Methods and compositions for the detection of microbial contaminants
The invention provides methods and compositions for the detection and/or quantification of a microbial contaminant, for example, a bacterial endotoxin or a glucan, in a sample. In particular, the invention provides a test cartridge useful in the practice of hemocyte lysate-based assays for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides methods of making and using such cartridges. In addition, the invention provides a rapid, sensitive, multi-step kinetic hemocyte lysate-based assay for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides a glucan-specific lysate that can be used in a variety of assay formats, including, for example, a test cartridge, optionally configured to perform a kinetic assay.
1 - 58 . (canceled)
59 . A method of preparing an amebocyte lysate depleted of Factor C activity, the method comprising:
(a) providing a preparation of amebocytes; and
(b) lysing the amebocytes in the presence of at least 0.15 M salt to provide an amebocyte lysate preparation depleted of Factor C activity.
60 . The method of claim 59 , comprising the additional step of after step (b) removing cellular debris from the amebocyte lysate preparation.
61 . The method of claim 59 , wherein the salt is comprises a monovalent cation.
62 . The method of claim 61 , wherein the salt is a sodium or potassium salt.
63 . The method of claim 62 , wherein the salt is sodium chloride or potassium chloride.
64 . The method of claim 59 , wherein in step (b) the amebocytes are lysed in the presence of salt at a concentration in the range from about 0.15 M to about 6 M.
65 . The method of claim 64 , wherein the salt is at a concentration from about 0.25M to about 4 M.
66 . The method of claim 65 , wherein the salt is at a concentration from about 1 to about 2 M.
67 . The method of claim 59 , wherein the lysate is substantially free of Factor C activity.
68 . The method of claim 59 , wherein the lysate retains Factor G activity.
69 . The method of claim 68 , wherein the lysate is a glucan-specific lysate.
70 . An amebocyte lysate substantially free of Factor C activity, wherein the lysate comprises at least about 0.25 M salt, and wherein the lysate is capable of reacting with glucan to produce a coagulin gel.
71 . The lysate of claim 70 , wherein the salt comprises a monovalent cation.
72 . The lysate of claim 71 , wherein the salt comprises a sodium ion, a potassium ion, or a combination of sodium and potassium ions.
73 . The lysate of claim 70 , wherein the lysate is substantially free of Factor C activity.
74 . The lysate of claim 70 , wherein the lysate comprises from at least about 0.25 M to about 6 M salt.
75 . The lysate of claim 74 , wherein the lysate comprises from about 0.5 M to about 4 M salt.
76 . The lysate of claim 75 , wherein the lysate comprises from about 1M to about 2 M salt.
77 - 78 . (canceled)