Antibody Detection

Dumbbell Routines and Exercises

Dumbbell Exercises and Lifting Routines

Get Instant Access

LATMAG can be applied for the detection of antibodies (Fig. 3) whose presence is researched, among numerous examples, in viral infections (anti-P24 antibodies) or for detecting pregnancy (anti-HCG antibodies). The strategy is to capture biotinylated IgG [56] between magnetic streptavidin particles and latex micro-spheres modified to exhibit affinity toward the targets. In Fig. 4 are reported images realized from microscope glass slides onto which are deposited preparations for different target IgG concentration. Only latex particles can be detected in these conditions because magnetic particles are too small to be optically resolved. An increase of the latex particle number per unit area as a function of antibody concentration is observed, proving that dumbbell complexes, bridged by IgG, are formed between latex and magnetic particles and that they can be separated from the medium by an appropriate magnetic field.

Dumbbells can also be studied more precisely by transmission electron microscopy (TEM) (Fig. 5). The commercial magnetic particles displays a highly heterogeneous structure, which makes them look like "corn flakes" [57]. On the other hand, size difference between magnetic and latex particle is very impres-

FIG. 3 LATMAG protocol for the detection of antibodies. Goat anti-mouse IgG latex particles are incubated with streptavidin magnetic particles and biotinylated mouse antibodies, to form dumbbells those number is a direct function of the antibody concentration.

sive, and it is difficult to imagine that such a small particle can carry the latex sphere load. Actually, one question remains in suspense: is it possible to magnetically attract one latex particle when only one magnetic bead is bound to its surface? TEM does not bring the answer because magnetic spheres hidden below latex are not imaged. If the answer is negative, the assay sensitivity is intrinsically limited by the fact that more than one target molecule should bind

FIG. 4 Images of microscopic analysis of dumbbells for increasing concentration of anti-TSH targeted antibodies (from 0 to 2.10 molecules/mL). The objective is X 40. Latex particles (1.1 |lm) are easily counted while magnetic particles (0.15 |lm) are not optically resolved.

Magnetic partiĆ³le adsorbed on the latex sphere

Magnetic partiĆ³le adsorbed on the latex sphere

magnetic particles

FIG. 5 TEM picture of a close-up view of the surface of a latex particle (1.1 |lm diameter) bound to a magnetic particle. Isolated magnetic particles that did not participate to the reaction are also present around the dumbbell. Their shape is that of irregular and polydisperse clusters whose mean diameter is about 150 nm.

a latex for having a positive signal, i.e., it is not possible to detect a unique molecule.

Even in the absence of targeted antibodies, a few latex spheres are found in the sample that can be free latex or magnetic complexes formed by the nonspecific adsorption of magnetic particle onto latex spheres. At high antibody concentration, chains of particles are observed with optical as well as with electron microscopy, due to the reaction of several antibodies on the same latex particle, each antibody being able to bind one magnetic particle. Thus, the separation of such complexes is much quicker thanks to their consequent size and the magnetic content (Fig. 6).

Dumbbells can be quantified per unit area (Fig. 7) after image treatment. While it is not possible to count all latex particles present on the microscope slide, surfaces analyzed are sufficiently large to be statistically representative of the sample. The dynamic range of the assay spread on 6 log10 units; the sensitivity threshold is estimated at 2 x 106 molecules/mL, equivalent to a molar concentration of 3 x 10-15 mol/L or a weight concentration of 5 x 10-10 g/L, which is sensitive enough for many assays. For example, the threshold for pregnancy is around 10 mUI/mL of HCG, approximately equivalent to 10-6 g/L. Also, LATMAG sensitivity is interesting compared to other dual-particle-based tests. As a matter of fact, Lim et al. [49] obtained a sensitivity of 1.6 x 10-2 g/L for the detection of anti-LPS antibodies by assaying the supernatant after magnetic sedimentation. In the case of the LATMAG assay, positive latex particles are

FIG. 6 Formation of chains of dumbbells for high analyte concentration. Several targets specifically bound on the same latex particle permit the formation of several bridges between both types of particles, leading to clusters whose size can reach 10 pm.

directly counted at the issue of magnetic separation, while Lim measured the spectrocolorimetric signal of negative latex particles.

Was this article helpful?

0 0
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.

Get My Free Ebook

Post a comment