美国ATCC细胞株（细胞系）ATCC 菌种（标准细菌菌株）ATCC细菌株 病毒 质粒 噬菌体 载体 藻类[介绍]

细胞株
细胞与培养基系统
动物血清
肿瘤及细胞株

ProSpect细胞因子
细胞因子(Cytokine Products)
化学(Chemokine Products)
激素(Hormone Products)
酶(Enzyme Products)
蛋白质(Protein Products)
抗体(Monochlonal Antibodies)

SUMMARY AND EXPLANATION OF TEST
In the 1950's, Frederik Bang observed that infection from gram negative bacteria resulted in intravascular
coagulation in Limulus polyphemus, the horseshoe crab (1). Levin and Bang demonstrated that the
coagulation was caused by the activation of a number of enzymes located in the blood cells (amebocytes)
of Limulus polyphemus, and that this activation was initiated by the endotoxin (lipopolysaccharide) in the
gram negative bacterial cell walls (2, 3, 4).
Subsequently, the Limulus Amebocyte Lysate (LAL) test using LAL reagent prepared from horseshoe crab
blood was shown to be the most sensitive and specific means of measuring bacterial endotoxins (5). The
chromogenic test, introduced in 1977 (6, 7), is a modification that enables endotoxin concentration to be
measured as a function of color intensity rather than by turbidity or gelation in the reaction mixture.
Results obtained by this modified method are generally comparable to those obtained by the gel-clot or
turbidimetric methods within the error of the tests.
In the Chromo-LAL test, co-lyophilized LAL and substrate reagent are mixed with test sample in a
microplate and incubated in a reader at 37±1°C. Absorbance measurements are collected with time after
addition of Chromo-LAL and analysed by suitable software. The time (onset time) taken for a sample to
reach a specified absorbance (onset OD) is calculated; and a standard curve, showing the linear
correlation between the log onset time and the log concentration of standard endotoxin, is generated. The
maximum range of endotoxin concentrations for the standard curve is 0.005 EU/mL - 50 EU/mL. The
sensitivity (λ) of the assay is defined as the lowest concentration used in the standard curve. The
maximum sensitivity of this test is 0.005 EU/mL.
BIOLOGICAL PRINCIPLE
LAL contains enzymes that are activated in a series of reactions in the presence of endotoxin. The last
enzyme activated in the cascade splits the chromophore, para-nitro aniline (pNA), from the chromogenic
substrate, producing a yellow color.
Endotoxin
1. Proenzyme Enzyme
Enzyme
2. Chromogenic Substrate Peptide + pNA
The amount of pNA released and measured photometrically at 405 nm is proportional to the amount of
the endotoxin in the system. The greater the endotoxin concentration, the faster the reaction.
REAGENTS
Reagents required to perform the Chromo-LAL Assay are listed below. Unopened reagents are stable at 2-
8°C until the expiration date printed on the container label. Before reconstitution, bring the reagents to
room temperature and tap the vials containing lyophilized material against a hard surface to cause loose
material to fall to the bottom of the vial.
1.Chromo-LAL, Limulus Amebocyte Lysate colyophilized with chromogenic substrate
This reagent is an aqueous extract of amebocytes of L. polyphemus, buffered at pH 7, and co-lyophilized
with the chromogenic substrate. Reconstitute Chromo-LAL immediately before use with 3.2 mL LAL
Reagent Water (LRW). This solution is stable 24 hours at 2-8°C or for two weeks at -20°C or colder if
frozen immediately after reconstitution and not contaminated. Chromo-LAL may be frozen and thawed
once. Contamination may be indicated by a dark yellow color that develops rapidly after reconstitution.
The reagent will turn yellow slowly under normal conditions of use.
2.Control Standard Endotoxin (CSE)
Control Standard Endotoxin (CSE) is not provided with Chromo-LAL and must be ordered separately.
CSE obtained from Associates of Cape Cod, Inc., is used to construct standard curves, validate product,
and prepare inhibition controls. Each vial contains a measured weight of endotoxin. USP Endotoxin