Informative prior distributions for ELISA analyses.

Immunoassays are capable of measuring very small concentrations of substances in solutions and have an immense range of application. Enzyme-linked immunosorbent assay (ELISA) tests in particular can detect the presence of an infection, of drugs, or hormones (as in the home pregnancy test). Inference of an unknown concentration via ELISA usually involves a non-linear heteroscedastic regression and subsequent prediction, which can be carried out in a Bayesian framework. For such a Bayesian inference, we are developing informative prior distributions based on extensive historical ELISA tests as well as theoretical considerations. One consideration regards the quality of the immunoassay leading to two practical requirements for the applicability of the priors. Simulations show that the additional prior information can lead to inferences which are robust to reasonable perturbations of the model and changes in the design of the data. On real data, the applicability is demonstrated across different laboratories, for different analytes and laboratory equipment as well as for previous and current ELISAs with sigmoid regression function. Consistency checks on real data (similar to cross-validation) underpin the adequacy of the suggested priors. Altogether, the new priors may improve concentration estimation for ELISAs that fulfill certain design conditions, by extending the range of the analyses, decreasing the uncertainty, or giving more robust estimates. Future use of these priors is straightforward because explicit, closed-form expressions are provided. This work encourages development and application of informative, yet general, prior distributions for other types of immunoassays.

In vivo therapy monitoring of experimental rheumatoid arthritis in rats using near-infrared fluorescence imaging.

An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund's incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.

Optical imaging of breast tumors and of gastrointestinal cancer by laser-induced fluorescence.

Optical imaging offers a high potential for noninvasive detection of cancer in humans. Recent advances in instrumentation for diffuse optical imaging have led to new capabilities for the detection of cancer in highly scattering tissue such as the female breast. We review recent developments in the detection of breast cancer in humans by fluorescent contrast agents. So far, the unspecific contrast agents indocyanine green (ICG) and omocyanine have been applied, whereas molecular probes for direct targeted imaging of this disease are still in preclinical research. We discuss recent improvements in the differentiation of malignant and benign lesions with ICG based on its enhanced extravasation in breast cancer. Whereas fluorescence imaging in thick tissue layers is hampered by strong light scattering, tissue surfaces can be investigated with high spatial resolution. As an example for superficial tumors, lesions of the gastrointestinal tract (GI) are discussed. In these investigations, protoporphyrin IX is used as a tumor-specific (due to its strong enhancement in tumor cells) target for spectroscopic identification and imaging. We present a time-gated method for fluorescence imaging and spectroscopy with strong suppression of tissue autofluorescence and show results on patients with Barrett's esophagus and with colitis ulcerosa.

Near-infrared fluorescence imaging of experimentally collagen-induced arthritis in rats using the nonspecific dye tetrasulfocyanine in comparison with gadolinium-based contrast-enhanced magnetic resonance imaging, histology, and clinical score.

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p<0.005) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, INF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81%) than for MRI (100%/91%). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.

State-of-the art comparability of corrected emission spectra. 1. Spectral correction with physical transfer standards and spectral fluorescence standards by expert laboratories.

The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods. The results, obtained for typical spectrofluorometric (0°/90° transmitting) and colorimetric (45°/0° front-face) measurement geometries, demonstrated a comparability of corrected emission spectra within a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based spectral correction when measurements and calibrations were performed under identical conditions. Moreover, the emission spectra of RMs F001 to F005, certified by BAM, Federal Institute for Materials Research and Testing, were confirmed. These RMs were subsequently used for the assessment of the comparability of RM-based corrected emission spectra of field laboratories using common commercial spectrofluorometers and routine measurement conditions in part 2 of this series (subsequent paper in this issue).

State-of-the art comparability of corrected emission spectra. 2. Field laboratory assessment of calibration performance using spectral fluorescence standards.

In the second part of this two-part series on the state-of-the-art comparability of corrected emission spectra, we have extended this assessment to the broader community of fluorescence spectroscopists by involving 12 field laboratories that were randomly selected on the basis of their fluorescence measuring equipment. These laboratories performed a reference material (RM)-based fluorometer calibration with commercially available spectral fluorescence standards following a standard operating procedure that involved routine measurement conditions and the data evaluation software LINKCORR developed and provided by the Federal Institute for Materials Research and Testing (BAM). This instrument-specific emission correction curve was subsequently used for the determination of the corrected emission spectra of three test dyes, X, QS, and Y, revealing an average accuracy of 6.8% for the corrected emission spectra. This compares well with the relative standard uncertainties of 4.2% for physical standard-based spectral corrections demonstrated in the first part of this study (previous paper in this issue) involving an international group of four expert laboratories. The excellent comparability of the measurements of the field laboratories also demonstrates the effectiveness of RM-based correction procedures.

In vivo tumor imaging using a novel RNAi-based detection mechanism.

A new concept of tumor imaging is introduced using a siRNA-based probe that is capable of amplifying a specific endogenous fluorescence emission in cancerous tissue. In previous studies, we demonstrated a significant downregulation of Ferrochelatase (FECH) mRNA-expression in colorectal carcinomas leading to the accumulation of protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis. In this article, we report on first in vivo experiments in xenografted nude mice using folate-coupled liposomes or dendritic polyglycerolamine nanoparticles carrying ferrochelatase-siRNA to enhance PpIX-derived fluorescence in the tumor tissue. Tiny tumor foci could be monitored by the emission of PpIX fluorescence in vivo. Due to the omnipresence of the heme synthesis pathway, targeted application of ferrochelatase-siRNA may provide a general means for molecular imaging. FROM THE CLINICAL EDITOR: A new concept of tumor imaging is presented in this paper using a siRNA-based probe detecting protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis previously demonstrated to accumulate in cancer tissue.

Cancer prevention in ulcerative colitis: long-term outcome following fluorescence-guided colonoscopy.

BACKGROUND: Patients with long-standing ulcerative colitis require repeated endoscopies for early detection of neoplasias, which, however, are frequently missed by standard colonoscopy. Fluorescence-guided colonoscopy is known to improve the detection rate but the long-term effects of fluorescence-guided colonoscopy are unknown. METHODS: Colitis patients with negative findings at index fluorescence-guided colonoscopy entered a prospective long-term study with conventional colonoscopies at 2-year intervals. Risk and time to progression were evaluated. The positive predictive value was assessed in patients with neoplasias at index fluorescence-guided colonoscopy who underwent immediate total colectomy. RESULTS: Thirty-one patients with negative fluorescence-guided colonoscopy were surveyed for a mean of 7.8 ± 0.9 years. Neoplasia was observed in only two of them (6%) after 7 and 8 years of follow-up, respectively. Neoplasia at index fluorescence-guided colonoscopy was observed in 10 patients. In all of them, multiple flat low-grade intraepithelial neoplasia was diagnosed. At immediate colectomy performed in eight of them, the diagnosis of flat low-grade intraepithelial neoplasia was confirmed, corresponding to a positive predictive value of 100%. However, synchronous more advanced neoplasia was detected in three of the eight patients (38%). All patients, those with and those without neoplasia, were alive at the end of the study. CONCLUSIONS: Fluorescence-guided colonoscopy misses, in contrast to standard colonoscopy, few, if any, patients with neoplasia. Most neoplasia-negative patients remain negative during prolonged follow-up. However, when low-grade dysplasia is diagnosed by fluorescence-guided colonoscopy, colectomy is recommended because more than a third of the patients harbor synchronous, more advanced neoplasia.

Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation.

We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ~70 sulfate groups) targets factors of inflammation (IC(50) of 3-6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1-3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

Detection of rheumatoid arthritis by evaluation of normalized variances of fluorescence time correlation functions.

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

Bayesian analysis of an international ELISA comparability study.

BACKGROUND: Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the sources of uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. METHODS: In contrast to the original publication for this international comparability study, we reanalyse the data using Bayesian inference. This provides a statistically coherent approach to estimate ELISA concentrations and their associated uncertainties. RESULTS: The Bayesian uncertainties of individual ELISAs and laboratory estimates are considerably larger than previously reported uncertainties. The average concentrations estimated here differ from the ones estimated by each study participant. In general, this leads to different conclusions about the study. In particular, the inter- and intra-laboratory consistency is increased, and repeatability problems occur for fewer laboratories. CONCLUSIONS: Decisions which are based on plausible ranges of measurements (such as credible intervals), are generally superior to those solely based on point estimates (such as the mean). Reliable uncertainties are thus vital, and not only in metrology. In this paper, a general method is developed to derive concentration estimates and valid uncertainties for ELISAs. Guidance on applying this Bayesian method is provided and the importance of reliable uncertainties associated with ELISAs is underlined. The applicability and virtues of the presented method are demonstrated in the context of an international comparability study.

Cyanine dyes as contrast agents for near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluorescence imaging.

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1(')-bis-(4-sulfobutyl) indotricarbocyanine-5,5(')-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

Detection of rheumatoid arthritis using non-specific contrast enhanced fluorescence imaging.

RATIONALE AND OBJECTIVES: The aim of this study was to develop a new tool for the early detection of inflammatory joint diseases using fluorescence imaging in the near-infrared (NIR) spectral range following the intravenous administration of an unspecific contrast agent. MATERIALS AND METHODS: A laser-supported system for fluorescence imaging of finger joints was designed and constructed. Five patients and a corresponding number of volunteers were examined using 0.1 mg/kg by weight of indocyanine green as an unspecific contrast agent. Fluorescence images were acquired continuously over a period of 15 minutes. As a control, 1 day before optical imaging, all patients and volunteers underwent contrast-enhanced magnetic resonance imaging (MRI) at 0.2 T. On the basis of MRI findings, all examined joints were divided into four groups: no change and mild, moderate, and severe synovitis. The emitted fluorescence photons were quantified in different regions of interest covering the finger joints and finger tips. The normalized fluorescence intensity of contrast agents was compared with MRI findings as a proven standard. RESULTS: NIR dyes of the cyanine class are enriched in inflammatory joints and show a different kinetic behavior compared to normal joints after bolus injection. These findings demonstrate clearly the capability of contrast-enhanced fluorescence imaging to detect early changes caused by rheumatoid arthritis in finger joints. The NIR results were correlated with MRI findings (r = 0.84). CONCLUSION: Contrast-enhanced fluorescence imaging provides adequate information for the evaluation of inflammatory involvement of finger joints comparable to low-field MRI.

Time-gated fluorescence spectroscopy improves endoscopic detection of low-grade dysplasia in ulcerative colitis.

BACKGROUND: Dysplasia in ulcerative colitis is frequently missed with 4-quadrant biopsies. An experimental setup recording delayed fluorescence spectra simultaneously with white light endoscopy was recently developed. OBJECTIVE: We compared detection of invisible flat intraepithelial neoplasia with protoporphyrin IX fluorescence and standard 4-quadrant biopsies. DESIGN: Prospective, crossover design without randomization of the order of procedures. SETTING: Gastroenterology Department, Humboldt University, Charité, Berlin, Germany. PATIENTS: Forty-two patients with extensive ulcerative colitis of more than 10 years' duration were included. INTERVENTIONS: Colonoscopy with 4-quadrant biopsies and targeted biopsies of macroscopic lesions and time-gated fluorescence-guided colonoscopy were performed 2 weeks apart by 2 blinded endoscopists. Three independent pathologists examined the biopsy specimens. MAIN OUTCOME MEASUREMENTS: The primary outcome criterion was detection rate of invisible flat intraepithelial neoplasia. RESULTS: Invisible flat intraepithelial neoplasia was detected in 3 (7%) patients by white light 4-quadrant biopsies and in 10 (24%) patients by fluorescence-guided endoscopy (P = .02). The sensitivity and specificity for differentiating patients with and without dysplasia were 100% and 81%, respectively. Dysplastic and nondysplastic mucosa could be discriminated with a sensitivity and specificity of 73% and 81%, respectively. LIMITATIONS: The trial was not randomized. CONCLUSION: The detection rate of intraepithelial neoplasia in patients with ulcerative colitis can be improved by fluorescence-guided colonoscopy.

Extra domain B fibronectin as a target for near-infrared fluorescence imaging of rheumatoid arthritis affected joints in vivo.

Abstract We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fibronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fluorescence imaging. The resulting AP39-TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fluorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

BACKGROUND: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. METHODS: The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories. RESULTS: Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty. CONCLUSIONS: Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.

Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer.

Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism. Previously, we demonstrated accumulation of the heme precursor protoporphyrin-IX (PpIX) in gastrointestinal tumor tissues. To elucidate the mechanisms of PpIX accumulation by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we studied expression of the relevant enzymes of the heme synthetic pathway. Here, we describe a significant down-regulation of ferrochelatase (FECH) mRNA expression in gastric, colonic, and rectal carcinomas. Accordingly, in an in vitro model of several carcinoma cell lines, ferrochelatase down-regulation and loss of enzymatic activity corresponded with an enhanced PpIX-dependent fluorescence. Direct detection of PpIX in minute amounts was achieved by a specifically developed pulsed solid-state laser dual delay fluorimetry setup. Silencing of FECH using small interfering RNA (siRNA) technology led to a maximum 50-fold increased PpIX accumulation, imageable by a specifically adapted two-photon microscopy unit. Our results show that in malignant tissue a transcriptional down-regulation of FECH occurs, which causes endogenous PpIX accumulation. Furthermore, accumulation of intracellular PpIX because of FECH siRNA silencing provides a small-molecule-based approach to molecular imaging and molecular therapy.

Real-time sonoelastography performed in addition to B-mode ultrasound and mammography: improved differentiation of breast lesions?

RATIONALE AND OBJECTIVES: The goal of the present study was to compare the sensitivity and specificity of elastography with that of B-mode ultrasound (US) and mammography. MATERIALS AND METHODS: A total of 300 patients with histologically confirmed breast lesions (168 benign, 132 malignant) were included. Evaluation was by means of the three-dimensional finite-element method. The data are color-coded and superimposed on the B-mode US scan. The images were evaluated by two independent readers. The results were compared with mammography, histology, and the data obtained by previous US investigations. Sensitivities and specificities were calculated. RESULTS: Sensitivity and specificity in the differentiation of benign and malignant lesions were 87% and 85%, respectively, for mammography and 94% and 83% for B-mode US. The two examiners were in very good agreement in their evaluation of the elastograms (kappa: 0.86). Elastography had a sensitivity of 82% and a specificity of 87%. Elastography was superior to B-mode US in diagnosing Breast Imaging Reporting and Data System (BI-RADS) 3 lesions (92% vs. 82% specificity) and in lipomatous involution (80% vs. 69% specificity). CONCLUSION: Elastography in breast lesions showed a higher specificity and a lower sensitivity in comparison with B-mode sonography. Elastography may be beneficial in BI-RADS 3 lesions and in lipomatous involution.

Assessment of unspecific near-infrared dyes in laser-induced fluorescence imaging of experimental arthritis.

OBJECTIVE: The aim of the study is to evaluate in vivo fluorescence imaging of experimental inflammatory joint disease by applying two different near-infrared (NIR) dyes in a model of Borrelia-induced Lyme arthritis. MATERIALS AND METHODS: Forty mice, 20 with Lyme arthritis and 20 controls, were examined. Two nonspecific NIR carbocyanine dyes, indocyanine green (ICG) and a hydrophilic carbocyanine derivative (1,1'-bis-[4-sulfobutyl] indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium salt [SIDAG]), were administered intravenously at two doses. Fluorescence images were acquired before and during 120 seconds after injection of cyanine dyes. For both dyes, the area under the curve (AUC) was determined for the interval between 40 and 80 seconds after injection. In addition, the slope of the signal decrease was compared among animal groups. Results were compared with histological findings. RESULTS: The general temporal fluorescence intensity course for ICG was characterized by a rapid increase, with a peak at 40-50 seconds followed by a decrease; conversely for SIDAG, by a slow increase. AUC analysis for both dyes showed that the fluorescence signal differed significantly between controls and arthritic animals (P < .05). Within these groups, there were significant differences between the two doses investigated. ICG differed significantly between control and arthritic animals in the slope of the signal decrease for both doses investigated (P < .05). Histological examination showed early stages of inflammation in arthritic animals. CONCLUSIONS: NIR fluorescence imaging based on the pharmacokinetic behavior of ICG or SIDAG is a promising approach to detect inflammatory joint changes of experimental arthritis. Moreover, SIDAG is suited to differentiate inflammatory and noninflammatory joints 24 hours after dye application.

Optical molecular imaging of lymph nodes using a targeted vascular contrast agent.

We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.