Method for detecting antibodies to thrombomodulin in patients

A method for monitoring a human patient with lupus anticoagulant for thrombotic risk, comprising the steps of:

• (a) providing in a reaction chamber a truncated soluble thrombomodulin reagent, said reagent comprising a protein having an amino acid sequence beginning from amino acid position 1 of SEQ ID NO:
• 1 and extending to an end amino acid position, said end amino acid position selected so that said reagent is capable of binding to known autoantibodies to thrombomodulin, said reagent lacking the cytoplasmic domain of SEQ ID NO:
• 1 and a sufficient amount of the transmembrane domain so that said reagent is soluble at a concentration sufficient to bind said autoantibodies which may be present in said patient's plasma or serum sample to said truncated soluble thrombomodulin reagent;
• (b) adding a first sample taken at a first collection time to said reaction chamber and incubating for a sufficient time to allow said autoantibodies which may be present in said sample to specifically bind with said truncated soluble thrombomodulin reagent thus forming a bound complex;
• (c) separating said bound complex from unbound materials remaining in said reaction chamber;
• (d) quantitating said bound complex, and correlating the amount of bound complex with the amount of said autoantibodies bound to said truncated soluble thrombomodulin reagent present in said sample;
• (e) repeating step (a)-(d) on a second plasma or serum sample obtained from said patient at a second collection time;
• and
• (f) comparing the amount of autoantibodies in said first sample to the amount of autoantibodies in said second sample and if said second sample has fewer autoantibodies than said first sample, concluding that there is a decrease in level of risk for thrombosis and if said second sample has more autoantibodies than said first sample, concluding there is an increase in level of risk for thrombosis.

A method for testing a human patient plasma or serum sample for autoantibodies to truncated soluble thrombomodulin as an indication of a propensity for a thrombotic disease or intimation, comprising the steps of:

• (a) providing in a reaction chamber a truncated soluble thrombomodulin reagent, said reagent comprising a protein having an amino acid sequence beginning from amino acid position 1 of SEQ ID NO:
• 1 and extending to an end amino acid position, said end amino acid position selected so that said reagent is capable of binding to known autoantibodies to thrombomodulin, said reagent lacking the cytoplasmic domain of SEQ ID NO:
• 1 and a sufficient amount of the transmembrane domain so that said reagent is soluble at a concentration sufficient to bind said autoantibodies which may be present in said sample to said truncated soluble thrombomodulin reagent
• (b) binding said truncated soluble thrombomodulin reagent to the surface of said reaction chamber;
• (c) washing said reaction chamber to remove reactants but so that said truncated soluble thrombomodulin reagent remains bound thereto;
• (d) adding a blocking buffer protein to said reaction chamber to prevent subsequent nonspecific binding of said autoantibodies and other proteins which may be present in the sample to be tested to said reaction chamber;
• (e) washing said reaction chamber to remove reactants but so that said truncated soluble thrombomodulin reagent and said blocking buffer protein remain bound thereto;
• (f) adding said sample to said reaction chamber and incubating for a sufficient time to allow said autoantibodies in said sample to specifically bind with said truncated soluble thrombomodulin reagent;
• (g) washing said reaction chamber to remove reactants but so that said autoantibodies specifically bound to said truncated soluble thrombomodulin reagent remain bound thereto;
• (h) adding a signal generating means to said reaction chamber, said signal generating means capable of binding to said bound autoantibodies to form a detectable end product;
• and
• (i) quantitating said detectable end product to determine the amount of said autoantibody,
• the presence of said autoantibodies indicating said propensity for said thrombotic disease or inflammation.

A method for monitoring a human patient with lupus anticoagulant for nephritis, comprising the steps of:

• (a) providing in a reaction chamber a truncated soluble thrombomodulin reagent, said reagent comprising a protein having an amino acid sequence beginning from amino acid position 1 of SEQ ID NO:
• 1 and extending to on end amino acid position, said end amino acid position selected so that said reagent is capable of binding to known autoantibodies to thrombomodulin, said reagent lacking the cytoplasmic domain of SEQ ID NO:
• 1 and a sufficient amount of the transmembrane domain so that said reagent is soluble at a concentration sufficient to bind said autoantibodies which may be present in said patient's serum or plasma to said truncated soluble thrombomodulin reagent;
• (b) adding a first sample of said patient's serum or plasma taken at a first collection time to said reaction chamber and incubating for a sufficient time to allow said autoantibodies which may be present in said sample to specifically bind with said truncated soluble thrombomodulin reagent, thus forming a bound complex;
• (c) separating said bound complex from unbound materials remaining in said reaction chamber;
• (d) quantitating said bound complex, and correlating the amount of said bound complex with the amount of said autoantibodies present in said sample;
• (e) repeating step (a)-(d) on a second sample of plasma or serum obtained from said patient at a second collection time;
• and
• (f) comparing the amount of said autoantibodies in said first sample to the amount of said autoantibodies in said second sample and if said second sample has fewer autoantibodies than said first sample, concluding that there is a decrease in level of risk for nephritis and if said second sample has more autoantibodies than said first sample, concluding that there is an increase in level of risk for nephritis.

A method for testing a human patient plasma or serum sample for autoantibodies to truncated soluble thrombomodulin as an indication of a propensity for a thrombotic disease or intimation, comprising the steps of:

• (a) providing in a reaction chamber a truncated soluble thrombomodulin reagent, said reagent comprising a protein having an amino acid sequence beginning from amino acid position 1 of SEQ ID NO:
• 1 and extending to an end amino acid position, said end amino acid position selected so that said reagent is capable of binding to known autoantibodies to thrombomodulin, said reagent lacking the cytoplasmic domain of SEQ ID NO:
• 1 and a sufficient amount of the transmembrane domain so that said reagent is soluble at a concentration sufficient to bind said autoantibodies which may be present in said sample to said truncated soluble thrombomodulin reagent;
• (b) adding said sample to said reaction chamber and incubating for a sufficient time to allow said autoantibodies which may be present in said sample to specifically bind with said truncated soluble thrombomodulin reagent thus forming a bound complex;
• (c) separating said bound complex from unbound materials remaining in said reaction chamber;
• and
• (d) quantitating said bound complex, and correlating the amount of said bound complex with the amount of said autoantibodies in said sample, the presence of said autoantibodies indicating said propensity for said thrombotic disease or information.