Ligand attachment and test formulation
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.
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
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
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.
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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.