• Title/Summary/Keyword: Incubation volume

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Radioimmunoassay Reagent Survey and Evaluation (검사별 radioimmunoassay시약 조사 및 비교실험)

  • Kim, Ji-Na;An, Jae-seok;Jeon, Young-woo;Yoon, Sang-hyuk;Kim, Yoon-cheol
    • The Korean Journal of Nuclear Medicine Technology
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    • v.25 no.1
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    • pp.34-40
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    • 2021
  • Purpose If a new test is introduced or reagents are changed in the laboratory of a medical institution, the characteristics of the test should be analyzed according to the procedure and the assessment of reagents should be made. However, several necessary conditions must be met to perform all required comparative evaluations, first enough samples should be prepared for each test, and secondly, various reagents applicable to the comparative evaluations must be supplied. Even if enough comparative evaluations have been done, there is a limit to the fact that the data variation for the new reagent represents the overall patient data variation, The fact puts a burden on the laboratory to the change the reagent. Due to these various difficulties, reagent changes in the laboratory are limited. In order to introduce a competitive bid, the institute conducted a full investigation of Radioimmunoassay(RIA) reagents for each test and established the range of reagents available in the laboratory through comparative evaluations. We wanted to share this process. Materials and Methods There are 20 items of tests conducted in our laboratory except for consignment tests. For each test, RIA reagents that can be used were fully investigated with the reference to external quality control report. and the manuals for each reagent were obtained. Each reagent was checked for the manual to check the test method, Incubation time, sample volume needed for the test. After that, the primary selection was made according to whether it was available in this laboratory. The primary selected reagents were supplied with 2kits based on 100tests, and the data correlation test, sensitivity measurement, recovery rate measurement, and dilution test were conducted. The secondary selection was performed according to the results of the comparative evaluation. The reagents that passed the primary and secondary selections were submitted to the competitive bidding list. In the case of reagent is designated as a singular, we submitted a explanatory statement with the data obtained during the primary and secondary selection processes. Results Excluded from the primary selection was the case where TAT was expected to be delayed at the moment, and it was impossible to apply to our equipment due to the large volume of reagents used during the test. In the primary selection, there were five items which only one reagent was available.(squamous cell carcinoma Ag(SCC Ag), β-human chorionic gonadotropin(β-HCG), vitamin B12, folate, free testosterone), two reagents were available(CA19-9, CA125, CA72-4, ferritin, thyroglobulin antibody(TG Ab), microsomal antibody(Mic Ab), thyroid stimulating hormone-receptor-antibody(TSH-R-Ab), calcitonin), three reagents were available (triiodothyronine(T3), Tree T3, Free T4, TSH, intact parathyroid hormone(intact PTH)) and four reagents were available are carcinoembryonic antigen(CEA), TG. In the secondary selection, there were eight items which only one reagent was available.(ferritin, TG, CA19-9, SCC, β-HCG, vitaminB12, folate, free testosterone), two reagents were available(TG Ab, Mic Ab, TSH-R-Ab, CA125, CA72-4, intact PTH, calcitonin), three reagents were available(T3, Tree T3, Free T4, TSH, CEA). Reasons excluded from the secondary selection were the lack of reagent supply for comparative evaluations, the problems with data reproducibility, and the inability to accept data variations. The most problematic part of comparative evaluations was sample collection. It didn't matter if the number of samples requested was large and the capacity needed for the test was small. It was difficult to collect various concentration samples in the case of a small number of tests(100 cases per month or less), and it was difficult to conduct a recovery rate test in the case of a relatively large volume of samples required for a single test(more than 100 uL). In addition, the lack of dilution solution or standard zero material for sensitivity measurement or dilution tests was one of the problems. Conclusion Comparative evaluation for changing test reagents require appropriate preparation time to collect diverse and sufficient samples. In addition, setting the total sample volume and reagent volume range required for comparative evaluations, depending on the sample volume and reagent volume required for one test, will reduce the burden of sample collection and planning for each comparative evaluation.

Review: Magnetite Synthesis using NanoFermentation (Review: NanoFermentation을 이용한 자철석 합성연구)

  • Moon, Ji-Won;Roh, Yul;Phelps, Tommy J.
    • Economic and Environmental Geology
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    • v.45 no.2
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    • pp.195-204
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    • 2012
  • Biomineralization has been explored for geochemical cycles and microbial tolerance mechanisms to metal toxicity. Here, we are introducing NanoFermentation which enables economic, environmentally friendly, requiring low input energy, and scalable manufacturing of nano-dimensioned magnetite. We are also focusing on controlling factors of crystallite size which can determine superparamagnetism and ferrimagnetism. Controlling factors are such as microbial species, temperature, incubation time, medium composition, substituted elements and their concentration, precursor type, reaction volume, precursor concentration density and their combinations. Crystallite size distribution of biomagnetite depends on the balance between nuclei generation and crystal growth. Biomineralization will elucidate elemental cycles on earth crust and microbial ecology as well as it will be applied to material sciences and devices via massive production of nanomaterials.

Effect of Sourdough Starter on the Characteristics of Rheological of Barley bread (Sourdough 첨가 보리식빵의 물성적 특성)

  • 홍정훈;김경자;방극승
    • Korean journal of food and cookery science
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    • v.16 no.4
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    • pp.358-362
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    • 2000
  • To investigate the effect of sourdough on the quality characteristics of barley bread, sourdough starlet with Lactobacillus plantarum, Lactobacillus sanfrancisco and Enterococcus mundtii was used for baking after 48 hours incubation at 37$\^{C}$. The specific volume of the bread containing sourdough starter was greater than that the control. Retarding of the firmness was observed in the bread with Lactobacillus sanfrancisco compared with the control and other breads. Above results suggest that sourdough starter fermented by Lactobacillus can be used as natural bread improver.

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Production of ${\delta}-Aminolevulinate$ by Using the Resting Cells of Rhodocyclus gelatinosus KUP-74 (Rhodocyclus gelatinosus KUP-74의 휴지균체를 이용한 ${\delta}-Aminolevulinate$의 생산)

  • Lee, So-Hee;An, Young-Chul;Lim, Wang-Jin;Hwang, Se-Young
    • Applied Biological Chemistry
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    • v.37 no.6
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    • pp.427-432
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    • 1994
  • The effectiveness of the resting cells of a photosynthetic bacterium, Rhodocyclus gelatinosus KUP-74, was investigated on the production of extracellular ${\delta}-aminolevulinate$ (ALA). The ALA generating system with 1.05 mg cells per milliliter required 6 hr-incubation to obtain maximal yield of extracellular ALA. Under this condition ALA inducers, i.e., levulinate or L-glutamate showed relatively low effects to increase extracellular ALA production. Instead, the volume of the system and proper cell density appeared to be important factors for effective production of the ALA. The life-span of the resting cells was remarkably extended to maintain stable production of the ALA by immobilization of cells.

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Identification of Lactobacillus ruminus SPM0211 Isolated from Healthy Koreans and Its Antimicrobial Activity against Some Pathogens

  • Yun Ji-Hee;Yim Dong-sool;Kang Jin-Yang;Kang Byung-Yong;Shin Eun-ah;Chung Myung-Jun;Kim Soo-Dong;Baek Dae-Heoun;Kim Kyungjae;Ha Nam-Joo
    • Archives of Pharmacal Research
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    • v.28 no.6
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    • pp.660-666
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    • 2005
  • The intestinal microbiota are important to the host with regard to resistance they impart against bacterial infections and their involvement in mediating metabolic functions. Lactic acid producing bacteria such as Lactobacillus play an important physiological role in these matters. The aim of the present study was to isolate Lactobacillus sp. that inhibits enteric pathogens. Initially, 17 isolates from healthy Koreans were collected on Lactobacillus selective medium. Resistance of the isolates to antibiotics including rifampicin, streptomycin, clindamycin and vancomycin was measured. One of the isolate was identified as Lactobacillus ruminus on the basis of bacterial cell morphology, cultural characteristic and biochemical characteristics, 16S rRNA sequence analysis and PCR-RAPD. Antimicrobial activity of the bacterium against Vancomycin Intermediate Resistant Staphylococcus aureus (VISA) and Vancomycin-Resistant Enterococci (VRE) was measured. About $10^4$ cells of VISA or VRE were mixed with 1, 5, and 9 mL of L. ruminus SPM 0211 and the final volume was adjusted to 10 mL with brain heart infusion (BHI) broth. The cell suspension was incubated for 3, 6, 9, and 24 h, serially diluted and then plated on BHI agar plates. As numbers of L. ruminus SPM 0211 were increased, viable cell count of VISA and VRE decreased. The strongest antimicrobial activity of SPM 0211 was observed after 9 h incubation in any mixture, almost completely inhibiting the growth of these two bacteria. The results suggest that the freshly isolated L. ruminus SPM 0211 may be used as a pro-biotic microbe that prevents the colonization of enteric pathogens and can thereby promote good gastrointestinal health.

Concentrations of Calcium-binding Protein and Bone Gla-protein in Culture Medium and CaBP mRNA Expression in Osteoblasts of Broiler Chickens

  • Guo, Xiaoyu;Yan, Sumei;Shi, Binlin;Feng, Yongmiao
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.2
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    • pp.239-245
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    • 2011
  • This study was conducted to determine the effects of excess vitamin A on alkaline phosphatase (ALP) activity, contents of calcium-binding protein (CaBP), bone gla-protein (BGP) in culture medium and CaBP mRNA expression in chicken osteoblasts in vitro. Osteoblastic cells in the tibia from 1-day-old Arbor Acre broiler chickens were isolated using enzyme digestion. The subconfluenced cells were divided into eight treatments with six replicates in each treatment and cultured in a medium containing either vehicle or different levels of vitamin A (0, 0.2, 0.6, 1.0, 2.0, 5.0, 10.0 and $20.0\;{\mu}g$/ml), and the control received an equivalent volume of ethanol. The incubation lasted 48 h. The results showed that vitamin A down-regulated ALP activity in the culture medium as well as CaBP mRNA expression of osteoblasts in a linear dose-dependent manner (p = 0.124 and p<0.10, respectively), and suppressed the contents of BGP and CaBP in the culture medium in a quadratic dose-dependent manner (p<0.05 and p<0.10, respectively) with increasing addition of vitamin A. The addition of 0-$0.2\;{\mu}g$/ml vitamin A to the culture medium increased ALP activity, BGP and CaBP contents as well as CaBP mRNA expression compared with other groups, but positive effects of vitamin A tended to be suppressed when vitamin A was increased to $1.0\;{\mu}g$/ml, and adverse effects occurred when vitamin A was increased to 10.0-$20.0\;{\mu}g$/ml. These results implied that there was a threshold level of vitamin A inclusion beyond which inhibitory effects occurred, and the mechanism by which overdose of vitamin A reduced bone growth in chickens was probably reduced osteoblastic cell activity, and inhibited expression of CaBP mRNA and CaBP secretion.

Characteristics of Protoplast Induction by Autolytic Enzyme of Rhizopus oryzae (Autolytic Enzyme을 이용한 Rhizopus oryzae의 원형질체 생성과 그 특성에 관한 연구)

  • Rhee, Young-Ha;Choi, Yong-Keel
    • The Korean Journal of Mycology
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    • v.14 no.4
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    • pp.273-280
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    • 1986
  • The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

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Production of polyclonal anti-$\beta$-adrenergic receptor antibody and it′s effects on receptor ligand binding

  • Kim, Hee-Jin;Shin, Chan-Young;Noh, Min-Su;Ko, Kwang-Ho
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1995.04a
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    • pp.86-86
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    • 1995
  • The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently by the use of specific anti-receptor antibodies. A 14-mer peptide (from Phe102 to Leu115 of ${\beta}$2-adrenergic receptor) was synthesized and this peptide was coupled to carrier protein Keyhole Limpet Hemocyanin(KLH) by glutaraldehyde method. A 0.5mg of KLH-coupled peptide was emulsified with equal volume of complete Freund's adjuvant and injected via popliteal lymph node to each of the three Newzealnd White rabbits. Booster injections were repeated at 4 weeks interval for three times with incomplete Freund's adjuvants. One week after the final injection, serum was prepared from ear artery. Nonspecific immunoglobulins were removed by passing the serum through KLH-Sepharose 6B affinity matrix and further by incubation with bovine lung aceton powder. The titer of the antibody for synthetic peptide which was determined by enzyme linked immunosorbent assay(ELISA) was about l/l,000. The antibody produced in this study revealed 67kDa protein band in the western blot of partially purified guinea pig lung ${\beta}$-adrenergic receptor preparation. The antibody inhibited ${\beta}$-adrenergic antaginist [3H] Dihydroalprenolol binding to soluble ${\beta}$-adrenergic receptor by 25% while control sera did not show any inhibitory effects, The result of this study suggests that the peptide sequence selected in this study may play some important roles in adrenergic receptor-ligand interaction.

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DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

  • Hawash, Yousry
    • Parasites, Hosts and Diseases
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    • v.52 no.3
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    • pp.263-271
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    • 2014
  • PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. $QIAamp^{(R)}$ DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume ($50-100{\mu}l$) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ${\approx}2$ oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

Antagonistic activity of Streptomyces apecies against Fusarium solani causing ginseng root rot (인삼뿌리 썩음 병균 Fusarium solane에 대한 Streptomyces species의 길한작용)

  • 정영륜;오승환;정후섭
    • Korean Journal of Microbiology
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    • v.27 no.1
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    • pp.56-62
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    • 1989
  • Antagonistic effects of Streptomyces species aganinst Fusarium solani causing ginseng root rot were investigated in terms of chitinase activity and growth inhibition in vitro. Among 131 isolates of streptomycetes obtained from ginseng cultivating soil, 9 isolates producing large clear zone around the colony on a chitin agar medium were selected for further study. All 9 isolates produced chitinase in a range from 0.10 to 0.38 U lysing cells of F. solani and inhibited germination of the conidia. In the ten-fold condentrated culture filtrate of S. alboniger ST59 and S. roseolilacinus ST129, the number of conidia of F. solane was reduced to about 20% of original count within 14 days. When S. alboniger ST59 and F. solani were grown simultaneously in the mineral saly medium, chitinase activity increased with incubation period, whereas mycelial volume of F. solani decreased. In a chitin added mineral salt medium, chitinase activity increased during the first four days and maintained steady level until the 8th day, and increased thereafter. S. alboniger ST59 lysed mycelia, conidia and even chlamydospores of F. solani. It is probable that the antagonistic activity of this streptomycete against F. solani is the lysis of fungal cell wall by streptomycete producing chitinase affected by antifungal substances.

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