Method for estimating immunostimulant activity of a polysaccharide
The ability of a polysaccharide to act as an immunostimulant may be assessed by comparing the degree of binding of a detection reagent, which contains a fluorescence-labeled version of the polysaccharide, with the binding of a reference reagent, which contains a fluorescence-labeled version of a different polysaccharide. The ability of a polysaccharide to act as an immunostimulant may also be assessed by comparing the degree of binding of a detection reagent, which contains a fluorescence-labeled version of the polysaccharide, with the binding of a different type of reference reagent, which contains both the same fluorescence-labeled version of the polysaccharide and an unlabelled version of the same polysaccharide.
1. A method for estimating an immunostimulant activity of a polysaccharide, which comprises:
(i) obtaining a peripheral blood sample which contains peripheral blood mononuclear cells from a mammal;
(ii) contacting a first portion of said blood sample with a detection reagent comprising a fluorescence-labeled immunostimulant polysaccharide, which contains a first polysaccharide moiety, to obtain a first fraction which comprises target peripheral blood mononuclear cells;
(iii) measuring fluorescent intensity of said first fraction to determine the amount (B) of said target peripheral blood mononuclear cells bonded to said fluorescence-labeled immunostimulant polysaccharide;
(iv) contacting a second portion of said blood sample with a reference reagent comprising a fluorescence-labeled polysaccharide, which contains a second polysaccharide moiety which is different from said first polysaccharide moiety, to obtain a second fraction which comprises target peripheral blood mononuclear cells;
(v) measuring fluorescent intensity of said second fraction to determine the amount (A) of said target peripheral blood mononuclear cells bonded to said fluorescence-labeled polysaccharide; and
(vi) correlating the difference (B−A) between the amounts (A) and (B) with the immunostimulant activity of said first polysaccharide moiety for said mammal.
2. The method of claim 1 , wherein said contacting of said first portion of said blood sample with said detection reagent is carried out at a temperature ranging from 0 to 50° C.
3. The method of claim 2 , wherein said immunostimulant polysaccharide is a β-glucan.
4. The method of claim 3 , wherein said immunostimulant polysaccharide is lentinan.
5. A method for estimating an immunostimulant activity of a polysaccharide, which comprises:
(i) obtaining a peripheral blood sample which contains peripheral blood mononuclear cells from a mammal;
(ii) contacting a first portion of said blood sample with a detection reagent comprising a fluorescence-labeled immunostimulant polysaccharide, which contains a first polysaccharide moiety, to obtain a first fraction which comprises target peripheral blood mononuclear cells;
(iii) measuring fluorescent intensity of said first fraction to determine the amount (B) of said target peripheral blood mononuclear cells bonded to said fluorescence-labeled immunostimulant polysaccharide;
(iv) contacting a second portion of said blood sample with a reference reagent which comprises:
(a) an immunostimulant polysaccharide which is not labeled with any fluorescent material and which has a polysaccharide moiety identical to said first polysaccharide moiety; and
(b) said detection reagent, to obtain a second fraction which comprises target peripheral blood mononuclear cells;
v) measuring fluorescent intensity of said second fraction to determine the amount (A) of said target peripheral blood mononuclear cells bonded to said fluorescence-labeled immunostimulant polysaccharide; and
(vi) correlating the difference (B−A) between the amounts (A) and (B) with the immunostimulant activity of said first polysaccharide moiety for said mammal.
6. The method of claim 5 , wherein said contacting of said first portion of said blood sample with said detection reagent is carried out at a temperature ranging from 0 to 50° C.
7. The method of claim 6 , wherein said immunostimulant polysaccharide is a β-glucan.
8. The method of claim 7 , wherein the said immunostimulant polysaccharide is lentinan.