Experimental study on low-detection limit for immunomagnetic reduction assays by manipulating reagent entities.

The low limit of detection (LLD) plays an important role in biomolecular assays, especially for early-stage assays. Biomolecular detections usually involve the use of two main elements: a reagent and an analyzer, which both greatly contribute to the LLD. In this work, the relationships among the LLD and reagent-related factors are investigated. The to-be-detected biomolecule is c-reactive protein (CRP) as an example. The assay method is immunomagnetic reduction (IMR). The components of reagent are Fe(3)O(4) magnetic nanoparticles bio-functionalized with antibodies against CRP, dispersed in pH-7.4 phosphate buffered saline solution. Several key factors of the reagent, such as particle concentration, volume ratio of reagent to sample, and particle size, are manipulated to optimize the LLD of detecting CRP.

New assay for old markers-plasma beta amyloid of mild cognitive impairment and Alzheimer's disease.

Although there is a consensus on the reduced levels of Aß1-42 in the CSF of patients with AD, studies of plasma Aß levels were inconsistent and have limited clinical value. We developed an immunomagnetic reduction assay (IMR) to determine the plasma levels of Aß. We surveyed patients with varying AD severity (CDR = 0.5, n=16; CDR ≥ 1, n=18) and controls (n=26). Significant group differences were apparent in the levels of Aß1-42 (F = 5.54, p = 0.002) and the Aß1-42/Aß1-40 ratio (F = 24.198, p < 0.001). Post-hoc analyses showed significant differences in the Aß1-42 levels of controls and AD patients (p = 0.001) and in the Aß1-42/Aß1-40 ratio of control, MCI and AD subjects (all p ≤ 0.001). Regression analysis of Aß1-42/Aß1-40 ratios on dementia severity showed an adjusted R2 of 0.553 (p = 0.001). We identified a cut-off of 16.1 pg/ml for Aß1-42 to differentiate control subjects from patients (both AD and MCI) with 85.3% sensitivity and 88.5% specificity. We also obtained a cut-off value of 0.303 for Aß1-42/Aß1-40 ratios with 85.3% sensitivity and 96.2% specificity. APOE 4 carriers had significantly higher Aß1-42/Aß1-40 ratios than the non-carriers (F = 4.839, p = 0.015). An independent group of case-control subjects validated both cut-off values for Aß1-42/Aß1-40 (100% sensitivity and 83.3% specificity) and for Aß1-42 (100% sensitivity and 75.3% specificity). In a subgroup of longitudinal follow- up study, we found that the plasma Aß was relatively stable with an interval of approximately 3 months. In conclusion, we found that the plasma Aß1-42 is a useful biomarker for AD. The Aß1-42/Aß1-40 ratio improves the diagnostic power of the plasma Aß biomarkers. The iron nanoparticles and IMR provides a novel method to measure plasma Aß and could serve as an important clinical tool for the diagnosis of neurodegenerative diseases.

Immunomagnetic reduction assay for nervous necrosis virus extracted from groupers.

Nervous necrosis virus (NNV) is the cause of viral nervous disease, which is a serious constraint on production for grouper aquaculture. Real-time PCR is commonly used to detect and quantify NNV, has the disadvantages of being expensive and technically demanding. In this study, an immunomagnetic reduction (IMR) assay was developed as a rapid and cost-effective alternative to real-time PCR. This method used magnetic nanoparticles conjugated with antibodies specific for viral surface antigens to detect NNV in grouper tissue samples. The association of NNV with the antibody-conjugated magnetic particles resulted in a reduction in magnetic signal, which was strongly correlated with the concentration of NNV, as determined by real-time PCR. Grouper larvae were prepared for testing using a viral extraction buffer which provided a rapid, 15-min method of extracting viral antigens and had an extraction efficiency of higher than 80%. In addition, this study proposes using magnetic nanoparticles as labeling markers and as an assaying reagent for NNV. The magnetic nanoparticles are functionalized with antibodies against the viral surface of NNV and are able to associate specifically with NNV. The reduction of the magnetic signals comes from the association between magnetic particles and NNV, and relates to the concentration of NNV. The results show that the detected concentrations of NNV are highly correlated to those detected by real-time PCR.

Non-invasive and high-sensitivity scanning detection of magnetic nanoparticles in animals using high-Tc scanning superconducting-quantum-interference-device biosusceptometry.

Although magnetic nanoparticles (MNPs) have been widely applied to animals in biomedicine, MNPs within animals should be examined in real time, in vivo, and without bio-damaged possibility to evaluate whether the bio-function of MNPs is valid or to further controls the biomedicinal process because of accompanying complex problems such as MNPs distribution and MNPs biodegradation. The non-invasive and high-sensitivity scanning detection of MNPs in animals using ac susceptometry based on a high-T(c) superconducting quantum interference device (SQUID) is presented. The non-invasive results and biopsy results show good agreement, and two gold-standard biomedicine methods, Prussian blue stain and inductively coupled plasma, prove the magnetic results. This confirms that the future clinical diagnosis of bio-functional MNPs could be operated by using scanning SQUID biosusceptometry as conveniently as an ultrasonic probe.

Exploration of the relationship between the tumor burden and the concentration of vascular endothelial growth factor in liver-cancer-bearing animals using immunomagnetic reduction assay.

To achieve early-stage diagnosis, a high-sensitivity assay method is needed. As a biomarker, vascular endothelial growth factor (VEGF) has played a growing role in diagnosing and treating hepatocellular carcinoma (HCC). In this work, an immunomagnetic reduction (IMR) through bio-functionalized magnetic nanoparticles and a high-temperature superconducting-quantum-interference-device magnetometer were utilized for quantitative detection of low-concentration VEGF in serum from rats with HCC. The precision and accuracy of IMR on VEGF were characterized. Further, the results of assaying VEGF in the serum of rats were compared with those of using enzyme-linked immunosorbant assay (ELISA). It was found the correlations between the detected VEGF concentration in the rat serum and tumor burdens were 0.99 and 0.90 for IMR and ELISA, respectively, within the range from 2 pg/ml to 8000 pg/ml of VEGF concentration.

In vivo and real-time measurement of magnetic nanoparticles distribution in animals by scanning SQUID biosusceptometry for biomedicine study.

Magnetic nanoparticles have been widely applied to biomagnetism, such as drug deliver, magnetic labeling, and contrast agent for in vivo image, etc. To localize the distribution of these magnetic particles in living organism is the first important issue to confirm the effects of magnetic nanoparticles and also evaluate the possible untoward effects. In this study, a scanning high T(c) rf-SQUID superconducting quantum interference devices (SQUIDs) biosusceptometry, composed of static SQUID unit and scanning coil sets, is developed for biomedicine study with the advantages of easy operation and unshielded environment. The characteristics tests showed that the system had the low noise of 8 pT/Hz at 400 Hz and the high sensitivity with the minimum detectable magnetization around 4.5 × 10(-3) EMU at distance of 13 mm. A magnetic nanoparticle detection test, performed by ex vivo scanning of the magnetic fluids filled capillary under swine skin for simulation of blood vessels in living bodies, confirmed that the system is feasible for dynamic tracking of magnetic nanoparticles. Based on this result, we performed further studies in rats to clarify the dynamic distribution of magnetic nanoparticle in living organism for the pharmacokinetics analysis like drug delivers, and propose the possible physiological metabolism of intravenous magnetic nanoparticles.

Magnetically enhanced high-specificity virus detection using bio-activated magnetic nanoparticles with antibodies as labeling markers.

This study describes magnetically driven suppression of cross-reactions among molecules. First, the magnetic nanoparticles are coated with bio-probes and dispersed in liquid. The bio-probes can then bind with homologous or heterologous bio-targets. When alternating-current (ac) magnetic fields are applied, magnetic nanoparticles rotate driven by ac magnetic fields. Thus, the bio-targets bound on the surface of magnetic nanoparticles experience a centrifugal force. The centrifugal force can be manipulated by adjusting the angular frequency of the rotating magnetic nanoparticles. The angular frequency is determined by the applied ac magnetic field frequency. Since the binding force for good binding is much higher than that of poor binding, frequency manipulation is needed for the centrifugal force to be higher than the poor-binding force but lower than the good-binding force. Therefore, poor binding which contributes to cross reactions between molecules can be suppressed efficiently by control of the ac magnetic field frequency.

Ultra-highly sensitive and wash-free bio-detection of H5N1 virus by immunomagnetic reduction assays.

A platform for assaying avian influenza H5N1 viruses that involves measuring the ac immunomagnetic reduction of a magnetic reagent mixed with a detected sample is developed. The magnetic reagent contained magnetic nanoparticles coated with antibodies. To achieve an ultra-high sensitivity assay, a system utilizing a high-transition-temperature superconducting quantum interference device was used to sense the immunomagnetic reduction of the reagents. The results confirmed the ultra-high sensitivity of the immunomagnetic reduction assay on H5N1.

Wash-free, antibody-assisted magnetoreduction assays of orchid viruses.

This study demonstrates the feasibility of wash-free magnetoreduction assays (MRA) of orchid viruses. A magnetic reagent, consisting of magnetic beads coated with antibodies and dispersed in water, was synthesized. By using a mixed-frequency alternative-current (ac) magnetosusceptometer, differences in the magnetic susceptibilities of the magnetic reagent before and after the addition of orchid solutions were measured. The results show significant advantages for MRA of orchid viruses.

Designing optical-fiber modulators by using magnetic fluids.

To reduce interface loss between optical fibers and devices in telecommunication systems, the development of an optical-fiber-based device that can be fused directly with fibers is important. A novel optical modulator consisting of a bare fiber core surrounded by magnetic fluids instead of by a SiO2 cladding layer is proposed. Applying a magnetic field raises the refractive index of the magnetic fluid. Thus we can control the occurrence of total reflection at the interface between the fiber core and the magnetic fluid when light propagates along the fiber. As a result, the intensity of the outgoing light is modulated by variation in field strength. Details of the design, fabrication, and working properties of such a modulator are presented.

Bone morphogenetic protein 9: a potent modulator of cartilage development in vitro.

Few publications describe the activity of bone morphogenetic protein-9 (BMP-9), but the consensus of these largely in vivo studies is that while BMP-9 can induce ectopic bone formation at relatively large concentrations, it is primarily active in non-skeletal locations--including the liver, nervous system and marrow. To study the effects of BMP-9 on chondrogenesis in a well-defined environment, calf articular chondrocytes were seeded onto biodegradable PGA scaffolds. The resulting cell-polymer constructs were cultured in either control medium or medium supplemented with 1, 10, 50 or 100 ng/ml of BMP-9. After 4 weeks of in vitro culture, all concentrations of BMP-9 increased the total mass of the constructs, and the amounts of collagen, glycosaminoglycans (GAG) and cells per construct. On a mass percentage basis, BMP-9 tended to increase GAG, to decrease the relative amount of collagen and had little effect on the relative amount of cells. BMP-9 elicited qualitatively similar responses as BMP-2, -12 and -13. However, in contrast to BMP-12 and -13, BMP-9 (at concentrations > or = 10 ng/ml) induced hypertrophic chondrocyte formation and was the only BMP tested to induce mineralization. Taken together, these data suggest that BMP-9 is a potent modulator of cartilage development in vitro.