Table 1.

Autoantibody (AAB) detection methods in routine diagnostics of systemic rheumatic diseases

Method	Principle	Advantages	Disadvantages	Application
Chip technique (Spot immunoassay) [142, 168, 172–175]	autoAb binding to purified native or recombinant proteins immobilized as a spot on an adsorbent membrane, measurement: see ELISA	•More autoAb per test detectable compared to DIA/LIA •Very low amount of autoantigens needed	•Optimal epitope presentation for each autoantigen difficult to achieve •Possible interferences (see ELISA) may lead to false positive reactions	Multiparametric determination of autoAb
Crithidia luciliae Immunofluorescence Test [108, 110–113, 191]	In situ autoAb binding to kinetoplast DNA of Crithidia luciliae, visualization of autoAb binding by fluorescence-labeled anti-human IgG	High diagnostic specificity for SLE	•Low diagnostic sensitivity for SLE •Semiquantitative analyses only	Determination of dsDNA autoAb in suspicion of SLE or in sera with homogeneous ANA pattern
DIA/LIA [90, 116, 140, 151–159]	autoAb binding to purified native or recombinant proteins immobilized as dot or line on an adsorbent membrane, measurement: see ELISA	•Allows the specific detection of numerous autoAb per test including vary rare autoAb •Low amounts of autoantigens needed	•Qualitative or semi-quantitative analyses only •Possible interferences (see ELISA) may lead to false positive reactions	Multiparametric determination of autoAb (e.g., myositis or SSc specific autoAb)
Double radial immunodiffusion (Ouchterlony technique) [14, 16, 17, 21, 31, 90, 113]	Precipitation of the autoAb with the corresponding soluble autoantigen in gel after radial immunodiffusion; determination of autoAb specificity by reference antibodies	High diagnostic specificity for CTD	•Low diagnostic sensitivity for CTD •Time-consuming (24–48 h)	Screening for autoAb against ENA in serum of patients with suspected CTD
ELISA [3, 22, 32, 37, 53, 62, 71, 80, 95, 101, 120–122, 184]	autoAb binding to solid-phase (multiwell plate) immobilized autoantigen, measurement of autoAb interaction by enzyme-labeled anti-human IgG (or IgA, or IgM): colorimetry by substrate conversion with proportional behaviour to the strength of immune reaction	•Versatile and sensitive analytical technique •Good quantification •Good automation •Quick and cost-effective •Differentiation of immunoglobulin classes possible	Interferences may lead to false positive reactions (cross-reactive autoAb, matrix effects, endogenic proteins, nonspecific binding, autoAb against blocking proteins)	Specific determination of autoAb (highly purified native or recombinant autoantigens are required)
Farr radioimmunoassay [7–9, 55, 57, 67, 96, 106, 205]	Precipitation of anti-dsDNA/DNA complexes; Measurement of the quantity of dsDNA autoAb by using radioactively labeled dsDNA	•High diagnostic specificity for SLE •Superior for monitoring lupus disease activity compared to ELISA	•Requires radioactive material •Higher effort compared to ELISA	Specific detection and quantification of dsDNA autoAb
IIF on HEp-2 cells [7–9, 55, 57, 67, 96, 106, 205]	In situ autoAb binding to antigens of HEp-2 cells, visualization of autoAb binding by fluorescence molecule labeled anti-human IgG	•High sensitive detection of most clinically relevant nonorgan-specific autoAb •Optimal combination of immunoassays for further evaluation of specific autoAb taking into account IIF pattern and suspected diagnosis •Detection of diagnostically relevant autoAb without further need of specific immunoassays (e.g., centromere autoAb) •Assessment of autoAb only detectable by this method since the autoantigenic targets have not been identified or commercial assays are not available yet	•Subjective assessment •Reliable results require qualified and experienced lab personnel •High intralaboratory and interlaboratory variance → Automatic image recognition and interpretation improves and standardizes results	autoAb screening in sera of patients suspected of having SARD or autoimmune liver disease
Microparticle based immunoassays [102, 138, 139, 142, 164, 170–172, 176–179, 206–208]	autoAb bind to antigens immobilized on beads; measurement by flow cytometry (suspension bead assay) or optical microscope (planar bead assay)	•More autoAb per test detectable compared to DIA/LIA •Very low amount of autoantigens needed •Better epitope presentation for each autoantigen compared to spot assay •Combination with IIF possible (CytoBead® assay)	•Possible interferences (see ELISA) may lead to false-positive reactions	Multiparametric determination of autoAb
Passive agglutination (Latex test: RF) [216]	Binding of RF to human IgG bound on the surface of biologically inactive latex particles leads to visible agglutination of the particles	•Easy to perform •No need of instruments •High sensitivity	•Qualitative or semi-quantitative analyses only •False-positive reaction if reaction time is surpassed •Intensity of agglutination does not correlate with RF titer •Low specificity	Screening for RF (only rarely used in routine diagnostic since introduction of CCP autoAb)
Passive hemagglutination (Waaler-Rose test: RF) [217]	Binding of soluble autoantigens coated on red blood cells leads to visible erythrocyte agglutination	•Easy to perform •No need of instruments	•Qualitative or semi-quantitative analyses only •Subjective assessment	Not used anymore in routine diagnostics (in the past used for detection of RF, dsDNA, and Sm/RNP autoAbs)
Radioimmuno-precipitation assay [124, 125, 129]	autoAb binding to autoantigens of radiolabelled cell extracts; analyses of bound antigens by autoradiography after gel electrophoresis of the immunoprecipitates	Allows the detection of numerous autoAb without purification of autoantigens	•Requires radioactive material •Higher effort	Not used in routine practice; may be used for assay comparison and to search for novel autoAb (specialized labs only)
Westernblot (Immunoblot) [81, 89, 113]	autoAb binding to electrophoretically separated proteins transferred to adsorbent membrane, measurement: see ELISA	Allows the detection of numerous autoAb without purification of autoantigens	•False-negative results due to destroyed (denaturation of proteins during electrophoresis) or masked epitopes •False-positive results due to comigrated proteins	Not used anymore in routine diagnostics; may be used to search for novel autoAb
Nephelometry [218]	The amount of antigen/antibody complexes were measured by light scatter	•Easy to perform •Less-time consuming •Greater precision compared to latex test (see passive agglutination)	•No discrimination between isotypes •Lower diagnostic sensitivity compared to ELISA	Quantification of RF

ANA antinuclear antibody, autoAb autoantibody, CCP cyclic citrulinated peptide, CTD connected tissue disease, DIA/LIA dot/line immunoassay, ELISA enzyme-linked immunosorbent assay, ENA extractable nuclear antigen, IIF indirect immunofluorescence, RF rheumatoid factor, SARD systemic autoimmune disease, SLE systemic lupus erythematosus, SSc systemic sclerosis