Detection of antibodies to Sendai virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sendai virus, a paramyxovirus, is described. The assay was found to be about 20-fold more sensitive than the hemagglutination inhibition assay. Differentiation between virus specific IgG and IgM is possible. The test appears to be especially useful in the study of early events in antibody formation in vivo as well as in vitro. Abbreviations: BSA, bovine serum albumin; CF, chorioallantoic fluid; ChHC, chicken host components; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HAU, hemagglutinating units; HAIU, hemagglutination inhibition units; HIA, hemagglutination inhibition assay; HRPO, horse radish peroxidase; i.n., intranasal; i.p., intraperitoneal; i.v., intravenous; M, molar; A, absorbance; PBS, phosphate buffered saline; RAM/Ig, rabbit anti-mouse-Ig; RAM/IgG, rabbit anti-mouse-IgG; RAM/IgM, rabbit anti-mouse-IgM (Fc); RIA, radioimmunoassay; T-cells, thymus derived cells; TCID50, 50% tissue culture infective doses; s.c., subcutaneous; SV, Sendai virus; w/v, weight per volum

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol

Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

Quantitative approach in screening the antiviral properties of Kandis Hutan in animal cell culture

This main aim of this study was to determine the concentration of Kandis Hutan leaf extracts that can inhibit the infectivity of pseudorabies virus (PrV) in Vero cells. The leaf extracts were crude and extracted with 3 organic solvents namely hexane, ethyl acetate and ethanol. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/ml in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the hexane extract (CC50 <1.25 µg/mL), followed by ethyl acetate extract (CC50 = 237.5 µg/mL), whilst minimal cell cytotoxicity was observed in ethanol extracts (CC50 = 555.0 µg/mL). There was a significant correlation between cell scoring system and MTT assay in term of cell cytotoxicity whereby the least toxic was ethanol extracts, followed by ethyl acetate extract and the most toxic hexane extract. Four antiviral assays were conducted for each extracts, namely plaque reduction assay, cytopathic effect (CPE) reduction assay, inhibition assay and virucidal assay. The most promising result was obtained from the inhibition assay, in which ethyl acetate extracts produced 75% viral inhibition at 125 µg/mL concentration. In virucidal assay, both ethyl acetate and ethanol extracts produced 100% viral inhibition at 250 µg/mL. For plaque reduction assay, there was a significant dose dependent inhibition for ethyl acetate extract but not for ethanol extract and hexane extract. In comparison to CPE reduction assay, findings from plaque reduction assay showed better viral inhibition by ethyl acetate extract (47%) at concentration of 300 µg/mL. The estimated selectivity index (ESI) calculated from the inhibition assay showed the highest antiviral response by ethyl acetate extract (2.7) in comparison with ethanol extract (1.8) and hexane extract (0.1). Therefore, the most promising antiviral activity was produced by ethyl acetate extract which showed consistent viral inhibition in all tested antiviral assays. In contrary, hexane extracts showed the least antiviral efficacy among the tested extracts

Phytochemical analysis and in-vitro antiurolithiatic properties of selected Malaysian herbs

The aim of this study was to investigate the phytochemical content of selected Malaysian herbs and their potential antiurolithiatic effects using in-vitro method. The herbs involved are Ceiba pentandra, Cymbopogon citratus, Euphorbia hirta, Melastoma malabathricum and Ortosiphon stamineus. Aqueous extracts of each herbs were prepared through decoction while Standard drugs Cystone, was used as positive control in comparison. Qualitative analysis was carried out to detect phytochemical presence and nucleation assay to investigate their inhibition effects towards calcium oxalate crystallization urolithiasis in vitro. Based on results, the same trends were observed between phytochemical content and inhibition rate of calcium oxalate crystallization. O. stamineus extract (73.48%) which showed the highest inhibition rate hold the most phytochemical content. while the lowest inhibition rate was occupied by C. citratus extract (45.45%) with the least phytochemical content. The high amount of phytochemicals particularly saponin followed by steroid and terpenoid in O. stamineus extract might contributes to the high inhibition activities of calcium oxalate crystallization as compared to low amount of phytochemicals observed in C. citratus extract. It can be concluded that O. stamineus possesses highest inhibition percentage against calcium oxalate which could be attributed to its saponins, tannins, steroid and terpenoid content

Quantitative in vitro assays for screening antiviral characteristics of Berembang Bukit leaf extracts

Berembang Bukit or Megawasih, is a native of our tropical rainforest and other ASEAN countries. In a preliminary review, a qualitative screening on the antiviral activities of its leaf extracts revealed various degrees of antiviral potential. Therefore this study was done to evaluate its antiviral characteristics in a quantitative approach. The leaf extracts were prepared using hexane, ethyl acetate and ethanol extractions and dissolved in DMSO. DMSO cytotoxicity was initially evaluated to ensure a safe working concentration. A negligible cytotoxicity was observed at a concentration of ≤ 0.1% DMSO. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/mL in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the ethyl acetate extract (CC50 = 218µg/mL), whilst minimal cell cytotoxicity was observed in both hexane (CC50 = 833µg/ml) and ethanol (CC50 = >1000µg/mL) extracts. However, there were no correlation between MTT and cell scoring for cytotoxicity in this study. A series of experiments including CPE reduction assay, plaque reduction assay, inhibition assay and virucidal assay were done to evaluate the total antiviral potential of the leaf extracts. The leaf extracts produced a dose-dependent antiviral response. Both ethyl acetate and ethanol extracts showed 100% plaque formation inhibition in plaque reduction assay, inhibition assay and virucidal assay. Hexane extracts showed absence of plaque inhibition in all the tested antiviral assays. In inhibition assay, the estimated selective index (ESI) for ethanol and ethyl acetate extracts were 8.3 and 1.9, respectively. Whilst in CPE reduction assay, the ESI for the respective extracts were 6.7 and 2.9. In conclusion, the ethanol extracts exhibited the highest antiviral efficacy among the tested extracts

POTENTIAL CYTOTOXIC DRUG EFFECTS OF SECONDARY METABOLITES DERIVED FROM SELECTED MEDICINAL PLANTS OF SAVANADURGA FOREST IN KARNATAKA

Objective: To evaluate the potential cytotoxic and antitumor activities of secondary metabolites of selected medicinal plants of Savanadurga forest, Karnataka. Methods: The soxhlet extracted crude methanolic leaf extracts of nineteen medicinal plants were assessed for their potential cytotoxic and antitumor activities by brine shrimp lethality assay and potato tumor inhibition assay at 100μg/ml respectively. Results: Kirganelia reticulata and Cissus quadrangularis showed highest cytotoxicity while Flacourtia indica failed to show any inhibitory activity in brine shrimp lethality assay. Kirganelia reticulata exhibited 100% antitumor activity while Albizzia amara failed to show any antitumor activity as tested by crown gall tumor inhibition assay. Conclusion: Both brine shrimp lethality assay and potato tumor inhibition assay indicated that Kirganelia reticulata seems to be the best anticancer plant

Effects of pharmaceutical wastes on growth of microalgae

The purpose of this work was to assay samples of waste material from Puerto Rican pharmaceutical industries for inhibition of growth of algae. Two samples (noted as I and II) supplied to us were tested for toxicity to six microalgae. The test organisms, two blue-green algae, two green algae, and two diatoms [r]epresent three major divisions of algae.University of Texas Marine Science InstituteMarine Scienc