Method and measurement kit for assay of normal agrecan, and method for evaluation of informations on the joint

A method of determining the concentration of normal aggrecan in a joint, comprising steps of:

• a) taking synovial fluid from a joint which is suspected to have arthritis;
• b) degrading or removing hyaluronan bound to normal aggrecan and free hyaluronan in said synovial fluid;
• c) contacting said synovial fluid treated by step b) with a solid phase having hyaluronan bound on said solid phase, thereby binding or adsorbing to said solid phase only normal aggrecan capable of binding to hyaluronan without binding or adsorbing to said solid phase proteoglycan, including aggrecan which has lost ability of binding to hyaluronan, other than normal aggrecan;
• d) separating said solid phase from said synovial fluid treated by step c);
• e) detecting normal aggrecan separated and bound or adsorbed to said solid phase in step d) by utilizing means for detecting keratan sulfate, chondroitin sulfate, or proteoglycan;
• and
• f) determining concentration of normal aggrecan in synovial fluid obtained in step a) from said normal aggrecan detected in step e).

A method of diagnosis of arthritis comprising steps of:

• a) taking synovial fluid from a joint which is suspected to have arthritis;
• b) degrading or removing hyaluronan bound to normal aggrecan and free hyaluronan in said synovial fluid;
• c) contacting said synovial fluid treated by step b) with a solid phase having hyaluronan bound on said solid phase, thereby binding or adsorbing to said solid phase only normal aggrecan capable of binding to hyaluronan without binding or adsorbing to said solid phase proteoglycan, including aggrecan which has lost ability of binding to hyaluronan, other than normal aggrecan;
• d) separating said solid phase from said synovial fluid treated by step c);
• e) detecting normal aggrecan separated and bound or adsorbed to said solid phase in step d) by utilizing means for detecting keratan sulfate, chondroitin sulfate, or proteoglycan;
• f) determining concentration A of normal aggrecan in synovial fluid obtained in step a) from said normal aggrecan detected in step e);
• g) measuring concentration B of total proteoglycan, including both normal aggrecan and other proteoglycan, in said synovial fluid obtained in step a);
• h) calculating ratio of said concentration A of normal aggrecan obtained in step f) to said concentration B of total proteoglycan obtained in step g), i.e., A/B;
• and
• i) diagnosing as an arthritis when said ratio A/B significantly decreases in comparison with corresponding value measured regarding synovial fluid from a normal joint.

A method of diagnosis of arthritis comprising steps of:

• a) taking synovial fluid from a joint which is suspected to have arthritis;
• b) degrading or removing hyaluronan bound to normal aggrecan and free hyaluronan in said synovial fluid;
• c) contacting said synovial fluid treated by step b) with a solid phase having hyaluronan bound on said solid phase, thereby binding or adsorbing to said solid phase only normal aggrecan capable of binding to hyaluronan without binding or adsorbing to said solid phase proteoglycan, including aggrecan which has lost ability of binding to hyaluronan, other than normal aggrecan;
• d) separating said solid phase from said synovial fluid treated by step c);
• e) detecting normal aggrecan separated and bound or adsorbed to said solid phase in step d) by utilizing means for detecting keratan sulfate, chondroitin sulfate, or proteoglycan;
• f) determining concentration A of normal aggrecan in synovial fluid obtained in step a) from said normal aggrecan detected in step e);
• g) measuring concentration B of total proteoglycan, including both normal aggrecan and other proteoglycan, in said synovial fluid obtained in step a);
• h) calculating difference, i.e. concentration B-A, between said concentration B of total proteoglycan obtained in step g) and said concentration A of normal aggrecan obtained in step f;
• i) comparing said concentration A of normal aggrecan and said concentration B-A of proteoglycan other than normal aggrecan with corresponding value measured regarding synovial fluid from a normal joint;
• and
• j) diagnosing as an arthritis when there is significant variation of A or B-A from the normal value of A or B-A, respectively.

A method of diagnosis of arthritis comprising steps of:

• a) taking synovial fluid from a joint which is suspected to have arthritis;
• b) degrading or removing hyaluronan bound to normal aggrecan and free hyaluronan in said synovial fluid;
• c) contacting said synovial fluid treated by step b) with a solid phase having hyaluronan bound on said solid phase, thereby binding or adsorbing to said solid phase only normal aggrecan capable of binding to hyaluronan without binding or adsorbing to said solid phase proteoglycan, including aggrecan which has lost ability of binding to hyaluronan, other than normal aggrecan;
• d) separating said solid phase from said synovial fluid treated by step c);
• e) detecting normal aggrecan separated and bound or adsorbed to said solid phase in step d) by utilizing means for detecting keratan sulfate, chondroitin sulfate, or proteoglycan;
• f) determining concentration A of normal aggrecan in synovial fluid obtained in step a) from said normal aggrecan detected in step e);
• g) measuring concentration B of total proteoglycan, including both normal aggrecan and other proteoglycan, in said synovial fluid obtained in step a);
• h) calculating difference, i.e. concentration B-A, between said concentration B of total proteoglycan obtained in step g) and said concentration A of normal aggrecan obtained in step f);
• i) comparing said concentration A of normal aggrecan and said concentration B-A of proteoglycan other than normal aggrecan with corresponding values, A' and B'-A', which respectively correspond to values A and B-A measured regarding synovial fluid from normal joint;
• j) diagnosing as an arthritis with slow cartilage destruction when A is significantly low but there is no significant difference in B-A;
• and
• k) diagnosing as an arthritis with rapid cartilage destruction when there is no significant difference in A but B-A is significantly high.