Single Tests for autoAb Analysis
The introduction of fluorescent dyes and the development of immunochemical methods for the labeling of antibodies on the one hand and fluorescence microscopy on the other hand paved the way for IIF as powerful tool for autoAb analysis in the 1950s [
75]. Thus, the detection of ANA by IIF employing first rodent liver tissue and later HEp-2 cells as autoantigenic substrate marks the beginning of autoAb detection in the serological diagnosis of CTD [
7,
9,
76,
77]. However, it turned out soon that the clinical need for disease-specific autoAbs was not appropriately addressed by ANA testing alone. The search for more specific autoAbs led to the introduction of immunodiffusion techniques which enabled the discovery of disease-specific autoAbs like the Sm autoAb in patients suffering from SLE [
16,
17,
21]. In particular, DID employing thymic extracts was used and clinicians learnt to appreciate the high specificity of this new parameter for CTD serology. Not surprisingly, autoAbs to Sm were included along with ANA in the diagnostic criteria for SLE later and are still considered as one of the most specific serological parameters for SLE [
11,
14]. However, DRID is a time-consuming technique and, thus, was replaced by CIE enabling a faster and more sensitive detection of precipitating autoAbs later on [
31]. Several other important autoAbs to the spliceosomal complex such as autoAbs to U1 ribonucleoprotein (U1-RNP) were identified in the quest for new CTD markers [
21]. Anti-U1-RNP was established as a specific serological marker for MCTD and found in patients with SLE as well [
78,
79]. The introduction of new assay techniques like radio- (RIA) and enzyme immunoassays as well as radio/immunoprecipitation paved the way for the development of autoAb detection assays with better assay performance [
32,
62,
80‐
86]. In particular, the progress in proteomics and the introduction of the immunoblot technique enabled the purification and identification of the distinct autoantigenic targets [
33,
87‐
89]. It turned out that Sm and U1-RNP consist of several autoantigenic components including U1-RNA with different characteristics regarding their performance as split autoantigens especially in solid-phase ELISAs [
79,
90,
91]. Furthermore, the SjS-specific autoantigens SS-A and SS-B form a complex interacting with yRNA [
92]. Of note, this confers only to the SS-A 60 kDa unit whereas the 52 kDa SS-A (TRIM21) does not bind to yRNA and is not related to this snRNP complex [
93,
94]. This raised the question of the best composition of these targets for the detection of the distinct autoAbs or the use of the target subcomponent with the best assay performance [
90]. In terms of U1-RNP consisting of components A, C, and a 68 kDa polypeptide, it was found that at least two of these three should be used as solid-phase antigens to set up an appropriate ELISA for the detection of autoAbs to U1-RNP [
19]. In contrast, SmD of the Sm complex with its six subcomponents mentioned earlier appeared to be the most specific and sensitive autoantigenic target in ELISA for the serology of SLE [
14].
In general, the introduction of solid-phase assays like ELISA was accompanied by four major aspects changing the understanding of autoAb testing for CTD diagnostics: (i) a better usability as assay platform, (ii) an increasing sensitivity compared with immunodiffusion techniques, (iii) the different assay performance of autoAbs recognizing conformational or nonconformational, linear epitopes, and (iv) the introduction of reference sera for standardized diagnostics. This was an essential step toward standardization and automation of autoAb testing addressing the growing demand thereof due to the inclusion of autoAb testing into diagnostic or classification criteria of more and more autoimmune diseases and changed the autoimmune laboratory environment dramatically [
80,
95]. Consequently, assay techniques like IIF, which have been prone to subjectivity and difficult to automate until recently, were subjected to a rising pressure to be substituted [
73,
96,
97]. In this context, several researchers were tempted by the advantages of the ELISA technique and in particular its higher sensitivity to develop assays employing cellular extracts of MOLT4 or HEp-2 cells [
98‐
101]. Furthermore, the elevated sensitivity of particularly anti-SS-A ELISAs revealed false-negative ANA sera of patients suffering from CTD [
102‐
105]. Indeed, this seems to be the only autoantigenic target which is not adequately presented even by HEp-2 cells and can result in false-negative ANA findings by IIF. To overcome this shortcoming of the appreciated IIF technique, genetically modified HEp-2 cells with a higher expression of the SS-A 60 kDA polypeptide were introduced in ANA testing [
103,
106].
Of note, the increased sensitivity of ELISA resulted in positive autoAb findings in nondiseased individuals, too, which started an intense discourse on the right method for cutoff determination [
80]. Finally, receiver operating characteristics curve analysis was approved for quantitative methods like ELISA as the best approach to do so [
39]. Part of the false-positive findings could be assigned to autoAbs occurring before the onset of disease as putative predictive markers thereof [
29,
107]. Nonetheless, false-positive findings in ELISA could be a result of autoAbs to less disease-specific nonconformational epitopes [
108]. These autoAbs often belong to the natural autoAb repertoire and display a low affinity to its corresponding targets [
109]. A very convincing example is the anti-double-stranded DNA (dsDNA) autoAb which was established as diagnostic marker of SLE [
110]. Of note, the SLE-specific dsDNA epitope is ill-defined and IIF assays employing kinetoplast dsDNA of
Crithidia luciliae (CLIFT) with its characteristic epitope structure appear to provide the best specificity for this important disease activity-associated SLE marker [
108,
111‐
113]. The replacement of CLIFT and the Farr RIA measuring mainly high-affinity anti-dsDNA autoAbs due to a high-salt reaction environment by ELISAs detecting autoAbs to both nonconformational and conformational dsDNA epitopes resulted in high numbers of false-positives particularly in patients with infectious diseases [
114].
A similar phenomenon was observed when recombinant or synthetic autoantigens were introduced into autoAb testing to overcome the difficulties related to antigen purification and standardization [
115,
116]. Not in each case, these nonnative polypeptides could replace the native autoantigenic targets for an appropriate autoAb analysis. Thus, the SmD polypeptide was dependent on the symmetric methylation of arginine to represent the SLE-specific epitope for the sensitive detection of anti-Sm autoAbs [
35,
36,
117]. Furthermore, the presence of yRNA for the autoantigenicity of the SS-A/SS-B complex on the one hand and of U1-RNA for the Sm/RNP unit on the other hand was obviously required for the sensitive analysis of the respective autoAbs [
118,
119].
Remarkably, specific ANCA testing demonstrated similar difficulties. Like for ANA testing, IIF was introduced as first assay technique on fixed neutrophils [
45]. However, the following identification of PR3 and MPO as the main ANCA targets and the subsequent analysis of respective autoAbs by ELISA were hampered by the nonsatisfactory sensitivity of anti-PR3 autoAb tests [
120‐
122]. Indeed, the conformational epitopes on PR3 were difficult to preserve on the solid phases of ELISAs. Recently, the third generation of PR3-ANCA ELISA has been introduced employing anchor molecules during adsorption of PR3 to the solid phase to preserve its confirmation and accessibility of vasculitis-specific epitopes [
121,
122]. Other attempts to develop highly sensitive PR3-ANCA ELISAs comprised the use of a mixture of native as well as recombinant PR3 [
123].
The close relation between sensitivity and specificity is presumably the reason that direct-ligand RIAs with their excellent sensitivity have not been used widely for the analysis of CTD- or AAV-specific autoAbs. Interestingly, this is in contrast to organ-specific autoimmune entities such as type 1 diabetes (T1D) where RIAs are appreciated hitherto due to their high sensitivity [
124,
125]. Of note, IIF on endocrine pancreas had also been the first technique used for autoAb analysis before the corresponding autoantigens were identified [
126]. The detection of islet-cell autoAbs by IIF is still in use; however, the impact of conformational epitopes for T1D autoAbs testing in conjunction with the increased sensitivity of RIAs and recently emerging ELISAs with similar assay performances have almost replaced IIF [
127].
After the discovery of TG2 as autoantigenic target of EmA for CD serology, a similar development was observed in the serological diagnosis of CD [
128]. To obtain a sensitive anti-TG2 autoAb assay, conformational epitopes of TG2 seem to be essential, too [
129]. In contrast to T1D autoAb testing, however, the detection of EmA by IIF is still the gold standard [
65].
As a fact, the higher disease specificity of autoAbs to conformational epitopes is probably the reason for today’s infrequent use of immunoblot assays for autoAb serology [
130]. Obviously, due to the poor presentation of such epitopes on the blot membrane as a result of the denaturing effect of sodium dodecyl sulfate during electrophoresis and the poor standardization of the method due to technical peculiarities, the immunoblot technique has lost its initial appeal for multiplex autoAb testing [
89,
131].
Notwithstanding, due to the progress in the identification of ever more autoAbs aiding in diagnosing, predicting and prognosing autoimmune diseases, the search for the most adequate strategy of autoAb testing fulfilling clinical needs and cost constraints has been in the focus of laboratory and clinical experts ever since [
5,
59,
132‐
134]. For instance, more than 100 autoAbs were found in SLE patients alone [
135]. This led to the introduction of fully automated random-access instruments employing fluorescence or chemiluminescence as read out for autoAb testing as well as screening [
136‐
139].
Remarkably, a two-stage strategy was recommended for both ANA and ANCA analyses by international consensus recommendations [
4,
54]. Thus, IIF is still considered a reliable screening test characterized by a high negative predictive value. Positive IIF findings should be confirmed by specific autoAb testing employing assay techniques with high specificity. For several other autoimmune disorders like for instance CD, IIF is still considered a gold standard [
65]. Thus, despite the introduction of assay techniques for the detection of specific autoAb reactivities, there is still a need for testing of autoAbs by various assay techniques.
Multiplex Assays for autoAb Testing
The rising number of autoAbs requested for the serology of one autoimmune entity as well as the growing demand for autoAb testing in general encouraged the development of multiplex testing [
3,
140‐
142]. Despite the fact that ANA assessment by IIF using HEp-2 cells as autoantigenic substrate is already a multiplex test revealing different patterns according to the autoAbs present in the serum investigated, the analysis of specific autoAbs is hardly achievable [
20,
96,
143,
144]. Even for such ready to detect ANA patterns like the centromere one with its more than 40 fluorescent dots spread in nuclei of interphase cells and densely aligned dots in the metaphase cells, several proteins could be recognized by autoAbs as autoantigenic targets (centromere-associated proteins A, B, and C) [
57,
76,
145‐
147].
As mentioned earlier, immunoblot was one of the first attempts to establish an appropriate multiplex test for the confirmation of ANA by using whole cell extracts with a similar autoantigen composition of HEp-2 cells [
98,
148]. However, due to technical challenges, poor reproducibility, and loss of the native conformational structure of the relevant autoantigenic epitopes, this method was not established as a standard for multiplex autoAb analysis [
19,
25,
149,
150].
As a result of improved purification methods for native autoantigens and progress in the expression of recombinant autoantigenic targets, the use of both molecule sources did not only enable the development of singleplex autoAb ELISAs but of multiplex dot or line immunoassays (D/LIAs), too [
116,
140,
151]. In daily laboratory routine, D/LIAs have been established as one of the standard tests for ANA and ANCA confirmation [
140,
152‐
154]. Moreover, D/LIAs appear to be an ideal solution for other serological diagnoses, where multiple autoAbs are required [
155‐
158]. This holds not only true for CD serology where even a simultaneous IgA deficiency can be conducted apart from the (auto)Ab testing but proved to be very effective for the serology of SSc, DM/PM, or autoimmune liver diseases [
140,
154]. Thus, D/LIAs with more than 20 autoantigenic targets have been introduced for the confirmatory diagnostics of ANA successfully [
159]. Of note, the miniaturization of the technique by deploying sophisticated nanoliter dispensing devices and pattern recognition software for optical density reading render this technique most potential for future multiplex autoAb testing [
160].
It should be noted in this context, that the attempts to employ the 96-well ELISA platform for autoAb multiplexing by using single wells for the immobilization of distinct antigens appear to be just an intermediate stage which was called into question very soon due to obvious shortcomings of the approach.
The progress in fluorescence reading as well as flow cytometry and microscopy paved the way for a new era in multiplexing [
161‐
169]. Thus, several multiplex assay developments employing surface-activated microbeads coded by fluorescent dyes, size, or shape on the one hand and fluorescence microscopy or flow cytometry as read-out on the other hand were reported [
170‐
172].
The intriguing biochip mosaic technology enabled multiplex autoAb IIF reading by using various cellular and tissue substrates on one solid phase [
173‐
175]. Further, the luminex technology deploying fluorescence-coded microbeads and flow cytometry enabled the development of an intriguing and very successful multiplex autoAb detection technique [
176,
177]. Very soon, this novel technology was commercialized by several companies. The possibility to detect several autoAbs and the high throughput led to the development of such multiplex autoAb systems like Athena and FIDIS or the fully automated BioPlex2000 system covering various serological autoimmune diagnoses [
172,
177‐
179]. The growing success and the ready automation of the luminex technology were very appealing especially for larger laboratories with ever growing sample volumes [
177]. Indeed, demand for autoAb testing started rising exponentially in the 1980s and this phenomenon called into question even the recommended two-tier strategy encompassing IIF as the ideal autoAb screening [
180‐
182]. As a matter of fact, laboratories in particular in the USA have begun replacing IIF due to its major shortcomings, namely lack of automation, standardization, modern data processing, and experts in IIF reading [
3,
43,
73,
170,
183,
184]. Although the newly developed luminex applications for autoAb testing helped to ease the pressure in terms of rising autoAb analyses, there was growing dissatisfaction among rheumatologists with the assay performance of the technology [
42]. Indeed, false-negative ANA findings leading to ill-defined diagnoses raised the concern of clinicians [
185,
186]. Consequently, the American College of Rheumatology (ACR) initiated a task force in 2009 investigating the issue [
42]. In conclusion, IIF was confirmed as standard method for ANA reading and laboratories requested to return to the two-stage strategy or to make sure that clinicians requesting ANA testing are aware of the different assay performance by multiplexing [
4].
Of note, despite the development of similar multiplex tests for ANCA testing, IIF was also not challenged as screening assay in the two-stage strategy yet.