Abstract Disclosed are chromogenic or fluorogenic peptide-type compounds adapted for use in determining endotoxin in a sample by a Limulus amebocyte lysate-type assay. The compounds have the general formula: R.sub.1 --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --B--R.sub.2 wherein R 1 represents hydrogen, a blocking aromatic hydrocarbon or acyl; A 1 represents an L or D-amino acid selected from either Ileu, Val or Leu; A 2 represents Glu or Asp; A 3 represents Ala or Cyst; A 4 represents Arg, B represents a linkage group selected from ester and amide linkage groups; and R 2 represents a chromogenic or fluorogenic group which is covalently attached to the C-carboxyl terminal of arginine through the B linkage group and which yields a chromophoric or fluorescent marker compound of the formula R 2 --B--H upon enzymatic hydrolysis from the remainder of the peptide-type compound by activated LAL pro-clot enzyme. Also disclosed are methods for the determination of endotoxin in a sample by contacting the sample with a pro-clotting enzyme from Limulus amebocyte lysate and one of the above chromogenic or fluorogenic peptide-type compounds.
A method for the determination of endotoxin in a sample comprising contacting the sample with a proclotting enzyme from Limulus amebocyte lysate and a chromogenic or fluorogenic compound of the formula: R.sub.1 --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --B--R.sub.2 wherein R 1 represents hydrogen, a blocking aromatic hydrocarbon or acyl; A 1 represents an L or D-amino acid selected from Ileu, Val or Leu; A 2 represents Glu or Asp; A 3 represents Ala or Cyst; A 4 represents Arg; B represents a linkage group selected form ester and amide linkage groups; and R 2 represents a chromogenic or fluorogenic group which is covalently attached to the C-carboxyl terminal of arginine through the B linkage group, the fluorogenic or chromogenic moiety being capable of being enzymatically cleaved from the remainder of the peptide-type compound in the presence of endotoxin and pro-clotting enzyme to form at least part of a marker compound, and detecting the presence or absence of the marker compound in the sample.
Claim 2
Independent Claim
162 words
A method for the determination of endotoxin in a sample comprising contacting the sample with a proclotting enzyme from Limulus amebocyte lysate and a chromogenic or fluorogenic compound of the formula: R.sub.1 --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --B--R.sub.2 wherein R 1 represents hydrogen, a blocking aromatic hydrocarbon or acyl; A 1 represents an L or D-amino acid selected from Ileu, Val or Leu; A 2 represents Glu or Asp; A 3 represents Ala or Cyst; A 4 represents Arg; B represents a linkage group selected form ester and amide linkage groups; and R 2 represents a chromogenic or fluorogenic group which is covalently attached to the C-carboxyl terminal of arginine through the B linkage group, the fluorogenic or chromogenic moiety being capable of being enzymatically cleaved from the remainder of the peptide-type compound in the presence of endotoxin and pro-clotting enzyme to form at least part of a marker compound, and detecting the presence or absence of the marker compound in the sample.
Claim 3
Dependent Claim
12 words
A method according to claim 1 where B is an amide linkage.
Claim 4
Dependent Claim
11 words
A method according to claim 2 where A 3 is Ala.
Claim 5
Dependent Claim
11 words
A method according to claim 2 where A 3 is Cyst.
Claim 6
Independent Claim
13 words
A method according to claims 3 or 4 wherein A 2 is Glu.
Claim 7
Independent Claim
13 words
A method according to claims 3 or 4 wherein A 2 is Asp.
Claim 8
Dependent Claim
11 words
A method according to claim 5 wherein A 1 is Ileu.
Claim 9
Dependent Claim
11 words
A method according to claim 6 wherein A 1 is Val.
Claim 10
Dependent Claim
11 words
A method according to claim 8 wherein R 2 is p-nitrophenyl.