Manual of Biological Markers of Disease

Section A: Methods of Autoantibody Detection.- The Editors.- 1. International cooperative activities in standardization of antinuclear antibodies.- 2. Detection of antinuclear antibodies by immunofluorescence.- 1. Introduction.- 2. The immunofluorescence assay.- 2.1 The commercial substrate.- 2.2 The culturing of HEp-2 cells.- 2.3 Fixation of the cells.- 2.4 Preparation of the serum sample.- 2.5 The conjugate antibodies.- 2.6 The incubation procedure.- 3. Interpretation of the results.- 3.1 Nuclear fluorescence patterns.- 3.2 Nucleolar fluorescence patterns.- 3.3 Spindle apparatus fluorescence patterns.- 3.4 Cytoplasmic fluorescence patterns.- 4. Solutions and suppliers.- 4.1 Solutions for culturing the cells.- 3. Counterimmunoelectrophoresis and immunodiffusion for the detection of antibodies to soluble cellular antigens.- 1. Introduction.- 2. Antigen extract preparations.- 2.1 Extraction from acetone powders.- 2.2 Whole cell extract.- 2.3 Spleen extract for Ro.- 2.4 Liver extract for aminoacyl-tRNA synthetases.- 2.5 Nuclear extract.- 2.6 Ion-exchange chromatography.- 3. Procedures.- 3.1 Immunodiffusion.- 3.2 Counterimmunoelectrophoresis (CIE).- 3.3 Staining with coomassie blue.- 3.4 Test planning and interpretation.- 4. Protein Blotting.- 1. Introduction.- 2. The antigens.- 2.1 Preparation of nuclear extracts.- 2.2 Preparation of cytoplasmic extracts.- 3. SDS polyacrylamide gel electrophoresis.- 3.1 Buffer selection.- 3.2 Power conditions.- 4. Protein blotting.- 4.1 Membrane selection.- 4.2 Blotting filter paper.- 4.3 Buffer selection.- 4.4 Semi-dry blotting.- 4.5 Tank blotting.- 4.6 Total protein staining.- 4.7 Blocking reagents.- 4.8 Serum samples.- 4.9 Immunoblot assay.- 5. Antigen appearance on the immunoblot.- 5.1 Nuclear antigens.- 5.2 Nucleolar antigens.- 5.3 Cytoplasmic antigens.- 5. Enzyme-linked immunosorbant assay in the rheumatological laboratory.- 1. Introduction.- 2. General notes.- 2.1 Solid phase.- 2.2 Conjugates.- 2.3 Substrates.- 2.4 Blocking agents.- 2.5 Antigens and antigen presentation.- 3. Assay methods.- 3.1 Solid phase ELISA.- 3.2 The chequerboard titration.- 3.3 Antigen capture (sandwich) ELISA.- 3.4 Dot immunobinding (dot blot) assay.- 4. Evaluation and improvement of the assay system.- 4.1 Evaluation.- 4.2 Improvement of the assay system.- 5. Validation of the assay system.- 6. Validation of a commercial assay system.- 7. Summary.- 8. Solutions and materials.- 8.1 Materials.- 8.2 Coating buffers.- 8.3 Dilution/washing buffer.- 8.4 Chromogenic substrate solution.- 8.5 Stop solutions.- 9. Laboratory notes on ELISA systems for the detection of antinuclear, anti-cytoplasmic and anti-phospholipid antibodies.- 9.1 Anti-nuclear antibodies.- 9.2 Anti-dsDNA.- 9.3 Anti-histone.- 9.4 Anti-Ro/SS-A.- 9.5 Anti-La.- 9.6 Anti-nRNP.- 9.7 Anti-Sm.- 9.8 Anti-ribosomal RNP (P protein).- 9.9 Anti-Sc170 (Topoisomerase 1).- 9.10 Anti-Ki.- 9.11 Anti-Jo-1.- 9.12 Anti-Cardiolipin.- 9.13 Anti-Centromere.- 6. Immunoprecipitation of labelled proteins.- 1. Introduction.- 2. Protein immunoprecipitation procedures.- 2.1 Preparation of 35S-methionine radiolabeled extract.- 2.2 Preparation of the gels.- 3. Interpretation.- 3.1 Sm and U1RNP.- 3.2 SS-B/La and SS-A/Ro.- 3.3 PCNA, Jo-1, rRNP and Ku.- 4. Materials.- 4.1 Reagents and equipment.- 5. Solutions.- 5.1 Tissue culture media.- 5.2 Acrylamide solutions and electrophoresis buffers.- 5.3 Other buffers and solutions.- 7. Analysis of autoimmune sera by immunoprecipitation of cellular RNPs.- 1. Introduction.- 2. Preparation of cellular extracts.- 2.1 Radioactive labeling of RNAs.- 2.2 Cell fractionation.- 3. Immunoprecipitation.- 3.1 Binding of antibodies to agarose beads and immunoprecipitation of RNP complexes.- 4. RNA gel electrophoresis.- 4.1 10% acrylamide/8.3 M urea gel.- 4.2 6-15% gradient acrylamide gels.- 4.3 Silver staining procedure.- 5. Reagents and suppliers.- 8. Measurement of antibodies to DNA.- 1. Introduction.- 2. Procedures.- 2.1 ELISA.- 2.2 IFT on Crithidia luci...