Aggregation phenomenon during microsphere labeling and its solutions

During the process of labeling antibodies with microspheres, different people often encounter various problems during practical operations. One of the most frustrating ones is the aggregation of microspheres, which occurs after cleaning, activation, and even after adding antibodies... Many people feel confused about this. The experiment is really tiring, and I followed the instructions clearly. Why does it still occur? Today, let's briefly talk about the reunions during the microsphere labeling process.

1. Agglomeration after washinga

Manufacturers of microspheres generally add a certain amount of surfactants, stabilizers, and other reagents to the preservation solution, and the presence of these components can have a certain impact on the labeled antibodies of microspheres. Therefore, before use, it is generally necessary to clean the microspheres, such as using PBS buffer for cleaning. However, due to the different synthesis processes of microspheres from different manufacturers, there are differences in the hydrophilicity and charge carried on the surface of microspheres. In this case, after removing the surfactant, the microspheres may aggregate or even precipitate due to the insufficient repulsive force of the surface charge to maintain stability in the system. When encountering this situation, the user can add a small amount of surfactant (such as 0.025% SDS) again. In addition, the phenomenon of microsphere sticking to the wall is often encountered after centrifugal separation of microspheres. When this phenomenon occurs, it can be solved from the following two aspects: firstly, increasing the centrifugal force, and secondly, using containers made of low adsorption materials

2. Agglomeration after centrifugation

The common method for microsphere separation during small volume labeling is centrifugation. Different particle sizes require different centrifugal forces. When the centrifugal force is too large, the microsphere adheres tightly to the bottom of the centrifuge tube, making it relatively difficult to resuspend; In this case, it is necessary to reduce the centrifugal speed or time appropriately to ensure that the microspheres exist in a loose, clustered structure as much as possible. In addition, during the process of resuspension of microspheres, a pipette can be used to forcefully blow the microspheres to achieve initial resuspension, and then ultrasound can be used to further disperse the microspheres (such as water bath ultrasound, high-power, recommended not less than500W, time not less than 2-5min).

3. Agglomeration after adding activator

When the microspheres are activated, the amount of EDC and other activators added can also cause the microspheres to agglomerate. When EDC is added, the first consideration for the agglomeration of microspheres is the excessive amount of activators. In addition, an appropriate amount of Sulfo NHS can be added before adding EDC, and thoroughly mixing the microspheres after adding EDC can also help with the re dispersion of the microspheres. The surface groups of the microspheres are activated by EDC to form an unstable intermediate, while the addition of Sulfo NHS transforms the intermediate into a relatively stable value, thereby avoiding agglomeration of the microspheres.

4. Agglomeration after antibody addition

When activated microspheres are added with antibodies, aggregation also occurs. In this case, we first need to determine whether the isoelectric point of the antibody is close to the pH value of the buffer solution. If so, we need to adjust the pH value of the buffer solution or replace it with another buffer system; On the contrary, the proximity between the isoelectric point and the pH value of the buffer solution causes.