• YAKHAK HOEJI
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• v.43 no.5
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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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• v.18 no.5
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• pp.429-433
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• 2003

LAL (Limulus amebocyte lysate) test to detect and quantity endotoxin is based on gellation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive requiring dedicated personnel, takes relatively long reaction time (approximately 1 hr), requires relatively large volume of samples and reagents, and its end-point detection method is rather subjective. To solve these problems, we attempted to develop a miniaturized LOC (lab-on-a-chip) prototype using PDMS and glass. Using the 62 mm (length) ${\times}$ 18 mm (width) prototype in which 2 mm (width) ${\times}$ 44.34 mm (length) ${\times}$ 100 $\mu\textrm{m}$ (depth) microfluidic channel was provided, we compared the various detection methods of gellation, turbidometric, and chromogenic assays to find the chromogenic method to be the most suitable for small volume assay. In this assay, kinetic point method was more accurate than end point method. We also found the PDMS chip thickness should be minimized to around 2 mm to allow sufficient light transmittance, which necessitated a glass slide bonding for chip rigidity. Through the miniaturization, the test time was reduced from 1 hr to less than 10 minutes, and the sample volume could be reduced from 100 ${\mu}\ell$ to 4.4 ${\mu}\ell$. In sum, this study revealed that the mini LOC could be an alternative for a semi-automated and reliable method for LAL test.

• Korean Journal of Clinical Laboratory Science
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• v.56 no.2
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• pp.171-180
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• 2024

The endotoxin test is based on the reaction between Limulus Amebocyte Lysate (LAL) and the lipopolysaccharides of Gram-negative bacteria. In this study, we sought to identify factors that interfere with the LAL testing of radiopharmaceuticals and evaluated acceptable ranges. A gel-clot LAL test and a chromogenic LAL test were used as endotoxin tests. We compared the performances of the Endosafe^Ⓡ LAL and recombinant Endosafe^Ⓡ Recombinant Cascade Reagent (rCR) cartridges for the chromogenic test. The factors that interfered with ⁶⁸Ga-DOTATOC injection were pH, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer, and organic solvents, especially ethanol. However, interference by these factors was overcome by diluting the ⁶⁸Ga-DOTATOC injection tenfold. In addition, no interference was observed at pH values between 4 and 8, at a HEPES concentration of 2,000 ㎍/mL, or an ethanol concentration of <1%. Furthermore, results showed that interfering factors had similar effects on the performances of the Endosafe^Ⓡ LAL and Endosafe^Ⓡ rCR cartridges. The results of this study are expected to be useful for evaluating factors that interfere with the endotoxin testing of new radiopharmaceuticals.

• Proceedings of the PSK Conference
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• 2003.10b
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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 Microbiology and Biotechnology
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• v.16 no.2
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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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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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 Pharmaceutical Investigation
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• v.16 no.2
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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.

• Korean journal of applied entomology
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• v.25 no.4 s.69
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• pp.229-233
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• 1986

By using the test fungus Macrophomina phaseolina, residues of triadimefon were found in straw collected after harvest from sprayed plants of wheat varieties Kharchia and Lal Bahadur but grains contain no such residues. Thin layer chromatographic method was developed to detect residues of the fungicide which was found to be present in straw of sprayed plants of both the varieties. No residues could be detected in grain samples. It was found that triadimefon was converted in triadimenol in/to host.

• The Korean Journal of Nuclear Medicine Technology
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• v.13 no.3
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• pp.156-158
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• 2009

Purpose: Since radiopharmaceuticals are intended for human administration, it is imperative that we should undergo quality control very strictly. Now almost all the PET laboratories have adopted Bacterial Endotoxin Test as the stand quality control method to monitor whether pyrogen is free or not in the product vial containing crude solution. The aim of this study is to find out the reason why false positive result is observed when using commercially available test vial. Materials and Methods: For this experiment, we used commercially available single test kit (Associates of Cape Code. Inc. USA) and we made pH samples by mixing each buffer whose pH ranges are 1.0 to 12.0. Otherwise we made Ethanol samples diluted with distilled water. After making test samples, it added 0.2 mL to the test vial. Assay mixture in the test vial was incubated in a water bath (Chang Shin Co. KOR) for 60 min at $35{\pm}2^{\circ}C$. Results: After incubation period ($60{\pm}1^{\circ}C$), we inverted the test vial about $180^{\circ}$ To know what pH and how many percentage of Ethanol (Fisher Scientific Korea. Ltd) will affect the reaction. With pH buffer, false positive result was observed at pH 1.0 to 5.0 and 7.7 to 12.6 but at pH 5.2 to.7.5, the test results show negative. It's very strange that we couldn't observe negative test result with Tris buffer at pH 8.4, 8.6, 8.8, 9.0. in other case Ethanol, the test result was seen with 5 to 10% Ethanol. But to my surprise we could see very thick gel formation with 100% Ethanol. Conclusions: In this study, we could notice that pH which is too much acidic or alkalic or high concentrated Ethanol would affect Bacterial Endotoxin Test result. As you know, LAL test is sensitive and very reliable method. Therefore, we are needed to elicit the accurate test result as possible as we can.