Journal of immunological methods最新文献_第10页

CpG-ODN from Streptococcus thermophilus as a tool for inducing extrinsic apoptosis via caspase-8 activation in mouse splenocytes 嗜热链球菌CpG-ODN通过caspase-8激活诱导小鼠脾细胞外源性凋亡

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-30 DOI: 10.1016/j.jim.2025.113999

Md. Rayhan Chowdhury , Takuma Okajima , Aito Murakami , Ariful Islam , Takeshi Shimosato

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the lacZ gene of Streptococcus thermophilus ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.

含有未甲基化CpG基序的合成寡脱氧核苷酸（odn）激活toll样受体9，从而引发多种免疫调节反应。尽管cpg - odn的免疫刺激特性已被广泛研究，但其作为细胞凋亡诱导工具的潜在应用仍未得到充分探索，特别是益生菌衍生序列。在这项研究中，我们建立了一种利用从嗜热链球菌ATCC19258的lacZ基因中鉴定出的CpG-ODN MsST诱导小鼠脾细胞外源性凋亡的方法。在基因组DNA片段分析中，MsST诱导中度但显著的核小体间DNA片段。使用膜联蛋白V/7-氨基放线菌素D染色的流式细胞分析证实，在MsST处理后，凋亡细胞群呈时间依赖性增加。此外，Western blot分析显示裂解的caspase-8水平升高，从而验证了外源性凋亡途径的激活。与具有代表性的CpG-ODN和细胞凋亡诱导剂放线菌素D相比，MsST表现出明显的细胞凋亡诱导特征。这些发现突出了MsST作为控制细胞凋亡诱导的潜在工具，为进一步探索cpg - odn在免疫学和治疗方面的应用提供了方法学基础，包括癌症免疫治疗、自身免疫性疾病和炎症性疾病的治疗。

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引用次数: 0

Advancing cervical cancer screening: A novel in-house ELISA assay for the simultaneous detection of p16 and Ki-67 推进宫颈癌筛查：一种同时检测p16和Ki-67的新型内部ELISA检测方法

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-29 DOI: 10.1016/j.jim.2025.113998

Sallam Al-Madhagi , Patricia Khashayar , Lieselotte Volckaert , Jan Vanfleteren , Mieke Adriaens

Immunostaining and cytology, commonly used to detect cervical cancer, are time-consuming and pathologist dependent. In this work, for the first time, we have developed an ELISA assay for the extraction and co-measurement of p16 and Ki-67 in both vaginal and cervical samples for cervical cancer screening. Novel custom-made antibodies were developed, and a competitive ELISA assay was developed and optimized based on the concentration and incubation time of different steps to improve the sensitivity and specificity of the final assay in assessing cervical samples. The as-developed ELISA assay shows high sensitivity and selectivity in different collection media. The LoD of the test is 0.00004 and 0.0058 μg/mL for p16 and Ki-67, respectively. The as-developed assay can be used in future studies to determine protein thresholds for different lesion grades of cervical cancer for samples collected in different settings. These assays can revolutionize the cervical cancer screening management process in the near future.

通常用于检测宫颈癌的免疫染色和细胞学检查耗时且依赖于病理学家。在这项工作中，我们首次开发了一种ELISA法，用于提取和共同测量阴道和宫颈样本中的p16和Ki-67，用于宫颈癌筛查。开发了新型定制抗体，并根据不同步骤的浓度和孵育时间，开发并优化了具有竞争力的ELISA检测方法，以提高最终检测方法评估宫颈样本的灵敏度和特异性。所建立的酶联免疫吸附试验在不同的采集介质中具有较高的灵敏度和选择性。p16和Ki-67的检测检出限分别为0.00004和0.0058 μg/mL。该试验可用于未来的研究，以确定在不同环境中收集的样本中不同病变级别宫颈癌的蛋白质阈值。这些检测可以在不久的将来彻底改变宫颈癌筛查管理过程。

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引用次数: 0

Preliminary evaluation of a whole blood dual-platform flow cytometry protocol for CAR-T cell quantitation: Toward accreditation and clinical routine application 用于CAR-T细胞定量的全血双平台流式细胞术方案的初步评估：走向认证和临床常规应用。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-22 DOI: 10.1016/j.jim.2025.113986

Rémi Pescarmona , Guillaume Monneret , Eleonore Micoud , Christophe Malcus , Emmanuel Bachy , Pierre Sesques , Lionel Karlin , Fabienne Venet , Morgane Gossez

Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects. This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes. Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0.5 μL CAR detection reagent compared to the 2 μL recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to >3000 circulating CAR-T cells/μL. The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results. Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 °C. In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.

嵌合抗原受体t细胞，通常称为CAR-T细胞，代表了癌症细胞治疗的重大科学突破，彻底改变了血液系统疾病的治疗。药代动力学研究强调了外周血CAR-T细胞监测对预测治疗结果和管理副作用的重要性。本研究旨在建立和评估三种不同的流式细胞仪（DxFLEX， Navios和Navios EX）在医院常规使用的情况下使用流式细胞仪进行CAR-T定量方法的双平台方案的性能。用生物素化CD19或BCMA重组蛋白和pe偶联抗生物素抗体对新鲜全血进行染色。消除双链后，在CD45+ CD3+细胞中鉴定CAR-T细胞，用对照管确定T淋巴细胞中CAR-T阳性表达阈值。结果以CD3+ T淋巴细胞中CAR - T细胞的百分比表示。Optilyse C和Versalyse红细胞裂解试剂（Beckman Coulter）在T淋巴细胞中的CAR - T细胞百分比方面给出了相当的结果，并且使用0.5 μL的CAR检测试剂与制造商推荐的2 μL相比。使用该方案，我们没有观察到CD19和BCMA CAR检测试剂之间的任何交叉反应性或信号降低，甚至高达3000个循环CAR- t细胞/μL。无论使用何种流式细胞仪、CAR试剂检测类型和循环CAR- t细胞的比例，重复性和中间精度的变异系数系统地低于10 %。所有三个流式细胞仪返回相关和可比的结果。最后，关于全血样本和染色细胞的保存时间，我们观察到，当取样后染色延迟超过一天，以及在4 °C下染色细胞保存三天后，CAR-T百分比显著下降。总之，我们提出了一种CAR-T细胞计数的双平台方案，该方案在BCMA和CD19 CAR-T细胞定量方面表现出一致和可靠的性能。

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引用次数: 0

Isolation of HPRT gene depleted human TK6 lymphoblastoid cell line through gamma irradiation γ辐照分离HPRT基因缺失的人TK6淋巴母细胞系。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-10 DOI: 10.1016/j.jim.2025.113985

P.R. Vivek Kumar , Anila Gopinathan , Deepak Sharma

Ionizing radiation induced DNA DSBs can cause large deletions in the human genome. This property may be utilized for gene disruption to create knockout mutants. The X-linked hypoxanthine–guanine phosphoribosyl transferase (HPRT) gene is commonly used as a reporter to evaluate the frequency and molecular nature of somatic mutations in the human population. Mutations at the HPRT locus are measured through a T lymphocyte cloning assay. This assay relies on lymphoblast feeder cells that support T lymphocyte proliferation via cell-to-cell contact and by helping in the expression of functional interleukin-2 receptors. However, the feeder cells must be HPRT-depleted to prevent feeder cell DNA from serving as substrate during molecular analysis of T lymphocyte mutations. To the best of our knowledge, the human lymphoblastoid TK6 cell line available in Indian cell repositories has an intact HPRT gene (HPRT+). In this study, the parent TK6 cell line was subjected to the clastogenic effects of a high dose gamma radiation to isolate TK6 cells with a total deletion of the HPRT locus. The HPRT-depleted cell line (TK6_LLrrL) was characterized by mPCR for the loss of exons and the extent of the deletion by flanking STS markers analysis. Further, the feeder cell function of TK6_LLrrL was evaluated by estimating the cloning efficiency of human peripheral blood lymphocytes. Thus, the study demonstrates the potential of low LET gamma radiation to generate gene knockout cell line. The method described here can be applied to various cell types to produce functionally null cell lines for gene-specific studies.

电离辐射诱导的DNA dsb可导致人类基因组的大量缺失。这一特性可用于基因破坏以产生敲除突变体。x连锁的次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）基因通常被用作评估人类群体中体细胞突变的频率和分子性质的报告基因。在HPRT基因座的突变是通过T淋巴细胞克隆测定测定。该试验依赖于淋巴母细胞饲养细胞，这些细胞通过细胞间接触和帮助表达功能性白细胞介素-2受体来支持T淋巴细胞增殖。然而，在T淋巴细胞突变的分子分析中，喂养细胞必须耗尽hprt，以防止喂养细胞DNA作为底物。据我们所知，印度细胞库中可用的人淋巴母细胞样TK6细胞系具有完整的HPRT基因（HPRT+）。在本研究中，将亲本TK6细胞系置于高剂量伽马辐射的致裂作用下，分离出HPRT位点完全缺失的TK6细胞。hprt缺失细胞系（TK6LLrrL）通过mPCR检测外显子的缺失和侧翼STS标记的缺失程度。进一步，通过对人外周血淋巴细胞的克隆效率评估TK6LLrrL的饲养细胞功能。因此，该研究证明了低LET γ辐射产生基因敲除细胞系的潜力。这里描述的方法可以应用于各种细胞类型，以产生用于基因特异性研究的功能空细胞系。

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引用次数: 0

Temperature stability of rabies neutralizing antibodies 狂犬病中和抗体的温度稳定性。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-08 DOI: 10.1016/j.jim.2025.113982

Nicholas J. Jones, Kim A. Appler, Jodie A. Jarvis, Alexander C. Keyel, April D. Davis

Rabies serology is most importantly done to evaluate immune response following vaccine administration in patients who have been exposed to rabies or are highly likely to be. When detecting antibodies after vaccination it is best to target rabies neutralizing antibodies specifically. Clinical laboratories performing this testing are required to uphold a strict set of standards laid out by federal and state guidelines. Sample integrity throughout the process of testing, from collection to results is of the utmost importance. Samples may experience a wide range of temperature variation depending on how each sample is shipped or handled by the submitter. In order to uphold quality clinical testing it is important to study the stability of rabies neutralizing antibodies across a wide range of temperatures.

狂犬病血清学最重要的是评估暴露于狂犬病或极有可能暴露于狂犬病的患者接种疫苗后的免疫反应。在疫苗接种后检测抗体时，最好针对狂犬病中和抗体进行特异性检测。进行这种测试的临床实验室必须遵守联邦和州指导方针制定的一套严格的标准。样品的完整性在整个测试过程中，从收集到结果是至关重要的。样品可能会经历很大范围的温度变化，这取决于每个样品的运输方式或提交者的处理方式。为了保证临床检测的质量，研究狂犬病中和抗体在广泛温度范围内的稳定性是很重要的。

引用次数: 0

Immunogenicity of tislelizumab: clinical summary of ADA incidence and feasibility of assessing the suitability of validation cut points using statistical methods tislelizumab的免疫原性：ADA发病率的临床总结和使用统计方法评估验证切入点适用性的可行性。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-02 DOI: 10.1016/j.jim.2025.113983

Boxiang Tao , Meng Wang , Yanfei Yang , Na Zhao , Meiling Tan , Xiaolei Xun , Chunxiang Zeng , Fan Wang

Anti-drug antibody (ADA) is a critical indicator of the immunogenicity of biotherapeutics. To investigate the immunogenicity of tislelizumab, a humanized anti-PD-1 (programmed cell death 1) antibody, 3815 patients were analyzed for ADA incidence. The incidence of ADA is reported for the first time as 19.2 %. For any immunogenicity study, appropriate cut points are essential to accurately distinguish true ADA-positive samples from varying backgrounds across populations. Apart from the standardized false positive rate (FPR) method, statistical methods may be more efficient and powerful in evaluating the appropriateness of cut points. However, a detailed protocol for this approach is not yet in consensus. Therefore, by utilizing extensive data from tislelizumab clinical trials, this study explored the feasibility of assessing the suitability of validation cut points (VCPs) using statistical methods. ADA response datasets from 21 clinical tumor populations were evaluated for distribution differences in comparison to the validation population using statistical methods. Fourteen datasets exhibited significant differences from the validation dataset in both medians and variances, with FPRs exceeding the 2–11 % range. Seven datasets only differed significantly in medians, with five having out-of-range FPRs and their positive rates narrowly affected by cut point alteration. The results showed that statistical methods can effectively assess the suitability of VCPs: in-study cut points are recommended for datasets with significantly different median and variance, while VCPs may be appropriate when only medians differ.

抗药抗体（ADA）是生物治疗药物免疫原性的重要指标。为了研究tislelizumab（一种人源抗pd -1（程序性细胞死亡1）抗体）的免疫原性，我们分析了3815例ADA患者的发病率。ADA的发生率首次报道为19.2 %。对于任何免疫原性研究，适当的切点对于准确区分不同背景的人群中真正的ada阳性样本至关重要。除了标准化的假阳性率（FPR）方法外，统计方法在评估切割点的适当性方面可能更有效和强大。然而，这种方法的详细协议尚未达成共识。因此，通过利用tislelizumab临床试验的大量数据，本研究探讨了使用统计方法评估验证切点（VCPs）适用性的可行性。采用统计学方法对21个临床肿瘤人群的ADA应答数据集与验证人群的分布差异进行评估。14个数据集在中位数和方差上都与验证数据集存在显著差异，fpr超过2-11 %的范围。7个数据集仅在中位数上有显著差异，其中5个数据集的fpr超出范围，其阳性率受切点改变的影响很小。结果表明，统计方法可以有效地评估vcp的适用性：对于中位数和方差有显著差异的数据集，建议使用研究中切点，而只有中位数不同时，vcp可能是合适的。

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引用次数: 0

Fluorescence immunochromatographic assay based on CdSe/ZnS quantum dots for the detection of OXA-48 CdSe/ZnS量子点荧光免疫层析法检测OXA-48。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-10-02 DOI: 10.1016/j.jim.2025.113984

Mengying Lun , Yumei Chen , Hongliang Liu , Yanhua Qi , Aiping Wang , Jingming Zhou

The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.

碳青霉烯酶的产生是细菌对碳青霉烯类抗生素产生耐药性的主要机制。这种耐药性严重影响了碳青霉烯类抗生素治疗感染的效果，对临床抗感染治疗提出了重大挑战。Oxacillinase-48 （OXA-48）是一种D类碳青霉烯酶，其基因通常位于可转移元件上，可以在不同菌株和属之间传播。因此，从感染控制的角度出发，对OXA-48进行有效的监测和预防势在必行。本研究将CdSe/ZnS量子点（QDs）与抗oxa -48单克隆抗体（mAb）偶联作为荧光信号探针，建立基于量子点的荧光免疫层析条带。试纸包含一条包被抗oxa -48单克隆抗体的试验线（t线）和一条包被葡萄球菌蛋白a （SPA）的控制线（c线）。基于双抗体夹心检测模式，可在10 min内完成OXA-48的高灵敏度检测。该试纸条对OXA-48重组蛋白的检出限为3.13 ng/mL，与其他常见碳青霉烯酶（IMP-1、NDM-1、KPC-2、VIM-2、OXA-23）无交叉反应。定位为一种快速可靠的OXA-48检测工具，为临床快速鉴定产生OXA-48耐药菌株提供了一种新的方法。

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引用次数: 0

Measurement of cleaved high-molecular-weight kininogen in patients with hereditary angioedema due to C1-inhibitor deficiency: preanalytical and analytical optimization c1抑制剂缺乏导致的遗传性血管性水肿患者的高分子量激肽原的测定：分析前和分析优化

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-09-30 DOI: 10.1016/j.jim.2025.113981

Chiara Suffritti , Roberta Gualtierotti , Andrea Zanichelli , Matteo Vidali , Massimo Cugno

High-molecular-weight kininogen (HK) is a plasma protein cleaved during the activation of contact system by the enzyme kallikrein, releasing the peptide bradykinin, potent angioedema mediator. Plasma measurement of cleaved HK allows evaluating in vivo contact system activation but, when its main natural inhibitor (C1-inhibitor) is deficient, conflicting results have been reported, possibly due to in vitro activation.

We collected blood samples from 18 patients with C1-inhibitor deficiency and 7 healthy controls using five different anticoagulant mixtures to find the one that could completely block in vitro activation of contact system, so that a snapshot of the in vivo scenario could be obtained. The ability to prevent contact system activation in vitro was evaluated in plasma samples measuring levels of cleaved HK by SDS-PAGE with precast gels and immunoblotting. Correlations between cleaved HK and functional C1-inhibitor were evaluated as well as assay reproducibility.

In healthy subjects, no differences in cleaved HK were found for the different anticoagulants. In patients with C1-inhibitor deficiency, we observed higher levels of cleaved HK in sodium citrate samples (median 60.5 %, IQR 51.9–61.6 %) than in samples collected in a mixture of protease inhibitors (median 49.2 %, IQR 48.1–52.0 %) (P = 0.001). Only in the latter, cleaved HK levels correlated with circulating functional C1-inhibitor levels (P = 0.005). Using precast gels, intra- and inter-assay coefficients of variation were < 5 %.

In conclusion, for measuring cleaved HK in plasma from patients with C1-inhibitor deficiency, the use of anticoagulant with protease inhibitors and precast gels allows reliable evaluation of in vivo contact system activation.

高分子量激肽原（high -molecular weight kininogen， HK）是一种在接触系统被缓激肽酶（kallikrein）激活时裂解的血浆蛋白，释放出缓激肽，是一种有效的血管性水肿介质。血浆测量裂解HK可以评估体内接触系统激活，但是，当其主要天然抑制剂（c1抑制剂）缺乏时，可能由于体外激活，报道了相互矛盾的结果。我们收集了18例c1抑制剂缺乏症患者和7名健康对照者的血液样本，使用5种不同的抗凝剂混合物，寻找一种可以完全阻断接触系统体外激活的抗凝剂，从而获得体内情况的快照。通过SDS-PAGE、预制凝胶和免疫印迹法测量血浆样品中裂解HK的水平，评估了体外防止接触系统激活的能力。我们评估了裂解HK与功能性c1抑制剂的相关性以及实验的重复性。在健康受试者中，不同抗凝剂的裂解性HK无差异。在c1抑制剂缺乏的患者中，我们观察到柠檬酸钠样品中裂解的HK水平（中位数为60.51 %，IQR为51.90-61.60 %）高于蛋白酶抑制剂混合物样品（中位数为49.21 %，IQR为48.10-52.05 %）（P = 0.001）。只有在后者中，裂解HK水平与循环功能c1抑制剂水平相关（P = 0.005）。使用预制凝胶，计算了测定内和测定间的变异系数

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引用次数: 0

A high-throughput multi-species platform using Biolayer Interferometry Immunosorbent Assay (BLI-ISA) as an alternative to indirect ELISA for vaccine development 使用生物层干涉免疫吸附测定（BLI-ISA）作为间接ELISA替代疫苗开发的高通量多物种平台。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-09-18 DOI: 10.1016/j.jim.2025.113980

Ti Lu, Md Shafiullah Parvej, Sean K. Whittier, Suhrid Maiti, Zackary K. Dietz, Debaki R. Howlader, Mst Nusrat Zahan, Satabdi Biswas, William D. Picking, Wendy L. Picking

In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.

在疫苗开发中，ELISA（酶联免疫吸附试验）通常用于比较来自几个治疗组的样品的抗体滴度。这通常需要大量的样品制备、手工劳动以及长时间的孵育和处理时间。生物层干涉法（BLI）已成为ELISA检测和定量生物样品中抗原特异性抗体的替代方法。然而，在疫苗开发中实施BLI作为ELISA的替代品，需要建立准确和可重复结果的实验参数。在这里，我们给出了一种生物层干涉免疫吸附测定（BLI-ISA）的一般方案，用于比较治疗组血清中抗原特异性抗体水平，使用二抗结合反应代替ELISA终点滴度。BLI-ISA提供抗原特异性抗体水平的快速相对测量（以nm结合位移为单位），产生与ELISA终点滴度（以ELISA单位表示）一致的结果，从而在传统ELISA所需的一小部分时间内实现跨治疗组的可靠比较。

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引用次数: 0

Peripheral blood and nasal swabs Type I and III IFNs signature in RSV positive infant with bronchiolitis. RSV阳性婴儿毛细支气管炎的I型和III型ifn特征。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-09-01 Epub Date: 2025-08-05 DOI: 10.1016/j.jim.2025.113918

Francesco Savino, Paola Montanari, Maddalena Dini, Anna Pau, Lorenzo Ferroglio, Sara Burdisso, Cristina Calvi, Anna Clemente, Stefano Gambarino, Ilaria Galliano, Massimiliano Bergallo

Bronchiolitis is an acute lower respiratory tract infection mainly affecting children under 24 months, primarily caused by Respiratory Syncytial Virus (RSV). Interferons (IFNs), cytokines central to innate and adaptive immunity, are secreted in response to viral infections. In RSV bronchiolitis, Type I and III IFN Signatures modulate antiviral responses and impact disease severity. This study aimed to evaluate the correlation between Type I and III IFNs transcriptional signatures in blood and nasal swabs with clinical presentation. We analyzed transcription levels of Type I and III IFN Signatures using a PCR real-time TaqMan assay in blood and nasal swabs from children hospitalized for RSV bronchiolitis and from healthy controls (HC). Interferon score I was significantly higher in bronchiolitis patients at admission than at discharge and compared to HC, in both blood and nasal swabs (p < 0.0001). A strong positive correlation for IFN score I between the two sample types was found (p < 0.0001). Patients with severe disease (requiring intensive care) had lower IFN score I than those with milder disease, in both blood (p = 0.0005) and nasal swabs (p = 0.0078). IFN score III was down-regulated in bronchiolitis patients compared to HC in blood samples (p < 0.0001), while in nasal swabs, this down-regulation was evident only at discharge (p < 0.0001 vs admission; p = 0.0546 vs HC). This study enhances understanding of IFN signature dynamics in RSV bronchiolitis, emphasizing the importance of sample type and signature specificity, and suggests their potential use as prognostic tools to improve patient management.

毛细支气管炎是一种急性下呼吸道感染，主要影响24 个月以下的儿童，主要由呼吸道合胞病毒（RSV）引起。干扰素（ifn）是先天免疫和适应性免疫的核心细胞因子，在病毒感染时分泌。在RSV细支气管炎中，I型和III型IFN信号调节抗病毒反应并影响疾病严重程度。本研究旨在评估血液和鼻拭子中I型和III型ifn转录特征与临床表现之间的相关性。我们使用实时TaqMan方法分析了RSV毛细支气管炎住院儿童和健康对照（HC）血液和鼻拭子中I型和III型IFN信号的转录水平。在血液和鼻拭子中，毛细支气管炎患者入院时干扰素I评分明显高于出院时和HC

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引用次数: 0