Composition for detecting β-1,3-glucan
The present invention relates to a composition for detecting an infinitesimal quantity of β-1,3-glucan, a preparation method thereof and a diagnostic kit detecting β-1,3-glucan. The composition of the present invention shows phenoloxidase activity by β-1,3-glucan in the presence of calcium ions. Using the composition of the present invention, a sample is gathered from a specimen, the composition of the present invention and calcium ions are added to the sample, and β-1,3-glucan is detected by measuring phenoloxidase activity.
1. A composition for detecting β-1,3-glucan in the presence of calcium ions, prepared by the method comprising:
(i) collecting a sample comprising a mixture of plasma and hemocyte lysate from Tenebrio molitor or Holotrichia diomphalia;
(ii) purifying said sample by column chromatography using a solvent or buffer solution containing a sufficient amount of a chelating agent to chelate calcium ions present in said sample to obtain fractions therefrom; and
(iii) selecting fractions exhibiting phenoloxidase activity by β-1,3-glucan in the presence of calcium ions from the obtained fractions.
2. A composition for detecting β-1,3-glucan according to claim 1 wherein the composition detects β-1,3-glucan concentrations as low as 20 pg/ml in the presence of calcium ions.
3. A composition for detecting β-1,3-glucan exhibiting phenoloxidase activity by β-1,3-glucan in the presence of calcium ions, said composition prepared by:
(i) treating Tenebric molitor or Holotrichia diomphalia plasma with a solvent or buffer solution containing a sufficient amount of a chelating agent to chelate calcium ions present in the plasma and
(ii) separating said chelated plasma by chromatography to obtain fractions from said chelated plasma;
(iii) adding hemocyte lysate or partially purified hemocyte lysate to said fractions to form lysate treated fractions, and
(iv) selecting lysate treated fractions exhibiting phenoloxidase activity by β-1,3-glucan in the presence of calcium ions.