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Troubleshooting protein binding in nitrocellulose membranes

Part 2: Common problems

Kevin D. Jones

Manufacturers can ensure good protein binding results if they perform carefully designed and controlled experiments during product development.

The first installment of this article (IVD Technology, March/April 1999) covered the basic principles behind protein binding.¹ This second installment discusses common problems and the most common solutions. For clarity, the problems have been broken down into six generic groups (see Table I). Because IVD technology is wide and varied, there are no universal answers. No amount of discussion can replace experimental results. The purpose of this article is, therefore, to suggest starting points for experiments.

Table I. Common defects associated with nitrocellulose membrane–based test development and the most likely causes.The decrease in nitrocellulose surface area available for protein immobilization can be seen as the membrane pore size increases from 0.45 (a) µm through 3.0 µm (b) to 8.0 µm (c). Increased pore size leads to a faster wicking rate, but also means that the protein being applied travels a greater distance before it comes into contact with the nitrocellulose membrane wall. The capture line will thus be wider on an 8-µm membrane than on a 0.45-µm membrane, because of both the increased lateral wicking rate and the increased distance traveled by the protein before immobilization.

Nonspecific Signal

Nonspecific signal in nitrocellulose-based assays is a significant problem for the diagnostic industry. It can appear either generally over the entire membrane surface or, more seriously, at the capture line.

Causes. Nitrocellulose will bind proteins, which is the mechanism behind the capture line. The reasons for this binding were discussed in Part 1.

Nonspecific protein binding. Nonspecific protein binding can be caused by an unwanted interaction between the sample or conjugate and the capture line. Such interactions at the capture line can be the result of any of the common causes of protein attachment, including charge attraction, hydrophobicity, disulfide bridging, or a genuine nonspecific immunogenic binding.

Conjugate binding. Since conjugate particles are covered by a protein layer, the causes of nonspecific conjugate binding are similar to the causes of nonspecific protein binding. Additional problems can occur. The conjugate particles themselves are often permanently charged. In the case of gold conjugate, the conjugate is very susceptible to interaction with sulfhydryl moieties on the surfaces of proteins and membranes. The causes of and remedies for the interaction of conjugates are discussed in literature available from conjugate manufacturers.^2–4

Solutions. Nonspecific attachment to the membrane can normally be reduced by blocking with a protein (e.g., bovine serum albumin [BSA]), surfactant (Tween 20, Triton X-100, or sodium dodecyl sulfate [SDS]). The effect of charge can be overcome by either changing the pH of the test or increasing the ionic strength of the system.

Disulfide bridging is rarely seen in diagnostic products, although nonspecific immunogenic interactions do regularly occur. Standard immunologic blocking techniques, such as use of a similar protein as a blocking reagent, can solve this problem. For example, if a nonspecific interaction occurs between a specific mouse IgG and a human sample, blocking the sample with mouse serum would remove the false-positive signal.^5–7

Weak or Diffuse Capture-Line Intensity

If the level of capture reagent per unit area of membrane surface is too low, the capture line may be weak or diffuse. The problem may be simply mechanical; for instance, too-rapid movement of the membrane through the application striping system. However, the cause may be more complex.

Causes. It is often impossible to determine the cause of this problem by merely examining the test strip. The only solution is to work through the potential causes and find by experiment where the major problem lies.

Concentration of the capture reagent in the application solution is too low. The application solution diffuses away from the point of application. The capture reagent spreads with the application solution to an extent defined by the partition coefficient between the solid phase and the solute. If attachment to the solid phase is preferred, the capture reagent will spread less. If solubilization in the solute is preferential, then the capture reagent will spread further (see Figure 1).

Figure 1. A weak capture line indicates that the amount of protein bound to the membrane is too low.

Figure 2. A diffuse capture line can result when the capture reagent is washed away by the passage of analyte proteins and surfactant solutions.

Capture reagent is washed off the membrane by the sample. If the strength of attachment between the capture reagent and the membrane is too low, or the surfactant level in the running buffer is too high, the capture reagent may be physically removed from the membrane surface by the sample (see Figure 2).

The lateral wicking rate of the membrane is too high, causing spread of the capture reagent after application. If the membrane used has a very high lateral wicking rate, the applied protein will diffuse very rapidly from the point of application. As in all physical interactions, there is a time-related function—the faster the protein is moving in a lateral direction, the wider the capture line will be. The protein solution will also penetrate vertically into the membrane, and most of the protein that penetrates will be wasted, as only the material trapped in the top 10 µm of the nitrocellulose membrane will be visible. This combination of lateral and vertical wicking can cause a weak capture line. The apparent concentration of the analyte in solution is a function of the lateral wicking rate of the test (apparent concentration 1/wicking rate).² This equation means that as the wicking rate increases, the apparent concentration of the analyte drops dramatically.⁸

A reduction in the lateral wicking rate (by use of a smaller-pore nitrocellulose or a viscosity modifier) results in a stronger test line. However, it may cause an increase in nonspecific signal, a change in the test sensitivity cut-off, or a significant increase in test time.

The capture reagent binds only weakly to the membrane. The binding of proteins to membranes can be influenced by optimization of the application buffer. However, some capture reagents are difficult to attach to membranes, either because of their size or their surface properties. In these cases where the binding is very weak, there are only a limited number of solutions. The most common is cross-linking the required reagent to a carrier protein.

The affinity constant between the capture reagent and the target analyte is too low to support efficient capture. The use of low-affinity antibodies is, fortunately, normally avoidable. Careful screening of antibodies during development is an absolute necessity. In cases where the use of a low-affinity antibody is the only option, then the use of a small-pore membrane coupled with a low lateral wicking rate will maximize the interaction between the sample and the analyte.

Solutions. Whatever the cause of weak or diffuse capture-line intensity, the solution lies in achieving a higher concentration of capture reagent at the desired point.

Use a smaller-pore membrane. The use of a smaller-pore membrane will increase line sharpness and intensity for two reasons. First, the smaller the pore, the greater the surface area of the material, and the greater the surface area, the higher the concentration of capture reagent that attaches to it. Second, the smaller the pore, the slower the wicking rate.

Use a different membrane. Different membranes have different binding characteristics for different capture materials. Selecting a membrane that has better binding characteristics for the particular capture reagent chosen improves the capture line appearance.

Use a higher protein concentration. Increasing the concentration of the capture reagent in the application buffer may cause a higher concentration of the capture reagent to attach to the membrane. This would allow a higher level of analyte to stick in the capture zone and hence improve signal intensity.

Change the application buffer. Modification of the application buffer adjusts the point of equilibrium between the amount of capture reagent attached to the membrane and the amount remaining in solution. Optimization of the buffer following the principles outlined in Part 1 of this article gives the maximum adsorption of the capture reagent to the membrane.¹

Reduce the lateral wicking rate. Slowing the lateral wicking rate by the introduction of a viscosity modifier or by the use of a smaller-pore membrane effectively increases the concentration of the analyte in solution and hence improves the appearance of the capture line.

Cross-link the capture reagents. If the molecular weight of the applied protein is low (or if there are unfavorable surface properties), the use of a cross-linking agent can significantly enhance the level of protein binding observed. The cross-linking itself can either precede or follow application of the sample to the membrane.

The most common method is to cross-link the capture reagent to a carrier protein that will not cause any nonspecific interactions prior to application. The proteins typically used for this purpose are BSA or keyhole limpet haemocyanin (KLH). It is possible to use a cross-linking agent after capture reagent application (e.g., a glutaraldehyde wash step). However, the developer should ensure that all active groups introduced to the membrane are efficiently blocked before performing the test (e.g., in the case of glutaraldehyde, an ammonium sulfate wash blocks any free aldehyde groups). More specific examples of cross-linking chemistries can be found in the literature or in suppliers' catalogs.^10–12

Uneven Capture-Line Wetting

The uneven rewetting of the capture line can have very serious consequences for the developers of rapid diagnostic assays. If the membrane rewets unevenly, the capture line will be seen to be striped. In the worst case, it is possible for the capture line to be more hydrophobic than the surrounding membrane (normally due to the removal of membrane surfactant in washes). In this case it is possible for "submarining" to occur—this is when the sample runs through the membrane until it reaches the capture line, the higher hydrophobicity of the capture line effectively stopping lateral flow. The sample may then run along the plastic support of the membrane (which is more hydrophilic), and then reenter the membrane above the capture line where the membrane is more hydrophilic. This can result in a capture line that has no or very little sample penetration, with obvious problems for test sensitivity and selectivity.

Causes. Capture-line wetting may be uneven because of a defect in the membrane itself or because of a flaw in the test-strip manufacturing process.

Membrane drying is uneven. The rewetting of a nitrocellulose membrane is usually dependent upon the degree of drying the membrane has undergone. If the relative rates of drying vary across a membrane due to variability of the drying conditions to which the membrane was exposed during manufacture, the rate at which the membrane rehydrates will vary across the membrane.

Table II. The efficiency of different blocking techniques.

The application of an aqueous sample can also affect the distribution by washing any water-soluble residues away from the point of application. Any variation in the character or concentration of these residues will affect the rewetting rate of the membrane.

Hydrophobicity of the membrane is uneven. Perhaps the most significant factor for uneven capture-line wetting is hydrophobicity variation in the membrane. The rate of membrane rehydration is strongly influenced by the presence of hydrophobic or hydrophilic residues on the membrane surface. These residues can be introduced by membrane posttreatments (e.g., the introduction of a rewetting agent), hydrophilic materials added during manufacture, or additives in the striping buffer. The distribution of these hydrophilic materials is a factor in the evenness of the initial application and any subsequent migration of the hydrophilic materials through the membrane during storage.

Membrane pore structure is inconsistent. The pore structure of nitrocellulose membranes is a function of the parameters in the casting machine during the casting process. Uneven airflow within the casting machine may cause a variation within the membrane. As nitrocellulose-casting machines normally use laminar airflow, uneven airflow is typically seen as a variation across the width of the machine. This can be seen as a variation in performance between adjacent rolls cut from the larger master roll.

The developer cannot solve this problem without performing 100% quality control checks on incoming materials. The best compromise is to ensure that across-machine variation is evaluated adequately during the development process.

Solutions. As mentioned above, if a defect in the membrane is the cause of uneven rewetting, the only recourse is more-rigorous inspection of raw materials. If the problem is a result of the test manufacturing technique, however, there are several possible solutions.

Change the application buffer. Introduction of a mild surfactant to the striping buffer ensures that the capture line is in an evenly hydrophilic environment. This encourages even rewetting of the capture line. The choice of surfactant and its concentration is critical; effective results can be obtained using a low-concentration (~0.1%) SDS or sodium dodecylbenzolylsulfonate solution.

Perform a membrane-blocking step. The most common way to ensure even wetting of the capture line is to use a blocking technique. Blocking the membrane with a material that promotes rewetting of the membrane ensures rapid and even membrane rewetting. The effect of these blocking agents has been evaluated in product support literature.^13,14 The developer should investigate a range of blocking agents to find the most efficient for any particular test.

The method used for membrane blocking is also a significant factor in the success or failure of the blocking step. There are three points where a blocking agent can be applied (see Table II). The choice of method for inclusion of the blocking agent depends on the efficiency of the blocking step coupled with the cost of achieving the solution. As a compromise, therefore, the second technique (inclusion of the blocking agent in the conjugate pad or in a sample application pad) is often chosen. While it is less efficient than blocking the entire membrane, the procedure is operationally simple.

Change the membrane. A membrane with a surfactant posttreatment is more likely to show uneven capture line rewetting than a membrane without one. Application of the capture reagent solution may wash the surfactant away from the membrane surface. The removal of the additional rewetting agent can significantly affect the rewetting properties of the capture line. When no additional rewetting agent is added to the membrane surface, the line rewetting is likely to be consistent. However, the overall speed of rewetting may be slow.

Capture Line Too Thick

If the capture line is too thick, test results may be difficult to interpret (see Figure 3). The capture line may well show significant intensity variation across its width, probably with an intense front and back edge. For an inexperienced user the potential variation across the width may be confusing.

Figure 3. Problems with protein binding are typically visible in the capture line of an assay's test result, as in these examples.

Causes. At first glance the cause of too thick a capture line may seem obvious: too thick a line is being applied. In reality, this is only one of many possible causes.

The capture line is spreading too far after its application. If the protein being applied favors remaining in solution rather than attaching to the solid phase, it is possible that the protein molecules will move with the solvent front of the application buffer. In such a case the capture line may be extremely wide or have sharp edges with a relatively diffuse middle portion. The latter effect (colloquially known as a "coffee ring effect") is caused by migration of the protein with the solvent and increasing concentration of the protein as the solvent evaporates.

The capture reagent is washed away when the sample is applied. If the physical attachment of the protein to the membrane is too weak, or if a surfactant is present in the system, the capture line itself can be washed away as the sample wicks up the membrane. The displaced protein can then reattach to the nitrocellulose above the capture line.

Too much protein is applied. If we assume that the protein saturates the available nitrocellulose, the effect of excess protein levels will be the spread of the protein capture line.

The application aperture is set too wide. The protein will normally attach to the membrane at the point of application. If the settings are such that the sample is applied over a wide area, then the capture line will be wide.

The membrane is dried insufficiently after capture line application. If the membrane is dried insufficiently, the capture reagent will not be efficiently immobilized.¹ The application of the test sample may wash the capture reagent away from the point of application. The capture line may therefore become significantly wider due to the spreading of the capture reagent.

Solutions. The possible means of achieving a line of the desired thickness involve varying many of the parameters already discussed. Changes in the membrane, the buffer, the blocking agent, or the manufacturing conditions may be appropriate.

Change the application buffer. Optimization of the application buffer, both to minimize protein stability in solution and to maximize the viscosity of the application buffer, will produce the sharpest capture line on the membrane. The slower the protein solution flows, the greater the chance that the protein will bind close to the point of application (see Figure 4).

Figure 4. Varied results from capture lines of 1mg/ml mouse IgG applied using different buffers: (a) 10 mmol phosphate, pH 7.2; (b) 10 mmol phosphate + 3% methanol, pH 7.2; (c) 10 mmol phosphate + 150 mmol NaCl + 3% methanol, pH 7.2; (d) 50 mmol phosphate + 150 mmol NaCl + 1% BSA, pH 7.2; (e) 50 mmol phosphate + 150 mmol NaCl, pH 7.2; (f) 50 mmol phosphate + 150 mmol NaCl, pH 6.0. All samples were detected by a 40 nmol gold-conjugated goat antimouse IgG antibody.

Use a smaller-pore membrane. Smaller-pore membranes have a greater surface area of nitrocellulose per unit area of membrane, and the wicking away from the point of application is therefore slower than in larger-pore membranes. The combination of these effects means that the protein line is relatively narrow.

Change the membrane. Selecting a membrane with higher binding capacity will improve line sharpness.

Change the membrane blocking conditions. The blocking agents used are normally chosen because they interfere with protein binding. If the blocking materials are present in high quantities, the capture line binding may experience some interference effects. Lowering the concentrations of the blocking agents and investigating alternatives may achieve a better result.

Use less protein. Reducing the protein content of the system will reduce the area of nitrocellulose occupied by the capture reagent. The protein binds to the first available unoccupied surface of nitrocellulose.

Set application aperture more narrowly. Applying the protein solution from a narrower aperture will initially apply the protein to a smaller area of membrane, encouraging the formation of a thinner line.

Increase drying of the membrane. The strength of protein binding to nitrocellulose has long been linked to drying. Drying the membrane more vigorously after the protein reagent has been applied may well therefore reduce the chance of the capture protein being washed away when the sample is applied.

Capture Line Too Thin

A capture line that is too thin may give a false-negative test result. This case may well be true for cases where there is significant proportion of eye disease in the target market, this would be especially prevalent where the target market is the elderly of many third world areas. A line that is very thin would be very difficult to read accurately. Perhaps the optimal line width would be in the region of 0.8 to 1 mm wide. If the capture line is of the order of 0.2-mm wide then reading even a strong positive result can be difficult.

Causes. The causes of too thin a capture line are mainly the opposites of those mentioned above.

Protein binds too rapidly after application. Binding of the protein to the membrane immediately after application results in a capture line that is very narrow, although the application-buffer front will be significantly wider than the capture line itself.

Protein in the application system is insufficient. If the protein shows good binding properties to the membrane surface, a low concentration of protein in the capture reagent gives narrow lines following application. All the protein will bind in a very small area before it has had a chance to diffuse away from the application point.

The application aperture setting is too narrow. With optimal protein binding, the protein binds immediately to the area to which it is applied and does not spread. If the capture reagent is applied over a very narrow area, the protein is unlikely to move away from the application point before binding.

Solutions. The solutions to the problem of a too-thin capture line are essentially the opposite of those to a too-thick line.

Add bulking protein. Introduction into the application buffer of a nonspecific protein, with membrane-binding qualities similar to those of the capture reagent, will provide competition for the protein-binding sites on the membrane and hence encourage formation of a wider line.

Use more protein. Increasing the protein content in the application buffer will cause a wider line. The excess protein will move farther to find empty binding sites on the nitrocellulose, leading to saturation of a larger area of membrane.

Use a larger-pore membrane. A larger-pore membrane accelerates lateral diffusion of the capture reagent and also has a lower available surface area of nitrocellulose than a smaller-pore membrane. The line will therefore be wider.

Change the protein buffer. If the buffer is adjusted to increase protein solubility, the protein will remain in solution longer and travel farther before binding to the membrane. The capture line width therefore increases with increasing protein solubility.

Change the membrane. A membrane that shows a lower binding affinity for the chosen protein will allow the formation of a wider protein line for a given level of protein applied.

Use a wider application aperture. Spraying a wider line of the protein solution onto the membrane will cause the protein to bind over a wider area.

Uneven Line Intensity

A test with uneven line intensity can be a quality-control nightmare. This problem also drives up cost, since many batches will be rejected. Unless 100% of the devices undergo QC testing, the problems may not be detected before the product reaches the customers. The least damaging effect may be wastage due to rejection of batches where the majority of the product works acceptably. A more significant problem will be lack of selectivity in test kits sold, which can lead to loss of market confidence or the removal of regulatory approval.

Causes. While a line of uneven intensity may not look much different from a line that is too thick or too thin, the possible causes of this problem are quite distinct from those already discussed.

Membrane hydrophobicity varies. Hydrophobicity in the nitrocellulose membrane, either before or after capture line application, can result in the formation of a variable capture line (see Figure 5). Depending upon the reason for the hydrophobicity, the problem can manifest itself as a striped or uneven capture line or as a significant variation in line widths between tests. Common causes of membrane hydrophobicity include poor storage of unblocked material, solvent interaction, or the washing away of the blocking agent during capture line application.

Pressure varies in application system. A pressure (or flow-rate) variation in the application system can often cause uneven line width (sometimes called "blobbing") or line intensity. This effect is caused by variation in the quantity of protein applied. Blobbing or differences in line intensity reflect the protein saturation limit for the membrane used. The result is a test with very poor within-batch reproducibility.

Protein solution is poorly mixed. Variations in the concentration of the protein solution applied result in problems similar to those caused by pressure variation. The line width and intensity vary along the length of the applied line.

Protein precipitates. Precipitation of the protein in the application system results in variation of the protein concentration (see effects mentioned previously) or clogging of the membrane by particulates (see next section). The particles can also cause blocking of small-bore print heads, such as those found in many piezoelectric printing systems.

Flow in the strip is interrupted. Flow problems in nitrocellulose membranes can be due to two major causes: clogging of pores by particulates and poor contact between components in the lateral flow strip.

Clogging of pores results in the membrane appearing striped, particularly at the capture line. Since the flow along a membrane strip is largely linear, blocked pores result in areas of the capture line where the sample does not penetrate and therefore no color develops. The formation of particulates (often by protein precipitation) should therefore be eliminated if possible.

The flow in a lateral flow assay is dependent upon continuous capillary contact among the various components of the test system. If contact among the test components is poor or nonexistent, the amount of sample passing through the affected area of the lateral flow device will be significantly reduced when compared with an area that has good contact. This problem can result in a capture line that appears striped.

Figure 5. Water present during the application of posttreatments can make sections of the membrane hydrophobic, resulting in striations or intensity variations in the capture line.

Storage conditions are suboptimal. The major effect of improper storage conditions is the loss of hydrophilicity from the membrane. This loss can result from any of several defects in the manufacturing process. Volatile rewetting agents may migrate (if added during manufacture), residual moisture may be lost from the surface (if no rewetting agent is added), or solvent, adhesive, or plasticizers may interact with the membrane. The exact cause can be hard to identify. The effect is often seen as a reduction in the lateral flow rate of the membrane or the appearance of hydrophobic spots on the surface.

Membrane pore structure is uneven. As mentioned previously, a variation of the pore size across the membrane will result in flow rate and surface area variations across the width of the membrane. Both of these factors can result in a significant variation in the line intensity seen at the capture line.

Solutions. The solution to this problem may be chemical, mechanical, or operational, depending on the cause. If the underlying cause is a variation in the membrane pore structure across the roll of membrane, there is no solution that can be practiced by the end user. The membrane will have to be replaced with a batch that has a more consistent pore structure.

Treat the membrane. Pretreating a nitrocellulose membrane with carefully selected rewetting agents can solve problems caused by hydrophobicity and membrane storage.¹³ The rewetting agents added by the blocking step will ensure an even capture line. Treatment of the membrane will result in more consistent performance throughout the roll, which will significantly improve the test consistency and performance after long-term storage.

Optimize the membrane storage. For nitrocellulose membranes that have no added rewetting agent, storing the membranes in controlled conditions, both before and after capture line striping, results in more consistent line intensity in the finished product. Suggested storage conditions for nitrocellulose membranes are 40 to 60% relative humidity and between 20° and 25°C. Under these conditions, nitrocellulose membranes are stable for several years. Equilibration of the membrane in a humid atmosphere before striping will result in more even and consistent line application.

Optimize the application system. For an even capture line across a membrane, the application system should have good pressure or volume control and allow mixing of the solution being applied to ensure consistency.

Minimize protein precipitation. If the buffer conditions used for protein application are too aggressive, the protein may precipitate. This event will result in the formation of particulates and reduce the total amount of protein in solution. While it is preferential to make the protein unstable in solution to ensure rapid and complete binding to the membrane, if the protein is too unstable the system becomes unusable. The buffer should be made more favorable for protein solubility (e.g., increase salt content slightly, adjust pH, or reduce the level of coprecipitating agent).

Ensure contact among system components. The level of contact among the various components is difficult to measure. However, the pressure in the system at the overlap between pads is critical. If the pressure is too high, the materials can be crushed, potentially blocking pores. If the pressure at the junction is too low, contact among the various pads may be poor. The amount of pressure required should be determined by experimentation, as differences in pad thickness and the relative compressibility of the materials will both be relevant in optimizing the system. Different tests therefore require reoptimization if any of the materials are changed.

Conclusion

While many problems can occur with protein application to a nitrocellulose membrane, it is possible to produce a highly consistent line that has very high intensity with no nonspecific interactions. The basic techniques for optimization of protein binding are straightforward. Good results can be obtained if a carefully designed and controlled series of experiments is performed.

One common mistake is use of the same conditions for a range of different proteins and tests. Most membrane-based rapid immunochromatographic assays on the market are unique, and the conditions required to give the optimal results are equally unique. A willingness to investigate the optimization of protein binding for each assay developed is key to obtaining the best results.

References

1. KD Jones, "Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 1: Principles," IVD Technology 5, no. 2 (1999): 32ם41.

2. The Latex Course, 1994 (Fisher, IN: Bangs Laboratories, 1994).

3. Immunogold Reagents, catalogue 2 (Cardiff, UK: BBInternational, 1996).

4. Gold Conjugates, Diagnostic Products Guide (Durham, UK: Jamare Biotest International, 1998).

5. E Harlow and D Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, NY: CSHL Press, 1988).

6. TG Wreghitt and P Morgan-Capner (eds.), ELISA in the Clinical Microbiological Laboratory (London, UK: PHLS, 1990).

7. JM Polak and S Van Noorden, Immunochemistry: Modern Methods and Applications (Bristol, UK: Wright, 1987).

8. Short Guide for Developing Immunochromatographic Test Strips (Bedford, MA: Millipore Corp., 1996).

9. KD Jones and AK Hopkins, "Protein Binding in Nitrocellulose Membranes 0.2 to 12 µm: A Comparison of Commercially Available Membranes for a Novel Flow-Through Immunoassay," poster no. 21 (presented at the 1998 Annual Meeting of the American Association for Clinical Chemistry, Chicago, August 2–6, 1998).

10. G Hermanson, Bioconjugate Techniques (San Diego: Academic Press, 1996).

11. Products Catalogue, 1999 (Rockland, IL: Pierce Chemical Company, 1998).

12. RP Haugland, Handbook of Fluorescent Probes and Research Chemicals (Eugene, OR: Molecular Probes Inc., 1996).

13. KD Jones and AK Hopkins, "Evaluation of the Efficiency of a Range of Membrane Blocking Agents for Nitrocellulose Membrane Based In Vitro Diagnostic Disease," poster no. 3 (presented at the 1998 Annual Meeting of the American Association for Clinical Chemistry, Chicago, August 2—6, 1998).

14. KD Jones, Technical Application Notes, nos. 1–3 (Maidstone, UK: Whatman International Ltd., 1997–1998).

Kevin D. Jones, PhD, is a research scientist for diagnostics at Whatman International Ltd. (Maidstone, Kent, UK).