Immunoassays for high positively charged proteins

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.

1 - 12 . (canceled) 
     
     
         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.   
     
     
         14 . The method according to  claim 13 , wherein the high positively charged protein is an FGF-18 protein. 
     
     
         15 . 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 
     
     
         16 . The method according to  claim 14 , wherein the FGF-18 protein is sprifermin. 
     
     
         17 . The method according to  claim 13 , wherein the dilution buffer of step b) is Rexxip HN. 
     
     
         18 . The method according to  claim 13 , wherein the high ionic force buffer has a salt concentration of at least 1M. 
     
     
         19 . The method according to  claim 18 , wherein the high ionic force buffer is 1.5M NaCl in 20% ethanol. 
     
     
         20 . The method according to  claim 13 , wherein the incubating time of step a) is 1 h. 
     
     
         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.   
     
     
         22 . The method according to  claim 21 , wherein the human synovial fluid sample is part of a set of samples. 
     
     
         23 . The method according to  claim 21 , wherein the column of step c) is one column in a set of columns 
     
     
         24 . 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.