Antiribonucleoprotein and anti-Sm - Sample
Serum
Specimen stable at 2-8°C for 1 week.

Antiribonucleoprotein and anti-Sm - Methods

Antiribonucleoprotein and anti-Sm - Indirect immunofluorescence assay
The immunofluorescence techniques used in autoimmune disease diagnosis can be divided into direct detection methods and indirect fluorescent antibody (IFA) assays, in the IFA methods a substrate (antigen) is immobilized on a solid phase (often a slide) and incubated with the patient's serum (containing the antibodies in question). After a washing step, specifically bound antibodies (usually IgG class) are detected using labeled anti-IgG antibodies. Fluorescein isothiocyanate (FITC) is generally used as the conjugate, the characteristic apple-green fluorescence then being observed under the microscope.
Early indirect immunofluorescence assays (IFA) used a fluoresceinated anti-immunglobulin to detect human ANA bound to Kidney Stomach Liver (KSL) tissue of mouse or rat. Since these first assays, which are nowadays as first line tool for the ANA diagnostics obsolete, several significant improvements have been made in the ANA assay. The single most important change was the replacement of mouse tissue substrate with HEP-2 cells for ANA screening.. The HEP-2 cells show greater sensitivity, have large nuclei, lower background staining, and can be manipulated to ensure the presence of a significant number of dividing cells.

Antiribonucleoprotein and anti-Sm - Radioimmunoasay
Radioimmunoassay (RIA) is based on competition of a known excess amount of radiolabeled antigen (or hapten) and an unknown amount of the same antigen in the The most frequently used procedure is the solid-phase RIA in which the antibody to the substance being assayed is bound to polystyrene tubes. The coupled antibody is still able to interact with the antigen, and after equilibrium is reached, separation of the bound from the free antigen is achieved by washing the tube. The bound antibody remains adsorbed to the tube surface.
The use of RIA methods has been hindered by the intrinsic hazard and instability of the isotopes and by the deluge of governmental regulations that control their manufacture and use. The search for nonisotopic labels has produced the enzyme immunoassay (EIA), which shares with RIA the specificity, sensitivity, and rapidity and has the advantage of safety. In many cases assays formerly performed by RIA are now done by EIA techniques.
The most common RIA techniques used in serology to detect the presence of antigen use a "sandwich principle", where plastic beads coated with antibody to the test antigen are incubated with serum or plasma and appropriate test controls. If the test antigen is present, it will bind to the solid phase antibody. After aspiration of the unbound material and washing of the bead, human 125I-antibody is added and allowed to react with the antibody-antigen complex on the bead. This forms the sandwich - "ab-ag-ab". The beads are washed to remove unbound 125I-antibody and the beads are read through the gamma counter. Specimens giving counts per minute (cpm) equal to or greater than the cutoff value are considered reactive for the test antigen.

Antiribonucleoprotein and anti-Sm - ELISA
ELISA can be performed with nuclear antigens isolated from tissue or cell sources,
although most preparations now are cloned recombinant products produced in bacteria. In an ELISA, an antigen is adhered to wells of a plastic microtiter plate and exposed sequentially to a serum, enzyme conjugated anti-immunoglobulin reagent, and a substrate, the breakdown product of which can be measured colorimetrically
Immunoblot and ELISA are less time consuming than immunodiffusion and show good interassay sensitivity without loss of specificity. The combination of immunoblot and enzyme immunoassay are reported to yield excellent assay sensitivity (100%) and specificity (99%) for detection of autoantibodies