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: 8150387
• 负责人: David Edward Kohne
• 金额: $ 15.72万
• 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/8150387
• 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. PUBLIC HEALTH RELEVANCE: This grant proposes to develop an ultrasensitive, inexpensive, easy-to-use, rapid diagnostic assay, that is specific for the detection of the ROR1 cell surface biomarker on B-CLL cells, for the detection and monitoring of B-cell Chronic Lymphocytic Leukemia (B-CLL) using a single drop of blood. Developing rapid, inexpensive and easy-to-use diagnostic tools that are better in sensitivity than the most sensitive flow cytometry or PCR methods, using a single drop of blood, presents a very broad public health benefit; for basic cancer research and for patient management worldwide. Furthermore, successful completion of this project is expected to result in a more useful method to differentially diagnose B-CLL from other clonal B cell malignancies such as mantle cell lymphoma and splenic marginal zone lymphoma. The SBIR funds requested in this proposal will accelerate the introduction of an important new technology for the detection of rare cell surface markers for use in both the research and medical communities.

描述（由申请方提供）：ROR 1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细胞或其他白色血细胞（WBC）上不表达。我们建议开发一种廉价的，易于使用的，快速的，超灵敏的检测特异性ROR 1的检测和监测B- CLL从一滴血。我们的目标是达到比现有方法更好的检测水平，最敏感的流式细胞术（MRD-FC）或等同于聚合酶链反应（ASO-PCR）方法可用于检测微小残留病（MRD）。MRD的早期检测对于管理治疗和检测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要方面是昂贵的。此外，FC方法无法充分区分B-CLL和其他克隆性B细胞恶性肿瘤，例如套细胞淋巴瘤和脾边缘区淋巴瘤。目前的测试需要静脉穿刺收集1-3毫升的血液和结果的时间是几个小时的FC和一个星期的ASO-PCR。JBI拥有一种平台技术来解决这些缺点。JBI首次使用其技术，推出了ZivaTM超灵敏BrdU细胞增殖检测试剂盒。仅使用50 μ L样品，Ziva在105个非增殖细胞中检测到1-4个增殖细胞，这是其他人没有达到的要求。将这种灵敏度水平应用于B-CLL的检测将使其成为最灵敏的方法ASO-PCR的有吸引力的竞争者。然而，与ASO-PCR需要一周的时间才能完成并且非常昂贵不同，Ziva检测可以在1小时内从一滴血中进行，并且价格低廉。与ROR 1（一种特异性生物标志物或B-CLL）的使用一起，如果成功的话，所得到的测定可以区分B-CLL和其他克隆性B细胞恶性肿瘤。初步数据显示，我们技术的早期适应产生的灵敏度比4色FC高出近2倍。我们预计使用我们的检测开发策略会有进一步的改进。当用于监测疾病进展的标准FC方法需要通过静脉穿刺收集1-3 mL血液时，JBI的测定提出使用手指针刺来收集血液样品，这代表了用于患者常规监测的更方便的样品收集方法。如果成功，拟议的测试将比FC更敏感和特异性，并减少患者管理的总体成本负担。我们的方法集中在三个具体的目标：目的1：开发细胞表面检测模型系统，使用已知的表面标志物的细胞。目的2：开发和优化ROR 1/CLL试验的分析性能，使用细胞系。目的3：使用临床相关样本初步评价ROR 1/CLL检测的性能特征。 公共卫生相关性：该资助计划开发一种超灵敏，廉价，易于使用的快速诊断检测方法，该方法专门用于检测B-CLL细胞上的ROR 1细胞表面生物标志物，用于检测和监测B细胞慢性淋巴细胞白血病（B-CLL）。开发快速，廉价和易于使用的诊断工具，其灵敏度优于最敏感的流式细胞术或PCR方法，使用一滴血，为基础癌症研究和全球患者管理提供了非常广泛的公共卫生益处。此外，该项目的成功完成有望产生一种更有用的方法来鉴别诊断B-CLL与其他克隆性B细胞恶性肿瘤，如套细胞淋巴瘤和脾边缘区淋巴瘤。本提案中要求的SBIR资金将加速引入一种重要的新技术，用于检测研究和医学界使用的罕见细胞表面标记物。