IVD Technology Magazine
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Originally published March, 1995

Microspheres, part 1:

Selection, cleaning, and characterization

Leigh B. Bangs and Mary Meza

Many diagnostic tests and assays use submicron-size uniform latex particles, or microspheres, as substrates or supports for immunologically based reactions. These range from the original "latex" agglutination tests to more recent particle-capture assays, particle immunoassays, the newest dyed-particle sandwich tests, and solid-phase assays using silica or magnetic microspheres. Before microspheres can be used in any test or assay, they must be prepared for binding and coated with a ligand (usually a protein). The microspheres' interaction with other test components, such as filters, membranes, and magnets, must also be factored into the choice of test format and microsphere.

The analyte involved partly determines the format. Molecules with molecular weights less than 6000, for instance, might be difficult to detect in a sandwich format, because many such small molecules do not have space for two antibodies. Small molecules require competitive assays and tests. Large analytes, like proteins, can be measured by either direct or inhibition tests and assays.

In approving assays, FDA is placing a high priority on process validation. Manufacturers need to address this requirement early in the development process and choose appropriate methods.

Test Components

Custom-made microspheres—with special chemical properties,^1,2 higher or lower density than polystyrene, or refractive indices above or below that of polystyrene (brighter or dimmer particles for turbidimetric assays)—are among those available. Because hydrophobic interaction is the most likely mechanism involved in adsorption of proteins, polystyrene (a hydrophobic polymer) surfaces often have high degrees of nonspecific protein adsorption. Polystyrene therefore might not be the best choice when microspheres that will not adsorb any protein are required. An example might be the covalent binding of antibodies to microspheres, in which it is essential that all coated protein be bound covalently. Microspheres made with high surface levels of hydrophilic monomers, like acrylic acid and acrylamide, come closest to being nonadsorbing.

Silica microspheres are naturally hydrophilic, so no protein should adsorb nonspecifically onto them. After covalent attachment, only the desired antigen-antibody reaction should occur. The difference in density between silica and polystyrene (1.96 g/ml for silica versus 1.05 for polystyrene) dictates a critical difference in settling velocity. Because settling in water depends on the difference in density between microspheres and water (1.96 – 1.00 = 0.96 for silica; 1.05 – 1.00 = 0.05 for polystyrene), the silica microspheres will settle about 19 times as fast as the polystyrene. This difference in settling velocity could be exploited in some interesting tests and assays based on differential settling times of agglutinated and unagglutinated microspheres.

Encapsulated superparamagnetic particles consist of a polymer/magnetite core sealed in a pure polymer shell or outer layer to protect sensitive enzymes from contact with iron oxide.

Microspheres are dyed either during or after polymerization in a rainbow of colors. Originally dyed for improved visibility and color discrimination, they are now also dyed with "fluorochromes" (used singly, or several on the same particle), "fluorophors" (fluorescent dyes with specific spectral properties), and scintillators (dyes that fluoresce when exposed to gamma or beta rays). Often only a small amount of these dyes is required to produce an intense signal.

Superparamagnetic microspheres are available in different mean diameters, size distributions, surface chemical properties, and levels of magnetite (for adjustable response to a magnet). Newer ones are core/shell encapsulated to prevent iron from coming into contact with sensitive enzymes or cells.

Will IgA, IgM, or IgG be used? Monoclonal (Mc) or polyclonal (Pc) antibody? Whole IgG or parts like F(ab')₂, Fab, or Fv? (Fc portions of IgG can be removed to avoid rheumatoid factor [RF] interference, which can lead to nonspecific binding and autoagglutination problems.) When screening PcAbs for an assay, request "nephelometry and turbidimetry grade" Abs, which are preselected as good precipitating Abs. This quality is a predictor that they will probably be good for adsorption onto polystyrene and perhaps good for agglutination as well.

Several factors weigh in favor of using monoclonal antibodies rather than polyclonals. Monoclonal antibodies, which usually have higher specificities than polyclonals, also normally have lower binding affinities. Monoclonals also do not normally cause immunoprecipitation. These characteristic differences between polyclonal and monoclonal antibodies should correlate with differences in antibody adsorption to microspheres.

Some special binding proteins are now available, including transducing antibody—a bifunctional Ab designed to recognize both an antigen and an enzyme.³ Binding an enzyme to an antigen could be the basis of some unusual tests. New "miniantibodies" are divalent Fv antibody fragments, grown in E. coli by recombinant techniques.⁴ Because chicken antibodies don't react with RF, complement interference is eliminated in tests using these special proteins.⁵

Recombinant polymeric IgG has been designed to combine the best properties of IgM and IgG for complement response.⁶ Oligomers, which gradually form in protein solutions (like bovine serum albumin and IgG) over time, adsorb onto microspheres more quickly and firmly than the monomers. If you want oligomers, either wait, or try to accelerate solution aging. It might be possible to create synthetic oligomers by cross-linking proteins or binding them to a synthetic polymer.

Researchers working with microspheres often assume that the water they have is clean. But even commercial deionized (DI) water can contain ionic and organic species that adsorb onto the microspheres; in these cases the microspheres clean the water instead of the reverse.

Cleaning

Before particles are coated with protein for use in various diagnostic tests and assays, surfactant and other solutes might have to be removed. Most uniform polystyrene microspheres are made by emulsion polymerization using surfactants (usually negatively charged alkyl sulfonates, sulfates, or carboxylates). The surfactants become adsorbed on the particle surface; there they give the particles a negative charge, which increases colloidal stability. In addition, surface-modified microspheres (those with COOH, NH₂, and other surface groups) may contain water-soluble polymer (WSP), which can interfere with the coupling of proteins to the surface. WSP, if present, supersedes protein in any coupling reaction designed to put protein on microspheres. Chloromethyl-modified particles (made with vinylbenzyl chloride monomer) come in highly acidic aqueous solutions (pH<3); cleaning to remove the acid before coupling might be desirable.

Highly uniform polymeric microspheres.

Although, early in the R&D process, it is probably a good idea to use thoroughly cleaned microspheres, it is often not necessary to remove all surfactant in the final formulation of a product. This decision depends on the type and concentration of the surfactant, the level of protein loading needed, and the type of test or assay being designed. Each system must be evaluated independently.

Uniform silica microspheres are made from pure Si(OC₂H₅)₄ reacted with water and ammonia. Because the resultant microspheres are pure SiO₂, they should have no surface-active impurities and therefore should need no cleanup before use.

Most polystyrene-based superparamagnetic microspheres are made by copolymerizing styrene with carboxylic acid—containing monomers in the presence of colloidal magnetite; they may contain some WSP. Sodium dodecyl sulfate (SDS) is added to ensure long-term colloidal stability.

The particle-cleaning method chosen should be capable of removing not only surfactant but also residual unbound protein after coating. Most methods work for removal of either surfactant or unbound protein.

Washing. Repetitive centrifuging, decanting, and resuspending in water is often the first cleaning method considered. The microspheres must be spun down to form a tight "button" to permit the clean separation (decantation) of liquid from solids. The smaller the particles, the more difficult this separation. If the brake is used to stop the centrifuge, the particles may be partially resuspended and some of them lost on decanting.

After decanting, fresh water or buffer is added, and microspheres should be fully resuspended. Effective washing must completely redisperse microspheres. Resuspension should be monitored by some reliable method, such as microscopic examination or fast instrumented size analysis, to verify that particles are primarily single, with only a few doublets. The more surfactant is removed, the more tightly the microspheres adhere to one another; buffers amplify this effect further. Larger particles (>0.8 µm) are more easily spun down, less likely to stick firmly together, and more easily resuspended. Hydrophilic microspheres and protein-coated microspheres are much less likely to stick together after centrifuging than are noncoated or hydrophobic microspheres.

Calculations of microsphere settling velocity and gravitational (G) forces generated by a centrifuge can be performed to determine the amount of time necessary to spin down microspheres of a specific size.⁷ Microspheres <50 nm (<0.050 µm) may require >300,000 G to sediment them efficiently (i.e., a 10-cm/hr settling rate).

Magnetic microspheres can be cleaned by washing with magnets to replace or assist gravity sedimentation. As the microspheres go through successive cleaning steps, however, they become more hydrophobic and, therefore, more difficult to resuspend and separate. An ultrasonic bath—but not a probe—can greatly assist resuspension. (Ultrasonic probes are notorious for introducing contamination. Even metal particles can come off the probe.) Ice added to the bath prevents sample heating.

Dialysis. A slow and unreliable method, dialysis may be used as a preliminary step. It is difficult to remove all impurities by dialysis because of the time required for the surfactant to desorb completely and diffuse through the dialysis tubing. Dialysis can, however, be very effective in one possible situation. In some cases, hydrophobic particles with low inherent surface charge are not colloidally stable without their surfactant; therefore, they might clump before they can be coated with protein. In such cases, one innovative idea is to mix protein with surfactant-containing particles and dialyze the mixture in a membrane chosen to let the surfactant diffuse out while holding the particles and protein in.

Dead-End or Bed Filtration. Standard filtration is generally unacceptable, because the small microspheres can easily plug any filter designed to catch them. Flow through a packed bed of submicron particles is extremely slow, and after filtration the redispersal problem remains.

Cross-Flow Filtration. Various mechanical means are employed to permit liquid to permeate the filter medium while preventing a particle layer build-up. The process could also be called "dialysis under pressure" or "filtration without a filtercake." Several filter manufacturers offer equipment to handle volumes from a few milliliters to many liters. The best method is one that can be easily scaled up from laboratory use to commercial production. Stirred beakers with a filter in the bottom and a stirrer just above the filter keep a filtercake from forming while the liquid is filtered under pressure.

In some devices, flat membranes are sandwiched between monofilament screens. The microsphere suspension is pumped horizontally through the screen, over and under the filaments of the screen, so that net flow is parallel to the plane of the screen. This flow path causes turbulent mixing of microspheres and liquid while a small amount of the total flow is allowed to pass perpendicularly through the filter elements, on either side of the screen. Water and soluble materials are removed, and the microspheres are cleaned and concentrated without clumping. Microspheres are, however, occasionally trapped in the corners of the screen.

Some hollow-fiber filter cartridges are designed to be disposable. Pore sizes can be as small as 0.05 µm, allowing cleanup of all but the smallest particles. The same fibers are used in small- and large-scale units, so the process is scalable. Cross-flow membranes that are tight enough to retain proteins are usually called ultrafiltration membranes. These membranes might also be useful for the simultaneous cleaning and coating method mentioned under "Dialysis." After protein coating, a separate step with cross-flow filtration would still be required to remove the unbound protein from the microspheres. After cross-flow filtration, the microspheres' surface ionic groups are only half neutralized: [-SO₄^–] [-SO₄H] and [-COO^–] [-COOH]. Microspheres cleaned this way should thus be more stable colloidally than those cleaned by ion exchange, because in the latter process all ionic groups are neutralized (all in -SO₄H and -COOH forms).

Mixed Ion-Exchange (IX) Resins. Used successfully for more than 25 years to remove all ionic species from latex particles, mixed IX resins consist of equal volumes of strong acid and strong base resins in the hydrogen ion and hydroxide ion forms, respectively. The mixed resins are added to the latex that is to be cleaned. All ionic surfactant and inorganic buffers are removed from the aqueous phase and quantitatively stripped off the particles' surfaces. Clean microspheres are then separated from the much larger IX beads by decantation and coarse filtering.

Uniform silica microspheres (1 µm diam, X 10,000).

Mixed ion exchange is the only cleaning process that rapidly and actively removes adsorbed surfactant. The other methods are passive—particles are cleaned as surfactant spontaneously desorbs from their surface and is subsequently removed from the aqueous phase.

Commercial IX resins (Dow or Rohm & Haas) often contain a variety of impurities and must be carefully cleaned before they are used. The resins do not need to be put into a "bed" or column; they can be mixed with the microspheres and later removed by coarse filtration. Prepurified IX resins can be purchased from BioRad (Hercules, CA). These strong acid and base resins are not designed to remove proteins; they may, in fact, denature some proteins.

Column Methods. The bed packing of the column should be as large as possible to ensure good fluid flow and to allow microspheres to percolate freely through the bed. Any hang-ups result in loss of microspheres and plugging of the bed. The packing bead porosity should be large enough to let the unbound solute enter easily, yet small enough to exclude the microspheres. Only the unbound protein and other water solubles should be caught within the pores.

Weak anion and/or cation columns can remove proteins. Many people recommend DEAE cellulose. Various affinity columns probably work well to remove unbound protein from microspheres. Several column manufacturers claim binding of a wide variety of proteins. Some columns contain genetically engineered binding agents, which bind various immunoglobulins selectively or comprehensively.

Gel-phase chromatography (GPC) or size-exclusion chromatography can be used to separate free surfactant, or unbound protein, from microspheres.⁸ As the microsphere suspension is poured or pumped through the bed, microspheres move quickly through the void volume between the beads, while dissolved surfactant (or unbound protein) diffuses into the pores of the beads, where it is detained briefly and exits the column after the microspheres. Sephadex G-25 columns have been used for this job; prepacked, disposable PD-10 columns (G-25 M) are available with bed volumes as small as 1.7 ml. Columns are available with a wide variety of porosities and gel-bead sizes.

Microsphere Characterization

It is advisable to test microspheres at various stages in their processing—before and after cleaning, protein coating, blocking, and final formulation (buffer adjustment). Attributes to monitor include the microspheres' monodispersity, colloidal stability, surface charge, percent solids, and changes in electrokinetic behavior (which relate to protein coverage).

Size/Monodispersity. Determining the level of monodispersity of the microspheres is vital. Should they be singlets or partly flocculated? Did clumping occur, and if so, when? Microsphere count (particles per gram or particles per milliliter) is an important piece of information to calculate and monitor.⁷

Surface Titration. Potentiometric or conductometric titrations on clean or coated microspheres document lot-to-lot reproducibility. Titration may provide clues for troubleshooting problems, such as microspheres' inability to adsorb as much protein as expected after a particular treatment, and can determine whether cleaning was thorough enough. The capacities of different lots of COOH-modified microspheres for covalent coupling (active surface COOH groups) can be compared by titration.

A "soap" (surfactant) titration is a standard colloid chemist's technique for determining the amount of open surface area on polymeric microspheres. The chemist titrates a known amount of clean or as-received microspheres with a standard soap solution, then measures the surface tension. Soap adsorbs onto the polystyrene microspheres, and surface tension remains steady, until each microsphere surface has a monolayer of soap molecules oriented perpendicular to the surface. Surfactant then goes to the water-air interface, and the surface tension starts to drop. The amount of soap added up to the surface tension break point is the surface capacity of the microspheres.

Other specialized titrations can be done, depending on the type of particles and the binding chemistry. For example, chloromethyl-modified poly(styrene/vinylbenzylchloride) [P(S/VBC)] microspheres can slowly form HCl in solution during long-term storage. Tracking the release of chloride ions is one way to monitor the shelf life of the microspheres.

Critical Coagulation Concentration. By a process that in some ways is the opposite of soap titration—titration with a standard salt solution—it is possible to predetermine stability against flocculation. This titration indicates whether the microspheres will flocculate in the buffer in which the particles may be coated, coupled, or stored.

Electrokinetics. There are several methods and appropriate instruments to monitor the progress of microsphere cleaning and coating. The most commonly used method measures the direction and speed of motion of individual microspheres in a standard setup.

Field Flow Fractionation (FFF). A family of flexible elution techniques capable of simultaneous separation and measurement, FFF measures component properties, including mass, size, density, charge, diffusivity, and thickness of adsorbed layers.

With these methods, chemists can determine whether their microspheres are clean and reproducibly and uniformly coated with protein. Without this validation, they are flying blind in their coupling processes.

References

1. Kapmeyer W, "Nephelometric Immunoassay with Shell/Core Particles," Pure & Appl Chem, 63:1135–1139, 1991.

2. Maehara T, Eda Y, Mitani K, et al., "Glycidyl Methacrylate-Styrene Copolymer Latex Particles for Immunologic Agglutination Tests," Biomater, 11(3):122–126, 1990.

3. Product Literature, Surface Active, Ltd., Dept. of Obstetrics, St. Michael's Hosp., Bristol BS2 8EG, UK.

4. "New Type of Antibody Can Bind Two Antigen Molecules," Chem Eng N (C & EN), 70(9):22, 1992.

5. Larsson A, and Sjoquist J, "Chicken Antibodies: A Tool to Avoid False-Positive Results by Rheumatoid Factor in Latex Fixation Tests," J Immunol Meth, 108:205–208, 1988.

6. Smith RIF, and Morrison SL, "Recombinant Polymeric IgG: An Approach to Engineering More Potent Antibodies," Bio/Tech, 12:683–688, 1994.

7. "Useful Equations, Tech. Note #49," Carmel, IN, Bangs Laboratories, 1994.

8. Vary CPH, "Triple-Helical Capture Assay for Quantification of Polymerase Chain Reaction Products," Clin Chem, 38:687–694, 1992.

Leigh B. Bangs, PhD, is president and Mary Meza is technical services manager at Bangs Laboratories, Inc. (Carmel, IN). Photos courtesy of Bangs Laboratories (Carmel, IN)

Continue to part 2 of this article.