US 12,474,329 B2
Immunoassays for high positively charged proteins
Elisa Bertotti, Strambino (IT); and Valeria Castagna, Pavone Canavese (IT)
Assigned to Ares Trading S.A., Aubonne (CH)
Filed by Ares Trading S.A., Aubonne (CH)
Filed on Feb. 3, 2023, as Appl. No. 18/164,275.
Application 18/164,275 is a continuation of application No. 15/547,080, abandoned, previously published as PCT/EP2016/051808, filed on Jan. 28, 2016.
Claims priority of application No. 15153117 (EP), filed on Jan. 29, 2015.
Prior Publication US 2023/0408529 A1, Dec. 21, 2023
Int. Cl. G01N 33/53 (2006.01); C12Q 1/40 (2006.01); G01N 33/543 (2006.01); G01N 33/68 (2006.01); G01N 35/00 (2006.01); G01N 35/10 (2006.01)
CPC G01N 33/5306 (2013.01) [C12Q 1/40 (2013.01); G01N 33/54306 (2013.01); G01N 33/54393 (2013.01); G01N 33/6887 (2013.01); G01N 35/0099 (2013.01); G01N 35/10 (2013.01); G01N 35/1095 (2013.01); G01N 2035/00495 (2013.01); G01N 2333/50 (2013.01); G01N 2800/102 (2013.01)] 9 Claims
 
1. A method for quantification of a highly positively charged protein in a human synovial fluid sample comprising the steps of:
a) pre-treating the human synovial fluid sample, the pre-treating step comprising adding hyaluronidase solution to the human synovial fluid sample, incubating said sample at room temperature (RT), and centrifuging the human synovial fluid sample;
b) diluting the pre-treated human synovial fluid sample with a buffer;
c) immobilizing a biotinylated antibody against the highly positively charged protein to a column;
d) washing the column to remove unbound antibody with a standard wash buffer;
e) contacting in the column the pre-treated and diluted human synovial fluid sample with the immobilized biotinylated antibody under conditions in which the antibody binds specifically to the highly positively charged protein, to produce an antibody-protein complex;
f) washing the column with a standard wash buffer;
g) adding to the antibody-protein complex in the column a fluorescent dye-labelled antibody specific for the highly positively charged protein to produce a measurable response, and washing the column with a standard wash buffer;
h) measuring the response produced; and
i) determining a quantity of highly positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard;
wherein the highly positively charged protein in the sample is fibroblast growth factor 18 (FGF-18) having an isoelectric point at or above 9.5, and
wherein the FGF-18 protein is selected from the group consisting of: a) a polypeptide comprising the amino acid residues 28-207 of SEQ ID NO: 1, b) a polypeptide comprising the amino acid residues 28-196 of SEQ ID NO: 1, and c) a polypeptide comprising SEQ ID NO: 2.
 
4. A method for automatic quantification of a highly positively charged protein in a human synovial fluid sample comprising the steps of:
a) pre-treating the human synovial fluid sample, the pre-treating step comprising adding hyaluronidase solution to the human synovial fluid sample, incubating said sample at room temperature (RT), and centrifuging the human synovial fluid sample;
b) diluting the pre-treated human synovial fluid sample with a buffer;
c) immobilizing a biotinylated antibody against the highly positively charged protein to a column;
d) washing the column to remove unbound antibody with a standard wash buffer;
e) providing an injection means for automatic transfer of the pre-treated and diluted human synovial fluid sample to the column;
f) washing the injection means with a high ionic force buffer that is 1.5M NaCl in 20% ethanol before the pre-treated and diluted human synovial fluid sample is transferred to the column;
g) transferring the pre-treated and diluted human synovial fluid sample to the column, thereby contacting the pre-treated and diluted human synovial fluid sample with the immobilized biotinylated antibody under conditions in which the antibody binds specifically to the highly positively charged protein, to produce an antibody-protein complex;
h) washing the injection means with the high ionic force buffer after the step g);
i) washing the column with a standard wash buffer;
j) adding to the antibody-protein complex in the column a fluorescent dye-labelled antibody specific for the highly positively charged protein to produce a measurable response, and washing the column a standard wash buffer;
k) measuring the response produced; and
l) determining a quantity of the highly positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard;
wherein the highly positively charged protein in the sample is fibroblast growth factor 18 (FGF-18) having an isoelectric point at or above 9.5, and
wherein the FGF-18 protein is selected from the group consisting of: a) a polypeptide comprising the amino acid residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising the amino acid residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising SEQ ID NO:2.