A Latmag Assay

Dumbbell Routines and Exercises

Dumbbell Exercises and Lifting Routines

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The common characteristic of all dumbbell assays described above is that they are intrinsically limited in sensitivity by the fact that a significant number of labeled particles is needed to reach a detectable signal. None of the assays allows the detection of the unique particle because detection systems are not sensitive or specific enough.

A new magnetic dumbbell system or LATMAG (latex and magnetic particle) assay tries to bring a solution to this challenge. The principle is as follows [51]: the analyte to be assayed (antigen, DNA, bacteria, etc.) is captured between a magnetic bead and a latex particle, these two colloids being functionalized by ligands specific to two different sites of the analyte (epitope or nucleotide sequence). Dumbbells are separated from reaction medium under a magnetic field to discard the excess of free latex, resuspended, and assayed to determine the amount of latex particles (Fig. 1).

The main originality of LATMAG is that magnetic particles are chosen so that they do not interfere during the detection. In the work of Lim [49], large Dynabeads magnetic particles (3 pm diameter) are used. The turbidimetric detection is processed on the supernatant after magnetic sedimentation, and a decrease of the optical density as a function of antigen concentration is observed, while the amount of latex particles forming a complex with magnetic beads increases. Generally, competition assays are known to be less sensitive and to display a shorter dynamic range than direct assays. The choice of small magnetic particles that cannot be detected permits one to carry a direct assay where the amount of latex involved in the dumbbell complex increases as function of the analyte concentration.

On the other hand, latex particles are chosen so that they can be individually counted under a microscope. Thus, the ultimate potential sensitivity of the assay is the detection of the unique particle, i.e., the unique molecule. Nevertheless, parameters such as capture yields between particles and analytes and nonspecific interactions between particles are likely awaited to corrupt the sensitivity.

LATMAG offers other potential interests. First, this assay is a one-step reaction wherein all relatively cheap reagents are incubated together, followed by a magnetic separation that can be easily processed without sophisticated instrumentation. The detection requires a basic microscope for counting latex when the utmost sensitivity is required. In other cases, the naked eye is sufficient to detect the presence of turbid latex particles after resuspension of the reaction medium. Second, LATMAG is a rapid test since the target capture occurs in a stirred homogeneous suspension of colloid particles, and it was shown above that capture on beads is much more favorable than that on flat support. Finally, displaying a large range of applications, LATMAG can be applied to the detection of nucleic acid targets, antigens, bacteria, cells, and, more generally, any analyte possessing at least two different specific sites that can be recognized by a ligand bound on a particle.

B. Magnetic Capture Particle

The first quality of a magnetic particle candidate for LATMAG utilization is the absence of interference during the detection of latex, thus offering two main possibilities:

The diameter should be significantly different from that of the latex particle for an easy distinction under the microscope. Small magnetic particles whose turbidity in the absence of latex is low are preferred in a view to enhance sensitivity in the case of a naked-eye detection. Furthermore, diffusion coefficient and, consequently, particle reactivity is higher for smaller particles.

The colloidal stability of the magnetic particle should remain excellent after binding of ligand, especially during magnetic separation steps. Formation of aggregates would reduce sensitivity by hiding reactive sites and lowering reaction yields. Also, the principle of a magnetic particle significantly smaller than the detection particle has not been applied so far and brings originality to the assay.

The magnetic particle should be chosen so that its tendency to adsorb nonspecif-ically on latex particle is as low as possible. Indeed, the formation of such a complex cannot be distinguished from those issued from the specific reaction. Detected even in the absence of the analyte, they are the main limitation for assay sensitivity.

Particle functionality, i.e., the density of active groups at the bead surface, is targeted to be as high as possible, so that the minimal number of particles can be added per trial to limit as far as possible nonspecific interactions between particles.

Last but not least, an accurate compromise must be found between particle size, which should be as small as possible as shown earlier, and the magnetic content of particles, which should be as high as possible for an efficient attraction of dumbbells under magnetic field. Ideally, one magnetic particle should be able to carry in a minimum of time—a few minutes—one latex particle. If several magnetic particles are needed to displace one latex bead, intrinsic sensitivity is dramatically degraded since at the final stage of detection one detected bead corresponds to several analytes captured on the surface.

TABLE 1 Some Commercially Available Magnetic Particles

Manufacturer

Particle diam. (nm)

No. reactive sites/^m2

Magnetic sedimentation speed (min)

Miltenyi [52] Immunicon [55] Seradyn [53] Dynal [54]

50 145 750 2800

Unknown 0.27 0.02 0.07

Several commercially available magnetic particles, whose main characteristics are summed up in Table 1, are evaluated. Colloidal stability of all these particles established by size measurement by light scattering is acceptable.

Miltenyi particles [52] are constituted by a ferric oxide core whose diameter is around 10 nm, surrounded by a thick layer of polysaccharide for a final diameter of 50 nm. Because of their low magnetic content, these particles cannot be separated by a common magnet. A specially designed column creating a strong magnetic gradient is available for this purpose. Although Miltenyi micro-spheres give positive results in the LATMAG process, their cumbersome and expensive separation step is an obstacle to their further exploitation.

Seradyn [53] and Dynal [54] display a very interesting magnetic separation speed and a satisfying surface functionality. But experimentally, it is observed that the dumbbell formation is limited by the particle diameter probably too large to lead to an efficient reaction rate. Finally, Immunicon [55] magnetic microspheres are chosen as favorite candidates for LATMAG.

FACING PAGE

FIG. 1 General LATMAG protocol. 1. Reagents for LATMAG. Magnetic particles coated with the first ligand are incubated with latex particles modified with the second ligand, in the presence of the target which bear receptors for both types of ligands. 2. Formation of dumbbells. Particles and targets react together to form magnetic complexes or dumbbells. 3. Separation of dumbbells from unreacted latex particles by magnetic sedimentation. In the presence of a magnet, free magnetic particles and dumbells are attracted against the test tube wall. Free latex particles are discarded with the supernatant. 4. Counting of latex particles after resuspension of magnetic dumbbells. Preparations are deposited onto a glass slide and analyzed under the microscope to estimate the number of latex particles, i.e., the number of dumbbells.

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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