• 약학회지
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• 제43권5호
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• pp.606-613
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• 1999

To survey the possibility of replacing the pyrogen test with Limulus Amebocyte Lysate(LAL) test and to find out a standard methods suitable to our blood products made in Korea, 100 samples of 20% human serum albumin were tested by commercial LAL test kits and results of those were compared with rabbit pyrogen test. The LAL test is used both dinetic-chromogenically and kinetic-turbidimetrically. Both methods equally showed broad detection range (5.0~0.005 EU/ml), excellent sensitivity ($\geq$ 0.005 EU/ml) and predominant recovery rate within valid dilution range, but kinetic-turbidimetric method seemed to be more reproducible than kinetic-chromogenic method(kinetic-chromogenic method : S.D. = 15.88, kinetic-turbidimetric method : S.D. = 8.12). After heating the sample at 75$^{\circ}C$ for 15 min, the results showed a little elevated recovery rate with both methods. After performing the test on 100 albumin samples with both kits, the results were analysed using the USP standard (1.33 EU/ml). 7% of samples in kinetic-chromogenic methods and 1% of samples in kinetic-turbidimetric method exceeded the limit of endotoxin levels regulated for blood products in USA. Because this phenomenon was not observed in both methods at the same time and both methods have high sensitivity ($\geq$0.005 EU/ml), these results seemed to depend on nonspecific reaction. Considering its sensitivity and reproducibility, we could assure that LAL test is proper to detecting pyrogenic with good sensitivity.

• KSBB Journal
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• 제18권5호
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• pp.429-433
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• 2003

LAL 측정용 chip을 제작하기 위해서 우선 시료의 부피 감소에 대한 비탁법과 비색법을 비교하였다. 비색법은 낮은 부피에서 높은 감도를 보여 주었으며 시료의 부피와 무관하게 같은 endotoxin의 농도에서는 같은 흡광도를 보인다는 결론을 얻었다. Endotoxin의 농도에 따른 표준곡선을 end point법과 kinetic point법을 비교한 결과 대한약전의 기준에 적합한 kinetic point법이 적합하였다. 이러한 기초 실험결과를 통해 PDMS LOC를 제작하여 LAL 시험을 수행하였다. LOC를 이용하여 더 짧은 시간과 더 작은 시료로 시험이 가능하도록 하였다. 특히 PDMS LOC는 복잡한 channel을 쉽게 만들 수 있을 뿐 아니라 mold를 이용하여 상용화를 위한 대량 생산이 가능하다. 따라서 PDMS를 이용한 LOC의 제작과 실험을 통해 기존의 수작업의 LAL 시험을 LOC를 이용한 다중시료 측정과 자동화의 가능성을 제시하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권2호
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• pp.132-136
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• 2004

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (lab-on-a-chip) prototype, 62mm (L) ${\times}$ 18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2mm (W) ${\times}$ 44.3mm (L) ${\times}$ 100 $\mu\textrm{m}$ (D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PDMS chip thickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 to ca. 4.4 ${\mu}$L. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.

• 핵의학기술
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• 제16권2호
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• pp.3-6
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• 2012

방사성의약품 제조 후 품질관리의 중요성이 점점 커지고 있다. 품질관리 항목에는 물리적 성상, PH, 핵종의 순도 측정, 화학적 순도 측정, 방사화학적 순도 측정, 내독소 검사, 크립토픽스 잔류량 측정 등이 있다. 내독소 검사는 발열 및 쇼크등을 일으킬 수 있는 그람음성간균의 리포다당질이 포함된 검체가 인체에 주입되는 것을 방지하기 위한 검사법이다. FASTlab module을 이용하여 합성된 $^{18}F$-FDG의 내독소 검사 시행 시 간섭요인이 발생하여 제대로 된 검사가 시행되지 못하여 희석하는 방법을 이용하였다. LAL free water를 이용하여 비색법은 25 ${\mu}l$, 겔화법은 200 ${\mu}l$를 최소단위로 하여 희석배수가 10배수를 초과하지 않는 범위내에서 희석배수를 10배수, 20배수, 30배수, 40배수로 하여 희석하였다. 희석된 시약을 겔화법은 STT와 PCT에 각각 200 ${\mu}l$를 주입하고 Incubator에 $37^{\circ}C$로 1시간동안 배양하여 겔형성 정도를 확인하고, 비색법은 25 ${\mu}l$를 최소단위로 하여 위와 같이 희석한다. 희석된 시약은 ENDOSAFE-PTS의 Dilution factor를 입력하고 배수 당 하나의 카트리지를 4곳의 주입구에 25 ${\mu}l$씩 주입하고 결과 데이터를 확인한다. 결과는 겔화법은 FASTlab의 경우 40배수에서만 겔이 형성되었고, TRACERlab Mx의 경우 거의 모든 배수에서 겔이 형성되었지만 간헐적으로 형성되지 않는 경우도 있었다. 비색법은 FASTlab의 경우 40배수에서만 Spike Rxn time CV, Sample Rxn time CV, Spike recovery 모두 정상범위 안에 포함되었다. TRACERlab Mx의 경우 10배수에서 Spike Rxn time CV, Sample Rxn time CV, Spike recovery 모두 정상범위 안에 포함되었다. FASTlab에서 생산된 방사성의약품은 중화제인 $H_3PO_4$가 PCT내에 겔 형성에 관여하는 $Mg^{2+}$이온과 결합하여 겔 형성을 하지 못하는 것으로 추측된다. 이에 희석을 할 경우 $H_3PO_4$ 양이 줄어 간섭효과를 제거할 수 있었다. 엔도톡신 검사에서 간섭인자들은 무수히 존재한다. 이 간섭인자들은 합성장치나 시약에 따라 달라질 수 있으므로 이와 같은 간섭인자들을 극복하기 위한 노력으로 품질검사의 정확성과 신뢰를 제공하도록 해야 할 것이다.

• 한국산업보건학회지
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• 제20권4호
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• pp.274-281
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• 2010

The objectives of this study are (a) to investigate the distribution patterns and exposure concentrations of biological agents in sawmill industries and (b) to compare sampling methods of biological agents. The representative processes of 5 sawmills were selected to measure total airborne bacteria, fungi, endotoxin as well as dust. Airborne bacteria and fungi were measured with one stage impactor, six stage impactor and gelatin filteration methods. Endotoxin was collected with polycarbonate filters and analysed by kinetic chromogenic Limulus Amebocyte Lysate method. Geometric mean levels of airborne bacteria, fungi, endotoxin and dust were 1,864 CFU/$m^3$, 2,252 CFU/$m^3$, 31.5 EU/$m^3$ and 2.4 mg/$m^3$. The ratios of indoor/outdoor concentrations were 3.7 for bacteria, 4.1 for fungi, 3.3 for endotoxin and 9.7 for dust. The respiratory fractions of bacteria were 68.0, 50.9, 49.2 and 45.1% in band-saw, table-saw, rip-saw process and outdoor air. The respiratory fractions of fungi were 78.7, 90.8, 87.5 and 84.8% in band-saw, table-saw, rip-saw process and outdoor air, respectively. There was no significant differences in bacterial concentrations among single stage, six stage impaction and filteration methods. But, fungal concentrations measured with filtration methods were significantly higher than those with impaction methods. Geometric mean levels of airborne bacteria and fungi were higher than the OSHA guideline values of 1,000 CFU/$m^3$. The respiratory fractions of fungi were above 75%. The concentrations of biological agents were significantly different among culture-based sampling methods. In the exposure assessments of biological agents, further studies are needed for the comparisons of diverse sampling methods and the investigations of environmental factors.