• 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.

• 대한임상검사과학회지
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• 제56권2호
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• pp.171-180
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• 2024

엔도톡신 시험은 투구게의 혈구세포 추출성분인 라이세이트(Limulus Amebocyte Lysate, LAL)가 그람음성균의 지질다당체와 반응하여 응고하는 원리를 이용한 in vitro 시험법이다. 엔도톡신이 혈액 내 노출될 경우 환자에게 심각한 부작용을 발생시킬 수 있으므로 진단용 방사성의약품은 제조 후 반드시 엔도톡신 시험을 통해 의약품 내 오염여부를 확인해야 한다. 진단용 방사성의약품은 유기용매와 완충용액 등을 사용하여 화학반응을 통해 제조되므로 다양한 반응간섭물질이 의약품 내 존재할 수 있다. 이에 본 연구에서는 방사성의약품에 존재하는 엔도톡신 시험의 반응간섭인자를 분석하고 이들의 허용범위를 평가하고자 하였다. 방사성의약품의 엔도톡신 시험을 위해 겔 응고 LAL시험과 비색 LAL시험을 실시하였고, 비색 LAL시험에서는 혈액 성분으로 만든 Endosafe^Ⓡ LAL cartridge와 재조합 단백질로 만든 Endosafe^Ⓡ rCR cartridge의 성능을 비교하였다. 방사성의약품 ⁶⁸Ga-DOTATOC 주사액의 엔토톡신 시험 반응간섭인자 평가 시 pH, 유기용매, 완충용액이 있었으며, 그 중 유기용매인 에탄올이 엔도톡신 시험에 간섭현상을 일으켰다. 하지만 ⁶⁸Ga-DOTATOC 주사액을 10배 희석할 경우 간섭현상을 극복할 수 있었다. 진단용 방사성의약품 내 존재할 수 있는 반응간섭인자들의 허용범위를 평가하였으며, pH의 경우 pH 4~8에서는 간섭현상이 없었고, 유기용매의 경우 에탄올 1% 이하에서는 간섭현상이 발생되지 않았다. 완충용액 HEPES는 2,000 ㎍/mL까지는 간섭현상이 발생되지 않았다. 본 연구를 통해 진단용 방사성의약품의 엔도톡신 시험에서 유기용매인 에탄올의 농도가 가장 중요한 반응간섭인자임을 확인하였고, 적절한 희석을 통해 1% 미만의 농도로 낮출 경우 엔도톡신 시험을 시행할 경우 간섭을 극복할 수 있음을 알 수 있었다. 또한 Endosafe^Ⓡ LAL cartridge와 Endosafe^Ⓡ rCR cartridge의 회수율도 비슷하게 측정되어 성능에 큰 차이가 없음을 확인할 수 있었다. 본 연구결과는 향후 새로운 진단용 방사성의약품의 엔도톡신 시험 시 반응간섭인자를 평가하는데 큰 도움이 될 것으로 기대한다.

• 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.

• 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.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.162.1-162.1
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• 2003

Endotoxin has been detected by the Limulus amoebocyte lysate (LAL) test. However, aluminum hydroxide used as an adjuvant and adsorbent for the recombinant protein antigen is known to increase efficacy of lipopolysaccharide vaccine in vivo thus interfering endotoxin test. The aim of this study is to determine effect of aluminum hydroxide on the LAL test using the hepatitis B vaccine as a model and to optimize the LAL test condition not to be interfered by aluminum gydroxide. (omitted)

• 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.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.219-225
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• 2006

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins present in vaccines. Currently, the rabbit pyrogen test is used to detect endotoxins in hepatitis B (HB) vaccines, even though the HB surface protein, which is the active ingredient, is overexpressed in and purified from eukaryotic cells that lack these endotoxins. Although the LAL clot assay is sensitive and reliable and can be used to replace the rabbit pyrogen test, its reaction is limited by the lack of responsiveness to the Gram-positive bacterial components. Furthermore, aluminum hydroxide in the HB vaccine can interfere with the LAL assay. In contrast, macrophages can detect the endotoxin as well as other pyrogens, and secrete $TNF-{\alpha}$. Therefore, this study was undertaken to examine the possibility of replacing the animal tests with a more efficient $TNF-{\alpha}$ secretion assay. With this in mind, we determined if aluminum hydroxide in the HB vaccines affects the $TNF-{\alpha}$ secretion assay. HB vaccines and the HB protein solutions spiked with lipopolysaccharide (LPS) produced the same level of dose-dependent $TNF{\alpha}$ secretion and temperature increase in rabbits, indicating that aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbits, nor does it interfere with $TNF-{\alpha}$ secretion. In addition, the $TNF-{\alpha}$ assay was found to be more sensitive than the LAL assay, and correlated well with the pyrogen test and the LAL assay. These results suggest that the $TNF-{\alpha}$ assay in RAW264.7 cells is a good substitute for the current pyrogen assays that are used for detecting LPS in HB vaccines as well as in other vaccines containing aluminum.

• Asian-Australasian Journal of Animal Sciences
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• 제33권7호
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• pp.1087-1095
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• 2020

Objective: The present study aimed to evaluate the effects of γ-aminobutyric acid (GABA)-producing bacteria (GPB) on in vitro rumen fermentation and on the growth performance and meat quality of Hanwoo steers. Methods: The effects of GPB (Lactobacillus brevis YM 3-30)-produced and commercially available GABA were investigated using in vitro rumen fermentation. Using soybean meal as a substrate, either GPB-produced or commercially available GABA were added to the in vitro rumen fermentation bottles, as follows: control, no additive; T1, 2 g/L GPB; T2, 5 g/L GPB; T3, 2 g/L autoclaved GPB; T4, 5 g/L autoclaved GPB; T5, 2 g/L GABA; and T6, 5 g/L GABA. In addition, 27 Hanwoo steers (602.06±10.13 kg) were subjected to a 129-day feeding trial, during which they were fed daily with a commercially available total mixed ration that was supplemented with different amounts of GPB-produced GABA (control, no additive; T1, 2 g/L GPB; T2, 5 g/L GPB). The degree of marbling was assessed using the nine-point beef marbling standard while endotoxin was analyzed using a Chromo-Limulus amebocyte lysate test. Results: In regard to in vitro rumen fermentation, the addition of GPB-produced GABA failed to significantly affect pH or total gas production but did increase the ammonia nitrogen (NH₃-N) concentration (p<0.05) and reduce total biogenic amines (p<0.05). Animals fed the GPB-produced GABA diet exhibited significantly lower levels of blood endotoxins than control animals and yielded comparable average daily gain, feed conversion ratio, and beef marbling scores. Conclusion: The addition of GPB improved in vitro fermentation by reducing biogenic amine production and by increasing both antioxidant activity and NH₃-N production. Moreover, it also reduced the blood endotoxin levels of Hanwoo steers.