Immunocapture-based measurements of mammalian pyruvate dehydrogenase complex

A method for measuring activity of PDH complex in a sample, said method comprising:

• a) contacting a sample comprising PDH complex with an isolated antibody that specifically bind to PDH complex under conditions to allow formation of an immunocomplex of the antibody and the PDH complex present in the sample;
• b) contacting the immunocomplex with a reaction mixture comprising a non-limiting amount of one or more substrates necessary for activity of the PDH complex;
• and
• c) detecting:
• (i) the amount of NADH produced in the reaction mixture, wherein the amount of NADH produced indicates the active state of the PDH complex;
• or
• (ii) the level of phosphorylation of immunocomplexed PDH complex in the in the sample as compared with that of an unphoshorylated PDH complex standard, wherein a level of phosphorylation greater than that in the standard indicates a lowered level of activity, and a level of phosphorylation substantially equal to that of the PDH complex in the sample indicates a normal level of activity of the PDH complex in the sample.

A method for screening patients to identify patients suspected of having a late onset mitochondrial disorder, said method comprising:

• a) contacting isolated antibodies that immunoprecipitate PDH complex with a patient sample comprising solubilized PDH complex so that the antibodies bind to solubilized PDH complex present in the sample to form an immunocomplex;
• b) separating the immunocomplex from the remaining sample contents;
• and
• c) detecting a decrease in the amount of PDH complex as compared with an amount in a corresponding normal sample, wherein the decrease indicates the patient is suspected of having the late onset mitochondrial disorder.