APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2021
·Vol. 105
·期 11
·4751-4759
·
IF 4.813
Chen Dongjie,Wang Di,Wang Caixia,Wei Fang,Zhao Hongyuan,Lin Xiangmei,Wu Shaoqiang
Abstract Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and “sandwiched” by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78–100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. Key points • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.
研究领域:
兽医学
·
分子诊断学
·
免疫学
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DOI
PubMed
高影响因子
RSC Advances
·
2015
·Vol. 5
·期 102
·84164-84171
·
IF 3.84
Guifang Du,Xiaomei Yang,Mu Hu,Chengcheng Hao,Yanan Gu,Xiuyi Zhi,Wen G. Jiang,Junqi He,Shan Cheng
NHERF1 might play a significant role in biological processes including oncogenic transformation and metastasis. Owing to the lack of highly sensitive and quantitative methods of NHERF1 in human plasma, there have been few reports on the plasma levels of NHERF1 and its correlation with cancer. Here, a novel amplified luminescent proximity homogeneous immunoassay (AlphaLISA) has been developed and validated for the quantification of NHERF1 in human plasma. This assay was based on an AlphaScreen detection technique with two different anti-NHERF1 antibodies coupled to donor and acceptor beads, respectively. The developed AlphaLISA assay was further optimized and validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, recovery, selectivity and interferences. The linear range of NHERF1 in human plasma was 5.00–100 ng mL−1, with an LOD of 2.00 ng mL−1. This AlphaLISA assay has been successfully applied to the quantification of NHERF1 in the plasma from 75 patients with non-small cell lung cancer (NSCLC). The levels of NHERF1 protein in plasma from patients with NSCLC were significantly higher than those in the healthy group (p = 0.0004). Based on the evaluation of the ROC curves, measuring the content of NHERF1 in human plasma could provide a potential diagnostic tool for NSCLC.
研究领域:
肿瘤学
·
分子生物学
·
临床化学
·
癌症生物标志物
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高影响因子
Analytical Methods
·
2024
·Vol. 16
·期 26
·4402-4408
·
IF 2.7
Yan Tang,Fuzhong Chen,Wei Lv,Zixuan Zhou,Yulin Fu,Yuan Qin,Xueqin Zhao,Junhong Wang,Biao Huang
Myocardial infarction occurs rapidly, and thus the rapid detection of cTnI levels is the key to its diagnosis. Most current assays take 10–30 min. In this study, we developed a method for accurately measuring cardiac troponin I (cTnI) levels in human sera with amplified luminescence neighborhood homogeneous assay (AlphaLISA). The method involves coupling two cTnI antibodies targeting different epitopes to the surface of carboxylated donor and acceptor beads. The final signal values were detected by the double-antibody sandwich method, and the best reaction conditions were obtained by optimizing the experimental conditions. The sensitivity, specificity, accuracy, and precision of the method were evaluated. Results showed that the method requires only 3 min to produce the results, the detection sensitivity is 27.06 ng L−1, and the measurement range is 34.56–62 500 ng L−1. cTnI-AlphaLISA has an intra-assay precision of 2.18–4.57% (<10%) and an inter-assay precision of 5.60–6.95% (<10%). The relative recovery rates are within reasonable limits. In addition, the serum assay results of the method were compared with chemiluminescence immunoassay, and the results are in agreement with one another (ρ = 0.8803; P < 0.0001). The method is expected to be developed as a routine method, but further studies and evaluations are needed.
研究领域:
医学诊断
·
生物化学
·
免疫学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2026
·
IF 7.3
Kao Shangbin,Tian Nannan,Zhang Yaping,Qin Yuan,Gu Bing,Fang Hongming,Zhao Xueqin,Huang Biao
Small extracellular vesicles (sEVs) have emerged as promising
biomarkers
for liquid biopsy; however, the limited specificity of single-marker
detection and the intrinsic heterogeneity of circulating sEVs hinder
their reliable clinical application. Herein, we develop a programmable
dual-aptamer amplified luminescent proximity homogeneous assay (AlphaLISA)
for rapid and wash-free detection of tumor-derived sEVs. In this strategy,
EpCAM and PD-L1 aptamers simultaneously recognize two membrane proteins
on the same vesicle, enabling proximity-induced interaction between
donor and acceptor beads to generate a chemiluminescent signal via
singlet oxygen-mediated energy transfer. This dual-recognition design
effectively improves analytical specificity by implementing a molecular
“AND” logic requirement for signal generation. The proposed
assay exhibits a broad linear range of 5.3 × 10 5 to
5.3 × 10 9 particles mL –1 with a
limit of detection of 1.63 × 10 4 particles mL –1 . Good analytical performance was demonstrated by
recovery rates of 93.9–107.5% with relative standard deviations
below 5%, indicating high accuracy and reproducibility. Notably, the
assay enables signal readout within 1 min under homogeneous conditions,
highlighting its operational simplicity and rapid response. Clinical
applicability was evaluated using serum samples from 20 lung cancer
patients and 20 healthy controls, achieving an area under the ROC
curve of 0.87 (95% CI: 0.75–0.97), with a sensitivity of 95%
and a specificity of 75%. Overall, this programmable dual-aptamer
AlphaLISA platform provides a rapid, homogeneous, and highly adaptable
strategy for tumor-derived sEVs analysis, offering promising potential
for noninvasive cancer diagnostics
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高影响因子
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