The main purpose of this article is to develop a new and reliable saliva-based clinical diagnostic method for the early detection of oral squamous cell carcinoma (OSCC). This study used an immunoproteomic approach which allowed the detection of immunogenic host proteins in patients' samples using pooled human antibodies. In an attempt to investigate potential biomarkers of OSCC, two-dimensional electrophoresis (2-DE) followed by immunoblotting of saliva from patients and controls were compared. The protein spots of interest were analyzed using 2-DE image analyzer and subsequently subjected to MALDI-TOF/TOF and then matched against NCBI database. The result showed that four protein clusters, namely Human Pancreatic Alpha-amylase (HPA), Human Salivary Amylase (sAA), keratin-10 (K-10), and Ga Module Complexed with Human Serum Albumin (GA-HSA), had exhibited immunoreactivity in western blot. The results are suggestive of the potential use of the differentially expressed saliva protein as tumor biomarkers for the detection of OSCC. However, further studies are recommended to validate this finding.
This study was aimed at gaining a quantitative understanding of the effect of protein quantity and membrane pore structure on protein immobilization. The concentration of immobilized protein was measured by staining with Ponceau S and measuring its color intensity. In this study, both membrane morphology and the quantity of deposited protein significantly influenced the quantity of protein immobilization on the membrane surface. The sharpness and intensity of the red protein spots varied depending on the membrane pore structure, indicating a dependence of protein immobilization on this factor. Membranes with smaller pores resulted in a higher color density, corresponding to enhanced protein immobilization and an increased assay sensitivity level. An increased of immobilized volume has a significant jagged outline on the protein spot but, conversely, no difference in binding capacity.
We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.
An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.
Helicobacter pylori is recognized as a major case of gastritis and peptic ulcer and a key factor in the development of gastric cancer, gastric lymphoma, and non-ulcerative dyspepsia in man. The detection of antibodies specific for strains of H. pylori has demonstrated the value of serology for providing evidence of infection. The present study was conducted to detect the antigenic proteins of excretory antigen of H. pylori with Western blotting and examine whether anti-H. pylori IgG and IgA antibodies from H. pylori positive patients cross-react with antigens from other common bacterial pathogens. By using SDS-PAGE, 20 different proteins were found in the excretory antigen. By Western blotting and absorption studies, there were indications that anti-H. pylori IgA antibodies directed against 54 kDa, 50 kDa and 27 kDa cross-reacted with antigens from other bacteria, and that H. pylori proteins of 99 kDa, 88 kDa and 81 kDa possibly shared similar epitope with antigens of other pathogens not tested in the absorption studies. The cross-reactivity occurred in this study was not significantly affect the performance of the in-house ELISA.
Statistical experimental design was employed to optimize the preparation conditions of polyvinylidenefluoride (PVDF) membranes. Three variables considered were polymer concentration, dissolving temperature, and casting thickness, whereby the response variable was membrane-protein binding. The optimum preparation for the PVDF membrane was a polymer concentration of 16.55 wt%, a dissolving temperature of 27.5°C, and a casting thickness of 450 µm. The statistical model exhibits a deviation between the predicted and actual responses of less than 5%. Further characterization of the formed PVDF membrane showed that the morphology of the membrane was in line with the membrane-protein binding performance.
This study investigated dexamethasone-treatment, shedding routes, tissue antigen distribution, and pathology of caprine Brucellosis. Eighteen non-pregnant goats were randomly grouped into A, B, and C. Group A was administered dexamethasone for 7 days at 2 mg/kg before inoculating 0.5 mL B. melitensis at 107 CFU ocularly while group B was inoculated 0.5 mL B. melitensis only, and C as control negative. Blood samples, ocular, nasal, and vaginal swabs were obtained for evaluation. Three goats were sacrificed from each group at days 21 and 42 post-inoculation (pi) and selected tissues collected for PCR, histopathology, and immunohistochemistry. Brucella melitensis was detected in the ocular swabs of group A significantly higher than group B. Shedding was prolonged in group A compared to B. The overall shedding was 22.2% in group A and 9.4% in group B. The uterus of both groups A and B revealed mild inflammation and microgranuloma, extensive necrotic lesions in lymph nodes. Liver showed multifocal necrosis predominantly in group A. Lesion scoring showed significantly higher scores in A compared to B. Strong immunostaining was observed in the liver, lungs, and spleen, predominantly at day 21 pi. This study demonstrated dexamethasone prolonged shedding, tissue antigen distribution, and pathology in dexamethasone-treated goats.
This study reports the influence of peste des petits ruminants (PPR) vaccination on the clinico-pathological outcomes of PPR in the face of an outbreak. Twenty-two West African dwarf goats procured for a different study started showing early signs of PPR during acclimatization. In response, PPR vaccine was administered either intranasally with phytogenic mucoadhesive gum (Group A; n = 6) or without gum (Group B; n = 6); subcutaneously (Group C; n = 6) or not vaccinated (Group D; n = 4) and studied for 21 days. The clinical scores, hematology, serology and pathology scores were evaluated. Clinical signs of PPR were present in all groups, presenting a percentage mortality of 33%; 33%; 64% and 100% for Groups A, B, C, and D, respectively. Polycythemia and mild leukopenia were observed in all groups, and all animals were seropositive by day 7 post-vaccination. The lung consolidation scores were low in Groups A and B, compared to Group C. Histopathological lesions consistent with PPR was observed in the lymphoid organs, gastrointestinal tract, and lungs with the presence of PPR antigen as detected by immunohistochemistry. The findings suggest that intranasal vaccination with or without mucoadhesive gum may influence the outcome of PPR infection more than the subcutaneous route in the face of an outbreak.
Brucellosis constitutes an infectious re-emerging zoonosis. Spread of diseases could be exacerbated by stress-induced immunosuppression. This study evaluated relationship between Brucella melitensis infection, shedding dynamics, dexamethasone-induced stress, pathological alterations and resveratrol ameliorative effects in goats. Twelve nonpregnant goats were divided into four groups A, B, C, and D of three animals each. Groups A and B were administered 107 CFU/mL of B. melitensis ocularly, 21 days prior to 7 days consecutive administration of dexamethasone (2 mg/kg). Group A was further administered resveratrol (5 mg/kg) intravenously for 5 consecutive days from day 31 post B. melitensis inoculation. Group C was administered similar dose of B. melitensis while group D was inoculated normal saline. Blood, nasal, ocular, and vaginal swabs were collected at intervals for analysis. The does were sacrificed at day 42 post inoculation (pi). Tissues were collected for tissue bacterial load determination, histopathology, and immunohistochemistry. Dexamethasone administration from day 21 pi increased the frequency in the shedding dynamics, tissue bacterial load, pathological alterations (frequency of microgranuloma and intensity of immunostaining) in group B while 5 days treatment with resveratrol following dexamethasone administration significantly reduced tissue bacterial load, decline in shedding dynamics, and ameliorate damage by dexamethasone administration/B. melitensis infection.
Early and accurate diagnosis of Brucella melitensis is essential for the treatment and control of brucellosis both in animals and humans. The thrust for the development of a rapid diagnostic technique to overcome the limitations of conventional microbiological and serological tests brought about this investigation on the development and application of dot-ELISA for antigen and antibody detection in infected goats. Fifteen apparently healthy Boer aged 2-3 years which tested negative for brucellosis using PCR and ELISA, were grouped into A (10 goats infected intraocularly with 10(7) CFU of B. melitensis) and B (5 goats) as control. Discharges (ocular, nasal, and vaginal) and blood were collected at days 3, 7, 10, 14, weekly until 42 post-infection (pi) for dot-ELISA, PCR, and RBPT. Dot-ELISA detected B. melitensis antigen and antibody in group A at day 3 and 7 pi, respectively with adequate sensitivity and specificity relative to PCR and RBPT. The bacteria shedding detected from discharges at day 3 pi in the nasal and ocular route with dot-ELISA. Group B were consistently negative. Values such as speed, simplicity, field adaptability, high sensitivity, and specificity make dot-ELISA a rapid and adequate technique for diagnosis of brucellosis in B. melitensis infected goats within few hours.