Dot Immunogold Filtration Assay (DIGFA) For The Rapid Detection Of Specific Autoantibodies Of Rheumatoid Arthritis

Dot immunogold filtration assay (DIGFA) and colloidal gold immunochromatography assay (GICA), two classical immunological techniques, were established along with the development of colloidal gold labeling technique. DIGFA, with features of being rapid, directed, convenient and stable, has been the basis for new rapid diagnostic reagents development and applied in multiple fields. Rheumatoid arthritis (RA) is an autoimmune disease with joint deformity in the late when it is difficult to cure. Early diagnosis is rather important to control the disease to the maximum extent. A lot of research work has shown that specific antibodies may arise several years before the onset of clinical manifestations, which can be very advantageous for the early diagnosis of RA through determination of these antibodies.A rapid DIGFA for specific serodiagnosis of RA was developed in this paper with intention to simplify operation procedure, reduce testing cost and carry out quickly in the case of small number of patients. Through comparison of serum detection results with that of the EUROIMMUN Anti-CCP ELISA kit and reagents adopted clinically, we screened better antigens against two specific autoantibodies of RA. Conjugates of a biotinylated cyclic peptide (CCP) containing 18 amino acids with streptavidin were used for determination of anti-CCP antibodies, and a Rabbit IgG with better biological stabilities was coated for detection of Rheumatoid factor (RF). Meanwhile, we optimize a more specific secondary antibody, F(ab')2 fragment of goat anti-Human lgM, for IgM isotype determination, and the diagnostic performances of several common types of antigens specific for RF were evaluated by ELISA established of our own.Then, colloid gold nano-probes against the two autoantibodies were prepared, for which several parameters were optimized.7 ?L K2CO3(5%) and 22 ?g antibodies for 1 mL colloidal gold were chosen as the optimal conjugation conditions. Better stabilizers for the nano-probes are BSA(immunoassay grade) and PEG-20K, and washing buffer for purification is Tris-HCl at pH 8.2. We also optimized the protective agent for lyophilization of nano-probes.Finally, the DIGFA was established using antigens and nano-probes prepared before, and multiple factors influencing the detection results were studied. Results showed that the best diluent for Rabbit IgG is Tris-HCl at pH 8.2, infiltration rate for serum is 1.4-3.3 ?L/s, blocking agent for NCMs is BSA and washing buffer is PBST with 1% Tween-20, and PEG or Tween can enhance the color of the dot to some extent. Detection of clinical serums with the DIGFA established showed some good results.