Rare Protein Detection on Cell Surfaces: Ultrasensitive Detection of B-Cell Chron

细胞表面稀有蛋白检测：B 细胞 Chron 的超灵敏检测

• 批准号: 8687881
• 负责人: David Edward Kohne
• 金额: $ 5.14万
• 依托单位: JADEN BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-28 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8687881
• 关键词: Address; Antibodies; Applications Grants; B lymphoid malignancy; B-Lymphocytes; Basic Cancer Research; Biochemical; Biological Assay; Biological Markers; Biological Models; Blood; Blood Cell Count; Blood specimen; Bromodeoxyuridine; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Line; Cell Proliferation; Cell surface; Cells; Characteristics; Chronic Lymphocytic Leukemia; Color; Communities; Data; Detection; Detection of Minimal Residual Disease; Development; Diagnosis; Diagnostic; Disease Progression; Drops; Drug resistance; Early Diagnosis; Fingers; Flow Cytometry; Funding; Goals; Grant; Health Benefit; Hour; Leukocytes; Lymphoma; MS4A1 gene; Malignant Neoplasms; Mantle Cell Lymphoma; Marketing; Medical Research; Methods; Monitor; Patient Monitoring; Patients; Performance; Polymerase Chain Reaction; Preparation; Proliferating; Proteins; Public Health; RORA gene; Reagent; Receptor Protein-Tyrosine Kinases; Reproducibility; Residual Neoplasm; Sampling; Sensitivity and Specificity; Small Business Innovation Research Grant; Staging; Surface; Technology; Testing; Time; Training; Venipunctures; assay development; base; clinically relevant; cost; instrument; new technology; public health relevance; sample collection; tool

DESCRIPTION (provided by applicant): The ROR1 cell surface biomarker is expressed on B-cell Chronic Lymphocytic Leukemia (B-CLL) cells but not on healthy B-cells or other white blood cells (WBCs) in B-CLL patients. We propose to develop an inexpensive, easy-to-use, rapid, ultrasensitive assay specific for ROR1 for the detection and monitoring of B- CLL from a single drop of blood. Our goal is to attain detection levels better than currently available methods, the most sensitive flow cytometry (MRD-FC) or equal to polymerase chain reaction (ASO-PCR) methods available to detect Minimal Residual Disease (MRD). Early detection of MRD is critical in managing therapy and detecting the emergence of drug resistant malignancies. Current methods are expensive with regard to instruments, reagents, and the need for highly trained technicians. Moreover, FC methods do not adequately differentiate between B-CLL and other clonal B-cell malignancies such as mantle cell lymphoma and splenic marginal zone lymphoma. Current tests require venipuncture to collect 1-3 mLs of blood and the time to results are hours for FC and a week for ASO-PCR. JBI possesses a platform technology to address these shortcomings. JBI's first use of its technology resulted in the ZivaTM Ultrasensitive BrdU Cell Proliferation assay. Using only 50 <L of sample, Ziva detected 1-4 proliferating cells among 105 non-proliferating cells, a claim others have not approached. Applying this level of sensitivity to the detection of B-CLL would make it an attractive competitor to ASO-PCR, the most sensitive method. However, unlike ASO-PCR that takes one week to perform and is very expensive, the Ziva assay can be performed in 1 hour from a single drop of blood and is inexpensive. Together with the use of ROR1, a specific biomarker or B-CLL, if successful the resulting assay could differentiate between B-CLL and other clonal B-cell malignancies. Preliminary data show that an early-stage adaptation of our technology yielded an almost 2-fold better sensitivity than 4-color FC. We anticipate further improvements using our assay development strategies. Where standard FC methods for monitoring disease progression require 1-3 mL of blood collected by venipunture, JBI's assay proposes using a finger prick to collect a blood sample which represents a far more convenient method of sample collection for routine monitoring of patients. If successful the proposed test will be more sensitive and specific than FC and reduce the overall cost burden of patient management. Our approach focuses on three specific aims: AIM 1: To develop cell surface assay model systems using cells with known surface markers. AIM 2: To develop and optimize the analytical performance of the ROR1/CLL pilot assay using cell lines. AIM 3: To preliminarily evaluate the ROR1/CLL assay performance characteristics using clinically relevant samples.

描述（由申请人提供）：ROR1细胞表面生物标志物在b细胞慢性淋巴细胞白血病（B-CLL）细胞上表达，但在B-CLL患者的健康b细胞或其他白细胞（wbc）上不表达。我们建议开发一种廉价、易于使用、快速、超灵敏的ROR1特异性检测方法，用于单滴血中B- CLL的检测和监测。我们的目标是达到比目前可用的方法更好的检测水平，最灵敏的流式细胞术（MRD- fc）或等于聚合酶链反应（ASO-PCR）方法可用于检测最小残留病（MRD）。MRD的早期检测对于管理治疗和发现耐药恶性肿瘤的出现至关重要。目前的方法在仪器、试剂和对训练有素的技术人员的需求方面是昂贵的。此外，FC方法不能充分区分B-CLL和其他克隆b细胞恶性肿瘤，如套细胞淋巴瘤和脾边缘带淋巴瘤。目前的检测需要静脉穿刺采集1-3毫升血液，FC检测需要数小时才能得出结果，ASO-PCR检测需要一周时间。JBI拥有解决这些缺点的平台技术。JBI首次使用其技术产生了ZivaTM超灵敏BrdU细胞增殖试验。仅使用50 <L的样品，Ziva在105个非增殖细胞中检测到1-4个增殖细胞，这是其他人没有提出的主张。将这种灵敏度水平应用于B-CLL检测将使其成为最敏感的方法ASO-PCR的有吸引力的竞争对手。然而，不像ASO-PCR需要一周的时间来完成，而且非常昂贵，Ziva分析可以在一滴血的1小时内完成，而且价格低廉。与ROR1（一种特定的生物标志物或B-CLL）一起使用，如果成功，所得到的检测可以区分B-CLL和其他克隆b细胞恶性肿瘤。初步数据显示，我们技术的早期适应产生了比4色FC高近2倍的灵敏度。我们期望利用我们的分析开发策略进一步改进。在监测疾病进展的标准FC方法需要通过静脉穿刺采集1-3毫升血液的情况下，JBI的检测建议使用手指刺破采集血液样本，这代表了一种更方便的常规监测患者样本采集方法。如果成功，提议的测试将比FC更敏感和特异性，并减少患者管理的总体成本负担。我们的方法侧重于三个特定目标：目标1：使用具有已知表面标记的细胞开发细胞表面测定模型系统。目的2：利用细胞系开发和优化ROR1/CLL中试试验的分析性能。目的3：利用临床相关样本初步评估ROR1/CLL检测的性能特征。