• 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.

• Journal of Pharmaceutical Investigation
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• 제16권2호
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• pp.85-88
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• 1986

To estimate the effect of some injectable antibiotics (ampicillin sodium, cefazolin sodium, cephaloridine, cefuroxime sodium and chloramphenicol sodium succinate) on pyrogen tests, the Limulus amebocyte lysate (LAL) test and an ultrafiltration technique were used. The rabbit pyrogen test was also used in the case of cafazolin sodium. At high antibiotic concentrations, these samples which were artificially contaminated with endotoxin inhibited the gelation reaction of LAL. But the gelation reaction occurred when most of the antibiotic was removed by ultrafiltration. Likewise, cefazolin sodium interfered not only with the LAL test but also with the rabbit pyrogen test. From these results it can be said that special modification to eliminate interference should be taken into consideration for valid method of pyrogen tests in the parenteral products containing these antibiotics.

• 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.

• 한국식품과학회지
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• 제52권1호
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• pp.109-112
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• 2020

본 연구에서는 6종의 E. coli 및 S. aureus 유래 박테리오파지 용액에 존재하는 내독소 함량 조사와 파지의 세포독성 여부에 대해 평가하였다. 대표적인 생물학적 내독소 시험법인 LAL assay을 시행하여 9-10 log PFU/mL 농도의 내독소 함량을 확인한 결과, 파지의 임상적용에는 부적합한 수치이나 식품에 존재하는 병원균의 살균 목적의 사용에는 매우 유해하지 않은 수치임을 확인할 수 있었다. 박테리오파지 용액의 세포독성평가를 확인하기 위해 MTT 분석을 시행하여 세포 생존율을 확인하였다. E. coli 파지 용액과 S. aureus 파지 용액 처리군 모두에서 98% 이상의 생존율이 관찰되어 파지용액에 존재하는 내독소 및 파지가 세포독성을 유발하지 않는다는 것을 확인하였다. 그러므로 일반적인 방법으로 증폭, 농축한 파지 용액은 인체에 대한 유해성이 적으며 식품에 살균소독제로 적용하더라도 문제가 없을 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제41권2호
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• pp.242-248
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• 2013

Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria and are continuously released during bacterial growth. Endotoxins typically induce severe sepsis and septic shock, which cause more than 50% of mortalities. Endotoxins are easily measured in the serum by the limulus amebocyte lysate (LAL) test. However, a nonspecific result is obtained, because the high concentration of serum proteins disturbs the enzyme reaction of the LAL test. In order to solve this problem, the LAL test was performed in this study after the centrifugation of the boiled serum samples to remove the impurities. As a result, among the various conditions examined, endotoxin measurement with the LAL test was the most accurate and repeatable after centrifugation of the boiled serum at $100^{\circ}C$. Moreover, the endotoxin was accurately and repeatedly measured from the prepared sera of mice that had been administered an intraperitoneal injection of purified lipopolysaccharides (LPS) or E. coli. Therefore, the application of centrifugation to remove impurities from boiled serum gives an accurate measurement of endotoxins in the sera of normal subjects or patients, and this will lead to the improved diagnosis and prevention of diseases caused by endotoxins. In addition, the centrifugation of boiled serum samples should be considered and included in the development of endotoxin test kits.

• 대한임상검사과학회지
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• 제48권2호
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• pp.124-129
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• 2016

세균내독소는 lipopolysaccharide (LPS)라고 불리우며, 그람음성 세균의 세포벽에 존재하는 물질로 어느 생체재료나 액체에 존재할 수 있다. 세균내독소가 혈액으로 침투되면 발열 및 염증을 유발시킨다. 본 연구에서는 장내 그람음성세균인 Escherichia coli O157:H7, Klebsiella oxytoca, Salmonella Typhi, Shigella sonnei, Morganella morganii의 LPS 양을 비교하려 하였다. 각 세균은 배양한 후 $1.5{\times}10^8CFU/mL$ 농도로 희석하여 동일한 농도로 하였으며 실험의 정당성을 위하여 LAL시약의 표시감도확인시험을 진행하였다. 10 fold dilution 후 1차 시험을 하여 각 균의 반응 end point를 확인하고 바로 윗단계 희석배수 사이에서 2 fold dilution하여 균의 최종 반응 end point를 확인하였다. 결과적으로 E. coli O157은 75~37.5 CFU/mL, K. oxytoca는 37.5~18.75 CFU/mL, M. morganii와 S. Typhi는 3.75~1.875 CFU/mL, S. sonnei는 7.5~3.75 CFU/mL에서 0.015 EU/mL 이상의 endotoxin이 검출되었다. 최종적으로 억제인자에 대한 확인시험을 진행하여 결과값을 보증하였다. 본 연구를 통하여 세균내독소에 대한 추가적인 연구가 지속되기를 기원한다.