Abstract
IntroductionClassic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.MethodsStandard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.ResultsBy testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.ConclusionsOur new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.
Highlights
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); no validated commercial assay is available
Autoantibody detection using quantitative real time PCR (qPCR) for the RNA component of the ribonucleoprotein autoantigen complex The sensitivity and linearity of the assay was evaluated for the detection of the Th and U3 RNA components, using serial 1:8 dilutions (1-1:4096) of cell lysate, RNA purified after IP and complementary DNA (cDNA) obtained after reverse transcription (RT) (Figure 1)
Linear dose response curves were noted for cell lysate, RNA, and cDNA dilutions, and the difference between positive samples and normal human serum (NHS) was clear even at 1:4096 dilution, which corresponded to a cell extract from 2.4 × 103 K562 cells (Figure 1A)
Summary
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies. Anti-Th/To and -U3RNP are anti-nucleolar antibodies that have been known for more than 25 years. Despite their clinical importance, these SSc autoantibodies have not been utilized clinically because of the unavailability of antibody testing [7,13]. The aim of our study is to establish a new method to detect anti-Th/To and -U3RNP antibodies based on quantitative PCR (qPCR) detection of the RNA components of the ribonucleoprotein autoantigens
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