Method for quantitative assessment in biological sample of the phosphorylated and non-phosphorylated form of rkip

A method for RKIP protein and p-RKIP protein quantitative measuring in a biological sample, comprising the steps of:

• a) coacting to two distinct supports:
• a1) an antibody recognizing a first amino acid sequence present in p-RKIP protein and
• a2) an antibody recognizing a first amino acid sequence present in RKIP protein;
• b) adding a blocking solution to both the supports,
• c) adding said biological sample;
• d) adding, in the respective supports
• d1) an antibody recognizing a second amino acid sequence present in p-RKIP protein,
• and
• d2) an antibody recognizing a second amino acid sequence present in RKIP protein;
• one of said first and second amino acid sequence being common between RKIP and p-RKIP, and the other one being a sequence containing the phosphorylation of serine 153, and so, different for p-RKIP and RKIP;
• e) adding a marker able to determine a variation of the optical properties proportional to the quantity of said antibody bound to said second amino acid sequence, when in contact with said antibody recognizing said second amino acid sequence;
• f) measuring an optical property of the two samples and comparing it with a calibration curve obtained by measuring the same optical property of two series of supports, in which there have been added, in the order:
• i) an antibody recognizing said first amino acid sequence present in p-RKIP protein for the first one of said series and said first amino acid sequence present in RKIP protein for the second one of said series;
• ii) a blocking solution;
• iii) known and increasing quantities of a recombinant hybrid standard comprising both said first and second amino acid sequences present in p-RKIP protein for the first one of said series and both said first and second amino acid sequences present in RKIP protein for the second one of said series;
• iv) antibodies recognizing specifically said second amino acid sequence present in p-RKIP or said second sequence present in RKIP, respectively;
• v) a marker able to determine a variation of the optical properties proportional to the quantity of said antibody bound to said second amino acid sequence, when in contact with said antibody recognizing said second amino acid sequence.