Method of preparing Limulus amoebocyte lysate

• US 5,401,647 A
• Filed: 05/27/1992
• Issued: 03/28/1995
• Est. Priority Date: 09/27/1990
• Status: Expired due to Term

• Current Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Original Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Inventors: Aketagawa, Jun, Tanaka, Shigenori, Shibata, Yuko
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Crawford, L. M.
• Application Number:
  US07/859,411
• Time in Patent Office: 1,035 Days
• Field of Search: 435/4, 435/18, 435/23, 435/176, 435/177, 435/178, 435/179, 435/180, 435/181, 435/184, 435/100, 435/101, 435/104, 436/175, 536/119, 536/120, 536/121, 536/117, 536/118
• US Class Current: 435/176
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials

Abstract

Disclosed is a method of preparing limulus amoebocyte lysate substantially free from factor G which comprises bringing limulus amoebocyte lysate into contact with an insoluble carrier on which a (1→3)-β-D-glucoside structural portion represented by the following formula [I] produced by depolymerizing and/or fractionating a carbohydrate chain is immobilized: ##STR1## wherein n represents an integer of 2 to 370.

Claims

1. A method of preparing limulus amoebocyte lysate substantially free from factor G which comprises bringing limulus amoebocyte lysate into contact with an insoluble carrier on which a (1→

3)-β

-D-glucoside structural portion represented by the following formula (I) produced by depolymerizing and/or fractionating a carbohydrate chain is immobilized by a method which comprises epoxy-activating an insoluble carrier with epichlorohydrin or bisoxiranes, reacting the resulting epoxy-activated insoluble carrier with hydrazine hydrate or a dihydrazide compound to obtain a hydrazine derivative or a dihydrazide derivative and bringing the (1→

3)-β

-D-glucoside into contact with the hydrazine or dihydrazide derivative;

##STR10## wherein n represents an integer of 2 to 370.

2. A method as claimed in claim 1, wherein the (1→

3)-β

-D-glucoside structural portion additionally contains at least one structural unit selected from the group consisting of structural units represented by the following formulae (II), (III), (IV), (V) and (VI);

##STR11## wherein at least one of R₁, R₂ and R₃ represents a group selected from the group consisting of a methyl group, a hydroxyalkyl group, a carboxyalkyl group, an acetyl group, a sulfate group, a phosphate group, an allyl group, a metal salt of any of said groups, an ammonium salt of any of said groups, and an organic amine salt of any of said groups, and the remaining groups represent a hydrogen atom.

3. A method as claimed in claim 1 or 2, wherein the (1→

3)-β

-D-glucoside structural portion is derived from natural sources selected from the group consisting of (1→

3)-β

-D-glucan derived from bacteria of the genus Alcaligenes, paramylon derived from Euglena, β

-glucan or callose derived from higher plants, laminatans derived from brown algae, chrysolaminarans derived from diatoms, pachyman derived from Poria, glucan derived from the cell wall of Phytophthora, sclerotan derived from Sclerotinia, schizophyllan derived from Schizophyllum, grifolan LE derived from Grifola frondosa, scleroglucans derived from Sclerotium, Corticium or Stromatinia, lentinan derived from Lentinus, β

-glucan derived from the cell wall of Saccharomyces (bakers'"'"' yeast), lichenans derived from Cetraria, Usnea or Evernia, and β

-glucan contained in barley endosperm.

4. A method as claimed in claim 3, wherein the (1→

3)-β

-D-glucoside structural portion is produced by depolymerizing a carbohydrate chain by a partial decomposition method selected from the group consisting of a hydrolysis using an acid, an alkali or β

-glucanase, an acetolysis and a sonication, and/or fractionating a carbohydrate chain by a fractional precipitation method using an organic solvent or salts, or a fractionation method using a molecular sieve agent or a molecular sieve membrane.

5. A method as claimed in claim 1, wherein the insoluble carrier is selected from the group consisting of cellulose, agarose, crosslinked dextran, polyacrylamide, porous glass, and a hydrazine or hydrazide derivative of a synthetic hydrophilic polyvinyl polymer.