Immunoassay methods

A method of detecting a disease state or disease susceptibility in a mammalian subject, the method comprising detecting an antibody in a test sample, wherein the test sample comprises a bodily fluid from the mammalian subject and wherein the antibody is a biological marker of a disease state or disease susceptibility, which method comprises:

• (a) preparing two or more different dilutions of the test sample and carrying out the following steps (i) and (ii) in respect of each test sample dilution in order to provide a cross-titration assay based on testing two or more dilutions of a test sample against two or more amounts of antigen to form a titration curve:
• (i) contacting each of the two or more test sample dilutions with two or more different amounts of an antigen specific for the antibody,
• (ii) detecting the amount of specific binding between the antibody and the antigen for each amount of antigen used in step (i),
• (b) plotting or calculating a separate curve of the amount of specific binding versus the amount of antigen for each of the two or more test sample dilutions used in step (a), and
• (c) determining the presence or absence of the disease state or disease susceptibility based upon the amount of specific binding between the antibody and the antigen for each test sample dilution at each amount of antigen tested
• wherein an increased amount of specific binding, when compared to a normal control having the same dilutions, indicates the presence of disease state or susceptibility.