different media supplements was assessed in a 96-well plate format using methylene blue staining assay. This technique was superior to manual counting method and allowed quick and accurate quantitative comparison bet-ween the response of proliferating BT cells to different growth conditions