Specific detection of chitin using chitin-binding domain
Methods and kits are provided for detecting chitin in biological samples using chitin-binding domain (CBD).
1 . A method for specifically detecting chitin and not cellulose in a sample, comprising the steps of:
(a) contacting the sample with a first reagent comprising a chitin-binding domain (CBD) and optionally fused to a maltose-binding domain (MBD); and
(b) detecting specifically whether chitin and not cellulose is present in the sample by the binding of CBD to chitin.
2 . A method as recited in claim 1 , wherein the CBD in the reagent is conjugated to a reporter.
3 . A method as recited in claim 2 , wherein the reporter is selected from the group consisting of a radioactive material, a fluorophore, a dye, an electron-dense compound, and an enzyme.
4 . A method as recited in claim 1 , wherein the sample comprises a plant tissue, an agricultural product, an animal tissue, a human tissue, a contact lens, a prosthetic device, or an air filter.
5 . A method as recited in claim 1 , wherein the sample comprises an animal body fluid, a human body fluid, a plant fluid, potable water, or a beverage.
6 . A method as recited in claim 1 , wherein the contacting step additionally comprises contacting the sample with a second reagent comprising an antibody to CBD or an antibody to a protein fused to CBD.
7 . A method as recited in claim 6 , wherein the first reagent additionally comprises a reporter.
8 . A method as recited in claim 7 , wherein the reporter is selected from the group consisting of a radioactive material, a fluorophore, a dye, an electron-dense compound, and an enzyme.
9 . A method according to claim 1 , wherein the CBD has a carbohydrate-binding module corresponding to CBM12.
10 . A method according to claim 1 , wherein step (a) is preceded by bleaching the sample.
11 . A method according to claim 1 , wherein the CBD is obtained from chitinase AI from Bacillus circulans.
12 . A kit, comprising: an immobilized CBD reagent.
13 . A kit according to claim 12 , further comprising instructions for use of the immobilized CBD reagent for detecting chitin.
14 . A kit according to claim 12 , further comprising a soluble CBD carrier protein fusion molecule linked to a reporter.
15 . A kit according to claim 14 , wherein the carrier protein is MBP.
16 . A kit according to claim 14 , wherein the reporter is a rhodomaine or fluorescein dye.
17 . A kit according to claim 13 , wherein the CBD is derived from chitinase AI.
18 . A method for detecting chitin in a sample, comprising:
(a) obtaining an immobilized first CBD;
(b) adding the sample and allowing any chitin in the sample to bind to the immobilized CBD;
(c) adding a second CBD for binding the immobilized chitin of step (b) wherein the CBD is optionally linked to a protein carrier and a reporter molecule or to reporter molecule only and wherein the first CBD and the second CBD are obtained from the same or different chitinase; and
(d) detecting the chitin in the sample.
19 . A method according to claim 18 , wherein the second CBD is linked to a carrier protein, wherein the carrier protein is MBP.
20 . A method according to claim 19 , wherein step (d) further comprises detecting the chitin by means of a labeled antibody.
21 . A method according to claim 19 , wherein the first CBD is immobilized by means of a chemical linker.
22 . A method according to claim 19 , wherein the first CBD is immobilized on a substrate selected from: a bead, a gel, a filter, a column and a reaction vessel surface.