Process for determining bacterial endotoxin and reagents...

C - Chemistry – Metallurgy – 12 – Q

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150/15, 150/3.3

C12Q 1/00 (2006.01)

Patent

CA 1130706

ABSTRACT OF THE DISCLOSURE This invention relates to a process for determining a bacterial endotoxin using an amoebocyte lysate of horseshoe crab and/or a pro- clotting enzyme separated from the lysate and (B) a peptide-type substrate of the formula R1 - Gly - Arg - R2 wherein R1 represents a member selected from the group consisting of an L-amino acid moiety whose N-terminal is protected by a protective group, a peptide moiety consisting of an L-amino acid and protected by a protec- tive group at its N-terminal, a D-amino acid substituted L-amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an L-amino acid, and is bonded to the amino group of the glycine moiety ex- pressed by Gly through a peptide bond; and R2 represents a moiety which is bonded to the C-terminal of an L-arginine moiety expressed by Arg through an acid amide bond and/or ester bond and can be enzymatically hy- drolyzed in the presence of the material (A) and the endotoxin to liberate R2H, and/or its mineral acid salt, and detecting the resulting R2H in which R2 is as defined above. According to the invention, various advan- tages such as improved reliability, improved measuring sensitivity, ease of measurement and rapid measurability can be achieved over conventional processes for endotoxin determination utilizing the above lysate or enzyme, and the measuring sensitivity of the conventional processes which is about 10-3 to 10-4 µg/ml can be increased to about 10-6 to 10-9 µg/ml.

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