Diagnostic assay
The present disclosure relates generally to the field of immunological-based diagnostic assays. Particularly, a method is contemplated herein for measuring cell-mediated immune response reactivity. The present disclosure further provides a method to reduce incidence of non-specific immune response reactivity in a cell-mediated immune response-based assay, comprising contacting a sample with a basic peptide structure capable of binding to lipopolysaccharides, such as polymyxin B or a sushi peptide.
1. A method for measuring cell-mediated immune response activity in a subject, said method comprising:
(a) contacting a sample to be assayed which comprises a source of immune cells that comprise lymphocytes from the subject, with (i) a basic peptide structure capable of binding to cell lipopolysaccharides, wherein the basic peptide is polymyxin B or a polymyxin B antibiotic family member that is a chemical analog of polymyxin B, or a sushi peptide, and (ii) an antigen to which a cell-mediated immune response by said immune cells is to be tested, wherein the source of immune cells is undiluted whole blood collected from the subject, wherein the whole blood comprises from about 50% to 100% by volume of the sample to be assayed, and wherein said step of contacting takes place within 24 hours of collecting the whole blood from the subject, thereby to obtain contacted immune cells;
(b) adding to said contacted immune cells (iii) an immune response stimulant which comprises a nucleic acid molecule that is polyinosinic acid-polycytidylic acid (polv(I:C)), to enhance the cell-mediated response to the antigen; and
(c) measuring, in plasma obtained from the whole blood subsequent to step (b), presence or elevation in a level of at least one immune effector molecule from the immune cells, wherein the presence or elevation in the level of the immune effector molecule is indicative of a level of cell-mediated immune responsiveness of the subject, said at least one immune effector molecule being selected from interferon-γ (IFN-γ), IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, TNF-α, TGF-β, (G)-CSF, and (GM)-CSF.
2. The method of claim 1 wherein the polymyxin B comprises N-[4-amino-1-[[1-[[4-amino-1-oxo-1-[[6,9,18-tris(2-aminoethyl)-15-benzyl-- 3-(1-hydroxyethyl)-12-(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10-,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-hydroxy-1-oxo- butan-2-yl]amino]-1-oxobutan-2-yl]-6-methyloctanamide, or the sushi peptide comprises the amino acid sequence set forth in SEQ ID NO:1 .
3. The method of claim 1 wherein the polv(I:C) is high molecular weight poly(I:C).
4. The method of claim 1 wherein the subject is a human.
5. The method of claim 1 wherein the immune cells are T-cells or NK cells.
6. The method of claim 1 wherein, the whole blood comprises from about 80% to 100% by volume of the sample to be assayed.
7. The method of claim 1 wherein at least one of (i) the whole blood is collected in a tube comprising antigen, (ii) the whole blood is collected in a tube comprising heparin, or (iii) the whole blood is collected in a tube comprising a simple sugar in dried form.
8. The method of claim 1 wherein the at least one immune effector molecule is IFN-γ.
9. The method of claim 1 wherein in the step of measuring, the immune effector molecule is detected by ELISA.