• Title/Summary/Keyword: microtitre assay

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Spectrophotometric microtitre assay for rapid screening of membrane-disrupting herbicides (분광분석을 이용한 막과산화작용 제초제의 신속한 검정법)

  • Kwon, Ok-Kyung;Cho, Kwang-Yun;Kim, Jin-Seog
    • The Korean Journal of Pesticide Science
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    • v.4 no.1
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    • pp.11-18
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    • 2000
  • This study was conducted to establish an efficient screening method of membrane-disrupting herbicides, based on the result that membrane leakage leading to evolution of carbonyl compounds and aldehydes could be colorimetrically assessed by formation of stable chromophores with MBTH and ferric chloride. Assay conditions including plant material, light intensity, irradiation time, concentrations of reagents for colorization, and reaction time were optimized, and activities of different types of known herbicides were investigated with cucumber cotyledon discs. That result indicated that this assay was sensitive to membrane-disrupting herbicides, such as protoporphyrinogen oxidase inhibitors (acifluorfen, oxyfluorfen, oxadiazon) and an electron interceptor from photosystem I(paraquat). With seven compounds newly synthesized, highly positive correlation was observed between herbicidal activities measured by greenhouse bioassay and MBTH method. Because only a single disc(4 mm diameter) from cucumber cotyledon in 60 ${\mu}L$ of incubation solution was required to assay, a large number of herbicides could be tested simultaneously using 96-well microtitre plates. But the assay efficiency is not likely to significantly increase because of the boiling step during the reaction.

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A Neutravidin-based Assay for Reverse Transcriptase Suitable for High Throughput Screening of Retroviral Activity

  • Brennan, Lyndall E.;Sune, Carlos;Klimkait, Thomas
    • BMB Reports
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    • v.35 no.3
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    • pp.262-266
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    • 2002
  • A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

Clq-Coated Microtitre Enzyme-linked Immunosorbent Assay for Measuring the Anticomplementary Activity of Intravenous Immunoglobulin Preparations (Clq-coated ELISA법을 이용한 정맥용 면역글로불린제제의 항보체성 측정)

  • 강혜나;김순남;신광훈;허숙진
    • YAKHAK HOEJI
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    • v.45 no.6
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    • pp.656-663
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    • 2001
  • The quality of an intravenous immunoglobulin preparation (IVIG) is reflected by the degree of nonspecific activation of complements, the so-called anticomplementary activity (ACA). ACA of aggregates in IVIG was investigated using method by the European Pharmacopoeia and Clq-coated microtiter enzyme-linked immunosorbent assay (ELISA). Both the EP method and the ELISA method showed a dose response curve with the amount of complements bound increasing with the percentage content of aggregates in immunoglobulin standard. The correlation between the two tests was good (r=0.96, r=0.99). However, the correlation was not found when the ACA (EP method) of IVIG product was compared with its aggregate percentage. These results emphasize that the method of aggregate formation affects ACA and that estimation of the percentage distribution of aggregates by HPLC may not reflect ACA. In analysing WIG product for Clq binding activity test with the ELISA, the result by using Protein A-HRP correlated with aggregate percentage (r=0.84). But the correlation decreased (r=0.48) when the result used Protein A-AP(having poorer sensitivity than HRP) was compared with aggregate percentage. As a result, some variation between the two methods, due to differences in assay principles, is to be expected. However, ELISA technique has the advantage in that it is easier to perform, more precise and less subject to reagent variability, and is the more suitable screening method than HPLC analysis.

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Characteristics and application of monoclonal antibody to progesterone II. Development of progesterone enzyme-linked immunosorbent assay(ELISA) (Progesterone의 단크론성 항체에 관한 특성 및 활용에 관한 연구 II. ELISA 기법의 개발)

  • Kang, Chung-boo;Kim, Jong-shu
    • Korean Journal of Veterinary Research
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    • v.31 no.4
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    • pp.403-409
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    • 1991
  • This experiment was carried out to develop a sensitive, rapid, solid-phase microtitre plate assay of progesterone using the monoclonal antibody to this hormone. Monoclonal antibody to progesterone was much higher titre and binding affinity about 10 times than conventional polyclonal antibody to progesterone. Dot-blot analysis of monoclonal antibody revealed a single precipitation band when reacted with anti-mouse IgM and anti-mouse K. A competitive reaction was used with a reaction time of 2 hours. The standard dose-response curve was linear through 1,000pg/well. This ELISA system approach is applicable to evaluation for the rapid assessment of luteal function and reproductive status in both clinical and research in a wide variety of species.

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Establishment of a Binding Assay System for Screening of the Inhibitors of $p56^{lck}$ SH2 Domain

  • Kim, Jyn-Ho;Hur, Eun-Mi;Yun, Yung-Dae
    • BMB Reports
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    • v.31 no.4
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    • pp.370-376
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    • 1998
  • Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

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Repellent activity of methanol extracts of native plants against Aedes albopictus and Blateria germanica (자생식물의 메탄올 추출물이 흰줄숲모기 및 바퀴에 대한 기피효과)

  • Kyung, Suk-Hun;Yoon, Young-Hee
    • The Korean Journal of Pesticide Science
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    • v.4 no.2
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    • pp.18-20
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    • 2000
  • Methanol extracts of nine native plants(Teraxacum platycerpum leaf, Pinus densiflora leaf, Artemisia prinseps, leaf, Allium tuberosum leaf, Cassia obtussifolia, whole plant, Sophora angestifolia root, Stemonae sessilifolia root, Lonicera japonica, stem, leaf, flower, and Clivia miniata leaf were tested for repellent activity against Aedes albopictus and Blateria germanica. The extracts of P. densiflora, T. platycerpum, A. tuberosum and L. japonica (flower) exhibited excellent repellent activity against A. albopictus, while only L. japonica (leaf) had some good activity against B. germanica.

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Time-resolved Fluoroimmunoassay for the Measurement of 17$\beta$-Estradiol using Anti-idiotypic Antibody (Anti-idiotype 항체를 이용한 17$\beta$-Estradiol 측정을 위한 Time-resolved Fluoroimmunoassay)

  • 김윤규;김창규;박성민;이치호;이원창;최영숙;김종배
    • Korean Journal of Animal Reproduction
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    • v.16 no.4
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    • pp.325-333
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    • 1993
  • A competitive type immunoassay method for 17$\beta$-estradiol(E2) based on the idiotypic anti-idiotypic antibody and time-resolved fluorescence is described. The anti-idiotypic antibody(Ab2) produced to E2 binding site of the primary idiotype antibody (Ab1) was labelled with europium and was allowed to compete with E2 standards or serum sample for the binding sites of Ab1 which was bound to 2nd antibody captured ontothe surface of microtitre plates. Fluorescence measured by time-resolved fluorometer was inversely proportional to the concentration of E2 over the range 5~500pg/well. The sensitivity of the assay was 5pg per well which was compatible with that ofradioimmunoassay using the same Ab1 and 3H-E2 as a tracer. One great advantage of this method described here was to enable antibodies to be labelled instead of haptens, and thus makes it easier to develop sensitive and robust immunoassay systems specially for haptens.

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Activity of Essential Oil from Mentha piperita against Some Antibiotic-Resistant Streptococcus pneumoniae Strains and Its Combination Effects with Antibiotics

  • Choi, Sung-Hee;Shin, Seung-Won
    • Natural Product Sciences
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    • v.13 no.2
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    • pp.164-168
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    • 2007
  • To investigate natural antibiotics from plant essential oils and to evaluate their synergism with current antimicrobial drugs in inhibiting antibiotic-resistant strains of Streptococcus pneumoniae. The minimal inhibitory concentrations (MICs) of eleven plant essential oils and their main components were established for two antibiotic-susceptible and two antibiotic-resistant strains of S. pneumoniae, using broth microdilution tests. Potential synergism with oxacillin, norfloxacin, or erythromycin was evaluated using a checkerboard microtitre assay. Among the tested oils, Mentha piperita oil and its main component, menthol, exhibited the strongest inhibitory activities against all of the tested strains. The activity of antibiotics against antibiotic-resistant strains of S. pneumoniae was enhanced significantly by combination with Mentha piperita oils and its main component, menthol. In conclusion, the combination Mentha piperita essential oil or menthol with antibiotics could be used to reduce the effective dose of antibiotic and to modulate the resistance of S. pneumoniae strains.

Assessment of free-radical-scavenging and antibacterial activities, and brine shrimp toxicity of Scutellaria pinnatifida (Lamiaceae)

  • Sauvage, Severine;Samson, Emilie;Granger, Melanie;Majumdar, Anisha;Nigam, Poonam;Nahar, Lutfun;Celik, Sezgin;Sarker, Satyajit D.
    • Advances in Traditional Medicine
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    • v.10 no.4
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    • pp.304-309
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    • 2010
  • Scutellaria pinnatifida A. Hamilt. (Lamiaceae) is an endemic Turkish herb. This plant is also endemic to Iran, and grows abundantly in other central and western Asian countries. Several species of the Scutellaria are known for their traditional uses in the treatment of hypertension, arteriosclerosis, inflammatory diseases, hepatitis, allergy, cancer and diarrhoea. Free-radical-scavenging property, antibacterial activity and brine shrimp toxicity of the n-hexane, dichloromethane (DCM) and methanol (MeOH) extracts of S. pinnatifida were assessed using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, the resazurin microtitre plate based assay, and the brine shrimp lethality assay, respectively. The DCM and MeOH extracts exhibited free-radical-scavenging property, with the $RC_{50}$ values of 0.362 and 0.127 mg/ml, respectively. Among the solid-phase extraction fractions of the MeOH extract, the 50% aqueous-MeOH fraction showed the highest level of free-radicalscavenging activity ($RC_{50}$ = 0.039 mg/ml). While the DCM extract showed low level of antibacterial activity against Bacillus subtilis and ampicillin-resistant Escherichia coli, the MeOH extract was active against B. cereus, B. subtilis, E. coli and ampicillin-resistant E. coli. However, the minimum inhibitory concentrations (MIC) of the MeOH extract against these bacterial strains were >10 mg/ml. None of the extracts showed any significant toxicity towards brine shrimps ($LD_{50}$ = > 1.00 mg/ml).

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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