Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2 N and S proteins: mass screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test "CoroSuchak," with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit "CoroSuchak" is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population. KEY POINTS: •Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences. •High sensitivity and specificity to detect variants. •Highly sensitive for mass screening.

Efficient ELISA for diagnosis of active tuberculosis employing a cocktail of secretory proteins of Mycobacterium tuberculosis.

Rapid and accurate diagnosis is important for preventing transmission of Mycobacterium tuberculosis. Currently available tuberculosis (TB) diagnostic methods lack desired sensitivity and specificity, and require sophisticated equipment and skilled workforce including weeks' long duration to yield results. In this study, extracellular proteins or secretory protein antigens of M. tuberculosis H37Rv have been isolated using ion exchange chromatography, immunocharacterized and exploited for the development of efficient enzyme-linked immunosorbent assay (ELISA) for diagnosis of active TB with enhanced specificity and sensitivity. Apparent molecular masses for purified proteins were found to be 6, 27, 30, 38 and 64 kDa. Out of five purified proteins, one (64 kDa) was found to be novel. Of the five proteins, four (6, 27, 30 and 38 kDa) were found significant to be used in the development of ELISA for pulmonary and extra-pulmonary TB. The immune responses of serum samples of TB patients and other healthy subjects against the above-mentioned antigens' cocktail were evaluated. Critical parameters of newly developed ELISA were optimized and it was observed that the cocktail antigens have a greater specificity (98.06 %) and sensitivity (98.67 %) as compared to other commercially available diagnostic tests. The present findings suggest that the developed ELISA is an effective tool for routine screening and early-stage diagnosis of TB.

Oxaliplatin-mediated inhibition of survivin increases sensitivity of head and neck squamous cell carcinoma cell lines to paclitaxel.

The present study deals with the evaluation of the efficacy of oxaliplatin and paclitaxel combination as a potential strategy in controlling HNSCC cell proliferation and the assessment of correlation between occurrence of apoptosis and changes in expression of survivin (IAP). The panel cell lines included two HNSCC cell lines (Cal27 and NT8e) and one normal cell line (293) with differential level of survivin expression in accordance with chemosensitivity. The cytotoxicity and effect of drugs on apoptosis was determined, separately and in combination. Combined treatment of cells with paclitaxel and oxaliplatin resulted in significantly higher cytotoxicity as compared to individual single drug treatment. Cytotoxicity was prominent in paclitaxel to oxaliplatin (pacl-oxal) sequence treatment with an approximate two-fold increase in apoptosis as compared to oxaliplatin to paclitaxel (oxal-pacl) sequence treatment. Paclitaxel treatment also caused increased survivin expression showing reduced apoptosis at low concentration. Oxaliplatin, when combined with paclitaxel, decreased the survivin level with increased cell death. Inhibition of survivin by a small interfering RNA (siRNA) method also increased the sensitivity of the cancer cell lines to paclitaxel whereas over-expression of survivin in the transfected 293-cell line provided resistance. In conclusion, the interaction between drugs was synergistic and schedule-dependent. Survivin played a critical role in paclitaxel resistance through the suppression of apoptosis, and a significant induction of apoptosis was observed when oxaliplatin was combined with paclitaxel at least in part by the down-regulation of survivin.

Rapid liposomal agglutination card test for the detection of antigens in patients with active tuberculosis.

SETTING: A total of 1360 subjects with clinically confirmed pulmonary and extra-pulmonary tuberculosis (TB) and other non-tuberculous conditions. OBJECTIVES: To develop a rapid, sensitive and specific diagnostic test for the detection of the glycolipid antigen of Mycobacterium tuberculosis in a variety of clinical samples. STUDY DESIGN: Affinity-purified rabbit anti-glycolipid antibodies (IgG) were coupled to liposome particles (0.2-0.4 microm) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinamide to prepare the working reagent of the TB/M card test. RESULTS: Antibody-conjugated liposomes, when determined with the glycolipid antigens present in the specimens, formed a dark blue agglutination within 4 min. No clumping was observed in samples from normal healthy subjects or patients with other diseases. The test was shown to be effective in detecting glycolipid antigens of M. tuberculosis in clinical samples from patients with active TB with as low as 1 ng/ml analytical sensitivity, 97.4% clinical sensitivity and 96.9% specificity. CONCLUSION: The TB/M card test was found to be comparatively economical (4 Indian Rupees or US$ 0.09/test), rapid (4 min) and seems fairly useful for mass testing of a variety of biological specimens (cerebrospinal, pleural and synovial fluids, serum, tissue biopsy extract) from patients with tuberculous meningitis, pulmonary TB and other extra-pulmonary TB in endemic countries.

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis.

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.

Phylogenetic relationships of three bacterial strains isolated from the pasture legume Biserrula pelecinus L.

Three bacterial strains (WSM 1283, WSM 1284, WSM 1497) isolated from root nodules of the pasture legume Biserrula pelecinus L. growing in Morocco, Italy and Greece, respectively, were studied in order to determine their phylogenetic relationship to the other members of the family Rhizobiaceae. A polyphasic approach, which included analyses of morphological and physiological characteristics, plasmid profiles, symbiotic performance and 16S rRNA gene sequencing, indicated that these strains belong to the genus Mesorhizobium.

Reversible ocular toxicity related to tamoxifen therapy.

A 42-year-old woman with metastatic breast cancer developed bilateral optic disc swelling, retinal hemorrhages, and visual impairment three weeks after starting treatment with low doses of tamoxifen. Neurologic evaluation failed to provide an explanation for the ocular findings which resolved completely after cessation of tamoxifen therapy. This case suggests that tamoxifen has the potential for causing serious ophthalmologic toxicity which may be reversible if recognized early.

Pseudotumor cerebri: an observation and review.

In five consecutive cases of pseudotumor cerebri we observed an elevation in the erythrocyte sedimentation rate that we were able to correlate with the changes in the degree of papilledema. In spite of an extensive medical workup, no other cause for the increased ESR was found. Our findings suggest that possibly serial ESR determinations might be of assistance in following cases of pseudotumor cerebri.