Immunoassays for high positively charged proteins

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.