HYBRID AMEBOCYTE LYSATE AND USES THEREOF
The invention relates generally to hybrid amebocyte lysate compositions (including both native and recombinant components) and their use in detecting and/or quantifying endotoxin in a sample.
1 . A hybrid amebocyte lysate composition, the composition comprising:
(i) a native horseshoe crab amebocyte lysate; and
(ii) a recombinant horseshoe crab factor B and/or a recombinant horseshoe crab proclotting enzyme.
2 . The composition of claim 1 , wherein the composition comprises a recombinant horseshoe crab factor B and a recombinant horseshoe crab proclotting enzyme.
3 . A hybrid amebocyte lysate composition, the composition comprising:
(i) a native horseshoe crab factor C;
(ii) a native horseshoe crab factor B;
(iii) a native horseshoe crab pro-clotting enzyme; and
(iv) a recombinant horseshoe crab factor B and/or a recombinant horseshoe crab proclotting enzyme.
4 . The composition of claim 3 , wherein the composition comprises a recombinant horseshoe crab factor B and a recombinant horseshoe crab proclotting enzyme.
5 . A hybrid amebocyte lysate composition, the composition comprising:
(i) horseshoe crab factor C;
(ii) horseshoe crab factor B; and
(iii) horseshoe crab proclotting enzyme;
wherein (a) the ratio of horseshoe crab factor B to horseshoe crab factor C is greater than the ratio of horseshoe crab factor B to horseshoe crab factor C in a native horseshoe crab amebocyte lysate, and/or (b) the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C is greater than the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C in a native horseshoe crab amebocyte lysate.
6 . The composition of claim 5 , wherein the ratio of horseshoe crab factor B to horseshoe crab factor C is greater than the ratio of horseshoe crab factor B to horseshoe crab factor C in a native horseshoe crab amebocyte lysate, and the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C is greater than the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C in a native horseshoe crab amebocyte lysate.
7 . The composition of any one of claims 1 - 6 , wherein the composition does not comprise a recombinant horseshoe crab factor C.
8 . The composition of any one of claims 1 - 7 , wherein the composition comprises from about 0.05 to about 1 U/mL of recombinant horseshoe crab factor B.
9 . The composition of claim 8 , wherein the composition comprises from about 0.1 to about 0.5 U/mL of recombinant horseshoe crab factor B.
10 . The composition of any one of claims 1 - 9 , wherein the composition comprises from about 0.05 to about 2,000 U/mL of recombinant proclotting enzyme.
11 . The composition of claim 10 , wherein the composition comprises from about 50 to about 200 U/mL of recombinant proclotting enzyme.
12 . The composition of any one of claims 1 - 11 , wherein the composition has substantially the same sensitivity in detecting endotoxin as native horseshoe crab amebocyte lysate.
13 . The composition of any one of claims 1 - 12 , wherein the composition has a greater sensitivity in detecting endotoxin than native horseshoe crab amebocyte lysate.
14 . The composition of any one of claims 1 - 13 , wherein the horseshoe crab amebocyte lysate is a Limulus polyphemus or a Tachypleus amebocyte lysate.
15 . The composition of any one of claims 1 - 14 , wherein the horseshoe crab factor B is a Limulus polyphemus or a Tachypleus factor B.
16 . The composition of any one of claims 1 - 15 , wherein the horseshoe crab proclotting enzyme is a Limulus polyphemus or a Tachypleus proclotting enzyme.
17 . The composition of any one of claims 1 - 16 , wherein the horseshoe crab factor C is a Limulus polyphemus or a Tachypleus factor C.
18 . The composition of any one of claims 1 - 17 , wherein the recombinant horseshoe crab factor B and/or the recombinant horseshoe crab proclotting enzyme are expressed in a mammalian cell.
19 . The composition of claim 18 , wherein the mammalian cell is a Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cell.
20 . The composition of claim 19 , wherein the recombinant factor B and/or the recombinant proclotting enzyme has different glycosylation than the factor B and/or the proclotting enzyme present in the native horseshoe crab amebocyte lysate.
21 . A method for preparing an endotoxin detection reagent, the method comprising adding a recombinant horseshoe crab factor B and/or a recombinant horseshoe crab proclotting enzyme to a native horseshoe crab amebocyte lysate.
22 . The method of claim 21 , wherein the method comprises adding a recombinant horseshoe crab factor B and a recombinant horseshoe crab proclotting enzyme to the native horseshoe crab amebocyte lysate.
23 . A method for increasing the endotoxin sensitivity of a native horseshoe crab amebocyte lysate, the method comprising adding to the native horseshoe crab amebocyte lysate a recombinant horseshoe crab factor B and/or a recombinant horseshoe crab proclotting enzyme, thereby to increase the endotoxin sensitivity of the native horseshoe crab amebocyte lysate.
24 . The method of claim 23 , wherein the method comprises adding a recombinant horseshoe crab factor B and a recombinant horseshoe crab proclotting enzyme to the native horseshoe crab amebocyte lysate.
25 . A method for reducing the amount of a native horseshoe crab amebocyte lysate required to detect endotoxin, the method comprising: (i) diluting the native horseshoe crab amebocyte lysate; and (ii) adding to the diluted native horseshoe crab amebocyte lysate a recombinant horseshoe crab factor B and/or a recombinant horseshoe crab proclotting enzyme to produce a hybrid amebocyte lysate.
26 . The method of claim 25 , wherein the method comprises adding a recombinant horseshoe crab factor B and a recombinant horseshoe crab proclotting enzyme to the diluted native horseshoe crab amebocyte lysate.
27 . The method of claim 25 or 26 , wherein the hybrid amebocyte lysate has substantially the same, or greater, sensitivity in detecting endotoxin as the native horseshoe crab amebocyte lysate.
28 . An isolated nucleic acid comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16.
29 . An expression vector comprising a nucleic acid of claim 28 operatively linked to a promoter.
30 . A host cell (e.g., a mammalian host cell) comprising the expression vector of claim 29 .
31 . A method of producing a recombinant horseshoe crab factor C protein comprising:
(a) expressing a nucleic acid sequence encoding the recombinant horseshoe crab factor C in a host cell engineered to remove a glycosyltransferase enzyme; and
(b) purifying the recombinant factor C expressed in the host cell.
32 . The method of claim 31 , wherein the host cell is a HEK293 or CHO cell.
33 . The method of claim 31 or 32 , wherein the glycosyltransferase is N-acetylglucosaminyltransferase.
34 . A recombinant horseshoe crab factor C protein produced by the method of any one of claims 31 - 33 .
35 . The recombinant factor C of claim 34 , wherein the factor C does not contain (α-2,3)-linked terminal sialic acid.
36 . A method of preparing an endotoxin detection reagent, the method comprising admixing the recombinant horseshoe crab factor C of claim 34 or 35 to a composition comprising recombinant horseshoe crab factor B and recombinant horseshoe crab proclotting enzyme.