It is seen from FIGURE that a solid support having affixed thereto an antibody specific for an antigen is contacted with a liquid suspected of containing the antigen, and conditions conducive to binding the antigen to the antibody are provided (Step 1). A second antibody (labeled A E b in the drawing), specific for a different determinant of the antigen and having enzyme conjugated thereto, is contacted under binding conditions to provide an antibody-antigen-antibody sandwich on the support (Ste 2). After a conventional separation of the support from the liquid, the sandwich on the support is contacted with a delta 5-3-ketosteroid substrate (Step 3). The enzyme component of the second antibody on support converts the substrate to a delta 4-3-ketosteroid, the extent of the conversion being proportional to the quantity of bound enzyme, which in turn is proportional to the quantity of antigen in the liquid. In Step 4, a tetrazolium salt is added. Any remaining delta 5 substrate reacts with the dye to give a color, but the delta 4-3-ketosteroid formed by the enzymatic reaction does not react with the dye and thus does not cause color formation. The color formed, or its rate of formation, may be measured to indicate the presence of antigen in the liquid.
The TSH receptor is formed as one polypeptide chain and inserted into the thyroid cell plasma membrane. It undergoes a processing that is reminiscent of that occurring with insulin. A segment of 30 or more amino acids is cut out of the receptor at approximately residue 320, forming a two peptide structure with the chains held together by disulfide bonds. It is thought that both the intact and the processed receptor are functional. The processing of the receptor is thought to involve a matrix metalloprotease-like enzyme cleaving the 120 kDa precursor to form the heterodimeric receptor. Subsequently, reduction of the disulfide bonds by a protein disulfide isomerase may separate the two molecules and lead to shedding of the “alpha” subunit. It is an interesting concept that shedding of the alpha subunit might be intimately related to onset of autoimmunity against the TSH receptor. Shedding of the receptor is augmented by TSH stimulation of thyroid cells (58). The amino-terminal ectodomain of the human TSH receptor has been expressed on the surface of CHO cells as a glycosylphosphatidylinositol-anchored molecule. This material can be released from the cells and is biologically active in that it binds immunoglobulins from serum of patients with Graves’ disease, and displays saturable binding of TSH (46), indicating that all of the “immunologic information” related to production of antibodies resides in the extracellular portion of TSH-R.
where n indexes ionization state of 4F d 2 (1 = neutral; 2 = anionic), I n KSI is the integrated intensity of a C-F peak in the protein ( Figure 2F ), and I n ref is the integrated intensity of the basis spectrum of neutral or ionized 4F d 2 ( Figure 2E ). We note that equation (2) assumes that the intensity of a transition is unperturbed by the active site environment. This assumption appears valid, as the sum of the two referenced intensities in the KSI D40N -bound spectrum ( Figure 2F , Table 2 ) is , quite close to unity. The doubly-peaked spectra of KSI D40N •4F d 2 ( Figure 4 ) were fit to two Gaussians using the OPUS software package with the Levenberg-Maquardt algorithm ( Figure 2F , red and blue traces). The Gaussian fits were integrated to give the integrated intensities, I n KSI . The integrated intensities were referenced (according to equation (2) ) to give relative populations of the ionization states, p n (1 = neutral; 2 = anionic). A simple ratio of the populations