Patent ID: 8563258

Claim:
A method for the determination of basophil activation induced by a test substance comprising contacting the basophils with a plurality of antibodies comprising anti-CD63 antibody, anti-IgE antibody and anti-CCR3 antibody, each of which is independently labeled with a fluorophore, wherein said anti-CCR3 antibody acts as a selection marker and said anti-CD63 antibody and said anti-IgE antibody each independently act as an activation marker; measuring, via a flow cytometer, a change in the mean or median fluorescence intensity (MFI) of the IgE antibodies bound to the F c εRI receptor on the cell surface of basophils (MFI-IgE) and the anti-CD63 antibodies bound to the CD63 antigen exposed on the cell surface of basophils after activation (MFI-CD63); and correlating the changes of the mean fluorescence intensities of said activation markers in the presence and absence of said test substance to obtain an Activation Index which is either the sum or the product of the activation index for parameter A (y(A)) and parameter B (y(B)), wherein A is the mean or median fluorescence intensity (MFI) of CD63 (MFI-CD63) and B is the MFI of IgE (MFI-IgE) and y for each parameter is calculated in terms of y = a - d ( 1 + ( x c ) b ) + d wherein i) a is the minimum value of the function curve, ii) b is the slope of the curve, iii) c is the turning point of the curve, iv) d is the maximum asymptotic value of the curve, and v) x is 1 minus the ratio between (A) of the negative control and (A) of the test substance, or 1 minus the ratio between (A) of the negative control and (A) of the positive control, or 1 minus the ratio between (B) of the negative control and (B) of the test substance, or 1 minus the ratio between (B) of the positive control and (B) of the negative control.