Limulus amoebocyte lysate

Endotoxin below specified level Limulus amoebocyte lysate method (see also Chapter 18)... 

However, the establishment of a new endotoxin standard by the World Health Organization is a recent example of successful international collaboration between the World Health Organization, the United Stated Pharmacopoeia and the European Pharmacopoeia (Poole et al. 1997). Thus this standard is available from any of these organizations to be employed as a reference in the harmonized Limulus Amoebocyte Lysate test. 

Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as lL-1 and TNF Chapter 5), themselves, induce a natural pyrogenic response. This rules out use of the rabbit-based assay for detection of exogenous pyrogens in such products. Such difficulties have led to the increased use of an in vitro assay the Limulus amoebocyte lysate (LAL) test. This is based upon endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other pharmaceutical preparations. 

U.S. Department of Health and Human Services (DHHS) (1987), FDA guidelines on validation of the Limulus amoebocyte lysate test as an end product test for human and animal parenteral drugs, biological products and medical devices, DHHS, Rockville, MD. 

Keywords Endotoxin Limulus amoebocyte lysate test LAL Molecular cloning Pyrogene assay Recombinant Factor C... 

Brunson, K.W., Watson, D.W. Limulus amoebocyte lysate reaction with Streptococcal pyrogenic exotoxin. Infect Immun 14 (1976) 1256-1258. 

Morita, T., Nakamura, T., Miyata, T., Iwanaga, S. Biochemical characterisation of Limulus clotting factors and inhibitors which interact with bacterial endotoxin. In Cate, J.W., Buller, H.R., Sturk, A., Levin, J. (eds). Bacterial endotoxin, structure, biomedical significance, and detection with the limulus amoebocyte lysate test. Alan R Liss Inc, NY (1985a) pp. 53-64. 

Novitsky, TJ. Limulus amoebocyte lysate (LAL) detection of endotoxin in human blood. J Endotoxin Res 1 (1994) 253-263. 

Table 1 Thermogravimetric (TG) and Karl Fischer Residual Moisture Data for Varicella Virus Vaccine Live and Limulus Amoebocyte Lysate...

When a clearly defined plateau is not in evidence for the residual moisture TG transition as is shown in the data for a limulus amoebocyte lysate (LAL) illustrated in Figure 12, TG/MS is employed to determine the endpoint of the evolution of the residual moisture. TG/MS has been shown to elucidate the TG transitions attrib-... 

Figure 12 Plot of TG/MS data for limulus amoebocyte lysate (LAL).

Two pharmacopoeial limit tests exist. That for pyrogens uses rabbits to assess pharmacological activity and therefore the presence of pyrogens of all kinds. The test for bacterial endotoxins uses lysed amoebocytes (blood cells) of the horseshoe crab and is therefore termed the Limulus amoebocyte lysate (LAL) test. This may be extended to many drug and device products and clearly will be developed in the future to assess the presence of endotoxins in biotechnology products. 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

EN 14569 2004 DIF using the limulus amoebocyte lysate test in conjunction with a gram negative bacterial count (LAL/GNB) Poultry meat... 

The quahty of the purified product is an important aspect, and must be ensured because it can influence the potency of dSLIM. Therefore, integrity is tested via exonuclease activity of T7-DNA-polymerase degrading residual DNA molecules with open ends and subsequent gel electrophoresis. Furthermore, the content of endotoxin is determined by an end-point Limulus amoebocyte lysate (LAL) test, and must meet strict standards (< 10 EU mg DNA). 

Limit of detection (LOD) 1568 Limit of quantification (LOQ) 1568 Limulus amoebocyte lysate (LAI) 197... 

Weary M.E., Donohue G., Pearson F.C. and Story K. (1980) Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests. Appl. Environ. Microbiol., 40, 1148-1151. 

The water used in the preparation of sterile products should be tested for bacterial endotoxin at least once per week and after any repair or disturbance to the system, using the limulus amoebocyte lysate test Sampling should include worst case situations, including start-up. 

The use of the limulus amoebocyte lysate test is valuable in monitoring bacterial endotoxin levels. 

One of the properties of P-glucans associated with their ordered structure is an ability to cause activation of coagulation factor G from Limulus amoebocyte lysate (LAL). The coagulation of amoebocyte lysate occurs in the presence of minor amounts of endotoxins... 

RP-HPLC, reverse phase HPLC GC, gas chromatography LAL, Limulus amoebocyte lysate SEC, size-exclusion chromatography lEF, isoelectric focusing lEC, ion exchange chromatography MS, mass spectrometry. 

A number of tests were also developed and further used routinely to detect other process-related impurities in clinical batches, such as a Limulus amoebocyte lysate test to quantify bacterial endotoxins (< 1 I.U./mg of BBG2Na) and a specific RP-HPLC assay to detect tetracycline (no trace detected) since this antibiotic was used for selection by drug pressure during the fermentation step. 

Magneto elastic sensors have also been used for the detection of endotoxin, which can cause sepsis in immunocompromised patients. The system was based on the use of magnetoelastic sensors to monitor gel formation (clot formation), which changes the viscosity of the limulus amoebocyte lysate (LAL) assay in response to endotoxin. By correlating different endotoxin concentration and the LAL assay clot rate, it was found that the maximum clot rate was directly proportional to the endotoxin concentration. Endotoxin concentrations as low as 0.0105 EU/mL have been detected in approximately 20 min (Ong et al. 2006). 

The limulus amoebocyte lysate (LAL) test with chromogenic substrate is faster than the gelation method, and it can be automated. The chromogenic substrate is attached to p-nitroaniline, that is released when reacted with the endotoxin-activated enzymes. The free p-aniline is read at 405 nm. 

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