de Carvalho Hianka J C, Seth Ayesha, Speidel Ruth, Abreu-Silva Ana L, de Andrade Hélida M, Miglino Maria A, Badu-Tawiah Abraham K
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43201, United States.
Department of Veterinary Surgery, Universidade de São Paulo, São Paulo, SP 05508-070, Brazil.
Anal Chem. 2025 Apr 8;97(13):7089-7097. doi: 10.1021/acs.analchem.4c05962. Epub 2025 Mar 24.
This work presents the first indirect immunoassay performed on a paper-based microfluidic platform for the diagnosis of canine visceral leishmaniasis (CVL). The IgG antibody biomarker, which signifies the presence of this infectious disease, was captured with a recombinant K39 antigen and detected with secondary antibodies that were conjugated with cleavable ionic probes. The use of ionic probes enabled direct analysis of the assay results through an on-chip paper spray mass spectrometry (MS) technology. This MS-based immunoassay was developed to allow for early detection of CVL in asymptomatic dogs. The sensitivity required for such a diagnostic method was demonstrated through internal standard calibration in which sample dilution as low as 1/4000 was achieved. Aside from high sensitivity, the ionic probes are stable, which allowed the paper device to be stored at room temperature and under ambient conditions for 30 days without affecting the diagnostic outcome. Our method was used to analyze 20 clinical canine serum samples, where we detected a 2 orders of magnitude higher signal for CVL-positive samples compared to negative samples. MS signal derived from the 10 CVL-positive serum clinical samples showed a strong correlation with antibody titers determined by immunofluorescence assay. This correlation was confirmed through Pearson's statistical analysis. Overall, the high sensitivity and positive results from stability tests observed for our platform are expected to enable large-scale CVL screening in asymptomatic dogs in remote areas, especially when combined with portable mass spectrometers.
这项工作展示了首次在纸基微流控平台上进行的用于诊断犬内脏利什曼病（CVL）的间接免疫测定。表示这种传染病存在的IgG抗体生物标志物，用重组K39抗原捕获，并用与可裂解离子探针偶联的二抗进行检测。离子探针的使用使得能够通过芯片上的纸喷雾质谱（MS）技术直接分析测定结果。开发这种基于MS的免疫测定是为了能够在无症状犬中早期检测CVL。通过内标校准证明了这种诊断方法所需的灵敏度，其中实现了低至1/4000的样品稀释。除了高灵敏度外，离子探针还很稳定，这使得纸质装置能够在室温及环境条件下储存30天而不影响诊断结果。我们的方法用于分析20份临床犬血清样本，我们检测到CVL阳性样本的信号比阴性样本高2个数量级。来自10份CVL阳性血清临床样本的MS信号与通过免疫荧光测定确定的抗体滴度显示出很强的相关性。这种相关性通过Pearson统计分析得到证实。总体而言，我们平台观察到的高灵敏度和稳定性测试的阳性结果有望在偏远地区对无症状犬进行大规模CVL筛查，特别是与便携式质谱仪结合使用时。
