Enzyme-linked Immunosorbent Assay Platform Research Articles

An electronic enzyme-linked immunosorbent assay platform for protein analysis based on magnetic beads and AlGaN/GaN high electron mobility transistors.

AlGaN/GaN high electron mobility transistor (HEMT) biosensors have attracted attention due to their high sensitivity, stability, and fast response characteristics. Some related studies have been explored but a Debye screening problem exists in physiological solutions hindering the detection of bio-macromolecules. Herein, a novel fast analytical platform for electronic enzyme-linked immunosorbent assay (e-ELISA) is proposed based on AlGaN/GaN HEMT with magnetic beads (MBs); MB-based e-ELISA decouples the modified area from the sensing surface to simplify the assay. Combining the advantages of e-ELISA and MBs, the resulting analytical platform presents a sensing capability beyond the Debye-screening limit and a novel ability to be reused. This platform offers a fast response toward prostate specific antigen (PSA) and the lowest concentration of detection is 1 fg mL-1. Compared with conventional AlGaN/GaN HEMT biosensors, it shows higher sensitivity (3.73 μA dec-1) in a linear range (1 fg mL-1 to 1 pg mL-1), which is within the constraints of emergency care applications. The platform's high sensitivity and fast repeatability endow it with great potential for early and rapid diagnosis.

Enzyme-induced in situ generation of polymer carbon dots for fluorescence immunoassay

Current fluorescence immunoassays generally employ synthetic fluorophore-labeled antibody and complicated fluorescence regulation process. We are dedicated to developing a conceptual fluorescent enzyme-linked immunosorbent assay (ELISA) based on enzyme-induced in situ fluorogenic reaction to resolve such issues. For the construction of novel fluorescent ELISA, discovering a moderate but rapid fluorogenic reaction and choosing a preferable substrate are both necessary and significant. In this study, we have demonstrated that 4-aminophenol (AP) can react with ethylenediamine at 37 °C for 2 h to produce strong green fluorescent polymer carbon dots (PCDs) for the first time. Inspired by this simple reaction and alkaline phosphatase (ALP)-triggered specific hydrolysis of 4-aminophenyl phosphate (APP) into AP, we have developed a strategy for detecting ALP activity via in situ formation of fluorescent PCDs. Furthermore, employing conventional ELISA platform and commercial antibodies to cardiac troponin I (cTnI), we construct a simple and convenient ALP-based fluorescent ELISA for quantitatively evaluating the cTnI level in human serum. Such original ELISA via in situ generation of fluorescent nanomaterials from scratch has great potential for the early diagnosis of disease markers in the near future.

Multicolor and Ultrasensitive Enzyme-Linked Immunosorbent Assay Based on the Fluorescence Hybrid Chain Reaction for Simultaneous Detection of Pathogens

Various pathogens may coexist in one sample; however, detection methods that rely on traditional selective culture media or immune agents designed specifically for a certain target are unsuitable for multiple targets. It is important to develop a simultaneous and sensitive detection method for multiple pathogens. Here, a multicolor and ultrasensitive enzyme-linked immunosorbent assay (ELISA) platform based on the fluorescence hybridization chain reaction (HCR) was developed. In the assay, multicolor fluorescence concatemers formed as signal amplifiers and signal reporters in the presence of target pathogens. When HCR occurred, Escherichia coli O157:H7, Salmonella serotype Choleraesuis, and Listeria monocytogenes were detected simultaneously with three different fluorescences. Additionally, the limits of detection for E. coli O157:H7, Salmonella Choleraesuis, and L. monocytogenes were 3.4 × 101, 6.4 × 100, and 7.0 × 101 CFU/mL, respectively. The assay achieved ultrasensitive, specific, and simultaneous detection of three pathogens and can be applied to the detection of pathogens in milk samples. Therefore, this multicolor and ultrasensitive ELISA platform has great potential in the application of simultaneous detection of pathogens.

Fluorescence Immunoassay Based on the Alkaline Phosphatase Triggered in Situ Fluorogenic Reaction of o-Phenylenediamine and Ascorbic Acid.

Inspired by the special reducing capability of ascorbic acid (AA), ascorbic acid 2-phosphate (AA2P) has been extensively utilized as a substrate in current alkaline phosphatase (ALP) activity assays owing to the ALP-triggered transformation of AA2P into AA. However, such assays usually require AA-related complicated and laborious synthesis and/or signal generation procedures. Herein, we report an interesting in situ fluorogenic interaction between o-phenylenediamine (OPD) and AA, which inspires us to put forward a novel and simple AA2P/OPD-participated fluorescence turn-on ALP activity assay for the first time, and then the corresponding ALP-based fluorescence enzyme-linked immunosorbent assay (ELISA) has also been developed by means of the conventional ELISA platforms. According to the convenient and facile detection process with clear response mechanism, our fluorogenic reaction-based assay exhibits good sensitivity, selectivity, and excellent sensing performance, which ensures fluorescence ELISA to potentially be applied in clinical diagnosis by employing a well-studied biomarker of hepatocellular carcinoma, α-fetoprotein (AFP) as the model analyte. Such original ELISA via in situ formation of fluorophore from scratch gives a new sight to develop other potential immunoassay platforms in early clinical diagnosis by controlling the target antigens in the near future.

Comparison of Serological Biomarkers in Rheumatoid Arthritis and Their Combination to Improve Diagnostic Performance.

The diagnosis of rheumatoid arthritis (RA) is based on a combined approach that includes serological markers such as rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA). The goal of this study was to evaluate the clinical performance of several RF and ACPA immunoassays for the diagnosis of RA, as well as the diagnostic value of a combinatory approach with these markers. The study cohort included 1,655 patients from the Swiss Clinical Quality Management registry with sera from 968 patients with RA and 687 disease controls, including patients with axial spondyloarthritis (n = 450) and psoriatic arthritis (n = 237). ACPA were determined by anti-CCP2 IgG enzyme-linked immunosorbent assay (ELISA), QUANTA Flash® CCP3 IgG [chemiluminescent immunoassay (CIA)], and QUANTA Lite® CCP3 IgG ELISA. RF was determined by ELISA (QUANTA Lite® RF IgM, RF IgA, and RF IgG) and with two research use only CIAs (QUANTA Flash® RF IgM and RF IgA). All three ACPA assays showed good discrimination between RA patients and controls and good clinical performance. Overall, CCP3 performed better than CCP2. More pronounced differences were observed between the RF assays. We observed that CIA platforms for both RF IgM and RF IgA showed better performance than the ELISA platforms. Excellent and good total agreements were found between ELISA and CIA for CCP3 (total agreement 95.3%, kappa = 0.90), and between CCP2 and CCP3 ELISA (total agreement 86.6%, kappa = 0.73), respectively. RF IgM CIA and ELISA had a good qualitative agreement (86.5%, kappa = 0.73); RF IgA CIA and ELISA showed a moderate total agreement (78.5%, kappa = 0.53). When combinatory analyses were performed, the likelihood of RA increased with dual positivity and triple positivity and combining different markers resulted in higher odds ratio than the individual markers in all cases. ACPA and RF showed good clinical performance in this large Swiss cohort of RA patients and controls. Overall, the performance of CCP3 was superior to CCP2. The combination of these biomarkers in an interval model represents a potential tool for the diagnosis of RA patients.

Plasma VEGF-A (pVEGF-A) level in efficacy analysis of metastatic colorectal cancer patients (mCRC) treated with mFOLFOX6/XELOX plus bevacizumab (BV) (WJOG7612GTR).

699 Background: Plasma levels of VEGF-A short isoforms (VEGF-A110 and -A121) measured by immunological multiparametric chip technique (IMPACT) were reported to be associated with clinical benefits from bevacizumab (BV) in advanced gastric and pancreatic cancer but not in metastatic colorectal cancer (mCRC). Negative results in mCRC studies might be caused by different sample handling: citrate instead of EDTA and repetition of freeze/thaw. Methods: Blood samples were collected in EDTA before the first-line treatment with BV+mFOLFOX6 or +XELOX for mCRC. Plasma samples were analyzed at Roche Diagnostics Ltd. (Penzberg, Germany) using IMPACT-2 (Roche proprietary multiplex enzyme-linked immunosorbent assay platform). A median value of pVEGF-A was used as a cut-off point to categorize patients (pts) into the low and high pVEGF-A groups. Progression free survival (PFS) and overall survival (OS) between the low and high pVEGF-A groups were compared, using Cox proportional hazards model. We hypothesized that BV-containing treatment extend shorter PFS of pts with high pVEGF-A to that with low pVEGF-A, and estimated a threshold hazard ratio (HR) between them as below 1.15. Results: Among 102 pts enrolled between January 2014 and April 2015, 100 (53 BV+mFOLFOX6 and 47 BV+XELOX) were eligible. Median PFS was 11.4 months [95% CI, 9.5-13.0] and response rate was 64.6 % [range, 53.3-74.9]. pVEGF-A was measured in 97 pts and the median value was 36.8 pg/ml [range, 6.5- 262.2]. The hazard ratios of PFS and OS between the high and low pVEGF-A groups were 1.23 [95%CI, 0.76-1.97, p = 0.40] and 2.47 [95%CI, 1.14-5.36, p = 0.02], respectively. Conclusions: mCRC pts with high pVEGF-A showed shorter PFS than those with low pVEGF-A beyond the predefined threshold (HR 1.15) in BV-containing chemotherapy, suggesting that pVEGF-A could not be a predictive marker for BV efficacy. Clinical trial information: UMIN000012442.

Plasma ICAM-1 (pICAM-1) and plasma IL-8 (pIL-8) level as biomarker of metastatic colorectal cancer patients (mCRC) treated with mFOLFOX6/XELOX plus bevacizumab (BV) (WJOG7612GTR).

670 Background: Bevacizumab (BV)-containing chemotherapy has been established as the standard first-line treatment for metastatic colorectal cancer (mCRC). However, no biomarkers have been established to predict the clinical benefit of BV for patients (pts) with mCRC. Methods: Blood samples were collected in EDTA before the first-line treatment with BV+mFOLFOX6 or +XELOX for mCRC. Plasma samples were analyzed at Roche Diagnostics Ltd. (Penzberg, Germany) using IMPACT-2 (Roche proprietary multiplex enzyme-linked immunosorbent assay platform). Median values of pICAM-1 and pIL-8 were used as cut-off points to categorize pts into the low and high groups. Progression free survival (PFS) and overall survival (OS) were compared between two groups, using Cox proportional hazards model. Results: Among 102 pts enrolled between January 2014 and April 2015, 100 (53 BV+mFOLFOX6 and 47 BV+XELOX) were eligible. Median PFS was 11.4 months [95% CI, 9.5-13.0]. Response rate was 64.6 % [53.3-74.9]. pICAM-1 and pIL-8 were measured in 99 pts and the median values were 190.0 ng/ml [range, 58.0- 1080.1] and 311.5 pg/ml [range, 83.2-4541.4], respectively. The hazard ratios of PFS and OS between the high and low pICAM-1 groups were 2.08 [95%CI, 1.28-3.40, p = 0.003] and 2.04 [0.92-4.50, p = 0.079], respectively. The hazard ratios of PFS and OS between the high and low pIL-8 groups were 1.63 [95%CI, 1.00-2.65, p = 0.048] and 3.42[1.57-7.44, p = 0.002], respectively. Conclusions: High pICAM-1 and pIL-8 were associated with short PFS and OS of mCRC pts treated with BV-containing chemotherapy, suggesting that pICAM-1 and pIL-8 could be predictive markers for prognosis of mCRC treated with BV. Clinical trial information: UMIN000012442.

Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization.

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA-DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

Fluorescence Immunoassay System via Enzyme-Enabled in Situ Synthesis of Fluorescent Silicon Nanoparticles.

The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique, which possesses several advantages in the research and clinical applications. However, current strategies for fluorescence immunoassay usually involve the routine fluorophore-labeled antibody and/or awkward signal generation procedure that may not be available in conventional enzyme-linked immunosorbent assay (ELISA) systems. Herein, we circumvent this problem by imparting an exquisite signal generation mechanism to commercially available alkaline phosphatase (ALP)-based ELISA platform and putting forward a conceptual fluorescent ELISA system based on an original ALP-enabled in situ synthesis of fluorescent nanomaterials. After adding target antigen, the presence of ALP labeled on antibody catalyzes the transformation of the substrate ascorbic acid 2-phosphate into ascorbic acid. Then the resultant ascorbic acid (i.e., ascorbate) interacts with amine-containing silane molecules (no fluorescence) to produce intense cyan fluorescent silicon nanoparticles. For the proof-of-concept, alpha-fetoprotein and human immunoglobulin G are chosen as the model antigen targets, and our proposed immunoassay (designated as the nanoparticles generation-based fluorescent ELISA) enables the detection with either fluorescence spectroscopy or naked-eye readout under the ultraviolet lamp. The convincing recognition mechanism and assay performance ensure fluorescent ELISA to quantitatively evaluate the alpha-fetoprotein level in serologic test and potentially apply in the clinic diagnosis of hepatocellular carcinoma.

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Development of Prototype Filovirus Recombinant Antigen Immunoassays.

Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.

Development of an automated on-chip bead-based ELISA platform.

We present a lab-on-a-chip and associated instrument for heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. We have previously described the instrument, which automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip reported here utilizes a high surface area bead bed to enhance capture efficiency and increase the dynamic range of the assay as compared to a standard plate-based ELISA. Its functionality is demonstrated using human IL-10 as a model antigen, but theoretically any sandwich ELISA could be ported onto this "open source platform." We show that our automated on-chip assays have greater sensitivities than the corresponding standard manual plate-based ELISAs, and that single samples can be assayed in a fraction of the time.

Micro-a-fluidics ELISA for Rapid CD4 Cell Count at the Point-of-Care

HIV has become one of the most devastating pathogens in human history. Despite fast progress in HIV-related basic research, antiretroviral therapy (ART) remains the most effective method to save AIDS patients' lives. Unfortunately, ART cannot be universally accessed, especially in developing countries, due to the lack of effective treatment monitoring diagnostics. Here, we present an inexpensive, rapid and portable micro-a-fluidic platform, which can streamline the process of an enzyme-linked immunosorbent assay (ELISA) in a fully automated manner for CD4 cell count. The micro-a-fluidic CD4 cell count is achieved by eliminating operational fluid flow via “moving the substrate”, as opposed to “flowing liquid” in traditional ELISA or microfluidic methods. This is the first demonstration of capturing and detecting cells from unprocessed whole blood using the enzyme-linked immunosorbent assay (ELISA) in a microfluidic channel. Combined with cell phone imaging, the presented micro-a-fluidic ELISA platform holds great promise for offering rapid CD4 cell count to scale up much needed ART in resource-constrained settings. The developed system can be extended to multiple areas for ELISA-related assays.

ELISA and SPR Studies of Ricin Binding to β-Galactoside Analogs

Early detection of ricin toxin, a biothreat agent, is of critical importance because it can be produced in large quantities rapidly. Ricin binds to β-galactoside analogs; therefore, these glycans can be used to capture and detect the toxin. Here, we have compared the binding affinities (Kds) of ricin with a panel of β-galactoside analogs using two different biophysical techniques, quantitative enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). We found that β-galactosides bind to ricin better than N-acetyl β-galactosamines on all surfaces. Second, ricin prefers to bind to densely packed glycans over sparsely packed glycans. Third, when the structure and density of the glycans are kept constant and only the spacer is varied, we found that the binding affinities of glycans attached via a hydrocarbon spacer are better than glycans attached via a short oligoethylene glycol spacer, presumably because the alkyl spacer provides a better spatial arrangement. We also observed that the ELISA platform is more sensitive than the SPR platform. Taken together, in addition to the glycan structure, glycan density, presentation, and assay conditions are important factors that modulate the binding affinities. These comparative quantitative studies revealed similarities and subtle differences in the binding of the same glycans attached to different surfaces and must be taken into consideration when developing glycan-based biosensors for ricin or other glycan binding proteins.

An Automated Method for Supporting Busulfan Therapeutic Drug Monitoring

Busulfan is a chemotherapeutic agent commonly used for myeloablative conditioning regimens such as in the treatment of chronic myelogenous leukemia. Busulfan dosing is complex due to wide interpatient variability in pharmacokinetics and a narrow therapeutic range. Although busulfan dose is normalized to body weight, therapeutic drug monitoring (TDM) using area under the plasma concentration curve is recommended after the first dose. A high busulfan area under the plasma concentration curve (>1500 μM·min) is associated with an increased risk for sinusoidal obstruction syndrome, and a suboptimal area under the plasma concentration curve (<900 μM·min) is associated with an increased risk for graft rejection or disease relapse. TDM of busulfan is not widely available due to the lack of commercially available and rapid methods to determine the area under the plasma concentration curve. The purpose of this study was to evaluate the Roche cobas c 111 instrument, a photometric automated chemistry analyzer, using the Busulfan PCM assay from Saladax Biomedical Inc. The assay using this instrument was compared with an enzyme-linked immunosorbent assay (ELISA) from Saladax Biomedical Inc and the Olympus AU400e. Linearity and accuracy were evaluated between 175 and 1750 ng/mL. Imprecision was determined by analyzing 5 concentrations of standards twice a day for 20 days. Linearity for the Roche method had a slope and y-intercept of 1.050 and -5.5, respectively, and percent recovery ranged between 95% and 105%. Correlation between the Roche and ELISA platforms was analyzed by linear regression on 26 frozen patient samples. The results from the comparison of the methods based on the Roche and ELISA platforms were as follows: coefficient of determination (R2) was 0.9684, with a slope and y-intercept of 0.752 and 108.41, respectively. Correlation between the Roche and Olympus instruments was analyzed by linear regression and Bland-Altman plots. The coefficient of determination (R2) was 0.9942, with a slope and y-intercept of 1.035 and -41.3326, respectively. Availability of TDM of busulfan can be improved by the use of commercially available reagents and automated platforms.

Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed.The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.

Quantification of Low-Picomolar Concentrations of TNF-α in Serum Using the Dual-Network Microfluidic ELISA Platform

For both research and diagnostic purposes, the ability to detect low levels of proteins in a cost- and time-effective manner is essential. In this study, the cytokine TNF-alpha (tumor necrosis factor-alpha), a widely used protein indicator of inflammatory response, was chosen to demonstrate the ability of the dual-network microfluidic ELISA (enzyme-linked immunosorbent assay) platform developed by the authors to rapidly quantify low concentrations of this biomarker in serum. Through the optimization of several experimental parameters, the system was shown to meet the requirements for fundamental and applied studies, while also being relevant for challenging clinical applications such as the diagnosis of septic patients. A sensitivity of 45 pg/mL (2.6 pM) in both culture medium and serum, with inter- and intravariations of less than 15%, was attained for the quantification of human TNF-alpha to a concentration of up to 500 pg/mL. The overall time for completion of the assay in eight parallel reactions was less than 1 h.

Screening of immunophilin ligands by quantitative analysis of neurofilament expression and neurite outgrowth in cultured neurons and cells

Immunophilins are protein receptors for the immunosuppressant drugs FK506, cyclosporin A (CsA), and rapamycin. Two categories of immunophilins are the FK506-binding proteins (FKBPs), which bind to FK506, rapamycin, and CCI-779 and the cyclophilins, which bind to CsA. Reports have shown that immunophilins are expressed in the brain and spinal cord, are 10–100-fold higher in CNS tissue than immune tissue, and their expression is increased following nerve injury, suggesting that their chemical ligands may have therapeutic utility in the treatment of neurodegenerative diseases. In this study, we report the development and utility of a rapid neurofilament (NF) enzyme-linked immunosorbent assay (ELISA) to quantify neuronal survival and the Cellomics ArrayScan platform to quantify neurite outgrowth following treatment with immunophilin ligands. Cultured neurons or F-11 cells were treated with various immunophilin ligands for 72 or 96 h and their promotion of neuronal survival and neurite outgrowth were determined. The results showed that all immunophilin ligands, in a concentration-dependent manner, significantly increased neuronal survival and neurite outgrowth, when compared to control cultures. Taken together, these results demonstrate the potential utility of the neurofilament ELISA and Cellomics ArrayScan platform to efficiently quantify neurotrophic effects of immunophilin ligands on cultured neurons and cell lines.

Relationships between factor VIII:Ag and factor VIII in recombinant and plasma-derived factor VIII concentrates.

A variety of plasma-derived (pd) and recombinant (r) factor VIII (FVIII) concentrates are used to prevent and treat bleeding in severe hemophilia A patients. A significant side effect of FVIII replacement is the development of FVIII neutralizing antibodies (inhibitors) in up to 30% of patients receiving FVIII concentrates. The FVIII protein content (FVIII:Ag) per unit of FVIII:C in FVIII concentrates, and how effectively the FVIII:Ag in FVIII concentrates binds to von Willebrand factor (VWF) may provide information relevant for the survival of FVIII:C in vivo and for estimating the risk for inhibitor development. The FVIII:Ag content of nine r-FVIII and nine pd-FVIII concentrates were quantified in this study using two enzyme-linked immunosorbent assay (ELISA) platforms. The two ELISA platforms were based on the use of a monoclonal anti-(FVIII light chain)-IgG and polyclonal anti-FVIII antibodies as capture antibodies and both ELISAs were equally able to detect > or =0.005 IU of FVIII:Ag. Measured in international units, the r-FVIII concentrates contained significantly higher FVIII:Ag per unit of FVIII:C than the pd-FVIII concentrates. The VWF-binding profiles of the r-FVIII and pd-FVIII concentrates were also determined by gel filtration chromatography. Unlike the plasma-derived products, the r-FVIII concentrates invariably contained a fraction of FVIII:Ag molecules (approximately 20%) which was unable to associate with VWF. Given that VWF regulates both factor VIII proteolysis and survival of FVIII:Ag in vivo, the fraction of FVIII:Ag unable to bind to VWF may have a reduced survival and be more susceptible to proteolytic degradation in vivo. The extent to which the fractions of FVIII:Ag in concentrates able and unable to bind to VWF contribute to inhibitor development in severe FVIII-deficient patients is unknown.