Patent
The present invention relates to a composition for selectively detecting an extremely small amount of peptidoglycan in sample, a preparation method of the composition, and a detection kit for peptidoglycan. It is possible to quantify a small amount of peptidoglycan contained in human blood, tissue, body fluid, water or food, and to diagnose an infection of microorganism with peptidoglycan as a component of cell wall using the composition and the detection kit. In addition, the composition can be applied for a diagnosis reagent of detecting an infection of Gram-positive bacteria in animal or human being in advance, and thus, can be used for the prevention or treatment of food poisonings and bacterial sepsis.
1. A composition for selectively detecting a peptidoglycan comprising an extract of insect body fluid which has a phenoloxidase activity on the peptidoglycan without the addition of divalent cations to activate the phenoloxidase activity on the peptidoglycan, wherein the extract of insect body fluid comprises a plasma solution and hemocyte lysate of insect body fluid derived from Galleria mellonella larvae.
2. The composition according to claim 1 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are prepared by lysing hemocyte contained in insect body fluid containing plasma and hemocyte of Galleria mellonella larva, and treating with solvent or buffer solution.
3. The composition according to claim 2 , wherein the solvent or buffer solution comprises a chelating agent in a sufficient amount for chelating calcium ion present in a sample and separation process.
4. The composition according to claim 2 , wherein the fraction is a fraction prepared through column chromatography.
5. The composition according to claim 4 , wherein the column is filled with a sugar resin or a vinyl resin.
6. The composition according to claim 1 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are prepared by adding hemocyte lysate or partially-purified hemocyte lysate to fractions obtained by treating plasma of Galleria mellonella larvae with solvent or buffer solution.
7. The composition according to claim 6 , wherein the solvent or buffer solution comprises a chelating agent in a sufficient amount for chelating calcium ion present in a sample and separation process.
8. The composition according to claim 6 , wherein the fraction is a fraction prepared through column chromatography.
9. A method of detecting a peptidoglycan comprising:
obtaining a sample from a test subject;
adding to the sample an extract of insect body fluid which has a phenoloxidase activity on the peptidoglycan without the addition of calcium to activate the phenoloxidase activity on the peptidoglycan, wherein the extract of insect body fluid comprises a plasma solution and hemocyte lysate of insect body fluid derived from Galleria mellonella larvae; and
measuring a phenoloxidase activity in the sample for detecting the peptidoglycan.
10. The method according to claim 9 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of the calcium or other divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are prepared by lysing hemocyte in insect body fluid containing plasma and hemocyte of Galleria mellonella larva, and treating with solvent or buffer solution.
11. The method according to claim 10 , wherein the solvent or buffer solution comprises a chelating agent in a sufficient amount for chelating divalent cations present in a sample and separation process.
12. The method according to claim 10 , wherein the fraction is a fraction prepared through column chromatography.
13. The method according to claim 12 , wherein the column is filled with a sugar resin or a vinyl resin.
14. The method according to claim 9 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of the calcium or other divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are prepared by adding hemocyte lysate or partially-purified hemocyte lysate to fractions obtained by treating plasma of Galleria mellonella larvae with solvent or buffer solution.
15. The method according to claim 14 , wherein the solvent or buffer solution comprises a chelating agent in a sufficient amount for chelating divalent cations present in a sample and separation process.
16. The method according to claim 14 , wherein the fraction is a fraction prepared through column chromatography.
17. The method according to claim 16 , wherein the column is filled with a sugar resin or a vinyl resin.
18. A detection kit for peptidoglycan comprising an extract of insect body fluid which has a phenoloxidase activity on the peptidoglycan without the addition of divalent cations to activate the phenoloxidase activity on the peptidoglycan, wherein the extract of insect body fluid comprises a plasma solution and hemocyte lysate of insect body fluid derived from Galleria mellonella larvae and instruction for the use thereof to detect the peptidoglycan.
19. The detection kit according to claim 18 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of the calcium or other divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are prepared by lysing hemocyte in insect body fluid containing plasma and hemocyte of Galleria mellonella larva, and treating with solvent or buffer solution.
20. The detection kit according to claim 18 , wherein the extract of insect body fluid is a fraction having a phenoloxidase activity on peptidoglycan without the addition of the calcium or other divalent cations to activate a phenoloxidase activity on the peptidoglycan, and the fraction is selected from fractions which are obtained by adding hemocyte lysate or partially-purified hemocyte lysate to fractions obtained by treating plasma of Galleria mellonella larvae with solvent or buffer solution.