The Elisa guidebook

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most usual regimes involve incubation at 37¡ãC for 1¨C3 h or overnight at 4¡ãC, or a combination of the two, or incubation (more vaguely) at room temperature for 1¨C3 h, (see ref. 2 for a typical study. There are many more variations, and, ultimately, each scientist must titrate a particular antigen to obtain a standardized regime. Increasing the temperature may have a deleterious effect on antigen(s) in the coating stage, and this may be selective, so that certain antigens in a mixture are affected whereas others are not. Rotation of plates can considerably reduce the time needed for coating by increasing the rate of contact between the coating molecules and the plastic.

1.3¡ª

Coating Buffer

The coating buffers most used are 50 mM carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mM phosphate-buffered saline (PBS), pH 7.2

(2). Different coating buffers should be investigated when problems are encountered or compared at the beginning of assay development. From a theoretical point of view, it is best to use a buffer with a pH value 1 to 2 units higher than the isoelectric point (pI) value of the protein being attached. This is not easy to determine in practice since antigens are often complex mixtures of proteins. By direct study of the effects of different pHs and ionic strengths, greater binding of proteins may be observed. An increase in ionic strength to 0.6 M NaCl in combination with an optimal pH was found to give better results for the attachment of various herpes simplex viral peptides (3). Proteins with many acidic proteins may require a lower pH to neutralize repulsive forces between proteins and the solid phase, as shown in ref. 3, in which the optimal coating for peptides was pH 2.5¨C4.6. PBS, pH 7.4, is also suitable for coating many antigens. Coating by drying down plates at 37¡ãC using volatile buffers (ammonium carbonate) and in PBS is often successful, particularly when relatively crude samples are available. Some antigens pose particular problems, including some polysaccharides, lipopolysaccharides, and glycolipids. In cases in which it proves impossible to directly coat wells with reagent, initial coating of the wells with a specific antiserum may be required. Thus, sandwich (trapping) conditions must be set up. Passive adsorption has several theoretical, although not necessarily practical, drawbacks. These include desorption, binding capacity, and nonspecific binding.

1.4¡ª Desorption

Because of the noncovalent nature of the plastic/protein interactions, desorption (leaching) may take place during the stages of the assay. However, if conditions are standardized, leaching does not affect the viability of the majority of tests. There are some reports that the vigorousness of washing at the

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various stages of assays (including that after coating) affects the assays through stripping of protein; however, I have not encountered this problem.

1.5¡ª

Binding Capacity

It is important to realize that plastic surfaces have a finite capacity for adsorption. The capacity for proteins to attach to microplate wells is influenced by the exact nature of the protein adsorbed to the specific plate used. Saturation levels of between 50 and 500 ng per well have been found valid for a variety of proteins when added as 50-µL volumes. The effective weight of protein per well can be increased if the volume of the attaching protein is increased, effectively increasing the surface area of the plastic in contact with the coating antigen. In cases in which there is an obvious discrepancy between the actual concentration of protein added (where known) and the values just given, then the titration of the ELISA should be re-examined. Thus, if concentrations of, e.g., 1¨C10 mg/mL (or greater) of sample are needed to coat wells, this is not have an ideal situation.

1.6¡ª

Nonspecific Binding

Unlike antigen/antibody interactions, the adsorption process is nonspecific. Thus, it is possible that any substance may adsorb to plastic at any stage during the assay. This must be considered in assay design because reagents may react with such substances. High levels of nonspecific binding can be alleviated through alteration of systems relying on direct adsorption of antigen and the use of sandwich techniques, in which specific antibodies capture and concentrate specific antigens.

1.7¡ª

Covalent Antigen Attachment

A variety of chemicals that couple protein to plastic have been used to prevent desorption, the antigen being covalently bound. These include water-soluble carbodiimines, imidoand succinimidylesters, ethanesulfonic acid, and glutaraldehyde. Precoating of plates with high molecular weight polymers such as polyglutaraldehyde and polylysine is another alternative (4,5). These bind to plates with a high efficiency and act as nonspecific adhesive molecules. This method is particularly useful for antigens with a high carbohydrate content since these normally bind poorly to plastic.

Generally, successful assays can be obtained without the need to link antigens to plates covalently. Specially treated activatable plates are now available. The use of covalently attached proteins does offer the possibility that plates could be reused. After an assay, all reagents binding to the solid-phase attached protein could be washed away after using a relatively severe washing procedure, e.g., low pH. The covalent nature of the bonds holding the solid-

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phase antigen would prevent this from being eluted. Provided this procedure did not destroy the antigenicity of the solid-phase attached reagent, the plates might be exploited after equilibration with normal washing buffers.

2¡ª Washing

The purpose of washing is to separate bound and unbound (free) reagents. This involves the emptying of plate wells of reagents followed by the addition of liquid into wells. Such a process is performed at least three times for every well. The liquid used to wash wells is usually buffered, typically PBS (0.1 M, pH 7.4), in order to maintain isotonicity since most antigen¨Cantibody reactions are optimal under such conditions. Although PBS is most frequently used, lower-molarity phosphate buffers (0.01 M) may be used, provided that they do not influence the performance of the assay. These buffers are also more cost-efficient.

In some assays tap water has been used for washing. This is not recommended because tap water varies greatly in composition (pH, molarity, and so forth). However, assays may be possible provided the water does not drastically affect the components of the test. Generally, the mechanical action of flooding wells with a solution is enough to wash wells of unbound reagents. Some investigators leave washing solution in wells for a short time (soak time) after each addition (1¨C5 min). Sometimes detergents, notably Tween-20 (0.05%) are added to washing buffers. These can cause problems in which excessive frothing takes place producing poor washing conditions since air is trapped and prevents the washing solution from contacting the well surface. When using detergents, care must be taken that they do not affect reagents adversely (denature antigen), and greater care is needed to prevent frothing in the wells. The methods used for washing are given next.

2.1¡ª

Dipping Methods

The whole plate is immersed in a large volume of buffer. This method is rapid but is likely to result in cross-contamination from different plates. It also increases the cost of washing solution.

2.2¡ª

Wash Bottles

Fluid is added using a plastic wash bottle with a single delivery nozzle, which is easy and inexpensive. Here the wells are filled individually in rapid succession and then emptied by inverting the plate and flicking the contents into a sink or suitable container filled with disinfectant. This process is repeated at least three times. Wells filled with washing solution may also be left for about 30 s before emptying.

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2.3¡ª

Wash Bottles Plus Multiple Delivery Nozzles

This is essentially as in Subheading 2. except that a commercially multiple delivery (usually 8) device is attached to the outlet of the bottle. This enable 8 wells to be filled at the same time.

2.4¡ª

Multichannel Pipets

The multichannel pipets used in the ELISA can be used to fill the wells carefully. The washing solution is contained in reservoirs .

2.5¡ª

Large Reservoir

The use of a large reservoir of washing solution is convenient. Here, a single or multiple nozzle can be connected to the reservoir via tubing so that the system is gravity fed. Care must be taken that large volumes of solution do not become microbially contaminated.

2.6¡ª

Special Hand-Washing Devices

Hand-washing devices are available commercially and involve the simultaneous delivery and emptying of wells by a handheld multiplenozzle apparatus. These are convenient to use but require vacuum creating facilities. In washing plates manually, the most important factor is that each well receive the washing solution so that, e.g., no air bubbles are trapped in the well, or a finger is not placed over corner wells. After the final wash in all manual operations, the wells are emptied and then blotted free of most residual washing solution. This usually is accomplished by inverting the wells and tapping the plate on to an absorbent surface such as paper towels, cotton towels, or sponge material. Thus, the liquid is physically ejected and absorbed to the surface, which is soft and therefore avoids damage to the plate.

2.7¡ª

Specialist Plate Washers

Specialty plate washers are relatively expensive pieces of apparatus that fill and empty wells. Various washing cycles can be programmed. These are of great advantage when pathogens are being examined in ELISA because they reduce aerosol contamination. Most of the methods involving manual addition of solutions and emptying of plates by flicking into sinks or receptacles must be regarded as potentially dangerous if human pathogens are being studied, particularly at the coating stage if live antigen is used. Also, remember that live antigens can contaminate laboratories where tissue culture is practiced. The careful maintenance of such machines is essential because they are prone to machine errors such as a particular nozzle being blocked.

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3¡ª

Addition of Reagents

Immunoassays involve the accurate dispensing of reagents in relatively small volumes. The usual volumes used in ELISA are in the range of 50 or 100 µL per well for general reagents, and 2¨C10 µL for samples. It is essential that the operator be fully aware of good pipetting technique and understand the relationships of grams, milligrams, micrograms, nanograms, and the equivalent for volumes, e.g., liters, milliliters and microliters. Thus, assays cannot be performed when there is no knowledge of how to make up, e.g., 0.1 M solutions. The ability to make accurate dilutions is also extremely important so that problems can be solved before you attempt ELISA or any other biological studies (e.g., having a 1/50 dilution of antiserum but needing to make up a 1.3500 dilution in a final volume of 11 mL).

3.1¡ª Pipets

The microtiter plate system is ideally used in conjunction with multichannel microtiter pipets. Essentially they allow the delivery of reagents via 4, 8, or 12 channels and are of fixed or variable volumes of 25¨C250 µL.

Single-channel micropipets are also required that deliver in the range of 5¨C250 µL. Samples are usually delivered by microtiter pipets from suitably designed reservoirs (troughs) that hold about 30¨C50 mL of solution.

General laboratory glassware is needed such as 5- and 25-mL glass or plastic bottles, and 10-, 5-, and 1-mL pipets.

3.2¡ª

Evaluating Pipet Performance

Pipetting errors are often a major cause of nonreliable test results in a diagnostic laboratory. A simple control technique is hereby proposed to circumvent this problem. At the beginning of each workday, the pipet should be checked for dust and dirt on the outside surfaces. Particular attention should be paid to the tip cone. No other solvents except 70% ethyl alcohol should be used to clean the pipet.

3.2.1¡ª

Short-Term Performance Evaluation:

Control of Pipet Calibration Using Graduated Tips

The exercise will take only a few minutes, but it will make you absolutely confident of preventing pipetting errors related to the function of the pipet.

1.Set the volume of the pipet as indicated in the accompanying manufacturer's instructions.

2.Place a graduated tip firmly on the tip cone.

3.Aspirate the specified volume of distilled water into the tip.

4.Hold the filled tip in a vertical position for a few seconds.

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Table 1

Example of Gravimetric Calibration Method Used for Three

Specified Volumes Pipet (40¨C200 µL)

		Nominal volume
	5:1	25:1	50:1
Measurement 1	5.12	25.08	50.21
Measurement 2	4.91	25.01	50.01
Measurement 3	5.07	25.27	50.19
Measurement 4	5.01	25.10	50.12
Measurement 5	4.98	24.89	50.00
Average	5.02	25.07	50.11
Accuracy (%)	0.36	0.28	0.21
Precision (%)	1.62	0.55	0.20

5.Check for leakage.

6.Check that the aspirated volume corresponds to the specified volume as indicated by tip graduation.

7.Repeat steps 1¨C6 at least five times.

8.Practice the reversed and nonreversed pipetting techniques.

3.2.2¡ª

Long-Term Performance Evaluation:

Control of Pipet Calibration Using Gravimetric Calibration Method

If the pipet is used daily, it should be checked every 3 mo. By means of the gravimetric calibration method (see Table 1), the pipet should be examined for leakage, accuracy, and precision.

Accuracy and precision can easily be calculated by the formulae in Subheadings 3.2.2.1. and 3.2.2.2. In contrast to commercial pipet calibration computer software, the conversion factor for calculating the density of water suspended in air at the test temperature and pressure are not considered. For calibration of multichannel pipets, examine each channel separately.

3.2.2.1¡ª Accuracy

(As Defined for This Exercise)

A pipet is accurate to the degree that the volume delivered is equal to the specified volume. Accuracy is expressed as the mean for replicate measurements:

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3.2.2.2¡ª Precision

(As Defined for This Exercise)

Precision refers to the repeatability of dispensed samples. It is expressed as the coefficient of variation (CV%).

in which S = standard deviation; and = mean weighing.

3.2.2.3¡ª Equipment Needed

The following equipment is needed:

1.Calibrated thermometer; to measure water temperature

2.Distilled water

3.Glass vessel with a volume 10 to 50 times that of the test volume

4.Analytical balance (calibrated?)

5.Pipet (labeled) and tips

Conduct the test on a vibration-free surface covered with a smooth, dark, nonglared material. Work in an area that is free of dust.

3.2.2.4¡ª Procedure

1.Set the volume of the pipet as indicated in the accompanying manufacturer's instructions.

2.Place a pipet tip firmly on the tip cone.

3.Aspirate the specified volume of water into the tip.

4.Hold the filled tip in a vertical position for a few seconds and check for leakage.

5.Dispense the distilled water into a preweighed beaker and record the weight to the nearest tenth of a milligram. Repeat at least five times.

6.Calculate the results (accuracy, precision).

7.Record the results over time.

In theory, optimum accuracy and precision values approach zero. Note that the smaller the specified volume chosen for evaluation, the greater the effect of volume variation on accuracy and precision. Therefore, it is good laboratory practice to plot the results of accuracy and precision for a pipet's specified volume on a data chart over time.

Finally, you must decide which level of accuracy and precision can be met in your laboratory. This depends on what the pipet is used for. For the preparation of aliquots of serum bank samples, the pipetting error will not significantly matter. For ELISA, however, pipetting of a small, e.g., 10-µL volume for preparation of working conjugate dilutions requires the best accuracy and precision; bear this in mind because if you are pipetting 5 rather than 10 µL, you will obtain a double-diluted working conjugate dilution and, consequently,

Table 2

Troubleshooting of Pipetting Errors

Problem	Cause
Leakage	Tips are not compatible.
	Tips are incorrectly attached.
	Foreign bodies are between
	the piston O-ring and cone.
	Insufficient grease is on the
	tip cone and O-ring.
Innaccurate	The pipet is incorrectly operated.
dispensing	Tips are incorrectly attached.
	High-viscosity fluids are
	not present.

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Solution

Choose compatible tips.

Attach firmly.

Clean the tip cone, and attach new tips.

Clean and grease the O-ring and tip cone.

Apply grease.

Follow instructions.

Attach tips firmly.

Recalibrate the pipet using highviscosity fluids.

an unreliable assay result. As a rule, a volume error of 1% for volumes ³10 µL is still acceptable in a serodiagnostic laboratory.

3.2.2.5¡ª Recalibrating of Pipets

To recalibrate pipets, refer to the manufacturer's detailed instructions or contact the service representative.

3.2.3¡ª

Pipet Troubleshooting

Table 2 provides the most common problems encountered with pipetting, possible causes, and solutions.

3.3¡ª Tips

After the microplate, the tips are the most important aspect of ELISA and also an expensive component. Many thousands of tips might be needed to dispense reagents. Many manufacturers supply tips, therefore care must be taken to find tips that fit the available microtiter pipets.

Multichannel pipet tips are best accessed by placing them in special boxes holding 96 tips in the microplate format. The tips can be purchased already boxed (expensive), and then the boxes can be refilled by hand with tips bought in bulk. Sterile tips are available in the box format. Generally, tips should not be handled directly by hand. When restocking boxes or putting the tips on pipets, plastic gloves should be worn to avoid contaminating the tips.

Tips for dispensing in single-channel pipets have to be carefully considered. In cases in which small volumes (5¨C20 µL) are pipeted, the pipet manufacturer's recommended tips should be used. It is essential that the tips fit securely on the pipets and that they can be pressed on firmly by hand (avoiding

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their end). Particular care is needed when multichannel pipets are used to pick up tips from boxes since often one or two tips are not as securely positioned as the rest, which causes pipetting errors. The operator should always give a visual check of the relative volumes picked up.

When cost-efficiency is a factor, tips may be recycled after washing. However, it is not recommended that tips that have been in contact with any enzyme conjugate be recycled and these should not be placed with other tips used for other stages in ELISA. The washing of tips should be extensive, preferably in extensive acid or strong detergent solutions, followed by rinsing in distilled water. The cost/benefit of washing must be examined carefully, because the production of sufficient quantities of distilled water can be expensive. Tips should be examined regularly and damaged ones should be discarded. Figure 3 illustrates some practical aspects of pipetting in ELISA. Note that the training in pipetting techniques is extremely vital to the successful performance of ELISA.

3.4¡ª

Other Equipment

Several manufacturers supply microtiter equipment to aid multichannel pipetting, including tube and microtip holders. The former consists of a plastic box that carries 96 plastic tubes with a capacity of about I mL. The tubes are held in exactly the same format as a microtiter plate so that samples can be stored or diluted in such tubes and multichannel pipets can then be used for rapid transfer from the tubes. The tip holders involve the same principle, whereby tips for the multichannel pipets are stored in the 96 well format so that they can be placed on to multichannel pipets rapidly in groups of 8 or 12. Various reservoirs with 8 or 12 channels for separation of reagents are also available. These are useful for the simultaneous addition of separate reagents.

4¡ª Incubation

The reaction between antigens and antibodies relies on their close proximity. During ELISA, this is affected by their respective concentrations, distribution, time and temperature of incubation, and pH (buffering conditions). In any interaction, the avidity of the antibodies for the particular antigen(s) is also important. Two types of incubation conditions are common: (1) incubation of rotating plates (with shaking) and (2) incubation of stationary plates. These conditions affect the times and temperatures required for successful ELISAs and therefore discussed separately.

4.1¡ª

Incubation of Reagents While Rotating Plates

The effect of rotating plates is to mix the reactants completely during the incubation step. Since the solid phase limits the surface area of the adsorbed

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Fig. 3.

Factors influencing proper dispensing of samples into microtiter plate wells.

reactant, mixing ensures that potentially reactive molecules are continuously coming into contact with the solid phase. During stationary incubation, this is not true and mixing takes place only owing to diffusion of reagents. Thus, to allow maximum reaction from reagents in stationary conditions, greater incubation times may be required than if they are rotated. This is particularly notable when highly viscous samples, e.g., 1/20 serum, are being examined. This represents 5% serum proteins, and diffusion of all antibodies on to the solid phase may take a long time. This can be avoided if mixing is allowed throughout incubation. Similarly, when low amounts of reactant are being assayed, the contact time of the possibly few molecules that have to get close

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Fig. 4.

Continuous mixing enables maximum contact of molecules in liquid phase with those on solid phase. This alleviates the following problems of: temperature¡ª the close approximation of antigen and antibody is necessary to allow binding. Increasing temperature under stationary conditions increases diffusion rate of molecules and variations in temperature will affect rate and variation in the test (e.g., stacking plates); variation in handling¡ª plates may be unequally moved

during incubation causing unequal mixing. Different operators use different techniques that are not controllable (e.g.., plates may be tapped or other operators may move the pates); viscosity effects¡ªwhen samples are of different viscosities, this may affect diffusion of molecules under stationary conditions; times of incubation¡ªthese can be reduced under rotationary conditions; and detection

of low concentrations¡ªthis is increased by rotation.

to the solid-phase reactant is greatly enhanced by mixing throughout incubation. Simple and very reliable rotating devices are available with a large capacity for plates. Shakers with limited capacity (e.g., four plates) are also available.

Figure 4 gives the advantages of rotation. Rotation allows ELISAs to be performed independently of temperature considerations. The interaction of antibodies and antigen relies on their closeness, which is encouraged with the mixing during rotation. Stationary incubation relies on the diffusion of molecules and thus, is dependent on temperature. Therefore standardization of temperature conditions is far more critical than when rotation is used.

The effect of temperature also has implications when many plates are stacked during incubation since the plates heat up at different rates depending on their position in the stack. The wells on the inside may take longer to equilibrate than those on the outside, which has a direct effect on the diffusion conditions, which, in turn, affects the ELISA. This is negated by rotation because there is the same chance of molecular contact in all wells.

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4.2¡ª

Stationary Incubation Conditions

Assays may be geared to stationary conditions, although the exact times and temperatures of incubation may vary. The temperatures for incubation are most commonly 37¡ãC, room temperature (on the bench), and 4¡ãC. Usually the time of incubation under stationary conditions reflects which incubation temperature is used. Therefore, at 4¡ãC, a longer incubation might be given (overnight). In general, most incubations for stationary assays involving the reaction of antigen and antibodies are 1¨C3 h at 37¡ãC. Sometimes these conditions are combined so that one reagent is added for, say, 2 h at 37¡ãC followed by one overnight at 4¡ãC, usually because this produces a convenient work schedule. When incubation is performed at room temperature, care must be taken to monitor possible seasonal variations in the laboratory, since temperatures can be quite different, particularly in nontemperate countries. Direct sunlight should also be avoided, as must other sources of heat, such as from machinery in the laboratory. As already stated, how the plates are placed during stationary incubation should be considered. Ideally, they should be separated and not stacked.

In assays, the plates should also be handled identically and there should be no tapping or shaking of the plates (including accidental nudging or movement by other personnel), because this will allow more mixing and interfere with the relative rate of diffusion of molecules in different plates. Regular handling can be a primary cause of operator-to-operator variation.

Under mixing conditions, most antigen/antibody reactions are optimum after 30 min at 37¡ãC, so that assays can be greatly speeded up with no loss in sensitivity. This is not true under stationary conditions. Care must be taken to consider the types of antibodies being measured under various conditions since ELISAs rarely reach classical equilibrium conditions. Figure 5 illustrates the factors affecting ELISA under stationary conditions.

5¡ª

Blocking Conditions and Nonspecific Reactions

Measures must be taken to prevent nonspecific adsorption of proteins to wells from samples added after the coating of the solid phase before, during, or as a combination of both times. Nonspecific adsorption of protein can take place with any available plastic sites not occupied by the solid-phase reagent. Thus, if one is assessing bovine antibodies in bovine serum, and bovine proteins other than specific antibodies bind to the solid phase, antibovine conjugate will bind to these and give a high background color.

Two methods are used to eliminate such binding. One is the addition of high concentrations of immunologically inert substances to the dilution buffer of the added reagent. Substances added should not react with the solid-phase antigen nor the conjugate used. Commonly used blocking agents are given in Table 3, and they act by competing with nonspecific factors in the test sample

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Fig. 5.

Effects when incubating stationary plates.

for available plastic sites. The concentration used often depends on the dilutions of the test samples; thus, if 1/20 serum is being tested (5% protein) then blocking agents have to be at high-concentration to compete successfully, or have an increased binding potential as compared to the nonspecific substance. Such blocking agents can also be added as a separate step before the addition of the sample; this increases the competing ability of the blocker. Nonionic detergents have also been used to prevent nonspecific adsorption. These are used at low concentration so as to allow interaction of antigen and antibody. Occasionally, both detergents and blocking substances are added together.

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