Although many methods for the Limulus tests have been reported, the following 3 methods are currently widely used:

　(1) Gel-clot Method ^{1, 2)}

　(2) Kinetic Turbidimetric Assay (Onset time method) ^{3, 4, 5)}

　(3) Synthetic Substrate Method ^{6, 7)} (Chromogenic Assay - KCA)

In the gel-clot method, LAL and samples are mixed in 10x75mm test tubes and allowed to react at 37℃ for 60 minutes. At the completion of the reaction time the tubes are then inverted 180 degrees and gel formation is judged visually. This method is semi-quantitative, relatively easy to perform, and economical because no special equipment is required.

In the Kinetic Turbidimetric Assay (KTA; onset time method), the LAL gelation process is recorded as the change in light transmittance intensity. The time until light transmittance changes to a certain fixed ratio is defined as the gelation time (Tg), and endotoxins are quantified based on the relationship between Tg and endotoxin concentration. Although this method requires specialized equipment, it is easy to operate and is objective because the device automatically detects gelation. In addition, it is very sensitive, quantitative, and economically efficient in terms of reagents, and has an extremely wide calibration range when compared with other methods. Our Toxinometer^® is one type of this specialized equipment. The kinetic chromogenic assay has recently been commercialized and applies principles similar to KTA, but for the synthetic substrate method.

In the synthetic substrate method, a chromogenic synthetic substrate (e.g. Boc-Leu-Gly-Arg-pNA) for the clotting enzyme is added to the reaction mixture. The synthetic substrate is hydrolyzed simultaneously with the reaction of LAL and endotoxin (refer Figure 1 in Episode 3), and endotoxins are quantified based on the amount of chromophore released from the substrate. The operation is slightly complicated and the reagent is expensive, but it is very sensitive, quantitative, and precise.

The gel-clot method was the first Limulus method to be established and it is the method listed in both the US Pharmacopoeia¹⁾ and the Japanese Pharmacopoeia²⁾. The kinetic turbidimetric assay and the synthetic substrate method are also listed in FDA guidelines⁸⁾, and have been officially adopted into the Pharmacopoeias.

Method selection depends on the purpose of the measurement and availability of equipment. The characteristics of each method should be taken into consideration: the gel-clot method requires no special equipment aside from an incubator; the kinetic turbidimetric assay enables a wide-range quantitative analyses; and the synthetic substrate mFethod has a high measurement precision.

Our evaluation of each method is shown in Table 1. Of course, this evaluation changes depending on the user. In any case, it is necessary to have a good understanding of the characteristics of each method and to select a measurement method suitable for the user's purposes.

Table 1 A comparison of the 3 methods of the Limulus test

◎: Excellent
○: Good
△: Slightly poor
×: Poor

	Gel-clot Method	Synthetic Substrate Method	Kinetic Turbidimetric Assay
Ease of operation	○	×	◎
Economic Efficiency	△	△	◎
Quantitative Precision	△	◎	○
Sensitivity	△	○	○
Processing Capacity	×	△	◎
Required Equipment	Incubator	Incubator Spectrophotometer	Specialized equipment

References

1. The United States Pharmacopeia 22th, The National Formulary 17th, p.1493-1495. Pharmacopeial Convention Inc., MD (1989).
2. Supplement B-11 to the Japanese Pharmacopeia 11th Edition, B-11 (1988).
3. Oishi, H., Hatayama, Y., Shiraishi, H., Yanagisawa, K., Sakata, Y., Journal of the Pharmaceutical Society of Japan, 105, 300-303 (1985)
4. Oishi, H., Takaoka, A., Hatayama, Y., Matsuo, T. and Sakata, Y., J. Parenter. Sci. Technol., 39, 194-200 (1985)
5. Novitsky, T. J., Remillard, J. F. and Loy, N., Detection of Bacterial Endotoxins With the Limulus Amebocyte Lysate Test (Eds. : Watson, S. W., Levin, J. And Novitsky, T. J.), 189-196, Alan R. Liss Inc., New York (1987).
6. Nakamura, S., Morita, T., Iwanaga, S., Niwa, M. and Takahashi, K., J. Biochem., 81, 1567-1569 (1977)
7. Iwanaga, S., Morita. T., Harada, T., Nakamura, S., Niwa, M., Takada, K., Kumura, T. and Sakakibara, S., Haemostasis, 7, 183-188 (1978)
8. Guideline on Validation of The Limulus Amebocyte Lysate Test as an End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, FDA (1987)