ABSTRACT
MicroRNAs (miRNAs) are potential clinical biomarkers for the detection of various diseases. However, their quantification has not been implemented in clinical practice given the inconsistencies in their variable recovery rate and accuracy of the results. Thus, we utilized a technique based on bioluminescent enzyme immunoassay (BLEIA) to perform fully automated miRNA quantification using magnetic particles conjugated with antibodies targeting DNA-RNA hybrids, biotinylated DNA probes specific to miRNAs, and firefly luciferase-labeled streptavidin. This method enabled direct use of diluted serum and automation of all processes within 1 hr. The results revealed a wide linear range between 10 fmol/L and 1 nmol/L, high sensitivity with a detection limit of 6.3 fmol/L or below, high specificity with a false positive rate under 2.4%, and high reproducibility in intra- and inter-experimental observations (under CV 10% and r = 0.9965, respectively). Furthermore, a significant correlation was revealed between quantitative reverse transcription-polymerase chain reaction and BLEIA assay for the quantification of synthetic miRNA (r = 0.9993) and endogenous miRNA in healthy serums (r = 0.8203), respectively. Overall, we developed a fully automated miRNA quantification method based on BLEIA, which can be adopted in a clinical setting. However, further studies are needed to assess its clinical performance.
Subject(s)
MicroRNAs , Biomarkers , DNA , DNA Probes , Immunoassay/methods , Immunoenzyme Techniques , Luciferases, Firefly , Reproducibility of Results , Sensitivity and Specificity , StreptavidinABSTRACT
Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.
Subject(s)
Single-Chain Antibodies , Enzyme-Linked Immunosorbent Assay , Ferritins , Immunoassay , Immunoenzyme Techniques , Luciferases/genetics , Peptide LibraryABSTRACT
Noroviruses (NoVs), which belong to the family Caliciviridae, are major causative agents of acute gastroenteritis worldwide. Thus, rapid and highly sensitive assays for detecting NoVs are required. Recently, a bioluminescent enzyme immunoassay (BLEIA) for detecting NoVs in fecal specimens was developed. This new assay was evaluated using fecal specimens obtained from acute gastroenteritis patients. Of the 107 specimens that were found to be NoV-positive by RT-PCR or RT-LAMP, 104 specimens produced positive results in the BLEIA (sensitivity: 96.3%). On the other hand, no false-positive results were observed during the testing of 176 NoV-negative specimens containing group A or C rotaviruses, astroviruses, sapoviruses, adenovirus type 41, bocaviruses, or parechoviruses. Furthermore, the BLEIA was able to detect many NoV genotypes in the tested specimens, including three genotypes from genogroup I (genotypes 1, 4, and 8) and ten genotypes belonging to genogroup II (genotypes 1, 2, 3, 4, 5, 6, 12, 13, 16, and 19). By quantifying the number of NoV genome copies in the clinical specimens tested with the BLEIA, its detection limit was estimated to be 10(6) genome copies per gram of stool and below. Furthermore, as the BLEIA can be performed with an automated device and does not involve complicated procedures it can be used to rapidly test many samples. Therefore, the BLEIA is a rapid and highly sensitive method and could be used as a diagnostic tool at hospitals and clinical laboratories that deal with large numbers of clinical specimens from acute gastroenteritis patients or food handlers. J. Med. Virol. 86:1219-1225, 2014. © 2013 Wiley Periodicals, Inc.
Subject(s)
Caliciviridae Infections/diagnosis , Clinical Laboratory Techniques/methods , Feces/virology , Gastroenteritis/diagnosis , Norovirus/isolation & purification , Humans , Immunoenzyme Techniques/methods , Luminescent Measurements/methods , Sensitivity and Specificity , Time FactorsABSTRACT
A novel GII.17 norovirus (NoV), Kawasaki 2014, has spread to several regions of the world. Rapid and reliable diagnostic tests are needed for the detection of this new NoV variant. In this study,analytical sensitivity of 7 different immunochromatographic (IC) test kits (6 are on the market in Japan and one in Europe) was evaluated by means of confirmed GII.17 NoV-positive stool samples. The stool samples were also tested by a bioluminescent enzyme immunoassay (BLEIA). Real-time RT-PCR served as a reference (gold standard) method. Among the 7 IC kits, RIDA QUICK was the most sensitive, with the limit of detection of 107 copies/ml, whereas the limits of detection of the other IC kits ranged from 108 to 109 copies/ml. It should be pointed out that the limit of detection of BLEIA was approximately 100- to 1,000-fold better (104-105 copies/ml) than that of RIDA QUICK. Nevertheless, the procedure of BLEIA took more time and required sophisticated equipment.