The cut-off point is as recommended by the manufacturers of the ELISA kit using the technique discussed in Collection Panel for anti-RA33. Results are considered positive at 25 U/mL or higher. 
Positive RA33 antibody is correlated with rheumatoid arthritis (RA) but can been found falsely elevated in patients with other autoimmune diseases, such as systemic sclerosis, mixed connective tissue disease, and juvenile idiopathic arthritis. [2, 3] Also, lupus patients with RA33 positive antibodies tend to have more erosive disease than lupus patients who present with arthralgia and nonerosive joint manifestations.
RA33 antibody is seen in initial phase of RA and is associated with severity of disease. [4, 2, 3] As compared to other antibodies, it is the only antibody with fluctuating levels correlating with disease activity. It decreases considerably after patients enters remission or respond to treatment. The only limiting factor in using this antibody in diagnostic criteria for RA (as suggested by EULAR and ACR) is its wide range of sensitivity, which was reported as low as 6% to as high as 75%.
The production of autoantibodies against specific or many self-antigens in the body is the diagnostic hallmark of autoimmune disorders. In the 1940s, the concept of autoimmunity in RA was proposed by Waaler, who shed light on the disturbances in the connective tissue metabolism involved in this disease. [5, 6] Waaler demonstrated that the autoantibody rheumatoid factor (RF) is elevated in patients with RA. Several years later, in 1970, Steffen hypothesized that RA could be a collagen autoimmune disease. [5, 6] Past clinical evidence has demonstrated the production of several autoantibodies, including RF and other anti-collagen antibodies, in synovial plasma cells in response to RA pathogenesis. These findings suggest local antigen activity and immune response activation in the synovial tissue. Despite its nonspecificity, RF is still widely employed in the diagnostic workup for RA. 
The need to explore other diagnostic markers with greater specificity for RA lead to further research revealing novel markers such as anti-RA33, anti-keratin antibody, and more recently, anti-citrullinated antibodies, which showed a satisfying specificity in the immunodiagnostic testing of RA. [7, 6, 8]
Two of these three antibodies (anti-citrullinated protein antibody and anti-keratin antibody) are found in the perinuclear region of the cell, whereas anti-RA33 antibody is found in the nucleolar region of the cell. Some of these antibodies were discovered in the 1970s but it took several years for researchers to establish the potential role of these antibodies in the pathogenesis of RA. Some of the research was done by Cordonnier et al, who tried to establish the role of anti-CCP, RF, and anti-RA33 in early RA.  Other groups mentioned that the frequency of positive RF, AKA and anti-CCP were lower in early RA but a similar trend was not noticed for the anti-RA33 antibody. This discovery drew the attention of researchers to anti-RA33 as it appeared to be a promising diagnostic test to establish the diagnosis of RA during the early stages of the disease.
Anti-RA33 antibody was discovered by Hassfeld et al in 1989 and was found to bind a specific nuclear protein in HeLa cells. Further studies done in arthritis mouse models established that this antibody is against a spliceosome, which is a heterogeneous ribonucleoprotein complex (36 KDa) A2 protein (hnRNP A2). However, hnRNP-A2 (and its alternatively spliced variants B1 and B2) is the sole epitope of RA33 that is reported to be potentially autoantigenic in RA patients. About 30 different epitopes of hnRNP have been discovered, each epitope referring to the specific protein sequence combined to pre-mRNA to form the hnRNP complex.
Beside hnRNP-A2, a few other members of the big family of hnRNPs have shown autoantigenic involvement, with more or less affinity, in systemic autoimmune rheumatic diseases. [6, 9] Even though HnRNPA2 is found in multiple body organs (skin, lymph nodes, brain, reproductive organs), it is found in abundance only in inflamed joints and lacks expression in the synovium of healthy joints. Hence, researchers believed this marker to be highly specific for RA. The diagnostic utility of anti-RA33 autoantibody in RA is still controversial, as it was recognized in a low proportion of RA patients. [6, 9, 10] Further research has demonstrated that anti-RA33 shows variable sensitivity ranging from 6-58% and specificity ranging from 69-96%. This might be attributed to either RA severity, ethnic origin, or the degree of purification of the RA33 that has been used as a recombinant autoantigen source in the ELISA methods.
Few authors have reported that anti-hnRNP B1 autoantibodies are significantly more prevalent in RA patient with combined systemic sclerosis and hypertension.  Anti-RA33 antibodies have a strong ethnic association and have been shown to be significantly elevated in Austrian and French populations with RA. No correlation is noticed with age or gender. 
Anti-RA33 has been found to be positive in the initial stages of RA and seems to correlate with disease activity. In contrast, anti-CCP antibodies always remain positive regardless of disease activity. [7, 4, 3] Positive anti-RA33 antibody is associated with less erosive disease than RF-positive RA, but patients who are seropositive for anti-RA33 antibody have been found to have mildly more severe disease than seronegative RA patients. [4, 2, 3] Anti-RA33 antibody levels vary with duration of disease and are usually lost once the patient enters remission or a quiescent phase of rheumatoid arthritis. Hence, researchers have suggested using a combination of RA33 antibody and CCP antibody in monitoring remission and relapses of disease. 
RA33 antibody has also been found to be positive in patients with juvenile idiopathic arthritis (especially its polyarticular subset/category) and systemic lupus erythematosus (SLE). Patients with SLE who are seropositive for RF and anti-RA33 have been found to have more erosive disease. [4, 2, 3]
Specimen required: Human serum, plasma, or cell culture supernatant and organizations in the natural and recombinant hnRNP/RA33 concentration.
Sample volume: 50-100 μL
Serum: Use a serum separator tube (SST) and allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2-8°C within 30 minutes of collection. Immediately assay or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Tissue Homogenates: Rinse 100 mg tissue with 1 × PBS, homogenize in 1 mL of 1 × PBS and store overnight at -20°C. After two freeze-thaw cycles to break the cell membranes, centrifuge the homogenates for 5 minutes at 5000 × g, 2-8°C. Remove and assay the supernate immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Preparation and storage: Samples to be used within 5 days may be stored at 2-8°C, otherwise samples must be stored at -20°C (≤1 mo) or -80°C (≤2 mo) to avoid loss of bioactivity and contamination.
Sample limitation: Grossly hemolyzed samples are not suitable for use in this assay.
Expiration date: 6 months
Test use: Anti-RA33 antibody was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit using double sandwich ELISA technique. Similar test has been used to diagnose RF and CRP.
Method of test : The assay reaction for anti-RA33 involves covalent immobilization of recombinant RA33 (hnRNP/A2) to the solid phase of microtiter strips and subsequent binding of anti-RA33 antibodies from patient serum. The bound antibodies are detected with a peroxidase-labelled secondary antibody that is directed against human IgG. After addition of the substrate solution, the antibodies are stained. The intensity of the color is proportional to the concentration and the avidity of the detected antibodies. After the addition of stop solution, the color changes from blue to yellow. The results were calculated from the standard curve obtained. 
Detection range: 10 ng/mL-0.156 ng/mL
Sensitivity of test used: The minimum detectable human hnRNP/RA33 up to 0.05 ng/mL
Precision of diagnostic test: 8% or less
Cross-Reactivity: Limited by current skills and knowledge. It is impossible to complete the cross-reactivity detection between the target antigen and all analogues for other species. Therefore, cross-reaction may still exist.