Introduction
There is an increasing desire in the medical community to develop rapid and personalized serum-based diagnostic tests to detect autoimmune [
1], neoplastic [
2] and infectious diseases [
3]. One major approach involves using antibody-based tests to diagnose and even predict the onset of various diseases [
1,
4]. However, most current quantitative immunoassays used to measure antibodies are impractical for rapid point-of-care testing because they are complex, time consuming, and difficult to standardize [
3]. As an alternative, rapid tests such as lateral flow immunoassays, which can more easily be integrated in point of care settings, are used for the diagnosis of several infectious agents such as HIV and HCV. However, one limitation of these assays is that they produce a qualitative (i.e. positive or negative) rather than a quantitative result. Currently there are no serological tests for rapidly detecting autoantibodies associated with autoimmune diseases that also satisfy the growing demand for high analytical sensitivity and reproducibility.
Sjögren's syndrome (SjS) is a common autoimmune disorder associated with epithelial inflammation and exocrine gland dysfunction [
5]. SjS is often associated with polyclonal B cell activation resulting in the presence of multiple autoantibodies including the well-known SSA and SSB antibodies. While positive SSA (Ro52 and Ro60) and SSB (La) autoantibodies are part of the diagnostic criteria, five other clinical signs including ocular and oral dryness and evidence of inflammation from minor salivary gland biopsy are required for the diagnosis of primary SjS [
6]. This is because antibodies to SSA and SSB are not specific to SjS, but are also found in other rheumatological diseases including systemic lupus erythematosus (SLE), and myositis [
7]. Nevertheless, in the 2002 classification standards for SjS diagnosis, positive SSA and SSB autoantibody tests were the only mandatory criteria if the salivary gland biopsy was negative [
6]. Current SSA and SSB ELISAs, which employ native antigen complexes isolated from calf thymus, show positive SSA and SSB serology in 50-70% and 40-45% of SjS, respectively [
8,
9].
Previously, luciferase immunoprecipitation assay systems (LIPS), which employs
Renilla luciferase (Ruc)-antigen fusions produced in mammalian Cos1 cells, was used to detect patient antibodies to a variety of pathogen antigens [
10‐
17] and also to detect human autoantibodies associated with several autoimmune diseases including Type 1 diabetes [
18], Stiff-person syndrome [
19] and Sjögren's syndrome [
20]. In the SjS studies, detection of anti-La/SSB antibodies by LIPS showed improved performance compared to existing ELISA and offered a highly sensitive, robust and high-throughput testing format [
20]. LIPS profiling of additional autoantigens revealed that certain SjS patients also showed positive immunoreactivity with Ro52, Ro60 and other extraglandular autoantigens including thyroid peroxidase, the aquaporin-4 water channel and the gastric H
+/K
+ ATPase.
A quicker version of LIPS (called QLIPS) has also been used to detect antibodies to several pathogen antigens associated with human infection [
14,
16], in which the two incubation steps of 1 hour were each reduced to 5 minutes. In the present study, we describe QLIPS tests for evaluating antibodies to the 3 major SjS recombinant autoantigens. Results from this study demonstrate that detection of anti-Ro52 antibodies by QLIPS was rapid, robust and has the potential to be used in the diagnosis of SjS and other rheumatologic diseases in point-of-care settings.
Discussion
Rapid and comprehensive serum-based diagnostic tests that can be used in point-of-care settings for diagnosis and even pre-symptom screening of autoimmunity are urgently needed. A significant challenge in the development of such assays is that, unlike antibodies associated with infectious agents, the detection of autoantibodies associated with autoimmunity requires more sensitive tests than ELISAs or other solid phase immunoassays such as protein arrays, which miss many conformational epitopes [
23]. Typically, liquid phase immunoprecipitation assays such as the radiobinding assay (RBA), which show much higher sensitivity, specificity and signal to noise ratios than ELISAs are needed for detecting autoantibodies in most autoimmune diseases [
23,
24]. However, a significant drawback of RBAs is the requirement for radioactively-tagged antigens. In this study we demonstrate that QLIPS, which utilizes a non-radioactive, luciferase enzyme-based tracer in a liquid phase assay, can rapidly and sensitively detect autoantibodies associated with SjS. The QLIPS format could easily be integrated into a point-of-care test because it requires only about 25 minutes of total processing time per 94 sera samples, which includes a 5 minute set-up, two five minute incubations steps, 10 minutes of washing and reading of the plate with a luminometer.
Due to the high signal to noise and large dynamic range of the LIPS assay, the coefficient of variation (CV) of approximately 20% for LIPS still provides remarkable diagnostic accuracy. One likely cause of the near 20% CV is due to the fact that the QLIPS sample processing (i.e. pipetting and washing) was preformed rapidly in less than 15 minutes with 84 or greater samples. With some of these high signals in the SjS positive samples, small pipetting errors can resulting in large changes in antibody titer (e.g. 10% pipetting error can result in over 100,000 LU differences). Despite the 20% CV, the SjS positive samples show 1000-fold higher anti-Ro52 antibody titers than the negative samples and evaluation of each of the two runs independently shows that show that the same samples are positive.
Previously, we found that all individuals have detectable anti-Ro52 antibodies by LIPS [
20]. However, unlike the normal range of anti-Ro52 antibody titers in healthy individuals, some patients with SjS or other rheumatological diseases have markedly higher anti-Ro52 antibody titers that can be detected by LIPS and other immunoassays. In this study, the QLIPS test for anti-Ro52-Δ2 antibodies had a higher diagnostic performance than the standard LIPS format. The reason for this increased performance is due to the loss of anomalously high signals in some of the control samples observed in the standard LIPS format. These anomalously high Ro52 antibody signals in several of the controls were no longer positive under the rapid, non-equilibrium conditions of QLIPS. Taken together these results also suggest that performing QLIPS and LIPS in parallel may allow a simple method of more accurately assessing antibody avidity in some situations. An analogous increase in specificity of QLIPS compared to LIPS has also been observed for distinguishing antibodies to
Loa loa and
Onchocerca volvulus antigenic proteins from antibodies to antigens from related filarial infections [
14,
16]. In contrast, the QLIPS tests for anti-La and anti-Ro60 antibodies showed a marked drop in test performance in the QLIPS format. For example, the LIPS test for detecting anti-La antibodies was 75% sensitive, versus the 49% sensitivity of QLIPS. The decreased detection of anti-La and anti-Ro60 seropositive antibodies under these rapid conditions compared to LIPS is likely due in part to the inability to detect the low affinity/low titer autoantibodies present in some of the SjS samples. However, we show that using QLIPS, the SjS patients can be distinguished from controls using the Ro52-Δ2 fragment alone.
While the standard LIPS format yielded 76% sensitivity and required two independent assays (anti-La and anti-Ro52 autoantibodies) to be performed [
20], QLIPS, with a single antigenic fragment of Ro52, showed approximately 70% sensitivity. Ironically, this C-terminal fragment of Ro52 used in QLIPS is the same antigenic fragment that shows no useful diagnostic immunoreactivity in ELISA and Western blotting [
25,
26]. The detection of diagnostically useful antibodies to the C-terminus of Ro52 by LIPS is supportive of the improved conformational epitopes using mammalian recombinant proteins in this liquid phase QLIPS/LIPS compared to ELISA. Both the LIPS and QLIPS formats are also as good as a conventional ELISA for measuring SSA and SSB. However, an ELISA requires significantly more time to complete (e.g. 5-24 hours). Furthermore, the QLIPS Ro52 test showed higher sensitivity in the validation cohort than an established RBA for SSA and SSB (66% vs. 56%).
The short incubation time, high performance and relative simplicity of the Ro52-Δ2 QLIPS test has practical implications for developing even simpler assay formats. The finding that the Ro52-Δ2 QLIPS test showed antibody titers that were 1000 times higher in the SjS positive samples compared to the control samples also provides a large diagnostic window for detecting seropositive samples. Furthermore, no other immunoassay format such as ELISA or RBA shows such a large signal-to-noise ratio. It is likely that additional assay modification, including reducing the volume of the reaction, may yield even more robust signals. We speculate that a microfluidic device configured for the QLIPS format might be suitable for point-of-care testing. Using such a microfluidics device, the addition of sera, Ruc-antigen mixture to immobilized protein A/G, washing, and the addition of coelenterazine luciferase substrate could all be automated and performed rapidly. The ability to stably freeze the Ruc-antigen reagents also has practical implications for point-of-care testing. Due to the highly scalable format of QLIPS, additional reagents for detecting anti-pathogen antibodies (e.g. HIV, HCV, and HSV-2) could also be employed for side-by-side diagnosis of these infections.
Conclusion
Ro52 autoantibodies are not only found in SjS, but are found in SLE, myositis and several other autoimmune disorders. These results suggest that the rapid and robust Ro52-Δ2 QLIPS test has the potential to aid in point-of-care evaluation of patients with SjS and other rheumatologic diseases. However, since not all SjS patients show anti-Ro52 positive antibodies, the addition of other autoantigens might improve the diagnostic performance of the QLIPS test. In particular, antigens that produce robust signals in Ro52-negative sera might be part of an antigen mixture used in the QLIPS format to further increase the sensitivity of this test. The ability of using QLIPS for screening for anti-Ro52 and other autoantibodies in early phases of the disease might make it possible to diagnose and even treat autoimmune diseases before more severe disease and/or substantial organ damage has occurred.
Competing interests
Two of the authors (P.D.B., and M.J.I.) have a patent application submitted using LIPS for detecting autoantibodies associated with Sjögren's syndrome.
Authors' contributions
PDB conceived of the study, developed the needed constructs, analyzed the sera by LIPS, analyzed the data, drafted the manuscript and made critical revisions; ATI, KHC and CL generated the needed constructs and/or lysates; YL and MS analyzed the sera by conventional immunoprecipitation assays; WHR, provided patient sera from cohort 2 with clinical information and was involved in critical revision; MJI helped develop the high-throughput assay and was involved in critical revision and final approval and all authors commented on and approved the manuscript.