There is a persisting need for lupus biomarkers that can diagnose active organ involvement during SLE disease flares. Ahearn et al. have recently highlighted this and the difficulties in identifying a specific biomarker to diagnose SLE [41
]. Among these difficulties, Ahearn highlighted they were required to aid in a) the under- and over-diagnosis of SLE, b) identification of lupus flares, c) stratification of patients with various organ involvements and d) monitoring of therapeutic interventions. To this end over 50 potential biomarkers have been investigated for monitoring SLE [43
]. Of these anti-DNA, anti-nucleosome, monocyte chemoattractant protein-1, neutrophil gelatinase-associated lipocalin, urinary tumor necrosis factor (TNF)-like weak inducer of apoptosis, soluble cellular vascular adhesion molecules, C4d levels on erythrocytes, biopsy positive C4d and anti-C1q autoantibodies have all been investigated as renal disease biomarkers. Of these renal biomarkers, anti-C1q has persisted over three decades as a means of monitoring LN in SLE patents in research studies. Autoantibodies against C1q were originally detected against the collagen-like tail region of C1q [29
], but more recently it has been shown that anti-C1q antibodies are also generated against the A, B and C chains of the globular heads of C1q [45
]. Our own studies have suggested oxidative modifications of common host proteins such C1q may lead to breakdown of immune tolerance [46
]. C1q has abundant cysteine, methionine and phenylalanines, which are susceptible to attack by reactive oxygen species that can lead to post-translational modifications and possible breakdown of immune tolerance by generating ‘foreign-appearing’ epitopes. Nitrating species such as peroxynitrite can also modify amino acids to form stable end products such as 3-nitrotyrosine that can be immunogenic [47
]. This may lead to the generation of unwanted anti-C1q antibodies that can be exploited to monitor disease activity in SLE patients [48
Many studies performed in the 1990s in northern Europe and the USA assessed anti-C1q as a biomarker for detecting LN in SLE patients. Most were enthusiastic, and whilst European centers continue to assess anti-C1q, those in the USA appear less keen in using anti-C1q to monitor LN. There has been a resurgence of interest in assessing anti-C1q as a biomarker of LN in Asia and South America, particularly in juvenile SLE patients, where renal involvement is a little more frequent than in adults [49
]. In our review of 28 studies measuring anti-C1q antibodies to detect a history of LN in SLE patients and 9 studies in which anti-C1q measurement was used to distinguish between active LN and inactive LN, the post-test probabilities after a positive test result were generally too low to be reasonably certain of the presence of the condition. Similarly the post-test probabilities after a negative test result were generally too high to rule out the condition with confidence. These findings apply across most of the range of hypothetical values for the prevalence of nephritis history/active nephritis and suggest the measurement of anti-C1q auto-antibodies as a ‘stand–alone’ biomarker is not diagnostically useful.
The sensitivity and specificity values were highly variable across the included studies (Figures 4 and 5). There are a number of possible reasons for this. Some of these factors were cited in a previous meta-analysis by Yin et al. [50
], and included detection methods employed (assay errors) and ethnicity (genetic/environmental) factors. In our analysis we included studies in which commercial and non-commercial ELISA methods were utilized, A frequently used commercial assay is the Bühlmann ELISA. One recent large study of 223 SLE patients monitored anti-C1q antibodies using this and another commercial assay [51
]. This individual study was not included in our current meta-analysis, but the corresponding author provided additional sub-cohort data (personal communication – H Julkunen) of their study. The sensitivity, specificity, positive predictive value, and negative predictive value for distinguishing active lupus nephritis versus inactive nephritis patients (n = 104) were 44%, 90%, 58% and 84% respectively. These values were similar to the values of other studies included in our analysis. Historically, the early studies, particularly in the 1990s had to make their own lab-based ELISAs (as they were not available commercially). Later studies, from 2003 onwards, frequently used commercially available ELISAs, but no single product has been adopted throughout the lupus-research community. For an immunoassay to be useful in routine clinical practice, clinical laboratories should adopt a single assay procedure. This is the case for measuring autoantibodies against cyclic citrullinated proteins (anti-CCP) as a diagnostic assay for rheumatoid arthritis in which selected commercial ELISAs are approved by the U.S. Food and Drug Administration (FDA).
The inclusion of both ‘lab-made’ and commercial anti-C1q diagnostic ELISAs in our analysis is justified since the assay has evolved with several stringent modifications over the past 30 years. A natural function of C1q is to bind non-specifically to immune complexes. Consequently, using an ELISA method employing whole purified C1q bound to a well of an ELISA plate is susceptible to binding to immune complexes present in the test sera, as well as to antibodies directed against C1q. In some of the earlier studies excluded from our analysis, this problem may have led to a higher number of false positive results. However, it was soon realized that raising the ionic strength of the test buffer to >0.15 M prevented the non-specific binding of immune complexes to solid-phase bound C1q [52
], but concerns have been raised that high salt buffers can also prevent anti-C1q autoantibodies binding to C1q [53
].Various other potential problems were raised by Siegert, including sera containing double-stranded DNA (dsDNA) that is known to bind to the collagen region of C1q, but increasing the salt concentration of test buffer also alleviates this problem [24
]. Another concern is the specificity of the antibodies that bind to C1q. The collagen-like region of C1q bears some homology to type II collagen, which is also a target for autoantibodies in many autoimmune diseases, especially SLE and rheumatoid arthritis. However, a study showed that the autoantibodies directed against type II collagen differed from those that bound to the collagen-like stalks of C1q [54
]. We have recently developed a more sophisticated form of ELISA that uses unmodified C1q and post-translationally modified forms of C1q as a target antigen for detecting anti-C1q autoantibodies in SLE sera. This variation may prove to be a more specific and sensitive alternative to current anti-C1q ELISA’s [46
Ten years ago Reveille indicated that the anti-DNA antibody test remained the ‘gold standard’ immunoassay marker for disease activity in SLE, particularly as an indicator of LN [55
] with a positive likelihood ratio of 4.41. He also suggested other tests including anti-C1q showed promise in monitoring renal disease in SLE patients. Our study reveals a lack of homogeneity in performing the anti-C1q assay. Despite this, measuring anti-C1q autoantibodies may be a useful diagnostic test for monitoring and detecting evidence of LN in SLE patients, but not as a ‘stand-alone’ assay, but as part of a panel of autoantibodies as has been the recommendation for many years [56
]. We would advocate that the anti-C1q immunoassay requires further refinement and development, with greater specificity and sensitivity in gauging LN before a single assay be adopted.