Differential Immunocapture Research Articles

Microfluidic chip for label-free removal of teratoma-forming cells from therapeutic human stem cells

Teratoma formation remains a safety concern in therapeutic cells derived from human-induced pluripotent stem cells (hiPSCs). Residual Teratoma forming cells are present in small numbers in differentiated hiPSC cultures and yet are of significant roadblock to the manufacturing and clinical translation of stem cell therapies. Rare cells are often difficult to remove with standard flow cytometry or magnetic bead sorting techniques. Here, we first characterized time-dependent expression of a teratoma marker, stage-specific embryonic antigen (SSEA)-5, which binds the H type-1 glycan during neural differentiation of hiPSCs. The fraction of cells SSEA-5+ remained high at 97% on day 3, dropped to 70% on day 4, 40% by day 6, and down to 1% on day 12 of differentiation, indicating successful differentiation. We engineered a microfluidic geometrically enhanced differential immunocapture (GEDI) technology to remove SSEA-5+ rare cells from hiPSC-derived neural progenitor cells (hiPSC-NPCs). The GEDI chip removed more than 95% of teratoma-forming cells and presents a facile tool to potentially functionalize with multiple antibodies and robustly enhance hiPSC-derived cell population safety prior to therapeutic transplantation. The approach is potentially amenable to generate a wide variety of high-quality therapeutic cells and can be integrated within the pipeline of cell manufacturing to improve patient safety and reduce the cost of manufacturing through early removal of undesirable cell types.

Microfluidic differential immunocapture biochip for specific leukocyte counting.

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes-e.g., for counting of CD4 and CD8 T cells in HIV/AIDS diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 μl of blood. This paper provides a comprehensive stepwise protocol to replicate our biosensor for CD4 and CD8 cell counts. The biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. Capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. Therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. The biosensor production requires 24 h, a one-time cell capture optimization takes 6-9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete.

Single-cell copy number analysis of prostate cancer cells captured with geometrically enhanced differential immunocapture microdevices.

Limited access to tumor tissue makes repeated sampling and real-time tracking of cancer progression infeasible. Circulating tumor cells (CTCs) provide the capacity for real-time genetic characterization of a disseminating tumor cell population via a simple blood draw. However, there is no straightforward method to analyze broadscale genetic rearrangements in this heterogeneous cell population at the single cell level. We present a one-step controllable chemical extraction of whole nuclei from prostate cancer cells captured using geometrically enhanced differential immunocapture (GEDI) microdevices. We have successfully used copy number profile analysis to differentiate between two unique cancer cell line populations of metastatic origin (LNCaP and VCaP) and to analyze key mutations important in disease progression.

Detection of Circulating Pancreas Epithelial Cells in Patients With Pancreatic Cystic Lesions

Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

Parametric control of collision rates and capture rates in geometrically enhanced differential immunocapture (GEDI) microfluidic devices for rare cell capture

The enrichment and isolation of rare cells from complex samples, such as circulating tumor cells (CTCs) from whole blood, is an important engineering problem with widespread clinical applications. One approach uses a microfluidic obstacle array with an antibody surface functionalization to both guide cells into contact with the capture surface and to facilitate adhesion; geometrically enhanced differential immunocapture is a design strategy in which the array is designed to promote target cell–obstacle contact and minimize other interactions (Gleghorn et al. 2010; Kirby et al. 2012). We present a simulation that uses capture experiments in a simple Hele-Shaw geometry (Santana et al. 2012) to inform a target-cell-specific capture model that can predict capture probability in immunocapture microdevices of any arbitrary complex geometry. We show that capture performance is strongly dependent on the array geometry, and that it is possible to select an obstacle array geometry that maximizes capture efficiency (by creating combinations of frequent target cell–obstacle collisions and shear stress low enough to support capture), while simultaneously enhancing purity by minimizing nonspecific adhesion of both smaller contaminant cells (with infrequent cell–obstacle collisions) and larger contaminant cells (by focusing those collisions into regions of high shear stress).

Abstract 1467: Development of a HER2-based GEDI microfluidic device for the molecular characterization of CTCs from metastatic breast cancer patients.

Abstract Microtubule targeting drugs (MTDs) constitute the mainstay of the most active chemotherapy regimens against breast cancer (BC) and have been standard therapy for locally advanced and metastatic disease for the last 20 years. However, metastatic BC (MBC) is still incurable mainly because of acquired drug resistance. The molecular basis of clinical drug resistance is not well understood which together with the lack of predictive biomarkers in the clinical setting prevents us from personalizing MTD-based therapy. Isolation of Circulating Tumor Cells (CTCs) from the blood of metastatic patients has emerged as a valid source of tumor tissue in the form of liquid biopsy that is being used as prognostic biomarker based on CTC enumeration. To be able to perform molecular CTC analyses we have developed a BC specific microfluidic device that operates on the principle of geometrically enhanced differential immunocapture (GEDI), using antibody-coated microposts. We selected HER2 as BC selective surface antigen to isolate CTCs from MBC patients as it is expressed in 90% of breast tumors and overexpressed in 30%, while it is minimally affected by epithelial-to-mesenchymal transition (EMT) often occurring in metastasis. We previously demonstrated that the ability of the HER2 GEDI microfluidic device to capture BC cells expressing high and intermediate to low levels of HER2 expression confirming its suitability for BC patients with highly heterogeneous HER2 levels. We have tested the HER2-based GEDI in MBC patients and have confirmed CTC capture. To test the performance of our device we are currently comparing it to the FDA approved CellSearch using blood from the same patient where CTCs are enumerated by both Cell Search and our device. Importantly, the retained viability of GEDI-captured CTCs provides us the opportunity to functionally test CTC response to chemotherapy directly on the device, in an effort to develop an assay predictive of clinical response so that we can tailor chemotherapy to the individual. In this assay, we are treating the GEDI-captured CTCs with physiologically relevant concentrations of MTDs and are subsequently assaying drug-induced target modifications. As a proof of principle, we have demonstrated that taxane sensitive BC cell lines captured on the GEDI device showed evidence of effective drug-target engagement by the presence of microtubule bundling following Docetaxel treatment. We are currently evaluating the response of GEDI-captured BC taxane-resistant cells to ex-vivo drug treatment to evaluate the dynamic range of our assay. We are currently conducting a clinical trial that characterizes GEDI-captured CTCs isolated from MTD treated MBC patients where we will monitor the molecular characteristics of the CTCs during chemotherapy treatment and at disease relapse. This approach will give us significant insight of the mechanisms of resistance of MTDs. Citation Format: Giuseppe Galletti, Brian Kirby, Linda T. Vahdat, Paraskevi Giannakakou. Development of a HER2-based GEDI microfluidic device for the molecular characterization of CTCs from metastatic breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1467. doi:10.1158/1538-7445.AM2013-1467

Abstract 3492: Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer.

Abstract Prostate cancer progression into castration-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR) signaling despite surgical and chemical androgen ablation. The taxanes represent the only class of chemotherapy that improves overall survival in CRPC patients. Despite their success, CRPC patients do progress on taxane treatment rendering taxane-resistant tumors. The molecular mechanisms underlying clinical taxane resistance in CRPC have not been well elucidated due to the lack of available tumor tissue to study. Circulating tumor cells (CTCs) represent a liquid biopsy of the original tumor and isolation of them can lead to their molecular characterization potentially revealing predictive biomarkers for taxane sensitivity or resistance. Here, we use a geometrically enhanced differential immunocapture (GEDI) microfluidic device that couples an anti-prostate specific membrane antigen (PSMA) antibody with optimized 3D geometry to capture and isolate live CTCs from whole blood of CRPC patients. The GEDI-microfluidic device was shown to have a 2-400 fold higher sensitivity for CTC capture than the FDA-approved CellSearch® system. We have previously shown that CRPC patient CTCs can be used to derive functional information that correlates to clinical response to taxane chemotherapy, namely AR subcellular localization status. We have developed a suite of other functional assays that can be performed on live GEDI-captured CTCs that enable their molecular characterization and allow us to test specific mechanistic hypotheses based on our extensive preclinical data. Included, and herein described, are the determination of AR subcellular localization, extent of effective drug-target engagement assessed by microtubule bundling, identification of RNA species relevant to the mechanism of taxane resistance and computer vision algorithms that will allow for enriched and automated analysis of high-volume image sets of GEDI-captured CTCs. In addition, we will be testing the hypothesis that distinct AR splice variants may affect patient sensitivity to taxane-based chemotherapy. This suite of assays are being rigorously applied in a phase II clinical trial in which chemotherapy-naïve CRPC patients will be initially treated with either docetaxel or cabazitaxel and clinically evaluated for an early switch to the other taxane following disease progression. This prospective, randomized, multi-site clinical trial will enroll 100 CRPC patients within one year. Patients will be followed until relapse and each patient will have 15 independent GEDI assays performed across five time points from baseline to chemotherapy crossover to relapse. The depth of coverage this suite of assays provides will offer unique insights for potential mechanisms of clinical taxane resistance and predictive biomarkers for taxane sensitivity in CRPC patient CTCs. Citation Format: Matthew S. Sung, Ada Gjyrezi, Guang Yu Lee, Alexandre Matov, Giuseppe Galletti, Matthew Loftus, Yusef Syed, Timothy Lannin, Atanas Hristov, Christopher Mason, Scott Tagawa, Brian Kirby, David Nanus, Paraskevi Giannakakou. Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2013-3492

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Abstract 3634: Molecular determinants of taxane activity using CTCs from CRPC patients

Abstract Androgen receptor (AR) signaling drives castration resistant prostate cancer (CRPC) disease progression. The taxanes (TXN) represent the only class of chemotherapy agents that improve survival in CRPC. Our recent work revealed that TXNs work by blocking AR nuclear accumulation and signaling downstream of microtubule (MT) stabilization. Moreover, we showed that AR cytoplasmic sequestration in CRPC patients’ circulating tumor cells (CTCs) significantly correlated with clinical response to TXN therapy. Our data, suggest that TXN-induced blockade of AR nuclear accumulation partly underlies the clinical activity of TXNs in CRPC. Recently several AR splice variants, lacking the ligand binding domain, have been identified in CRPC. These variants are constitutively active in the nucleus and likely to be insensitive to current therapies targeting either ligand biosynthesis or ligand-receptor interactions. Currently, the role of AR splice variants on TXN chemotherapy response in CRPC is unknown. Our preliminary data in preclinical PC models suggest that certain AR variants depend on MTs for nuclear accumulation and are TXN sensitive while others are MT-independent and TXN-insensitive. In this study we are prospectively evaluating whether the presence of AR splice variants and the integrity of the MT-AR axis in CTCs can predict clinical response to TXN chemotherapy in CRPC. CTCs are captured by the geometrically enhanced differential immunocapture (GEDI) microfluidic device that uses PSMA-based immunocapture and size-selective cell transport for maximum specificity of CRPC-derived CTCs. We captured viable CTCs from CRPC patients (median 54 CTC/ml) and performed molecular and functional analyses. Comparison of CTCs captured by CellSearch or GEDI, using same day blood draw from 30 CRPC patients, revealed a 2-400 fold increase in CTC numbers with the GEDI. GEDI-capture of live CTCs allowed us to evaluate ex-vivo TXN sensitivity and determine drug-target-engagement (DTE) by MT bundling. CTCs isolated from docetaxel (DTX)-resistant CRPC patients did not show any evidence of DTX activity in this assay suggesting that it can be used to monitor patient's response in real time. In addition, we have miniaturized RNA sequencing (RNA-Seq) to enable molecular analyses of GEDI-captured CTCs. RNA-Seq of 50 GEDI-captured LNCap 86.2 cells spiked into 1 ml healthy donor blood, detected both the T868A AR point mutation and ARv567 splice variant. In an ongoing prospective clinical study, we are capturing CTCs from CRPC patients treated with TXNs and are evaluating the integrity of the MT-AR signaling axis, in relation to clinical response to therapy. Our CTC analyses include RNA-Seq for the detection of AR splice variants, alterations in tubulin isotypes and MT related proteins, and multiplex confocal microscopy of DTE and AR localization. These analyses are performed longitudinally and may identify biomarkers predictive of clinical TXN efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3634. doi:1538-7445.AM2012-3634

Abstract 2376: Development of a novel microfluidic device for the capture and molecular characterization of circulating tumor cells from metastatic breast cancer patients

Abstract Breast cancer (BC) is the most commonly diagnosed malignancy and the second leading cause of cancer death among women in the United States. Microtubule targeting drugs (MTDs) represent the most active chemotherapy against BC and are standard therapy for locally advanced and metastatic disease. However, metastatic BC (MBC) remains an incurable disease due to acquired drug resistance. The molecular basis of clinical resistance to MTD therapy is not well understood, primarily due to the lack of readily accessible BC tissue during therapy and at the time of relapse precluding the development of rational approaches to overcome it. Circulating Tumor Cells (CTCs) have emerged as a reliable and accessible source of tumor tissue enabling molecular analysis of CTCs during disease progression. However, the extremely low purity associated with the use of the commercially available EpCAM-based CTC capture technology, precludes accurate CTC molecular analyses. To overcome this limitation, we are developing a novel BC-specific microfluidic device that operates on the principle of geometrically enhanced differential immunocapture (GEDI), using antibody-coated microposts. This innovative design achieves capture of viable CTCs using only 1 ml of blood and minimizes leucocyte contamination. We selected HER2 as an alternative to EpCAM-based capture, as HER2 is expressed in 90% of breast tumors and overexpressed in 30%, whereas it is absent from leucocytes. In addition, our preliminary data suggest that EpCAM can be downregulated during epithelial-to-mesenchymal transition (EMT) that often precedes dissemination of cancer cells to the circulation. HER2, on the other hand, is expressed in EMT cells further indicating its suitability for CTC capture from most of the clinically relevant BC subtypes. We tested the performance of various HER-2 antibodies using immunoblot and confocal microscopy analyses of 10 different BC cell lines with varying HER2 expression levels. Surprisingly, the antibody that provided the best dynamic range of HER2 detection in these assays, exhibited poor capture efficiency and lack of discrimination between HER2-positive and negative cell lines. Next, we tested antibody performance using flow cytometry and live-cell imaging in order to preserve the native conformation of HER2 extracellular epitope. These assays identified a different HER2 antibody that showed high capture efficiency and specificity for HER2 positive cells when applied to the GEDI microfluidic device. Currently, we are using the HER2-based GEDI to isolate CTCs from MBC patients receiving MTD-based chemotherapy. In this prospective study, we are characterizing the GEDI-captured CTCs isolated from MBC patients at baseline (before chemotherapy), during the course of therapy and at disease progression in order to gain insights into mechanisms of MTD-resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2376. doi:1538-7445.AM2012-2376

Abstract 4891: Molecular and functional analysis of circulating tumor cells in castrate resistant prostate cancer using a geometrically enhanced microfluidic device based on PSMA immunocapture

Abstract Prostate cancer (PC) is the most common type of cancer in males and the second leading cause of male cancer deaths in the United States. Circulating tumor cells (CTCs) are commonly found in the blood of metastatic patients and the capture of these cells is important in the study of disease progression. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device coated with the prostate-specific membrane antigen (PSMA) which captures prostate CTCs with high purity and efficiency. This device is designed with a staggered obstacle array in order to maximize the capture efficiency (>80%) and purity (70%) of CTCs. We have used this device to capture, enumerate and molecularly characterize CTCs isolated from 1 ml of blood from patients with castrate resistant prostate cancer (CRPC). First, we compared the capture efficiency from 16 CRPC patients’ blood and subjected it to CTC enumeration by either the GEDI device or the FDA approved CellSearch technology (EpCAM-based immunocapture). Our results showed a 2-350 fold enrichment in CTC counts by the GEDI (range 15-1200 CTCs/ml, median 54). In addition, there was minimal false positive CTC detection in healthy donor blood analyzed by the GEDI. Next we sought to characterize the captured CTCs using multiplex immunofluorescence for proteins specific for PC like androgen receptor (AR), PSMA, TMPRSS2-ERG, EpCAM and other markers involved in epithelial to mesenchymal transition (EMT). We observed low or lack of EpCAM expression in the captured PSMA+/CD45- CTCs, concomitant with high vimentin expression, suggesting that our device may be preferentially capturing CTCs that have undergone EMT. Using the GEDI device, we are able to detect a known single point mutation in the AR using cDNA extracted from approximately 50 C4-2 cells spiked into 1 ml of blood from a healthy donor. Importantly, we have captured CTCs from different CRPC patients and treated them ex vivo with docetaxel to assess effective drug-target engagement by determining the extent of microtubule (MT) stabilization and apoptotic cell death. Our results showed non-uniform CTC response to drug treatment, such that a subset of PSMA+/CD45- CTCs showed MT bundling, indicating tumor heterogeneity and further suggesting that we can potentially use this device to predict patient response to taxane-based chemotherapy. In summary, the GEDI microfluidic device is a novel and specific technology to isolate and characterize PC CTCs at the molecular level. We plan to use this device in prospective clinical studies in order to understand the molecular basis of disease progression and response to taxane-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2011-4891

Capture and analysis of prostate cancer circulating tumor cells (CTCS) using geometrically enhanced differential immunocapture (GEDI).

53 Background: EpCAM-based immunocapture of prostate cancer (PC) CTCs yields relatively low purity and specificity. We developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that incorporates flow dynamics and utilizes a mAb to prostate-specific membrane antigen (PSMA) to optimize isolation and analysis of CTCs from PC patients. Methods: GEDI microfluidic silicon chips, fabricated using standard photolithography techniques, were functionalized by chemical cross-linking ending with a neutravidin terminated surface to which anti-PSMA biotinylated-mAb J591 was bound. C4-2 (PSMA+) and PC-3 (PSMA-) cells were used for chip optimization. 1 mL of peripheral blood from PC patients was flowed (1mL/hour) over functionalized chips. Captured cells were washed with PBS × 30 min, fixed with 3.7% formaldehyde, immunofluorescently stained for DAPI, androgen receptor (AR), tubulin and EpCAM and analyzed by high resolution point-scanning confocal microscopy. PSMA+, DAPI+, and CD45- cells were manually scored. RNA was extracted from unfixed captured CTCs using lysis buffer flowed thru the chip. Results: ∼80% capture efficiency was achieved from 26 PSMA positive C4-2 cells spiked into 1 mL blood flowed through the GEDI chip. RNA extracted from 50 C4-2 cells in 1 mL blood flowed thru the chip detected a known AR point mutation by RT-PCR and Sanger sequencing. Immunofluorescence staining of PSMA+ cells captured on the chip detected changes in AR subcellular localization and microtubule structure following treatment with DHT or paclitaxel, respectively. 10 patients with metastatic PC were analyzed by CellSearch (range 0-201 cells/7.5 mL) and GEDI chip (range 35->1200 cells/mL) yielding a 7->350 fold enrichment using GEDI. Captured PC cells isolated from PC patient incubated in 50 nm paclitaxel (ex vivo) overnight demonstrated microtubule bundling, indicative of drug-target engagement. Conclusions: PSMA based GEDI microfluidic CTC capture is highly specific and sensitive in capturing PSMA positive PC CTCs; and allows detailed CTC analysis including protein expression and subcellular localization, mutational analysis and drug sensitivity assessment. [Table: see text]

Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody

Geometrically enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.

Determination of serum neuron-specific enolase by differential immunocapture

A new method for the determination of serum neuron-specific enolase is presented. It consists of two steps: first, an immunocapture of γ-subunit containing isoenzymes by absorption on immobilized anti-γ antibodies; second, bioluminescence assay of enolase activities in untreated control samples and in the supernates of antibody treated samples. Total and αα activities are obtained, from which the neuron-specific enolase activity (αγ + γγ) can then be calculated by difference. As compared to the procedures currently in use, the immunocapture method is very rapid (30 min) and is more suitable for small series of determinations as needed in clinical chemistry applications. Reference interval values for serum found by this method agree with published data. When tested with samples from patients suffering from neuroblastoma or small cell lung cancer, it confirms the specific elevations in neuron-specific enolase activity previously described for these cancers, using other analytical approaches.