- Pasteurization: 24 hours at 78-80º C
- Gamma irradiation: 0.03 megarads for 24 hrs
Hydrophobic latex products are usually stable in systems free of biological molecules. In biological systems, however, including applications for immunoassays, the microspheres can easily be coated with various proteins or polysaccharides that greatly reduce their capacity to absorb biomolecules non-specifically.
Specific irreversible adsorption of protein molecules such as avidin, streptavidin, and antibodies is accomplished by simply mixing the latex and protein together for a specified period of time, then separating the bound from the unbound protein through centrifugation and removal of the supernatant.
To reduce nonspecific binding—coat the particles with BSA or dextrans. To further reduce nonspecific binding—covalently couple proteins, nucleic acids, and other biomolecules to the particles.
Covalent coupling requires more effort than passive adsorption, but can result in conjugates with greater specificity that remain active longer. Carbodiimide-mediated coupling to CML latexes is the method of choice for conjugating low molecular weight peptides and oligonucleotides.
Choosing the Right Buffers
- Buffer choice is dependent on the type, size, and density of surface charge groups.
- Smaller particles generally have fewer surface charge groups for stabilization and need more stringent conditions to prevent aggregation.
- Avoid cationic buffers like Tris when using sulfate, carboxyl, or CML (negatively-charged) latex particles.
- Negatively-charged particles are also sensitive to low concentrations of multivalent cations such as Ca++ and Mg++ salts—buffers containing these cations should be avoided if at all possible. If multivalent cationic buffers are necessary for an experiment, then cationic latex particles which are not as sensitive to Ca++ and Mg++ ions are a better choice.
- Avoid phosphate or borate buffers when using cationic (amidine and AML) latex particles.
- The ionic strength of the buffer should be kept as low as possible, especially when particles are very small or have a low charge density.
- The particles should be used at a pH > 5.0. If these conditions are not followed, the charge groups may be neutralized, leading to aggregation. If aggregation does occur, the particles can usually be redispersed by adjusting the pH to the correct range, followed by gentle sonication.
- Water used in buffer preparation should be as pure as possible to prevent scavenging of impurities by the hydrophobic particles.
Controlling non-specific binding of proteins
Nonspecific binding is probably the most common problem encountered in working with latex particles—it is often the major reason for abandoning an otherwise well-conceived experiment.
Latex particles are generally hydrophobic, and although various modifications tend to make them less so, the particles always retain some hydrophobic characteristics because they are polystyrene-based. In biological systems, most of the nonspecific binding problems are a result of hydrophobic interactions. However some of the problems may also be caused by charge-based interactions, such as a positively charged molecule binding to a negatively-charged latex surface.
The best way to minimize these nonspecific binding events:
- Coat the particle with a large molecule such as a protein or polysaccharide which reduces nonspecific binding by blocking the hydrophobic or charged binding sites on the particle surface. Although many types of blocking agents may be used, the most frequently used are BSA, egg albumin, and whole serum. Egg albumin should be avoided in biotin-avidin systems.
- When using hydrophobic latexes, add the desired protein at a concentration of 250-500 µg/ml of latex suspension at a concentration between 0.5 and 2.0% solids.
- Dextran (40k MW at 2% w/v) can be used as a blocking agent in place of, or in addition to proteins. Unlike proteins, dextrans bind reversibly to latex microspheres, to form a layer on the surface, creating a more hydrophilic environment and reducing nonspecific interactions. When a hydrophilic CML particle is used, the blocking agent may not bind as strongly. In this case, covalently coupling the protein may solve the problem.
- Specific binding proteins such as immunoglobulins or avidin can be mixed with BSA and simultaneously coupled covalently, resulting in a specifically active latex with covalently-bound BSA coating. In situations where detergents are acceptable, a nonionic surfactant such as Triton X-100 or Tween 80 can be coated onto the particle at concentrations ranging from 0.01 to 0.1%. The exact concentration should be determined experimentally for each application.