Journal of immunological methods最新文献

英文中文

Cell-based potency assay for anti-CD3-anti-CD19 diabody 抗cd3 -抗cd19抗体的细胞效价测定。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-12 DOI: 10.1016/j.jim.2025.114004

Tania L. Weiss , Justine Paniagua , Tonny Johnson , Timothy P. Cripe , Peter Ralph

A cytotoxicity assay was developed to measure the potency of GP101, a single chain diabody. GP101 is comprised of two linked Fv domains, with one that binds CD3 (expressed on T cells), and the other that binds CD19 (expressed on B cells). GP101 directs CD3 positive T lymphocytes to CD19 positive B lymphocytes. This immunotherapy redirects CD3+ T cells to CD19-expressing B-cell malignancies, enabling T-cell-mediated tumor cell lysis. We developed a GMP cell-based potency assay to satisfy the FDA requirements. The potency assay uses a cytotoxic T cell line, and a B cell lymphoma cell line as the target. The target lymphoma B cell line is prelabeled with a fluorescent dye, and upon T cell mediated killing, the fluorescence dye is released and detected using a fluorometer. The emitted fluorescence is proportional to the dose of GP101. Greater than 90 % of the target B cells were killed within two hours exposure in vitro with the lowest amount of detectable killing at 60 pg/mL GP101. The assay is suitable for measuring purified GP101, or GP101 expressed by cells transduced by GP101 plasmid or AAV preparations, and bioactivity in animal or human blood. This novel assay met GMP/GLP compliance, allowing a quantifiable and reproducible measure of efficacy, ensuring batch-to-batch consistency, and met safety and effectiveness regulatory requirements. This potency assay may be applicable for testing other CD3-CD19 T cell engagers and suitable for developing other diabody mediated potency assays with appropriate antigens.

采用细胞毒性测定法测定单链糖蛋白GP101的效力。GP101由两个相连的Fv结构域组成，一个结合CD3（在T细胞上表达），另一个结合CD19（在B细胞上表达）。GP101将CD3阳性T淋巴细胞导向CD19阳性B淋巴细胞。这种免疫疗法将CD3+ T细胞重定向到表达cd19的b细胞恶性肿瘤，使T细胞介导的肿瘤细胞裂解成为可能。我们开发了一种基于GMP细胞的效价测定来满足FDA的要求。效价测定使用细胞毒性T细胞系和B细胞淋巴瘤细胞系作为靶标。用荧光染料预先标记目标淋巴瘤B细胞系，在T细胞介导的杀伤后，释放荧光染料并使用荧光仪检测。发出的荧光与GP101的剂量成正比。在体外暴露两小时内，超过90% %的靶B细胞被杀死，最低的可检测杀伤量为60 pg/mL GP101。该方法适用于测量纯化GP101，或GP101质粒或AAV制剂转导细胞表达的GP101，以及动物或人血液中的生物活性。这种新型检测方法符合GMP/GLP要求，允许定量和可重复的有效性测量，确保批次间的一致性，并满足安全性和有效性监管要求。该效价测定方法可用于检测其他CD3-CD19 T细胞接合体，并适用于开发其他具有适当抗原的糖尿病介导的效价测定方法。

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引用次数: 0

Detection of human IgM and IgG antibodies reactive to Dengue virus using magnetic bead immunoassay 磁珠免疫分析法检测人对登革病毒反应的IgM和IgG抗体。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-07 DOI: 10.1016/j.jim.2025.114002

Marcelo S. Conzentino , Thais A. Silva , Ana Carolina A. Gonçalves , Fabiane G.M. Rego , Vera J.L. Chicora , Stephanie M.S. Lo , Fernanda L. Martiny , Fabio O. Pedrosa , Luciano F. Huergo

Here we describe a magnetic bead-based indirect ELISA that enables the rapid and scalable detection of human IgM and IgG antibodies reactive to dengue virus (DENV). The assay can be performed in 12 min in the chromogenic format to IgM or IgG detection. The system can also handle simultaneous detection of IgM and IgG within less than 8 min when combined with fluorescent-labelled secondary conjugated IgM and IgG detection antibodies. The sensitivity and specificity of the assay are in the same range as described for commercially available ELISA kits. This simple and ultrafast assay for dengue seroconversion detection is an efficient alternative for the rapid track dengue cases.

在这里，我们描述了一种基于磁珠的间接ELISA，能够快速和可扩展地检测对登革热病毒（DENV）有反应的人IgM和IgG抗体。该试验可在12 min内以显色形式进行IgM或IgG检测。当与荧光标记的二联IgM和IgG检测抗体结合时，该系统还可以在不到8 min的时间内同时检测IgM和IgG。该检测的灵敏度和特异性与市售ELISA试剂盒描述的范围相同。这种简单、超快速的登革热血清转化检测方法是快速追踪登革热病例的有效替代方法。

引用次数: 0

Long-term cell storage of RAW 264.7 cells in a deep freezer dampens M2 marker expression raw264.7细胞在冷冻室中长期保存可抑制M2标记物的表达。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-05 DOI: 10.1016/j.jim.2025.114001

Tomonori Makiguchi , Akio Nakane , Sadatomo Tasaka

RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at −80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a − 80 °C deep freezer for a few years (defined as “long-stored cells”) and newly purchased cells (referred to as “new cells”), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (p < 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (p = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.

RAW 264.7是一种成熟的细胞系，广泛用于研究；然而，在环境条件的细微变化或重复通道上，它很容易两极分化。由于细胞储存条件和时间对其表型变化的影响尚不清楚，我们研究了-80 °C长期储存对IL-4刺激后RAW 264.7细胞M2标记物表达的影响。在 - 80 °C深度冷冻室中储存数年的细胞（定义为“长期储存细胞”）和新购买的细胞（称为“新细胞”），通过流式细胞仪比较cd68阳性naïve巨噬细胞（M0）和M2状态下三种M2标记物CD206-FITC、CD206-PE和Arginase-1-Alexa Fluor 488的表达。CD68阳性在M0型巨噬细胞间相似。IL-4刺激后，cd206 - fitc阳性细胞在长存细胞和新生细胞中的比例分别为5.0 ± 0.7 %和61.5 ± 3.1 % (p

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引用次数: 0

Impact of oral Bacillus Calmette–Guérin on B220 receptor expression in Peyer's patches of lactating mice: Analysis of receptor percentiles and cell frequencies 口服卡介苗-谷氨酰胺芽孢杆菌对泌乳小鼠Peyer斑中B220受体表达的影响：受体百分位数和细胞频率分析。

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-05 DOI: 10.1016/j.jim.2025.113995

Ivana Vieira de Mello Loureiro, Claudia Andrea Alves de Araujo

The oral administration of the Bacillus Calmette–Guérin (BCG) vaccine aims to replicate the earliest inoculation route. We investigated the impact of this method by analyzing major Peyer's Patches (PP) cell lineages in mice. Oral BCG altered T/CD3, B/B220, double-labeled CD3–B220, double-labeled IgMB220, and triple-labeled CD49b–CD3–B220 cells. Both immunized (IL) and non-immunized (NIL) lactating mice served as controls in the flow cytometry analyses. The findings suggest that oral BCG vaccination induces changes in specific cell lineages, which may have implications for the development of novel vaccination strategies. Since population percentages vary according to source, frequencies based on fluorescence intensity were calculated according to percentiles, and subpopulations were subsequently analyzed per percentile. A robust statistical analysis assessed whether immunization influenced either the percentage of B220-labeled cells or the cellular expression of B220 per bin. The Thy1.2–depleted population revealed B220 as a receptor shared by both B and T lymphocytes. Statistical evaluation by receptor type and cell difference distance revealed subpopulations in transitional phases and showed that, among polychromatic receptor cells such as B220–IgM (CD11b⁻), oral BCG selectively increased B220 expression. Oral BCG also induced CD3 cell activation, evidenced by a decrease in CD5 within the double-labeled CD3–CD5 cluster, despite an overall increase in CD3 receptors. Receptor analysis in regularly turning-over populations, such as PP lymphoid tissue, highlighted the double-labeled B220–CD3 cells. Notably, oral BCG influenced the development of double-labeled B220–CD3 cells in NKT-like populations.

口服卡介苗（Bacillus calmette - gusamrin， BCG）疫苗旨在复制最早接种途径。我们通过分析小鼠主要的Peyer’s Patches （PP）细胞系来研究这种方法的影响。口服BCG改变T/CD3、B/B220、双标记CD3-B220、双标记IgMB220和三标记CD49b-CD3-B220细胞。在流式细胞术分析中，免疫（IL）和未免疫（NIL）的泌乳小鼠作为对照。研究结果表明，口服卡介苗接种可诱导特定细胞系的变化，这可能对开发新的疫苗接种策略具有重要意义。由于种群百分比因来源而异，基于荧光强度的频率按百分位数计算，随后按百分位数分析亚种群。一项强有力的统计分析评估了免疫接种是否影响B220标记细胞的百分比或每箱B220的细胞表达。在thy1.2缺失的人群中发现B220是B淋巴细胞和T淋巴细胞共有的受体。通过受体类型和细胞差异距离的统计评估，揭示了过渡时期的亚群，并表明在B220- igm （CD11b-）等多色受体细胞中，口服卡介苗选择性地增加了B220的表达。口服卡介苗也诱导CD3细胞活化，证据是双标记CD3-CD5簇中的CD5减少，尽管CD3受体总体增加。受体分析在定期翻转人群中，如PP淋巴组织，突出了双重标记的B220-CD3细胞。值得注意的是，口服卡介苗影响了nkt样人群中双标记B220-CD3细胞的发育。

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引用次数: 0

Challenges limiting the establishment of standardized clinical cutpoints for antibody tests used in the evaluation of hypersensitivity pneumonitis 限制建立用于评估过敏性肺炎的抗体测试的标准化临床切入点的挑战

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113954

Bethany Schuder, Anne Tebo

引用次数: 0

Donor selection for basophil activation testing 嗜碱性粒细胞激活试验的供体选择

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113944

Renee K. Johnson, Amir A. Sadighi Akha, Attila Kumánovics

引用次数: 0

Validation of a live cell based assay for the detection of MOG antibodies by flow cytometry 流式细胞术检测MOG抗体的活细胞检测验证

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113938

Conlan Tran

引用次数: 0

Comparative evaluation of pretreatment techniques for enhancing specificity in solid-phase bead-based immunoassays 增强固相珠基免疫测定特异性的预处理技术的比较评价

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113948

Dharmendra Jain , Denise Hurst , Andre Cruz Cruz Delgadillo , Eszter Lázár-Molnár

引用次数: 0

Machine learning can identify an antinuclear antibody pattern that may rule out systemic autoimmune rheumatic diseases 机器学习可以识别抗核抗体模式，可能排除系统性自身免疫性风湿病

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113967

May Y. Choi

引用次数: 0

Determining reference values for the ALZPath plasma p-tau217 assay in a large cohort of elderly individuals with normal cognitive function 确定ALZPath血浆p-tau217测定在一大群认知功能正常的老年人中的参考值

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jim.2025.113930

Pankaj Kumar , Anna Mammel , Ali Mousavi , Mary Encarnacion , Donald Biehl , Hans Frykman

引用次数: 0

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