The Elisa guidebook

We can examine sera for antibodies by using full dilution ranges or at a single dilution (spot test). This approach has already been described for the indirect ELISA (see Subheadings 3.6. and 3.7.4.). The methodology for capture assays is similar except that the antigen is presented to the test sera after being captured by an antibody coating the microtiter wells. The optimum amounts of capture antibody and antigen are determined as in Subheadings 5.1.5.1.¨C5.2.5. After plates have been prepared with an optimal amount of antigen, the stages following this are very similar to the indirect ELISA.

Set up the capture assay to present optimal amounts of guinea pig IgG. After washing the plates, use the same already examined in Subheading 3.6. (rabbit anti-guinea pig sera) in a similar way as described from stages 6¨C15 in Subheading 3.6. This entails making dilution ranges of sera, incubation, washing plates, addition, and incubation with anti-rabbit conjugate, washing, and addition if substrate/chromophore. Compare the data obtained with that in

Subheading 3.6., Table 4.

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Repeat the same procedure but this time making spot dilutions of the various rabbit sera as in Subheading 4.3. Compare the data with that in Subheading 4.4., Table 6.

5.4¡ª

Problems Using Capture Assays

1.Care must be taken to examine whether any of the reagents interact. Unexpected crossreactions can be found with immunological reagents. For example, the conjugated antibodies might react with other species other than which they were prepared. There are crossreactions between certain species so that conjugates against cow proteins will react with sheep and goat proteins. Thus, in such a system the use of sheep or goat Ig as a capture antibody precludes the use of antibovine conjugates to detect the reaction of bovine antibodies with a particular antigen.

2.When relatively crude antigens are captured, contaminating proteins may also be trapped, which interfere with the assay. As an example, when purified FMDV is injected into an animal there is a specific response against the virus, but also a response against contaminating bovine serum proteins that are present in extremely low amounts, which comes from the tissue culture medium. Such sera used as capture reagent will capture not only virus but also bovine proteins. Thus, in typing exercises using tissue culture or bovine epithelial samples, a high quantity of bovine protein is captured. The use of antibovine conjugates to detect bound bovine serum in a trapping assay therefore also binds to the trapped bovine protein giving high backgrounds. In the typing assay proper, guinea pig sera are prepared as the second typing detecting sera. These also bind bovine proteins and therefore detect bound bovine protein to the capture antiserum. Again specific typing is affected. However, the second antibody can be treated to remove the crossreactivity either by adding a high concentration of the crossreactive protein to the reagent (in this case 1 mL of normal nonimmune bovine serum is added to 1 mL of typing guinea pig serum), or by using affinity reagents in which bovine serum is attached to a solid phase (e.g., agarose beads), which can be incubated with the serum so that the crossreactive antibodies are removed after incubation and separation of the beads by centrifugation. Or, as is most common, the test may be made using blocking buffers containing high levels (around 5%) of the crossreactive protein.

6¡ª

Competitive ELISA

The direct, indirect, and capture ELISAs have now been examined. You should be able to optimize the conditions of the tests and be able to use them to measure antigen or antibody in a variety of formats. Competitive ELISAs involve the principles of all these types of assay.

Basically, they involve methods that measure the inhibition of a reactant for a pretitrated system. The degree of inhibition reflects the activity of the unknown. We can therefore measure antibody or antigen, and even compare

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small differences in the binding of antigens or antibodies so that antigenic subtyping may be performed by comparing the relative avidity of one antiserum for two antigens in the same system. As a reminder, let us consider the competitive assays based on the indirect test and the trapping test for the detection of antigens or antibodies in a diagrammatic way.

6.1¡ª

Indirect Assay:

Antigen Detection by Competition

6.1.1¡ª

Reaction Scheme

A pretitrated indirect assay with optimal Ag1, AB, and conjugate, is competed for by Ag2, as a dilution range in the liquid phase. If Ag2 can bind AB, it will prevent AB binding that would normally react with Ag on the plate. The maximum expected OD for the pretitrated system without competitor (Ag2) is therefore reduced in the presence of the competitor Ag2. The degree of inhibition of the pre-titrated reaction is proportional to the relative amount of the competitor.

6.2¡ª

Indirect Assay:

Antibody Detection by Competition

6.2.1¡ª

Reaction Scheme

A pretitrated system is challenged by a dilution range of Ab. The competing antibody is from a species that is not the same as that of the AB in the optimized system, and obviously the Anti-AB*E should not react with the Ab. The degree of inhibition of the pretitrated system depends on the concentration and interaction of the Ab competitor with the Ag, this time on the solid phase.

The direct assay could also be used for both these assays. Note that in the direct assay, any species of competing antibody can be used since the AB is labeled with conjugate. Such assays are becoming increasingly relevant when mAbs are used.

6.3¡ª

Capture Assay:

Antigen Detection by Competition

6.3.1¡ª

Reaction Scheme

The capture assay is optimized to detect the Ag I trapped on the plates using Ab. The competition is achieved in which Ag2 is mixed with the Ab in the liquid phase. If this reacts, the amount of Ab available for reaction with the trapped Ag1 is reduced.

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6.4¡ª

Capture Assay:

Antibody Detection by Competition

6.4.1¡ª

Reaction Scheme

The capture antibody is optimized to bind Ag, which is detected by a constant amount of AbX (from animal species X). The competition involves the reaction of the Ag with antisera from species Y (which should not interact with the conjugate anti-AbX), in the liquid phase. The remaining Ag is then trapped and titrated with the AbX and the conjugate. A reduction in the expected OD for the system without any AbY represents competition.

Next, we discuss the following assays:

1.Direct competition assay¡ªantigen detection and quantification.

2.Indirect assay¡ªantigen competition.

3.Indirect competition assay¡ªantibody detection.

a.Full titration curves.

b.Spot test assessment of sera.

7¡ª

Direct Competitive ELISA for Antigen Detection and Quantification

The direct assay for antigen detection and quantification has assumed an increased importance with the development of mAbs. A single mAb can be the one reagent that dominates a diagnostic assay, and therefore mAbs are worth labeling for use in an assay. The specificity of the assay is ensured and relatively crude antigenic preparations can be coated for use in a direct test format (provided enough antigen attaches). This is also relevant to polyclonal antibodies. The demonstrated assays involve IgG/anti-IgG systems.

7.1¡ª

Learning Principles

1. Optimizing of homologous system.

2. Understanding competition curves.

7.2¡ª

Reaction Scheme

+= addition and incubation steps

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Table 11

Data from CBT of Guinea Pig IgG and

Anti-Guinea Pig Enzyme Conjugate in Subheading 1.1.

This exercise will most simply demonstrate the principles involved with competitive assays.

7.3¡ª

Materials and Reagents

1.Ag1: guinea pig IgG at 1 mg/mL for attachment to solid phase.

2.Ag2: Two samples: (a) guinea pig IgG (known concentration) and (b) rabbit IgG at 1 mg/mL.

3.Ab*E: rabbit anti-guinea pig IgG conjugated to HRP.

4.Microplates.

5.Multichannel, single-channel 10and 1-mL pipets.

6.Carbonate/bicarbonate, pH 9.6, 0.05 M.

7.PBS 1% BSA, 0.05% Tween-20.

8.Solution of OPD in citrate buffer.

9.Hydrogen peroxide.

10.Washing solution.

11.Paper towels.

12.Small-volume bottles.

13.1 M sulfuric acid in water.

14.Multichannel spectrophotometer.

15.Clock.

16.Graph paper.

17.Calculator.

7.4¡ª

Practical Details

Repeat the exercise in Subheading 1.1., involving the CBT of antigen and enzyme-linked antibody. You should obtain a similar picture. Compare your results to those in Table 11.

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Fig. 28.

Data from Table 11 relating antigen titrations at different concentrations of conjugate.

The labeled conjugate dilutions are made from A to H, IgG is diluted 1¨C11, 12 has no antigen. Figure 28 presents a plot of a CBT of guinea pig IgG against rabbit anti-guinea pig conjugates.

7.4.1¡ª

Assessment of Data and Choice of Conditions for Competition

We are trying to compete the same antigen (guinea pig IgG) and a different antigen (IgG from the rabbit), for a pretitrated homologous solid-phase reaction. The ultimate sensitivity of the assay depends on the exact relationship of the antibody and antigen attached to the solid phase. If we use too much antibody, so that it is in excess of that required to saturate the antigen, we will have a quantity of free antibody that may bind to the competitor, and there will still be an amount left to react with the solid-phase IgG. Thus, competition will only occur when extremely high concentrations of competitor are used.

This can be illustrated by examination of the titration curves in Fig. 29. Note that the plateau region represents excess antibody for any given antigen concentration. The OD and extent of these plateau regions vary according to the exact amount of antigen attached to the solid phase. As we reduce the antigen the plateau height values decrease. At the highest concentrations of antigen, the titration curves are similar for different antibody concentrations,

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Fig. 29.

Illustration of regions of conjugate excess and nonexcess when titrating conjugate against constant concentration of antigen.

indicating that the antigen and antibody are behaving at maximum saturating levels. On dilution of the antigen, we see that the plateau height is reduced, even where we know that the antibody is available for higher OD values (curves 3 and 4). Here, the antigen is the limiting factor in color development. In the competition assay, a maximum plateau height dependent on the amount of antigen attached of around 1.0¨C1.5 OD should be selected. In other words, find out which dilution of antigen produces serum titration curves giving a maximum plateau of these values, e.g., curves 3 and 4. From this titration curve, we need to estimate the dilution of antibody yields about 70% of the maximum plateau OD. Using curve 1, we can illustrate this, as shown in Fig. 30.

7.4.2¡ª

Estimation of Antibody Dilution to Be Used in Competition Assay

The conditions are now set for competition. We have: (1) the antigen dilution as for curve, and (2) the antibody dilution estimated as shown in Fig. 30.

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Fig. 30.

Estimation of conjugate dilution for use in competition stage.

7.5¡ª

Competition Assay Proper

1.Prepare the optimum antigen coated with guinea pig IgG.

2.Wash the plates.

3.Dilute the guinea pig Ig (homologous competitor) and the rabbit Ig (heterologous competitor) to 40 µg/mL in blocking buffer.

4.Add 50 µL of blocking buffer to each well of the Ig-coated plate.

5.See the plate design in Fig. 31. Make a twofold dilution range of the guinea pig and rabbit Ig by adding 50 µL of the Igs to the first row 1. Do this in triplicate (three rows for the guinea pig Ig¡ª1A, B, and C; and three rows for the rabbit Ig¡ª1D, E, and F).

6.Double dilute the IgGs across the plate (1¨C11).

7.Dilute the anti-guinea pig conjugate (pretitrated) in blocking buffer. Make up 6 mL.

8.Add 50 µL of the diluted conjugate to rows A¨CG. Do not add to row H. Mix the contents of the plates by gentle tapping. Add 50 µL of blocking buffer to row H.

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Fig. 31.

Plate design for performance of competition assay.

9.Incubate for 1 h at room temperature (or under conditions you used to titrate the conjugate). Rotate the plate to mix reagents every 10 min.

10.Wash the plates.

11.Add OPD/H2O2 solution.

12.Stop the reaction after 10 min by addition of 50 µL of 1 M H2SO4.

13.Read the OD in spectrophotometer at 492 nm.

7.5.1¡ª

Data:

Typical Results

Table 12 presents the results from the spectrophotometer readings of the plates. Figure 32 relates the OD values to the various concentrations of the competitors added. The results are processed intially as the mean OD for the triplicate estimations.

7.5.2¡ª

Further Processing of Data

1.Calculate the mean OD reading of row G. This represents the OD resulting from the reaction of the conjugate with only the solid-phase Ig and the conjugate. This value should be similar to that obtained when you titrated the conjugate. It represents the 0% competition OD, where most color is obtained.

2.Calculate the mean of the OD from row H. This represents the 100% competition level, i.e., where there is a total inhibition of the binding of antibody (not a strictly true 100% control since the conjugate was excluded from the test, but it approximates very well). Thus we have the 100% competition (degree of inhibition) and the 0% competition OD values.