Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Patent
1992-05-27
1995-03-28
Warden, Robert J.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
435 23, 435179, 435180, 435181, 436175, 536117, 536118, 536119, C12N 1106, C12N 1108, C12N 1114, C12Q 137
Patent
active
054016479
DESCRIPTION:
BRIEF SUMMARY
SUMMARY OF THE INVENTION
The present invention relates to a method of preparing limulus amoebocyte lysate.
BACKGROUND OF THE INVENTION
It has been widely known that limulus amoebocyte lysate (hereinafter referred to as LAL) reacts with an endotoxin which is a bacterial pyrogen (hereinafter referred to as endotoxin) to cause a coagulation.
On the basis of this reaction, various methods of assaying endotoxin have been developed.
Recently, the above-described coagulation reaction mechanism has been elucidated, i.e., coagulogen is converted to coagulin to cause a coagulation (gelation) according to a stepwise reaction as shown in FIG. 1 [S. Iwanaga et al., The hemolymph coagulation system in invertebrate animals, J. Protein Chem., 5, 255-268 (1986)]. It can be understood that the reaction mechanism includes two coagulation systems: a system initiated by endotoxin (factor C system) and a system initiated by (1.fwdarw.3)-.beta.-D-glucan (e.g., curdlan, partially carboxyfnethylated (1.fwdarw.3)-.beta.-D-glucan) (factor G system).
The present inventors already found that the (1.fwdarw.3)-.beta.-D-glucan structural portion having a particular molecular weight inhibits the activation of the system of LAL initiated by (1.fwdarw.3)-.beta.-D-glucan (factor G system), and filed the patent application as a limulus amoebocyte lysate factor G activation inhibitor (Japanese Patent Application No. 63-216341 and WO 90/02951).
However, when this inhibitor is added to LAL in order to obtain LAL specific to endotoxin, a complex comprising the inhibitor and factor G remaining in LAL is possibly dissociated with a sample added and the liberated factor G is activated by a factor G-activating substance.
DETAILED DESCRIPTION OF THE INVENTION
An object of the present invention is to provide LAL free from factor G which is first activated with (1.fwdarw.3)-.beta.-D-glucan in the LAL coagulation mechanism.
Thus, the present invention provides a method of preparing limulus amoebocyte lysate substantially free from factor G which comprises contacting limulus amoebocyte lysate with an insoluble carrier on which (1.fwdarw.3)-.beta.-D-glucoside structural portion having formula [I] is fixed. ##STR2## wherein n represents an integer of 2 to 370.
The (1.fwdarw.3) -.beta.-D-glucoside structural portion used in the present invention is a polyglycoside having, in one molecule, at least one poly-(1.fwdarw.3)-.beta.-D-glucoside structural portion [hereinafter referred to as a poly(1.fwdarw.3)glucoside moiety] comprising 2 to 370, preferably 3 to 310, and more preferably 4 to 180 consecutive (1.fwdarw.3)-.beta.-D-glucoside structural units (molecular weight: 162) represented by the following formula. ##STR3##
Thus, the polyglycoside used in the present invention is required to have at least one poly(1.fwdarw.3)glucoside moiety in one molecule. For example, the polyglycoside used in the present invention may consists substantially of one poly(1.fwdarw.3)glucoside moiety, for example, poly-(1.fwdarw.3)-.beta.-D-glucoside represented by the following formula ##STR4## wherein n is an integer of 2 to 370, preferably 3 to 310, and more preferably 4 to 180. Alternatively, it may be of a structure in which one poly(1.fwdarw.3)glucoside moiety has a carbohydrate chain, which may be bound to the above-described poly(1.fwdarw.3)glucoside moiety as a branched chain, composed of one or more (1.fwdarw.4)-.beta.-D-glucoside structural units represented by the following formula; ##STR5## one or more (1.fwdarw.6 )-.beta.-D-glucoside structural unit represented by the following formula; ##STR6## or one or more modified .beta.-D-glucoside structural unit represented by the following formulae; ##STR7## wherein at least one of R.sub.1, R.sub.2 and R.sub.3 represent(s) a chemically introducible group(s), i.e., a methyl group, a hydroxyalkyl group such as a hydroxymethyl group, a carboxyalkyl group such as a carboxymethyl group, an acetyl group, a sulfate group, a phosphate group, or an allyl group, a metal salt of any of the above-mentioned groups, an ammonium sal
REFERENCES:
patent: 4270152 (1990-11-01), Ashida et al.
patent: 4454315 (1984-06-01), Sasaki et al.
patent: 5155032 (1992-10-01), Tanaka et al.
Tanaka et al., "Activation of a Limulus Coagulation Factor G by (1-3)-.beta.-D-flucans.", Carbohydrate Research., 218(1991)., pp. 167-174.
Tanaka et al., "Inhibition of High Molecular Weight (1-3)-.beta.-D-glucan Dependant Activation of a Limulus Coagulation Factor G by Laminaran Oligosaccarides & Curdlan Degradation Products"., Carbohydrate Research., 244 (1993)., pp. 115-127.
Biological Abstracts, vol. 91, No. 10, May 15, 1991, Abstract No. 108553.
N. Ohno et al., "Reactivity of Limulus Amoebocyte Lysate Towards (1-3)-.beta.-D-glucans," Carbohydrate Research, vol. 207, No. 2, Oct. 25, 1990, pp. 311-318.
Aketagawa Jun
Shibata Yuko
Tanaka Shigenori
Crawford L. M.
Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation)
Warden Robert J.
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