Granulocyte-based methods for detecting and monitoring immune system disorders

1. An ex vivo method for determining immunoglobulin-E (IgE)-mediated food allergy in a subject independent of exposing a whole blood sample from the subject to a stimulating allergen and regardless of whether the subject has a known allergy or predisposition to allergy, and for treating the subject if the subject is determined to have an allergic disease comprising:

(1) determining whether the subject has a food allergy by:

(a) collecting from the subject a whole blood sample containing a live granulocyte population containing a live basophil population whose in vivo activation status has been preserved by not exposing the whole blood sample to a stimulating allergen;

(b) labeling a portion of the whole blood sample of (a) with a labeling agent, wherein the labeling agent is an antibody coupled with a fluorophore, a chemical stain, a radioisotope, or a combination thereof, that is effective to label an intracellular signaling marker expressed by the live basophil population whose in vivo activation status has been preserved, thereby forming a labeled population of live basophils,

wherein the intracellular signaling marker is phospho-SRc;

(c) measuring an expression level of the phospho-SRc protein in the labeled live basophil population of granulocytes in (b) whose in vivo activation status has been preserved, wherein the measuring is carried out via flow cytometry or fluorescence-activated cell sorting (FACS);

(d) collecting from a healthy control subject a whole blood sample containing a live granulocyte population containing a live basophil population whose in vivo activation status has been preserved;

(e) labeling a portion of the whole blood sample of (d) with a labeling agent, wherein the labeling agent is an antibody coupled with a fluorophore, a chemical stain, a radioisotope, or a combination thereof, that is effective to label an intracellular signaling marker expressed by the live basophil population whose in vivo activation status has been preserved, thereby forming a labeled population of live basophils,

wherein the intracellular signaling marker is phospho-SRc;

(f) measuring an expression level of phospho-SRc in the labeled live basophil population of granulocytes in (e) whose in vivo activation status has been preserved, wherein the measuring is carried out via flow cytometry or fluorescence-activated cell sorting (FACS); and

(g) determining if the subject has an immunoglobulin-E (IgE)-mediated food allergy by comparing the expression level of the intracellular signaling marker protein in the labeled live basophil population measured in (c) with the expression level of the intracellular signaling marker protein in the labeled live basophil population measured in (f), wherein the expression level of the intracellular signaling marker protein corresponds to an increase in number and/or activation state of the basophil population;

the expression level of one who has an allergic disease being characterized by an increased level of the intracellular signaling marker protein in the labeled live basophil population in the whole blood sample collected from the subject compared to the expression level of the intracellular signaling marker protein in the labeled live basophil population in the whole blood sample collected from the healthy control subject;

(2) once the subject is determined to have an immunoglobulin-E (IgE)-mediated food allergy in (g), identifying from a panel of food-based allergens one or more specific food-based allergens to which the subject produces an allergic response by repeating steps (a)-(g), wherein steps (a)-(g) further comprise (a′) contacting the population of granulocytes in the whole blood sample with the one or more specific food-based allergens prior to labeling step (b); and

(3) treating the subject with a therapeutically effective allergy treatment regimen, based on the specific food-based allergens identified in (2).

2. The method according to claim 1 , further comprising monitoring effectiveness of the allergy treatment regimen in the subject, wherein the subject

(i) has an allergic disease; and

(ii) has been or is being treated with the allergy treatment regimen;

wherein, in collecting step (a), the whole blood sample collected before the allergy treatment regimen serves as a baseline control; and the expression level of one responsive to the allergy treatment regimen being characterized by a decreased level of the intracellular signaling marker protein in the live basophil population whose in vivo activation status has been preserved compared to the expression level of the intracellular signaling marker protein of the live basophil population in the baseline control.

3. The method according to claim 1 , wherein collection step (a) is carried out via venipuncture.

4. The method according to claim 1 , the basophil being characterized as expressing cell surface marker CD16 and negative for or expressing a low level of cell surface marker CD66b.

5. The method according to claim 1 , wherein steps (b) through (g) are performed at 4° C.