Abstract

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reagent reacts with bacterial endotoxin and lipopolysaccharide (LPS), which is a membrane constituent of Gram-negative bacteria. This reaction is the base on the LAL reagent, which is then used for the finding and quantification of bacterial endotoxins. The Gel Clot LAL test provides very simple positive or negative result and is most often mentioned in international pharmacopeia monographs as the official test. Gel Clot assay is a qualitative LAL test for detection of Gram-negative bacteria endotoxins. The Gel Clot assay is run in tubes that are placed in a water bath or in dry heated oven at 37°C. After a one-hour incubation period, the tubes are flipped 180°. A firm clot that stays in the bottom of the tube indicates a positive reaction. If the liquid flows down the side of the tube, the result is negative for endotoxins.

Keywords: Limulus amebocyte lysate, lipopolysaccharide, endotoxin, blood, bacteria, detection, horseshoe crab, pharmacopeias, delta, toxin, gel, chromogenic, acetic acid, Gram-negative

### 1. Introduction

Endotoxins, a type of pyrogen, are natural compounds found in the outer cell membrane of Gram-negative bacteria and can impact over 30 biological activities. Endotoxin can lead to cell death by initiating complement activation. The Limulus amebocyte lysate (LAL) test was commercially introduced in the 1970s. LAL is derived from the blood cells, or amebocytes, of the horseshoe crab, Limulus polyphemus. Frederick Bang and Jack Levin observed that blood cells from horseshoe crabs were found to clot in the presence of endotoxin, and this technology was used in the development of endotoxin detection assays. Today, endotoxin tests are performed on raw and in-process materials, and for the final release of products in the pharmaceutical and medical device industries.

Limulus amebocyte lysate test is an aqueous extract of blood cells (amoebocytes) which obtain from the horseshoe crab (Limulus polyphemus). LAL reagent reacts with the bacterial endotoxins or lipopolysaccharide (LPS). LAL test is recommended in all international pharmacopeias as the method for finding

bacterial endotoxins. Gram-negative bacteria produce endotoxins (pyrogen). Exceptionally Bacillus thuringiensis, a Gram-positive bacteria produce delta toxin as endotoxins [1] (Figures 1 and 2).

2. Applications of the LAL test in the pharmaceutical industry

lasting problems after the extraction.

DOI: http://dx.doi.org/10.5772/intechopen.81331

peptide-chromogen complex.

UV/visible spectrophotometers.

4. Test performance

95

nous substrate.

Figure 2. Horseshoe crab [1].

2.1 Limulus amebocyte lysate (LAL) test types

Gel Clot technique: based on gel formation [4].

3. Methods to determine the pyrogen in pharma products

End point method: 0.005 endotoxins units (EU) per ml. Kinetic method: 0.001 endotoxins units (EU) per ml.

Chromogenic method: based on the producing color after cleavage of a synthetic

Turbidimetric method: based on forming turbidity after cleavage of an endoge-

Kinetic method: time taken to reach a specific absorbance at 405 nm (onset time) is determined. The assay requires specialized instrumentation. Take optical density readings at regular intervals. The greatest sensitivity, λ, of lysate is 0.001 EU/ml. Endpoint chromogenic method [5]: the released amount of pNA can be calculated after a fixed incubation period. A standard curve, consisting of measured optical density plotted against known standard endotoxin concentration. Later used to determine concentrations in the product. The greatest sensitivity, λ, is 0.005 EU/ml

Add volume of lysate to a volume of product dilution. Incubating the reaction mixture at 37.5°C. Endotoxin in the reaction would activate the LAL reagent. Cleave

Among the most well-known and important applications of the LAL test are the ones related to the pharmaceutical industry. It can be said that the most common pyrogens in pharmaceutical products are endotoxins, which is why the pyrogen tests on rabbits have been replaced by the LAL test according to the recommendations of the international pharmacopeia. One of the reasons that has made the LAL test prevail in the pharmaceutical industry is the careful avoidance by the LAL manufacturers of bringing harm to live animals during both production and testing. It is important to clarify that the crabs, from which part of the hemolymph used for the LAL test was extracted, are returned to alive to their natural habitat with no

What Is Limulus Amebocyte Lysate (LAL) and Its Applicability in Endotoxin Quantification…

#### Figure 1.

Activation of inflammation in body [1]. Note: LPS, lipoglycan. LAL test used according to the U.S. Food and Drug Administration (FDA) [2] guidelines Substituted for the U.S. Pharmacopeia (USP) pyrogen test (rabbit fever test) European Pharmacopeia (EP) Japanese Pharmacopeia (JP) [3]. LAL is used for human injectable drugs, animal injectable drug, medical devices, raw materials used in production, in process quality control.

What Is Limulus Amebocyte Lysate (LAL) and Its Applicability in Endotoxin Quantification… DOI: http://dx.doi.org/10.5772/intechopen.81331

Figure 2. Horseshoe crab [1].

bacterial endotoxins. Gram-negative bacteria produce endotoxins (pyrogen). Exceptionally Bacillus thuringiensis, a Gram-positive bacteria produce delta toxin as

Activation of inflammation in body [1]. Note: LPS, lipoglycan. LAL test used according to the U.S. Food and Drug Administration (FDA) [2] guidelines Substituted for the U.S. Pharmacopeia (USP) pyrogen test (rabbit fever test) European Pharmacopeia (EP) Japanese Pharmacopeia (JP) [3]. LAL is used for human injectable drugs, animal injectable drug, medical devices, raw materials used in production, in process quality control.

endotoxins [1] (Figures 1 and 2).

Growing and Handling of Bacterial Cultures

Figure 1.

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