Western blotting using semi-purified hnRNP A / B as antigen is the best detection method. Nuclear extracts can also be used when they are not pure, but you may encounter difficulties in identifying protein bands, especially the serum of patients with SLE. The hnRNP preparation can be obtained by heparin agarose chromatography, and after 12% SDS-polyacrylamide gel electrophoresis, it is transferred onto a nitrocellulose membrane. Serum was diluted 1:25 with 20mm01 / LTris-HCl or pH 7.4 phosphate buffer containing 3% skimmed milk powder, and incubated with the antigen on the membrane for 40min. Avoid containing detergents such as TritonX-100 or Tween20 in the incubation solution, because detergents are presumed to cause non-specific binding of DNA-anti-DNA immune complexes of SLE sera to cause false positive results. It is recommended to use alkaline phosphatase-labeled anti-human IgG antibodies in immunoassays, because certain peroxidase-labeled antibodies can cause non-specific staining, especially in the hnRNP-A2 band. Because the protein migrates into a doublet in the SDS gel, double banding can occur at about 33 / 34kD with anti-hnRNP-A1 staining. Anti-hnRNP-A2 /: RA33 was stained into a band at about 36kD, in addition to 37k8kD corresponding to hnRNP-B1 / B2 also has a doublet. The appearance of these staining features is believed to better explain the Western blot results.

E1 using high purity natural hnRNP-A2. ISA is more sensitive than protein: the sensitivity of the blotting method is high, especially in the detection of serum of patients with SLE and MCTD. However, the results of ELISA and Western blot are sometimes inconsistent, which may be due to the difference of exposed antigenic determinants or the recognition of different epitopes and / or the low solubility of the antigen.