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.

Assessment of materno-foetal transmission of SARS-CoV-2: A prospective pilot study.

BACKGROUND: The issue of vertical transmission of SARS-CoV-2 infection to the foetus has not yet been resolved. Its main reason is lack of a bigger study to analyse this question. The evidence of the affection of the foetus during antenatal or intrapartum period is limited to some anecdotal reports. To look for the possibility of vertical transmission of Severe Acute Respiratory Syndrome - Corona Virus-2 (SARS-CoV-2) infection to the foetus, this prospective pilot study was conducted at a tertiary health care COVID-19 designated centre of Armed Forces. METHODS: This study was conducted during 01 June 2020 and 15 October 2020 and included 54 covid-positive pregnant mothers. During delivery, amniotic fluid and cord blood samples were collected in a sterile manner. Amniotic fluid samples were not collected during vaginal deliveries as chances of contamination was very high. These samples were tested for the presence of SARS-CoV-2 gene by Reverse Transcriptasee Polymerase Chain Reaction (RT-PCR) test, and the results were analysed. Newborns were allowed to room in with mother, and they underwent throat and nasal swab RT-PCR testing of covid within 24-48 h of delivery. RESULTS: A total of 1520 pregnant mothers underwent RT-PCR test during the study period. Total positivity rate among our pregnant women was 2.8%. Out of 54 covid-positive women during the study period, amniotic fluid RT-PCR tests were carried out for 43 women, and cord blood was tested for 45 women. CONCLUSION: RT-PCR test of amniotic fluid, cord blood and nasal and throat swab of all newborns delivered by SARS-CoV-2-positive pregnant women were negative. Based on our study, the possibility of intrauterine vertical transmission of SARS-CoV-2 infection appears to be unlikely.

Remarkable preservation of terpenoids and record of volatile signalling in plant-animal interactions from Miocene amber.

Plants produce and release a large array of volatile organic compounds that play many ecological functions. These volatile plant metabolites serve as pollinator attractants, herbivore and pathogen repellents and protect plants from abiotic stresses. To date, the geological evolution of these organic compounds remains unknown. The preservation potential of these metabolites in the fossil record is very poor due to their low boiling points. Here we report a series of volatile sesquiterpenoids, including δ-elemene, α-copaene, ß-elemene, ß-caryophyllene, α-humulene, germacrene D, δ-cadiene and spathunenol, from early Miocene (~17 million year) amber from eastern India. The survival of these unaltered bioterpenoids can be attributed to the existence of extraordinary taphonomic conditions conducive to the preservation of volatile biomolecules through deep time. Furthermore, the occurrence of these volatiles in the early Miocene amber suggests that the plants from this period had evolved metabolic pathways to synthesize these organic molecules to play an active role in forest ecology, especially in plant-animal interactions.

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.

Correlation of radon anomalies with seismic events along Mat Fault in Serchhip district, Mizoram, India.

Radon emanation from the soil gas was studied using a deep red coloured cellulose nitrate LR-115 type II film. The study was carried out from March 2012 to February 2013 at Mat Bridge (23°18΄ N, 92°48΄ E) along Mat Fault in Serchhip district, Mizoram (India). Changes in radon concentrations have been observed. Effects of meteorological parameters on radon emission were also studied. The measured radon data shows a moderate positive correlation with relative humidity but no specific relation with air temperature and rainfall. Data obtained have been correlated to the earthquakes that occurred around the measuring sites.

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi.

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.

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.

Diagnosis of tuberculosis: available technologies, limitations, and possibilities.

Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections.

Microbial alkaline pectinases and their industrial applications: a review.

The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes. Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations. The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment. It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential. The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology.

Factors affecting haemolysin production and congo red binding in Salmonella enterica serovar Typhimurium DT 98.

Differences in haemolysin expression were observed in a strain of Salmonella enterica serovar Typhimurium definitive phage type (DT) 98 cultured under various conditions. Haemolysin expression was optimal in cultures grown micro-aerobically. The zones of haemolysis were wider after longer periods of incubation. Haemolysin production varied after growth in the following media (greatest to least): brain heart infusion (BHI) broth > nutrient broth (NB)>trypticase soy broth (TSB)> M-9 glucose medium. Haemolysin production correlated directly with Congo red binding in nutrient broth. On Congo red blood agar, colonies were smaller, with dark centres and wider zones of haemolysis. Culture-cell-free haemolysin activity was higher, but cell-bound haemolysin activity was very low in growth medium supplemented with Congo red. Boiled tea extract at 25% v/v (of 25% w/v tea infusion) in PBS and nutrient broth was bactericidal to S. Typhimurium DT 98. The addition of boiled tea extract to growth medium inhibited haemolysin production by S. Typhimurium DT 98 at higher concentrations (6-12.5% v/v) but stimulated haemolysin production at lower concentrations (1.5-3% v/v). The pre-treatment of bacterial cell suspensions with lower concentrations of tea extract (1.5-3% v/v) also altered the Congo red binding, which showed an inverse correlation in nutrient broth.

Modulatory effects of Salmonella LAP-LPS on murine macrophages.

PURPOSE: To study the modulatory effects of Salmonella lipid associated protein - lipopolysaccharides (LAP-LPS) on murine macrophages as the intracellular survival within the host macrophages is an important feature for a number of gram-negative pathogens like S. typhi. METHODS: Macrophage functions were studied in two groups of mice immunized with either LPS or LAP-LPS. RESULTS: Comparison of protective efficacy of mice preimmunized with LPS based preparations, against challenge infectious doses, showed higher protection in LAP-LPS complex immunized mice group as compared to the mice group immunized with LPS alone. Aggregation of S. typhi cells was lesser with intestinal mucus extracted from LAP-LPS immunized mice as compared to LPS immunized challenged group. A significant increase in the number of macrophages in LAP-LPS immunized mice was also observed in comparison to control and LPS immunized mice groups. Nitric oxide (NO) and superoxide dismutase (SOD) production were also more in macrophages derived from LAP-LPS immunized mice group. Phagocytic uptake studies showed that there was enhanced uptake of bacteria in the LAP-LPS immunized animals in comparison to LPS immunized and controls. Similar trend was observed in intracellular killing of bacteria by the macrophages. CONCLUSIONS: The results indicated the involvement of protein moiety in LAP on modulation of effects of LPS on macrophages.

Correlation between Congo red binding and contact haemolysin production in Shigella species.

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.

Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8.

An extracellular lipase isolated from Pseudomonas sp. AG-8, had an optimal activity at 45 degrees C and pH 8.0-8.5. It retained more than 80% of its initial activity after keeping for 1 h at 65 degrees C. The enzyme was stable in 5 M NaCl and 6 M urea. Triton X-100 increased the lipase activity by 2.4 fold. Ca2+ ions activated the enzyme, while Zn2+, Fe2+, Fe3+ strongly inhibited its activity. Ethanol, methanol and acetone at 20% (v/v) enhanced the lipase activity by 2.9, 3.6 and 4.5 fold respectively. Dimethylsulphoxide at 90% (v/v) enhanced the enzyme activity up to 5.7 fold.

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.

Legume root nodule bacteria and acid pH.

In 1984 the Australian Wool Research Trust Fund called for expressions of interest in projects directed at using the developing techniques of molecular biology for application to agricultural problems. With our interests in legume root nodule bacteria and their physiology, we felt that the problems for legume nodulation and N2 fixation posed by soils which were already acid, or which were rapidly acidifying, required just such attention. Further, the finding body's request coincided with the highly successful introduction into Western Australian agriculture of acid-tolerant strains of the medic-nodulating bacteria Sinorhizobium meliloti originating from acid soils on Sardinia (see below). The existence of such strains made it obvious that acid tolerance was a genetically determined trait, and provided invaluable biologically diverse material with which to work. The biological bases for that trait of acid tolerance were totally obscure, and many remain so, but the following account provides some light in the darkness. The research that we have done since in pursuit of explanations for acid tolerance have been funded first by the Wool Research Trust Fund and the Rural Credits Development Fund, and later by the Australian Research Council, and we here record our appreciation for their support.

Salmonella. typhi OMPs induced immunomodulation in peritoneal macrophages.

The immunomodulatory properties of outer membrane proteins (OMPs) from S. typhi Ty2 were studied in mouse model at 72 hr and 20 days post-infection. Inspite of reduction in the number of macrophages and their protein content observed in the immunized group vis-à-vis infected group, OMPs activated macrophages showed significant upregulation of NO. At 20 days post infection, the level remained almost the same suggesting the prolonged cytotoxic and cytostatic activity due to the long lasting effects of OMPs activated macrophages. Higher activity of SOD in these aged cells pointed out towards the protective efficacy of OMPs to keep the macrophages themselves away from the noxious effects of O2-. Lower level of acid phosphatase in the macrophages from immunized mice group indicated the involvement of oxygen dependent rather than oxygen independent killing process. The enhanced uptake of organisms and their killing could be related to the production of oxygen and nitrogen radicals in the OMPs immunized group.

Purification & characterisation of haemolysin from Serratia marcescens B-91.

BACKGROUND & OBJECTIVES: Serratia marcescens an opportunistic human pathogen, is frequently encountered in a variety of debilitating diseases. Relatively little is known about its virulence traits though most clinical isolates secrete a distinct haemolysin which is considered as a useful marker for pathogenicity of Serratia. In this study purification and characterisation of S. marcescens B-91 haemolysin have been attempted. METHODS: S. marcescens B-91 haemolysin was purified to homogeneity from the growth medium using ammonium sulphate fractional precipitation and gel filtration through Sephadex G-75 column. Homogeneity was determined by gel electrophoresis and purified haemolysin was tested for its stability and other characteristics. RESULTS: The haemolysin was characterised to be a 45 kDa molecular weight protein on SDS-polyacrylamide gel electrophoresis. It was inactivated at 60-100 degrees C within 30 min, and on overnight treatment with 2 per cent formaldehyde. It was also susceptible to the action of pronase, protease and trypsin. INTERPRETATION & CONCLUSIONS: The results indicate that the fragile stability of S. marcescens haemolysin is dependent on the storage temperature. The purified haemolysin can be used for understanding the role of haemolysin in the pathogenesis of S. marcescens and also for evaluation of immunoprophylactic activity.

Resistance to antibiotics, metals, hydrophobicity and klebocinogeny of Klebsiella pneumoniae isolated from foods.

Milk samples and milk products (69 in toto) were screened for the presence of Klebsiella pneumoniae (52%), and maximum isolations (77%) were from ice cream samples (13). The isolates were hydrophobic, non-haemolytic and possessed both mannose resistant (MR) and mannose sensitive (MS) pili or only MR pili when tested with human or sheep blood, respectively. All isolates were resistant to one metal at least whereas about 98% exhibited resistance to two or more metal ions. The resistance frequency of 93%, 90% and 66.7% was observed against silver (20 micrograms/ml), cadmium (20 micrograms/ml) and mercuric ions (20 micrograms/ml), respectively. Multiple drug resistance (MDR) was observed in 10% of the isolates only. A direct correlation between the metal ion and antibiotic resistance was found in MDR strains. The klebocin typeability of 53% and 61% was observed with 153-158 and 153-156, U-5 and U-6 groups, respectively. The most common typing patterns involved strains 424 (21%) and 442 (31.8%). Only 61% of the isolates showed enterotoxigenicity by the coagglutination test.