Immunochemical and PCR analysis of Staphylococcus aureus Enterotoxin B (SEB) in milk and fruit juices collected in Lahore, Pakistan

Enterotoxins secreted by S. aureus are known as a food-poisoning agent that is associated with various gastro-intestinal pathological conditions. In this study, a one-step immunodetection method was devised for routine checking of SEB in milk and fruit juices available locally. Antibodies against recombinant SEB were raised, purified, and cross reactivity was checked against clinically important bacteria (Shigella flexneri, Streptococci, Salmonella typhi, Klebsiella and Bacillus subtilis). Purified anti-SEB antibodies were conjugated with gold nanoparticles (Ab-GNPs) for direct detection of SEB in samples. SEB (33%, 4.76% and 15%) was found in non-sterilized milk (118), sterilized milk (42) and juices (60), respectively. Coagulase, MSA tests and PCR amplification of 725 bp of the SEB gene confirmed the presence of S. aureus in the collected samples positive for SEB. Immunoassay is easy, reliable and less time consuming and will be helpful to detect the SEB in food samples at local level

Microbial Contamination and Antibiotic Resistance in Food and Water: Assessing the Threat of Staphylococcus aureus in Lahore Metropolitan

Background: The research project in Lahore, Pakistan, seeks to detect S. aureus in drinking water, raw milk, and yogurt samples due to health concerns. S. aureus is a pathogenic bacterium with potential risks if present in food and water sources.Method: In this study, 300 samples of raw milk, yogurt, and drinking water were collected in Lahore. The presence of S. aureus was determined through morphological, microscopic, and biochemical methods. The biochemical analysis included testing for specific features of S. aureus. Disk diffusion technology was employed to assess the antimicrobial susceptibility of the isolates, following the recommendations of the Clinical Laboratory Standards Institute. Molecular confirmation was achieved through 16S rRNA sequence analysis using universal and specific primers.Results: The investigation uncovered that 6% of drinking water samples, 9% of yogurt samples and 58% of raw milk samples were tainted with S. aureus. These findings were further validated through 16S rRNA sequence analysis, affirming their reliability. S. aureus exhibited notable resistance rates, with 100% resistance to penicillin and 95% resistance to erythromycin. Conversely, resistance to ciprofloxacin and gentamicin was lower, at 10% and 5% each, indicating the potential efficacy of these antibiotics in treating S. aureus infections.Conclusion: The study emphasizes the risk of S. aureus infection from raw milk consumption in Lahore, Pakistan, due to inadequate sanitary practices. It stresses the necessity of implementing stricter measures in dairy production and water treatment to ensure public safety and reduce multidrug-resistant bacteria prevalence. Continuous monitoring and preventive actions are vital for safeguarding public health.Keywords: S. aureus; Antibiotic resistance; Food and water; Lahore, Pakistan; Sanitary measures 

Engineering Quantum Dot (Cadmium Sulfide) on Antibodies for Fluoroimmunoassays

Antibodies are good escorts for a therapeutic and diagnostic tool against many diseases as well as for targeting drug delivery and in immunohistochemistry. Cysteine-capped cadmium sulfide (quantum dot) nanoparticles have wide applications in immunohistochemistry due to their unique physical, chemical, and fluorescent properties. In this study, polyclonal antibodies against transferrin (as ligand), doxorubicin (anticancer drug), and H. pylori (cancer-causing agent) were developed in rabbits and purified. Purified antibodies were labeled with cadmium sulfide (CdS) quantum dot (QD) and used as a fluorescent-labeled marker for one-step identification in diagnosis and therapeutics. Cysteine-capped CdS QDs were synthesized in the presence of taurine (antioxidant) and characterized by FTIR, VSM, DLS, and TEM. CdS nanoparticles are monodisperse with a narrow size range below 7 nm and showed an increase of almost 30 nm after conjugation to IgG. The binding of QD-labeled antibodies was observed and confirmed by binding on MDA-MB 231 cancer cells, mouse liver tissue, mouse tumor tissue, and H. pylori under a fluorescent microscope. QD-labeled antibodies gave sharp fluorescence after binding with their respective targets. Intensity of fluorescence with quantum dot enhances many folds as compared to that of traditional fluorescent-labeled compounds. Experiments are underway to target transferrin and doxorubicin and to analyze the role of H. pylori in a mouse model of gastric cancer

Ovarian Cancer among Women of Lahore Metropolitan; a Survey

Abstract The symptoms, causes and different risk factors related to ovarian cancer (OC) in Lahore Metropolitan are studied. Information was collected from 52 patients (having mean age 45±10 years) referred to the Institute of Nuclear Medicine and Oncology Lahore (INMOL), Pakistan. The risk was found relevant in the patients having positive family history of any cancer other than OC and that's why 21.1% patients faced this dilemma. Marital status was not found a cause of OC as unmarried and married patients both were affected. Birth control pills were not a cause of OC as 96.6% patients in Lahore did not used them. High parity and Brest feeding were the cause of OC as 59.6% patients have high parity and 78.8% have breast fed their children for more than one year. Fallopian tube tide (94.2%), hysterectomy (86.5%) and smoking (98.1%) were not the cause of OC. Irregularity in menstrual cycle (30.8%), eating disorders (38.5%), abdominal pain or bloating (90.4%) and natural menopause (50%) were the symptoms of OC. Hormone replacement therapy (HRT) is not found a cause of OC in Lahore as all the selected patients under study never used HRT

Development of Diagnostic Dip Strip Immunoassay Using Antibodies of PreS 2 Region of Hepatitis B Surface Antigen

Abstract.-An immunoassay based on immunochromatographic lateral flow is developed for qualitative determination of HBsAg in serum samples using anti-preS 2 antibodies. The diagnostic test strip consists of four components i) sample application pad ii) conjugate pad iii) nitrocellulose membrane and iv) absorbent pad. Affinity purified anti-HBsAg antibodies and goat anti-rabbit IgG antibodies were coated onto nitrocellulose membrane as a test line and control line, respectively. The affinity purified anti-Pre-S 2 antibodies conjugated to colloidal gold particles (20 nm) were coated on polyester conjugate pad. The conjugate pad was attached to nitrocellulose membrane and the sample application pad was attached to conjugate pad. The absorbent pad comprising 3mm Whatman paper was attached to the nitrocellulose strip opposite to the sample pad. Purified HBsAg (1.4µg/µl in PBS), 100 µl, was loaded on sample pad and allowed to flow over the strip for 10 minutes. Pink colour test line indicates the correct binding of gold labeled antibodies to antigen. The staining of control line confirms the accuracy of the strip. The sensitivity and specificity of dip strip was measured by using serum samples of one hundred clinically confirmed HBV positive individuals and twenty HBV negative individuals. The test line appeared colored in the case of HBV positive individuals and remained blank in the case of negative individuals. The results of indigenous dipstrip were compared with the results of imported dipstrips. The results indicated that indigenous dipstrip is as good as the imported one, therefore, recommended for use in clinical trials

A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells’ surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future