# US 2018/0024142 A1 · Immunoassays for high positively charged proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0024142 A1 - Document ID: US20180024142A1 - Status: Published application - Kind: A1 - Application number: 15/547,080 - Filed: 2016-01-28 - Published: 2018-01-25 - Earliest claimed: 2015-01-29 - Assignees: Ares Trading S.A. - Applicants: Ares Trading S.A. - Inventors: ELISA BERTOTTI; VALERIA CASTAGNA - Web page: https://patentgrep.com/patent/US20180024142A1 ## CPC - G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding} - C12Q 1/40: involving amylase - G01N 33/54306: {Solid-phase reaction mechanisms} - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - G01N 33/6887: {from muscle, cartilage or connective tissue} - G01N 35/0099: {comprising robots or similar manipulators (robots per se B25J)} - G01N 35/10: Devices for transferring samples {or any liquids} to, in, or from, the analysis apparatus, e.g. suction devices, injection devices {(G01N35/0099 takes precedence)} - G01N 35/1095: {for supplying the samples to flow-through analysers (for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00)} - G01N 2035/00495: {Centrifuges} - G01N 2333/50: Fibroblast growth factors [FGF] - G01N 2800/102: Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to immunoassays for quantification of a high positively charged protein, such as a FGF-18 protein, in human synovial fluid sample. ## Claims ### Claim at position 1 1-12. (canceled) ### Claim 13 A method for quantification of a high 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), 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 high 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 high 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 high positively charged protein to produce a measurable response, and washing the column with a standard wash buffer, h) measuring the response produced, i) determining a quantity of high positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard. ### Claim 14 (depends on 13) The method according to claim 13, wherein the high positively charged protein is an FGF-18 protein. ### Claim 15 (depends on 14) The method according to claim 14, wherein the FGF-18 protein is selected from the group consisting of: a) a polypeptide comprising or consisting of the amino acid residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising or consisting of the amino acid residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising or consisting of SEQ ID NO:2 ### Claim 16 (depends on 14) The method according to claim 14, wherein the FGF-18 protein is sprifermin. ### Claim 17 (depends on 13) The method according to claim 13, wherein the dilution buffer of step b) is Rexxip HN. ### Claim 18 (depends on 13) The method according to claim 13, wherein the high ionic force buffer has a salt concentration of at least 1M. ### Claim 19 (depends on 18) The method according to claim 18, wherein the high ionic force buffer is 1.5M NaCl in 20% ethanol. ### Claim 20 (depends on 13) The method according to claim 13, wherein the incubating time of step a) is 1 h. ### Claim 21 A method for automatic quantification of a high 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) 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 high 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 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 high positively charged protein, to produce an antibody-protein complex, h) washing the injection means with a 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 high positively charged protein to produce a measurable response, and washing the column a standard wash buffer, k) measuring the response produced, l) determining a quantity of the high positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard. ### Claim 22 (depends on 21) The method according to claim 21, wherein the human synovial fluid sample is part of a set of samples. ### Claim 23 (depends on 21) The method according to claim 21, wherein the column of step c) is one column in a set of columns ### Claim 24 (depends on 21) The method according to claim 21, wherein all the steps are repeated as often as needed to automatically quantify a high positively charged protein in a set of human synovial fluid samples to be analysed.