Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-18
2002-05-21
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S184100, C424S234100, C424S278100, C424S282100, C435S004000, C435S007200, C435S034000, C435S184000, C435S962000, C514S023000
Reexamination Certificate
active
06391570
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to an amebocyte lysate preparation for use in the detection and/or quantification of a bacterial endotoxin in a sample, and more particularly to an endotoxin-specific amebocyte lysate preparation having reduced Factor G activity for use in the detection and/or quantification of a bacterial endotoxin in a sample.
BACKGROUND OF THE INVENTION
Bacterial endotoxins, also known as pyrogens, are the fever-producing byproducts of Gram negative bacteria and can be dangerous or even deadly to humans. Symptoms of infection may range from fever, in mild cases, to death. In order to promptly initiate proper medical treatment, it is important to identify, as early as possible, the presence of an endotoxin and, if possible, the concentration of the endotoxin in the subject of interest. Similarly, the U.S. Food and Drug Administration (USFDA) requires certain manufacturers to establish that their products, for example, parenteral drugs and medical devices, are free of detectable levels of Gram negative bacterial endotoxin.
To this end, a variety of methods have been developed for use in the detection of bacterial endotoxins. A currently preferred method involves the use of amebocyte lysate (AL) produced from the hemolymph of a horseshoe crab, for example, a horseshoe crab selected from the group consisting of
Limulus polyphemus, Tachpleus gigas, Tachypleus tridentatus
, and
Carcinoscorpius rotundicauda
. Amebocyte lysates produced from
Limulus, Tachpleus
, and Carcinoscorpius maybe referred to as LAL, TAL, and CAL, respectively.
Presently, LAL is employed in bacterial endotoxin assays of choice because of its sensitivity, specificity and relative ease for avoiding interference by other components that may be present in a sample of interest LAL, when combined with a sample containing bacterial endotoxin, reacts with the endotoxin to produce a product, for example, a gel or chromogenic product, that can be detected, for example, either visually or by the use of an optical detector.
The endotoxin-mediated activation of LAL is well understood and has been thoroughly documented in the art. See, for example, Levin et at. (1968) Thromb. Diath. Haemorrh. 19: 186, Nakamura et al. (1986) Eur. J. Biochem. 154: 511, Muta et al. (1987) J. Biochem. 101: 1321, and Ho et al. (1993) Biochem. & Mol. Biol. Int. 29: 687. When bacterial endotoxin is contacted with LAL, the endotoxin initiates a series of enzymatic reactions, referred to in the art as the Factor C pathway, that involve at least three serine protease zymogens called Factor C, Factor B and pro-clotting enzyme (see FIG.
1
). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor, Factor C is activated. Activated Factor C thereafter hydrolyses and activates Factor B, whereupon activated Factor B activates proclotting enzyme to produce clotting enzyme. The clotting enzyme thereafter hydrolyzes specific sites, for example, Arg
18
-Thr
19
and Arg
46
-Gly
47
of coagulogen, an invertebrate, fibrinogen-like clottable protein, to produce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.
Although the clotting cascade of LAL initially was considered specific for endotoxin, it was later discovered that (1→3)-B-D glucans also activate the clotting cascade of LAL through a unique enzymatic pathway, referred to in the art as the Factor G pathway (see FIG.
1
). Upon exposure to (1→3)-B-D glucan, Factor G is activated to produce activated Factor G. Activated Factor G thereafter converts the proclotting enzyme into clotting enzyme, whereupon the clotting enzyme converts coagulogen into coagulin, similar to the case with endotoxin. Accordingly, the coagulation system of LAL, like the mammalian blood coagulation system, consists of at least two coagulation cascades which include an endotoxin-mediated pathway (the Factor C pathway), and a (1→3)-B-D glucan-mediated pathway (the Factor G pathway). See, for example,
Morita
et al. (1981) FEBS Lett. 129: 318-321 and Iwanaga et al. (1986) J. Protein Chem. 5: 255-268.
In view of the Factor C and Factor G pathways of LAL, the detection of bacterial endotoxin in a sample can, under certain circumstances, become ambiguous. As a result, attempts have been made to increase the specificity of LAL for endotoxin, i.e., to produce an endotoxin-specific amebocyte lysate preparation.
In one approach, polysaccharide based Factor G inhibitors are combined with amebocyte lysate to reduce or eliminate clotting induced by (1→3)-B-D glucan present in the biological sample, i.e., inhibit the Factor G cascade. See, for example, U.S. Pat. Nos.: 5,155,032; 5,179,006; 5,318,893; 5,474,984; and 5,641,643.
In an alternative approach, several groups have attempted to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate that is insensitive to (1→3)-B-D glucan. For example, Obayashi et al. (1985) Clin. Chim. Acta 149:55-65 disclose a method for fractionating coagulation enzymes in LAL and then recombining only those factors involved in the endotoxin induced coagulation cascade (i.e., the Factor C cascade) to produce a Factor G depleted amebocyte lysate. The resulting lysate, however, may not only lack Factor G but also other components required for a complete Factor C cascade. The reconstituted lysate produced by this procedure, apparently does not produce a natural coagulin type clot and can be used only with synthetic chromogenic substrates.
U.S. Pat. No. 5,401,647 discloses a method for removing Factor G from LAL by combining LAL with (1→3)-B-D glucan immobilized on an insoluble carrier. Once bound to the carrier via the (1→3)-B-D glucan moiety, the Factor G can thereafter be removed from the LAL to produce a Factor G depleted lysate. Similarly, U.S. Pat. No. 5,605,806 discloses an immunoaffinity based method using a Factor G specific antibody to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate.
There still exists, however, a demand for an endotoxin-specific amebocyte lysate that can be produced economically in commercial quantities. A method for producing such an amebocyte lysate should be rapid, reproducible, inexpensive, simple to conduct, and preferably should result in an amebocyte lysate that can be used in a reliable, and quantitative determination of endotoxin in a sample of interest.
SUMMARY OF THE INVENTION
The invention features improved amebocyte lysate preparations having reduced Factor G activity, methods of making such lysate preparations, and methods of using such lysate preparations in the detection and/or quantitation of one or more bacterial endotoxins in a sample of interest.
In one aspect, the invention provides a method of producing an endotoxin-specific amebocyte lysate preparation for use in the detection of bacterial endotoxins in a sample. The amebocyte lysate preparation is rendered endotoxin-specific by the reduction and/or elimination of Factor G activity in the preparation. The amebocyte lysate preparation of the invention is produced by (a) admixing crude amebocyte lysate, i.e., amebocyte lysate reactive with both endotoxin and (1→3)-B-D glucan, with a surfactant in an amount sufficient to produce a solution containing a precipitate; and (b) separating the precipitate from the solution thereby to produce an amebocyte lysate preparation which is less reactive with a (1→3)-&bgr;-D glucan than is the crude amebocyte lysate. The precipitate produced by addition of surfactant to crude lysate may contain any component necessary for a complete Factor G cascade, however, the production of a precipitate actually containing Factor G is preferred.
The amebocyte lysate preparation produced by the methodologies described herein comprises all the components necessary for a complete Factor C cascade, i.e., is still capable of producing a coagulin gel via the endotoxin-mediated pathway. Accordingly, the resulting amebocyte lysate preparation is capable of reacting with a bacterial endotoxin, e.g., a bacterial endotoxin produced by Gram negative b
Chiang Hui-Ti
Cooper James F.
Jordan Foster T.
Wainwright Norman R.
Charles River Laboratories
Swartz Rodney P
Testa Hurwitz & Thibeault LLP
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