Note: This is the second part of a two part article. If you have not already done so, you might want to read part one of this article first.

Once scientists have chosen, characterized, and cleaned the microspheres for a test or assay based on information in part 1 of this article, they must attach ligand to them and include them in a test or assay. Before attaching protein, the scientist must determine the microspheres' surface capacity for it. One gram of 1-µm polystyrene microspheres, for example, can adsorb 18 mg bovine serum albumin (BSA) or 15 mg IgG.¹ This amount represents a monolayer of protein and is close to the maximum that can be attached to the microspheres, by either adsorption or covalent attachment.

Adsorption

Simply adsorbing protein, especially polyclonal IgG, on the surface of polystyrene microspheres is successful more than 95% of the time. For maximum surface coverage (up to a monolayer), buffer pH should be at, or slightly more basic than, IgG's isoelectric point (that is, pH 8), where the protein is in its most relaxed, compact form ( , not ). Gibbs and colleagues (Costar Corp., Kennebunkport, ME) say, "Binding is favored when pH is close, but not equal to, the pI [isoelectric point] of the protein being immobilized," and "IgG binds best at a slightly basic pH which exposes hydrophobic groups due to partial denaturation."² Tris buffer (pH 8.0) and phosphate buffer (pH 7.4) work well. The Fc and Fab portions of IgG adsorb differently in response to pH changes. A slightly alkaline pH optimizes adsorption of the Fc portion and ensures relative suppression of Fab adsorption.³

Using a dilute microsphere suspension (1% solids) ensures coating of single microspheres. While a final protein concentration of 0.1 mg/ml is enough to achieve a monolayer of protein, addition of 3 to 10 times that amount ensures favorable stoichiometry, a good driving force for adsorption, and crowded, upright positioning (, not ). Excess (unbound) protein is removed by one of the methods for removing surfactant described in part 1.

Many practical, experienced microsphere users do not add a large excess of their antibody (Ab), perhaps because of the extra expense of using (and losing) precious Ab. One recipe uses 1 mg IgG/m², a fraction of the amount of Ab that could be bound, and does not call for removal of any unbound protein in solution. We feel that this practice is dangerous, because of the unknown orientation of the adsorbed Ab and the unadsorbed Ab left in solution.

As an alternative to simple adsorption, IgG and serum albumin (human or bovine) can be mixed and then adsorbed simultaneously. One commercial protocol calls for a weight ratio of 1 IgG to 10 albumin in the coadsorption mixture. Adsorption can be followed by glutaraldehyde cross-linking of the mixed proteins on the microsphere surface.

Haptens and other low-molecular-weight labels, which on their own might not adsorb well or remain attached, can be covalently bound to proteins (such as BSA), dextran, polylysine, or other polymers that adsorb well. Another alternative is to adsorb the polymer on the particles and then couple the hapten or other label.⁴ These polyhaptens are used commercially. Another novel idea is to adsorb peptide onto the microspheres and then covalently link more peptide onto the surface.⁵

Goat antimouse polyclonal antibodies (PoAb), which adsorb well, attach to microspheres to form generic microspheres. These then capture any of several poorly adsorbing monoclonal antibodies (MoAb). In theory, a manufacturer can make a series of tests (or assays) from one PoAb preparation. In descriptions of magnetic microsphere assays run on the Access instrument, Peterson and colleagues (Sanofi Pasteur, Inc., Chaska, MN) cite this technique.⁶

Hemmes (private communication, July 1994) compared activities of primary adsorbed Ab with those of secondary Ab (bound by an adsorbed primary Ab). He reports better activity, or recognition of antigen (Ag), by an Ab if it is the second Ab away from the surface, perhaps because it is freer to move around. This binding strategy even improves the performance of PoAb-based assays.

Adsorbed protein A or G binds various immunoglobulins. Protein A enables positioning of IgG with the binding sites directed outward. Vendors claim, however, that dimethyl pimelimidate (DMP) or dimethyl suberimidate (DMS) should be used to cement the IgG to the protein A. Sikkema asserts that a genetic fusion of protein A and protein G binds IgG better than either A or G alone.⁷

Although avidin and streptavidin will adsorb onto polystyrene, they are often covalently coupled to ensure more secure binding. Prozyme, Inc. (Richmond, CA), claims to offer improved, more-tightly adsorbing streptavidin, which may make covalent attachment unnecessary. After attachment, avidin or streptavidin binds tightly to biotinylated ligands. Molecular biologists use the streptavidin-biotin system with paramagnetic particles (Promega Corp., Madison, WI) for mRNA isolation, covalently linking the streptavidin to the microspheres. In the reverse of this technique, the scientist adsorbs biotinylated BSA onto polystyrene particles and later reacts these microspheres with any other streptavidin-labeled ligand.⁴

Lectins, like concanavalin A and other hemagglutinins, bind carbohydrates of molecular weight greater than 2000 daltons. Thus, they can bind immunoglobulins via the carbohydrate moieties linked to their Fc portions. One lectin, jacalin, binds IgA specifically.

Why Attach Covalently?

Adsorption seems to be more than adequate to attach IgG to polystyrene microspheres for most assay systems. Why then would anyone consider covalent attachment?

1. Some evidence indicates that one can attach 10–40% more protein covalently than by adsorption.⁸

2. When the desired protein coverage is low, covalent coupling may provide more-precise control of the coating level.

3. Covalent coupling binds protein more securely, an asset in production of tests or assays that are so sensitive that they would be influenced by minute quantities of IgG that might leach off the particles over time.

4. The covalent bond is more thermally stable. In one study, after 1 hour at 56°C, 99.7% of covalently linked IgG remained bound, compared with only 70% of adsorbed IgG. This property could be essential if the microspheres are to be used in polymerase chain reaction or other applications requiring thermocycling.

5. Covalent coupling conserves costly reagent because it does not require the large excess of protein necessary for crowded adsorption.

6. Hydrophilic molecules must be covalently linked to the microsphere surface. Unless bound to the surface, they will surely desorb when the equilibrium is disturbed by removal of unbound soluble molecules from solution. The smaller, specialized antibody pieces, such as the F(ab')₂, Fab, or Fv portions or the new "miniantibodies," do not normally adsorb well.

7. Covalent introduction of a spacer arm permits secure but flexible attachment of many different molecules. Covalent attachment of hetero- or homo- bifunctional and trifunctional cross-linkers (many available from Pierce Chemical Co., Rockford, IL) facilitates coupling of ligands with unusual available chemical groups.

8. Directional binding (e.g., periodate oxidation of vicinal hydroxyls on the carbohydrate portion at the Fc end of IgG and binding of the oxidized IgG to hydrazide microspheres) ensures that recognition sites are pointed outward and accessible.

9. Covalent attachment at relatively few sites may overcome the "Gulliver effect," whereby large, well-adsorbing protein molecules become tightly adsorbed over so wide an area or at so many contact points that they become distorted or denatured.

10. Some MoAbs have isoelectric points at 4. At this pH, some microspheres flocculate in suspension. In these cases, covalently coupling MoAbs to microspheres may be easier than adsorbing them.

11. Working with very small polystyrene microspheres (<100 nm) is difficult. Obtaining them surfactant-free is difficult to impossible because of colloidal instability, and cleaning them is not easy. They tend to flocculate when added to the buffer used in protein coating. There are ways to minimize these problems, but it may be easier to covalently bind to surface-modified particles instead. Their hydrophilic surface groups (–COOH, –OH, etc.) make these microspheres more stable.

12. After covalent attachment, protein will not come off the surface of a microsphere. Anecdotal reports suggest it is possible to add as much surfactant as necessary (perhaps up to 1% Tween) to eliminate nonspecific binding. Adding this much surfactant to adsorbed protein/particles could displace unbound protein, but not adding the surfactant could result in significant interference from serum effects in samples. This may be the most important reason for covalent coupling.

13. Adsorption of DNA to microspheres by multiple site attachment may hinder its hybridization. The best method for attachment of DNA to microspheres is probably covalent coupling, at only one point of attachment, preferably at the 5' or 3' end. DNA can be bound to either polymeric or silica microspheres.

14. Protein does not adsorb to hydrophilic silica microspheres; it must therefore be linked covalently to any of several coupling groups.

Surface-modified polymeric microspheres are often made by copolymerizing styrene with a small amount (<5%) of a functional monomer, such as acrylic acid, which yields microspheres covered with –COOH groups. Other monomers are used for microspheres with different surface chemistries.

Native silanol groups on the surface of silica microspheres are readily reacted with aqueous or solvent-based silane coupling agents to yield preactivated silica microspheres with a large variety of surface functional groups. Examples include chloromethyl, carboxyl, and amino groups. DNA and RNA are isolated from serum by adsorption onto silica in the presence of chaotropic agents.⁹ Oligonucleotides can be covalently bound to surface-modified silica via the 5'-amino end. Lipids can be bound via the -carboxyl group on the fatty acid chain and propylamine surface groups on the silica.¹⁰

Optimizing Coating

As noted in part 1 of this article, whether to coat particles with protein completely or incompletely depends on the type of assay. For microsphere-capture ELISAs and tests, dyed-microsphere sandwich tests, solid-phase assays, and DNA probes, coating the microspheres as heavily as possible (a monolayer coating) is desirable. In a monolayer coating, Ab will bind the maximum amount of Ag and second Ab (with whatever tag it carries), maximizing the signal.

Determination of proper coating for microsphere agglutination tests and immunoassays and filter-separation agglutination tests and assays is more critical than in the applications just described and requires careful calculation. When Ag is bound to the microsphere surface, for example, desired agglutination begins when the two Ab recognition sites on an IgG molecule react with Ags on separate microspheres, linking them. If Ag is packed too closely on the surface, Ab can bridge between adjacent Ags on a single microsphere, rather than linking separate microspheres. If Ab is too sparsely distributed on the surface, or the microspheres are too dilute, agglutination is also less likely to occur.

Too much Ab in the sample can prevent agglutination, too. If there were one Ab for each Ag site on the microspheres, no bridging would occur. This phenomenon is called the "hook" or "prozone" effect. To determine the proper coating level for agglutination tests, we recommend the box titration, or checkerboard, approach,^11,12 adapted for slide agglutination. This technique is designed to optimize coverage of the microspheres and ensure the proper dilution, or ratio of microspheres to sample, for highest agglutination sensitivity.

Many materials (called blocker molecules) can be coadsorbed with hard-to-adsorb proteins, such as MoAbs, to promote their attachment. Blocker molecules space out the Ags or Abs bound to the microsphere surface, to ensure optimum coverage and preserve upright orientation. They also fill in any unoccupied sites on the polystyrene surface to prevent, or block, unwanted proteins from interfering with, or causing agglutination of, the particles. Depending on the ratio of blocker molecules to desired protein and the strategy for their use, blocker molecules may be put on the particles before, during, or after coating with the primary protein.

BSA, casein (or nonfat dry milk), gelatin, and Tween seem to be the most popular blockers, although many others are also used. BSA need only be used once, after IgG coating; its effect seems to be permanent. Tween is more labile and will desorb if the equilibrium solution concentration is changed. It must therefore be added to every rinse and buffer if it is to be kept on the surface. Effective concentrations are 1% for BSA, 0.1% for casein, and 0.01–0.05% for Tween. If Ag or Ab has been covalently coupled to microspheres, some users recommend concentrations >1% of Tween or Triton to prevent nonspecific reactions.

Final Formulation of Reagents

In the formulation of microsphere agglutination tests, the pH, electrolyte, and stabilizer content must be optimized to obtain sensitive yet stable reagents that agglutinate only in the presence of the target Ag or Ab. One approach involves first coating the microspheres with Ag or Ab, then adjusting the pH to make the coating as highly charged as possible without reducing its binding. Electrolyte content is then adjusted to the level just below that at which the microspheres flocculate. A particular combination of format, microspheres, protein, and analyte may require a custom protocol. We urge users to develop their own recipes.

Consideration must be given to the timing of the agglutination reaction in a turbidimetric assay. In an automated instrument, reagent and sample must be mixed and turbidity measured at zero time. If this measurement is delayed by even 5 seconds, the agglutination of very small (100 nm), fast-moving microspheres may already be well advanced. The manufacturer may therefore need to slow down the reaction to allow an accurate zero-time reading. Dilution of the microspheres keeps the particles apart, and sucrose or polyethylene glycol (PEG) adds viscosity, to literally slow them down. PEG content has also been found to influence aggregate size.¹³ Salt (100 mM) and 3% PEG are both important ingredients of some turbidimetric assays. PEG's effect may be the result of exclusion of analyte from hydrated PEG domains, a phenomenon that effectively increases concentration.³

Filters

The choice of filter material depends on the intended use. Are the particles to be captured on the filter permanently, or are they to migrate through the filter, as on a chromatographic strip? Costar recommends cellulose acetate (a nonbinding membrane) for general filtration, microsphere concentration, membrane capture assays, and bead washing.² For binding of protein-coated microspheres to a membrane, nitrocellulose or nylon is appropriate.

Hybritech (San Diego) apparently chose an inert-fiber filter for the Icon to capture 1-µm protein-coated microspheres. Scanning electron micrographs from early work at Hybritech show physical entrapment of the microspheres in the intersections of the filter fibers. Abbott Laboratories (Abbott Park, IL) chose a glass-fiber filter to which their 0.2– 0.5-µm coated microspheres adhere—a very different capture mechanism.

Porex Technologies Corp. (Fairburn, GA) offers a device (like the Hybritech Icon) that combines a membrane designed to capture microspheres physically with an absorbent base designed to wick reagents and wash solutions away from the membrane and microspheres. To catch the microspheres on the surface of the membrane, they recommend that the ratio of microsphere diameter to membrane porosity be significantly greater than 1 (i.e., with a 1-µm membrane, one might need a microsphere with diameter greater than 1.3 µm). Similarly, Costar recommends 0.3-µm microspheres to be caught on a 0.2-µm membrane. If a filter is used to entrap the microspheres physically, we recommend microspheres approximately 50% larger than the filter porosity, to ensure their capture at the top of the filter.

Figure 1. Basic chromatographic strip test. A. Dry strip. B. Sample (with antigen) added. C. Sample flow moves microspheres; antigen forms sandwich. D. Dyed microspheres form colored lines for positive test and control.

 

Strip Test Assembly

For most basic chromatographic-type strip tests (see Figure 1), cellulose nitrate (nitrocellulose) or nylon membranes are recommended to immobilize the capture proteins in the windows. The idea is to immobilize Ab₂ (anti-hCG) in a line placed in the first (test result) window. This line must survive drying and rehydration, remaining in place and holding the microspheres coated with Ab₁ and antigen (hCG). In the second (control) window, Ab₃ (polyclonal anti-mouse) is placed in a line to capture monoclonal antibody–coated microspheres that get past the test window. Meanwhile, the dyed Ab₁-coated microspheres are dried on the nitrocellulose or nylon strip. They are supposed to remain in place until the strip is wet with sample. Then they must rehydrate readily, bind to the antigen (hCG), and move freely through the membrane, stopping only when they encounter the Ab₂ or Ab₃ lines in the windows downstream. One might expect, however, that the Ab₁-coated microspheres would also naturally stick to the nitrocellulose (just as the Ab₂ and Ab₃ lines stick). What ensures that the Ab₂ and Ab₃ lines stick, while the Ab₁-coated microspheres remain free?

Pall Corp. (Glen Cove, NY) suggests protecting the Ab₁-coated microspheres from sticking permanently by pretreating the microspheres, and/or the membrane where they will be dried, with a hydrophilic substance. This release agent could be surfactant and/or saccharides, such as sucrose or trehalose (see Figure 2). Then the sample, when added, will readily rehydrate the microspheres; released from the surface, they will move freely along the strip with the liquid flow.

Figure 2. Pall scheme for depositing nonstick microspheres on strip.

 

Deposition of dyed Ab₁-coated microspheres in or on the hydrophilic absorbent pad will also prevent adherence. When sample is added to the pad, the microspheres will be released from the pad and flow onto the membrane and along it to the immobilized Ab stripes.

The chromatographic strip test relies on dyed microspheres that move freely through the membrane. The ratio of microsphere diameter to membrane porosity should be much less than 1, perhaps 0.1, for good flow (i.e., with a 5-µm membrane, one might need microspheres with diameter < 0.5 µm). Because the color signal of small microspheres is not as intense as that of larger, darkly dyed microspheres, the manufacturer must choose the best compromise between small particles for mobility and large dark ones for sensitivity.

Many variations on the basic strip test technique are used. One option is to prevent drying of the small, dyed Ab₁-microspheres on the strip and subsequent binding. The Ab₁-microspheres are used as a liquid reagent or reconstituted lyophilized reagent. Sample is mixed with the microspheres and the mixture is added to the strip, where the microspheres immediately start to migrate to Ab₂, which is bound to the strip.

Alternative membranes are used when the membrane is only the conduit for moving sample and dyed marker microspheres. Cellulose acetate, for instance, does not bind protein or protein-coated microspheres well. The capture proteins (Ab₂ and Ab₃) are immobilized in their respective windows by large, colorless Ab-coated microspheres. These large microspheres, which are too big to move through the membrane, are sprayed or printed on the membrane. The ratio of microsphere diameter to membrane porosity should be much greater than 1, perhaps greater than 2, so that, like boulders in a stream, the microspheres cannot move with the liquid flow. Small dyed microspheres coated with Ab₁, which were dried in place, are easily rehydrated by sample and move downstream toward the large colorless microspheres with Ab₂ (and Ab₃).

Some Japanese researchers use both of the above ideas for plant disease tests (cucumber mosaic virus and tobacco mosaic virus).¹⁴ They dry large Ab₁-coated microspheres onto a strip, then dip the strip into a mixture of Ab₁-coated, dyed microspheres and sample. The dyed microspheres migrate up the strip and form a colored line if Ag is present in the sample.

Whether designing a simple agglutination test with protein adsorbed to the microspheres or a sophisticated turbidimetric assay with antibody covalently bound by some complicated chemistry, take the time to monitor each step of the process. All too often, we hear from users who tell us that our microspheres did not work in their assay, but they have no idea at which point problems began to occur. Proper planning and periodic monitoring can ease microsphere use.

References

1. Cantarero LA, Butler JE, and Osborne JW, "The Adsorptive Characteristics of Proteins for Polystyrene and Their Significance in Solid-Phase Immunoassays," Anal Biochem, 105:375–382, 1980.

2. Gibbs J, Brown C, Root D, et al., "ELISA Optimization," presented to the American Association for Clinical Chemistry (AACC) Northern California Regional Meeting, Napa, CA, September 1991.

3. Kawaguchi H, Sakamoto K, Ohtsuka Y, et al., "Fundamental Study on Latex Reagents for Agglutination Tests," Biomat, 10:225–229, 1989.

4. Shah D, Chandra T, Chang A, et al., "Acridinium-Labeling to Latex Microspheres and Application in Chemiluminescence-Based Instrumentation," Clin Chem, 40:1824–1825, 1990.

5. Leahy DC, Shah DO, and Todd JA, "A Method for Attachment of Peptides to a Solid Surface with Enhanced Immunoreactivity," BioTechniques, 13(5):738–743, 1992.

6. Peterson T, Kapsner K, Liljander B, et al., "A Chemiluminescent Immunoassay for the Determination of Liver Ferritin," Poster 624 presented to the 44th National Meeting of the AACC, Chicago, July 1992.

7. Sikkema WD, "An Fc-Binding Protein," Am Biotech Lab News, 7:4A, 1989.

8. Douglas AS, and Monteith CA, "Improvements to Immunoassays by Use of Covalent Binding Assay Plates," Clin Chem, 40:1833–1837, 1994.

9. Boom R, Sol CJA, Salimans MMM, et al., "Rapid and Simple Method for Purification of Nucleic Acids," J Clin Microbiol, 28:495–503, 1990.

10. Haggin J, "New Applications Touted for Immobilized Artificial Membranes," Chem Eng News, 72:34–35, 1994.

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

12. Matsuzawa S, Itoh Y, Kimura H, et al., "Microtiter Latex Antiglobulin Test for the Detection of Antibodies to DNP, Digoxin, HCG, and Similar Antigens," J Immunol Meth, 60(8):189–196, 1983.

13. Cheng ML, Shah N, Obremski RJ, et al., "Effects of PEG and Analyte Concentration on the Size of Aggregated Complexes in a Microparticle Enhanced ASO Immunoassay," in "Abstracts of Posters at CLAS 20th National Meeting, Orlando, FL, April 1994," J Clin Immunoassay, 17:59, 1994.

14. Tsuda S, Kameya-Iwaki M, Hanada K, et al., "Novel Detection and Identification Technique for Plant Viruses: Rapid Immunofilter Paper Assay (RIPA)," Plant Dis, 76:466–469, 1992.

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

Photos courtesy of Bangs Laboratories, Inc. (Carmel, IN)

Return to Part 1 of this article.

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)

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