A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a “microscope” based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 μm from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Neel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 ± 2) × 104 magnetic nanoparticles to register a reproducible signal.

https://www.pnas.org/content/pnas/97/26/14268.full.pdf

Ultrasensitive magnetic biosensor for homogeneous immunoassay

We are developing a biosensor system, the Bead ARray Counter (BARC), based on the capture and detection of micron-sized, paramagnetic beads on a chip containing an array of giant magnetoresistive (GMR) sensors. Here we describe the design and performance of our current chip with 64 sensor zones, compare its performance with the previous chip design, and discuss a simple analytical model of the sensor micromagnetics. With assay-ready Dynal M-280 microbeads (2.8 μm diameter), our threshold for detection is approximately 10 beads per 200 μm-diameter sensor. Single beads made of solid Ni 30 Fe 70 can easily be detected, but they must be made biocompatible. The relatively large size of our sensors helps to improve their practical sensitivity compared with other microsensor-based magnetic particle detectors.

https://www.nve.com/Downloads/Rife_Sensors_Actuators_A_revised_final.pdf

Design and Performance of GMR Sensors for the Detection of Magnetic Microbeads in Biosensors

Understanding of ferritin biology has traditionally centered on its role in iron storage and homeostasis, with low ferritin levels indicative of deficiency and high levels indicative of primary or secondary hemochromatosis. However, further work has shown that iron, redox biology and inflammation are inexorably linked. During infection, increased ferritin levels represent an important host defense mechanism that deprives bacterial growth of iron and protects immune cell function. It may also be protective, limiting the production of free radicals and mediating immunomodulation. Additionally, hyperferritinemia is a key acute-phase reactants, used by clinicians as an indication for therapeutic intervention, aimed at controlling inflammation in high-risk patients. One school of thought maintains that hyperferritinemia is an 'innocent bystander' biomarker of uncontrolled inflammation that can be used to gauge effectiveness of intervention. Other schools of thought maintain that ferritin induction could be a protective negative regulatory loop. Others maintain that ferritin is a key mediator of immune dysregulation, especially in extreme hyperferritinemia, via direct immune-suppressive and pro-inflammatory effects. There is a clear need for further investigation of the role of ferritin in uncontrolled inflammatory conditions both as a biomarker and mediator of disease because its occurrence identifies patients with high mortality risk and its resolution predicts their improved survival.

/pdf/hyperferritinemia-and-inflammation-5go9h6q61g.pdf

Hyperferritinemia and inflammation

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(09)00106-1.pdf

Biomedical applications of distally controlled magnetic nanoparticles

Immunotherapies such as checkpoint-blocking antibodies and adoptive cell transfer are emerging as treatments for a growing number of cancers. Despite clinical activity of immunotherapies across a range of cancer types, the majority of patients fail to respond to these treatments and resistance mechanisms remain incompletely defined. Responses to immunotherapy preferentially occur in tumors with a preexisting antitumor T-cell response that can most robustly be measured via expression of dendritic cell and CD8+ T cell-associated genes. The tumor subset with high expression of this signature has been described as the T cell-"inflamed" phenotype. Segregating tumors by expression of the inflamed signature may help predict immunotherapy responsiveness. Understanding mechanisms of resistance in both the T cell-inflamed and noninflamed subsets of tumors will be critical in overcoming treatment failure and expanding the proportion of patients responding to current immunotherapies. To maximize the impact of immunotherapy drug development, pretreatment stratification of targets associated with either the T cell-inflamed or noninflamed tumor microenvironment should be employed. Similarly, biomarkers predictive of responsiveness to specific immunomodulatory therapies should guide therapy selection in a growing landscape of treatment options. Combination strategies may ultimately require converting non-T cell-inflamed tumors into T cell-inflamed tumors as a means to sensitize tumors to therapies dependent on T-cell killing. Cancer Immunol Res; 6(9); 990-1000. ©2018 AACR.

/pdf/t-cell-inflamed-versus-non-t-cell-inflamed-tumors-a-596ln9f9tx.pdf

T Cell–Inflamed versus Non-T Cell–Inflamed Tumors: A Conceptual Framework for Cancer Immunotherapy Drug Development and Combination Therapy Selection

A system is developed to magnetically measure biological antigen-antibody reactions with a superconducting quantum interference device (SQUID) magnetometer. In this system, antibodies are labeled with magnetic nanoparticles of γ-Fe2O3, and the antigen-antibody reactions are measured by detecting the magnetic field from the magnetic nanoparticles. A setup of the system is described, and the sensitivity of the system is studied in terms of detectable weight of nanoparticles. Magnetic particles as small as 600 pg can be detected at present. An experiment is also conducted to measure antigen-antibody reaction with the present system. It is shown that the sensitivity of the present system is better than that of the conventional optical method. A one order of magnitude improvement of sensitivity will be realized by the sophistication of the present system.

https://iopscience.iop.org/article/10.1143/JJAP.38.L1102/pdf

Detection of Magnetic Nanoparticles with Superconducting Quantum Interference Device (SQUID) Magnetometer and Application to Immunoassays

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.

/pdf/recent-advances-in-the-generation-of-human-monoclonal-4xsfe9jx0j.pdf

Recent advances in the generation of human monoclonal antibody.

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4+ and CD8+ T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4+ and CD8+ T cells, as well as in CD19+ B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation ...

https://www.tandfonline.com/doi/pdf/10.1271/bbb.68.2279

IL-10 Augments Antibody Production in in Vitro Immunized Lymphocytes by Inducing a Th2-Type Response and B Cell Maturation

In the present study, we clarified that transforming growth factor beta (TGF-beta) induces cellular senescence in human normal diploid cells, TIG-1, and identified protein kinase Cs (PKCs) as downstream mediators of TGF-beta-induced cellular senescence. Among PKCs, we showed that PKC-delta induced cellular senescence in TIG-1 cells and was activated in replicatively and prematurely senescent TIG-1 cells. The causative role of PKC-delta in cellular senescence programs was demonstrated using a kinase negative PKC-delta and small interfering RNA against PKC-delta. Furthermore, PKC-delta was shown to function in human telomerase reverse transcriptase (hTERT) gene repression. These results indicate that PKC-delta plays a key role in cellular senescence programs, and suggest that the induction of senescence and hTERT repression are coordinately regulated by PKC-delta.

Protein Kinase C δ Plays a Key Role in Cellular Senescence Programs of Human Normal Diploid Cells

An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, Fc(epsilon)RI(alpha), using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with Fc(epsilon)RI(alpha)-expressed KU812F cells. Subsequently a combined phage antibody library 6 x 10(3) in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant Fc(epsilon)RI(alpha) and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against Fc(epsilon)RI(alpha). These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.80640

Makiko Yamashita

Papers

Detection of Magnetic Nanoparticles with Superconducting Quantum Interference Device (SQUID) Magnetometer and Application to Immunoassays

Recent advances in the generation of human monoclonal antibody.

IL-10 Augments Antibody Production in in Vitro Immunized Lymphocytes by Inducing a Th2-Type Response and B Cell Maturation

Protein Kinase C δ Plays a Key Role in Cellular Senescence Programs of Human Normal Diploid Cells

Production of Human Monoclonal Antibodies against FcεRIα by a Method Combining in Vitro Immunization with Phage Display