The importance of the development of a convenient assay for anti-Th/To and -U3RNP antibodies cannot be underestimated because these two classic anti-nucleolar autoantibodies are associated with particular SSc variants and clinical features [1
]. In fact, anti-Th/To antibodies are frequently detected in lcSSc patients with pericarditis and mild lung involvement [13
], while anti-U3RNP antibodies are associated with severe pulmonary, renal and muscular disease, mainly in African-American dcSSc patients [1
]. Thus, the aim of our study was to establish a new method that can be used widely and easily to detect anti-Th/To and -U3RNP antibodies to replace complicated RNA analysis by gel electrophoresis.
Our results clearly show that qPCR is highly efficient and reliable to detect the RNA components of the SSc nucleolar autoantigens, when data are compared with the gold standard IP. Advantages of this new method include easy application without technically demanding RNA analysis by urea-PAGE and without the use of toxic chemicals (for example, acrylamide, silver staining) or 32P radioisotopes. The results obtained by our IP-qPCR assay are consistent and reproducible, and are expressed as semi-quantitative data, while the silver staining gel only detects the presence or absence of specific bands. The high sensitivity of our new method is demonstrated by requiring cell extract from only one million cells and 1 µl of serum to obtain a signal that is at least 128- to 512-fold more sensitive than standard IP-silver staining method. Another important advantage, when considering its use in a large laboratory setting, is the high number of samples that the new assay can handle at a time. In fact, a 96-well plate can be used for qPCR, while only 10 to 16 samples can be tested in a standard gel for RNA analysis by urea-PAGE.
A minor limitation of the IP-qPCR assay is the lack of information on other RNAs immunoprecipitated by the same serum sample. Examples are co-IP of 7-2 and 8-2 RNA by La antigen [18
], and U3 RNA by rare autoimmune sera that recognize the TMG cap structure of U1-U5RNA [17
]. In practice, it will be reasonable to apply this qPCR method for anti-Th/To and U3RNP antibodies only to SSc patients who are negative for anti-topo I, RNAPIII, and ACA, since the coexistence of multiple SSc autoantibodies is uncommon [1
]. Also, nucleolar immunofluorescence staining in antinuclear antibody (ANA) screening will be helpful to decide which patients should be tested by this IP-qPCR method, since the majority of anti-Th/To- and U3RNP-positive patients show clear nucleolar staining [13
], in contrast to anti-RNAPI/III sera which often do not show a nucleolar pattern [7
]. It should be noted that the specificity of the IP-qPCR assay is nearly 100% in SSc samples and that non-SSc sera that are expected to be, or potentially are, false positives (such as anti-La, anti-TMG) were included in the study to clarify the potential limitations of the assay. The SSc sera we tested were not randomly selected, however, the proportion of each specificity is not far from a randomly selected SSc cohort, and it should give us a reasonable idea of the specificity of the assay in general SSc patients. Thus, the positive results obtained by the new IP-qPCR assay in the presence of rare antibodies in SSc, such as anti-La and anti-TMG, do not represent a clinically significant problem.
The new method can be readily applied to detect other autoantibodies, such as autoantibodies to aminoacyl-tRNA synthetases and signal recognition particle (SRP) in polymyositis/dermatomyositis patients, just by using appropriate specific primers for the RNA species of interest. With the IP-qPCR method, each tRNA specificity can be readily defined without the complicated aminoacylation assay [24
]. Although clinical manifestations of many anti-synthetase antibodies appear similar and they are known as the 'anti-synthetase syndrome', it is possible that fine specificity still has clinical significance as there are some suggestions of different clinical manifestations in patients with different anti-synthetase antibodies [15