Affinity purification and characterisation of human ACPAs
Elena OssipovaCátia CerqueiraEvan ReedNastya KharlamovaAilbhe ComynLena IsraelssonAnca I. CatrinaLars KlareskogPer‐Johan JakobssonKarin Lundberg
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Abstract:
Backgroundand objectives
Autoimmunity in rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins/peptides (ACPA). These antibodies, present in 60–70% of patients, antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression. With this study, the authors aimed to develop an efficient method for the purification of human ACPA, and to characterise their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients.Materials and methods
SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the American college of rheumatology criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulphate, dissolved and further dialysed against PBS, before the IgG fractions were purified on Protein G columns (GE Healthcare, Uppsala, Sweden).Plasma samples (n=10) were diluted in PBS before applied to the Protein G column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP immunoglobulin G (IgG) were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using inhouse ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1).Results
Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13.6 mg/ml) compared to SF (4.2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 µg/ml of purified antibodies), demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients.Conclusions
The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments.Objective:To study the characteristics of autoantibodies in patients with primary hepatocellular carcinoma(HCC).Methods:83 patients with primary hepatocellular carcinoma(HCC)were studied.Different autoantibodies were detected by indirect immunofluorescent assay(IIF,Euroimmuno,Germany).Results:31 of 83 cases were found autoantibodies(37.3%),in whom 64 patients were in HBV infection,with 23 autoantibodies positive(35.9%).8 were autoantibody-positive in 16 patients with HCV infection(50.0%).There was no significant differences between the two groups(P0.05).24.1%(20/83)or 12.1%(10/83)or 1.2%(1/83)patients were positive for one,two or three kinds of autoantibodies respectively.Anti-nuclear antibody(ANA)was the most popular(24.1%)than others.Anti-cytoskeleton(CS)was 13.3%,whereas anti-smooth muscle antibody(SMA)was 9.6% and anti-mitochondria antibody(AMA)4.8% respectively.It was shown that anti-Ro-52 or anti-CENP-B was positive by ANA analysis.Comparing AFP normal with abnormal patients,antoantibody rate(22.2% vs 36.8%)had no significant difference.There were no remarkable difference between autoantibodies positive group and autoantibodies negative group for HBV-DNA.Conclusion:Non-organ-specific autoantibodies—ANA,CS,SMA and AMA can be detected in 37.3% patients with HCC.The highest detection rate is ANA,the next is CS and SMA,and the lowest is AMA.All of detected autoantibodies are shown with low titer(1∶100).Concentration of AFP and autoantibodies is not shown significant correlation.There is no remarkable difference in autoantibodies between HBV-DNA positive group and negative group.
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