-Contents-
Establishment of Limulus test
Endotoxin-specific assays
Glucan-specific assays
Pretreatment methods of plasma
Plasma endotoxin levels in spsis and SIRS
Plasma endotoxin levels in other diseases than sepsis
Plasma glucan levels in depp mycoses and in some clinical situations
Why we should use endotoxin-specific limulus tests for assaying endotoxin in clincal samples
★Endotoxin activity assay (EAA)
REFERENCES

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The name of "Limulus" is derived from a scientific name of the American horseshoe crab; Limulus polyphemus. Four species of horseshoe crabs exist in limited regions of the world. L. polyphemus is living in the East coast of America. Tachypleus tridentatus is living in coasts of Japan and China. T. gigas and Carcinoscorpius rotundicauda are found in the Western Pacific and Indian Ocean.

In 1956, Frederick B. Bang discovered that Gram-negative bacteria caused the blood of a horseshoe crab, L. polyphemus, to clot (Ref. 1). He, with Jack Levin, found that the lysate of amebocyte (circulating blood cells, hemolymph) obtained by osmotic shock clotted on addition of purified endotoxin (Ref. 2).


Prof. S. Iwanaga and his colleagues contributed to the development of quantitative limulus test. They have found that the clotting enzyme and its substrate, coagulogen, are involved in the gel fotmation. A quantitative limuls test by adding a synthetic chromogenic substrate into the lysate was establised by them (Ref. 3). The quantiative limulus test kit (Toxicolor) was commercialized by Seikagaku Corporation Tokyo, Japan (Fig. 1).



Endotoxin-specific assays
As Prof. S. Iwanaga and his colleagues found the endotoxin and glucan involved pathway of the coagulation system (Ref. 4, 5, Fig. 1), Seikagaku Corporation (Tokyo, Japan) commercialized Endotoxin-specific test by removing factor G from the lysate of T. tridentatus (Fig. 1, Ref. 6). The lysate was fractionated by a chromatography and reconstituted fractions other than factor G-containning fraction. This endotoxin-specfic assay, Endospecy, is a chromogenic quantitative test, like the conventional Toxicolor test.








Glucan-specific assays
Glucan-specific limulus tests was developed by deleting factor C fraction from the lysate: Gluspecy, Seikagaku Corporation (Ref. 10).

Wako Pure Chemical Industries Ltd. developed an unique (β-(1, 3)-glucan test by inhibiting the endotoxin activity in test specimen with polymyxin B (Ref. 11) (Fig. 3).

Maruha Corporation, Tokyo, Japan also established a glucan-specific test (B-G Star)(Ref.12).

Recently, FDA approved glucan-specific chromogenic limulus test (Glucatell, ACC) for the diagnosis of fungemia.

We, with Immunology Institute, Tokyo, Japan, have developed monoclonal antibodies against activated factor C. The monoclonal antibodies were produced by a fusion with myeloma cells and mouse spleen cells immunized with LPS-factor C complex. Monoclonal antibodies which reacted with the complex but not with LPS or factor C were selected. When an appropriate amount of the monoclonal antibody was added to the lysate, the lysate responds only to glucan but not to LPS (Ref.13).

Table 　Major Limulus tests available in Japan
Kit	Company	Specificity1)	Method2)
Toxicolor	Seikagaku3)	E/G	C
Endospecy	Seikagaku	E	C
Gluspecy#	Seikagaku	G	C
HS-test Wako	Wako4)	E/G	T
ES-test Wako#	Wako	E	T
β-D-glucan Test Wako#	Wako4)	G	T
B-G Star#	Maruha5)	G	C
QCL-1000, Kinetic QCL	Ronza Japan6)	E/G	C
Pyrogent5000	Ronza Japan	E/G	T
PyroGene	Ronza Japan	E	F
Endosafe-PTS	Charles River7)	E/G	C

1) E: Specific for endotoxin, G: Specific for glucan, E/G: Reacts with both endotoxin and glucan
2) C: synthetic chromogenic method, T: turbidimetric kinetic assay, F: fluorscence method
3) Seikagaku Corporation
4) Wako Pure Chemical Industries Ltd.
5) Maruha Corporation
6) Ronza Japan
7) Charles River
8) This test uses a conventional limulus lysate (HS-Test Wako). A test sample is pretreated with polymyxin B to inhibite endotoxin activity.
#These tests are authorized by the Ministry of Health and Welfare of Japan as being diagnostic tools for the health insurance adjustment.

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Interfering factors, i.e., inhibitors and non-specific amidase activity for limulus reaction, are present in human plasma, and it must be removed before testing.

For this aim, a perchloric acid (PCA) method was devised for Toxicolor and Endospecy (Ref. 14): PCA is added to plasma and the precipitate (proteins) is discarded after centrifugation, and the supernatant is used for Endospecy test.
However, we found that this method can not offer to measure protein-bound endotoxin in plasma. Then we developed a devised perchloric acid method ( (New PCA method, Ref. 15, 16). This new method allows us to detect the total content of endotoxin in the plasma. About 8 to 10 times the endotoxin content and 1.4 times the glucan content can be measured by the new PCA method which is more than by the PCA method.

A new method using an alkali reagent was thereafter developed for pretreatment of plasma endotoxin (Ref. 17). This method was reported to be shown comparable result with New PCA method. However, we pointed out the occurrence of false positive reaction by the generation of turbidity at early time during incubation in this method (Ref. 18). This method is no longer used subsequently for endotoxin assay.

Dilution and heating method has been widely used for limulus tests. Wako Pure Chemical Industries Ltd. devlpoed an improved dilution and heating method, for a turbidimetric kinetic assay. In this method 0.02% Triton X-100 is used instead of water for the 10-times dilution of plasma, and heating condition was set at 70 degree C for 10 min (Ref. 19).


We investigated the plasma endotoxin levels in many situations, using endotoxin-specific limulus test (Endospecy) with the new PCA method. Cut-off value wa set at 9.8 pg/ml by the measurement of endotoxin levels of volunteer's plasmas (Ref. 20).

The plasama endotoxin value often exceeded the cut-off value in a sepsis, septic shock, and the typhoid fever (Ref. 20, 21).

As a conclusion, we have found that high plasma endotoxin levels are related to local or systematic infection due to gram-negative microrganisms.

SIRS (systemic inflammatory response syndrome) is a proposed concept for systemic inflammation in infectious and noninfectious conditions (Ref. 22). SIRS is thought to be due to the high cytokinemia induced by some bacterial components. We concluded that endotoxemia was related to a part of SIRS (Fig. 4).





Plasma endotoxin levels in other diseases than sepsis
It has been reported that major stresses such as burns, massive trauma,or hemorrhagic shock produce ischemia of the intestinal tract, which allows endotoxin and gram-negative bacteria to enter the blood and gives rise to endotoxemia.

There are reports as to endotoxemia in the early stages of burn injury, and as the burned area becomes larger the blood endotoxin level apparently increases.

However, we hardly found endotoxemia in severely burned patients at the time of admission and in the first week after the burn injury whe using the endotoxin-specific limulus test (Ref. 23). In 42 patients with burns covering more than 20 percent of the body surface area, only one patient had a plasma endotoxin level above 9.8 pg/ml at the time of admission, and only 6 among 956 blood samples (0.70 %) showed endotoxemia at most 12 pg/ml.

Endotoxin levels increased more than 1 week after the burn injury when infection had become established. It seems possible that infected burns, and not the intestinal tract provided the source of endotoxemia.

We have never found a patient with endotoxemia or a positive blood culture in the early stages of hemorrhagic shock (Ref. 24). During and after a cardiopulmonary bypass operation, endotoxemia was hardly found. On the otherhand, β-D-glucan or glucan-like activity was found in those plasmas (Ref.25, 26). Fig. 5 shows that β-D-glucan level (Toxicolor - Endospecy) rises during the operation. As this situation was not related to fungal infection, it is thought to be due to the generation of endogenous β-D-glucan-like activity.

Fig. 5 Endotoxemia does not occure in CBP

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