The Elisa guidebook

The basis of this assay is to titrate antibodies that have reacted with an antigen by using an antispecies conjugate. The indirect aspect therefore refers to the fact that the specific antiserum against the antigen is not labeled with an enzyme, but a second antibody specific for the particular species in which the first antibody was produced is labeled. Such assays offer flexibility and form the bases of other ELISAs. In principle, the optimization of reagents is similar to the direct ELISA. However, three factors have to be considered:

1.The optimal dilution of antigen.

2.The optimal dilutions of antisera.

3.The optimal dilution of conjugate.

The third factor was dealt with in the direct ELISA. You should now be able to titrate the conjugate (anti-rabbit in this case). The major use of indirect ELISA is to titrate antibodies against specific antigens. In this case, a constant amount of antigen is adsorbed to wells, and antisera are titrated against this as dilution ranges. Any reacting antibody is then detected by the addition of a constant amount of antispecies conjugate. Such assays can be evaluated fully from the diagnostic point of view in which numbers of field and experimental antisera (known history) are available. Therefore, they can be used to assay single dilutions of antisera, and tests can be adequately controlled using stan-

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dard positive and negative antisera. Thus, the indirect ELISA has found many applications in epidemiological studies assessing disease status.

2.4¡ª

Materials and Reagents

1.Ag: guinea pig IgG at 1 mg/mL (1 g/L).

2.Ab: rabbit anti-guinea pig serum.

3.Anti-antibody*E: Sheep anti-rabbit serum linked to HRP (rabbit IgG needed if conjugate titration not done, as for titration of anti-guinea pig conjugate).

4.Microplates.

5.Multichannel and single-channel pipets.

6.10 mL and 1 mL pipets.

7.Carbonate/bicarbonate buffer, pH 9.6, 0.05 M.

8.PBS containing 10% BSA and 0.05% Tween-20.

9.Solution of OPD in citrate buffer.

10.Bottle of hydrogen peroxide (30% w/v).

11.Washing solution (PBS) in a bottle or reservoir.

12.1 M sulfuric acid in water.

13.Paper towels.

14.Small-volume bottles.

15.Multichannel spectrophotometer.

16.Clock.

17.Graph paper.

2.5¡ª

Protocol for Indirect ELISA

The first stage in this assay involves the titration of the antispecies conjugate under the conditions described in the direct ELISA. Remember that the antigen used to titrate the conjugate must be appropriate; for example, if an antibovine conjugate is to be used, then use BSA as the antigen in the original CBT. If detection of an antibovine IgG is required, then use bovine IgG as the antigen in the direct ELISA CBT.

The anti-rabbit conjugate needs to be titrated so that we know the dilution to use in the indirect assay in order to detect any reacted rabbit serum (the optimal dilution of conjugate may be given in class if this procedure has not been carried out):

1.Titrate the anti-rabbit conjugate (optimal dilution may be given).

2.Take a microtiter plate with A1 at the top left-hand corner. Add 50 µL of carbonate /bicarbonate buffer to each well using a multichannel pipet.

3.Make a dilution range of the guinea pig IgG from 5 µg/mL from column 1 (eight wells) to 11. This is made exactly as described for the direct ELISA. Add 50 µL of the guinea pig IgG at 10 µg/mL (or 1/50 if the concentration is unknown) to column 1. Mix (pipet up and down eight times with a multichannel pipet), and then transfer 50 µL of dilution to column 2. Mix and continue transferring to

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column 11. Discard 50 µL remaining in tips after mixing in column 11. Thus, we have a twofold dilution range of IgG in each row A to H, excluding column 12 wells.

4.Incubate at room temperature or 37¡ãC for 2 h.

5.Wash the wells in PBS (fill and empty the wells four times).

6.Blot the plates.

7.Dilute the rabbit anti-guinea pig serum to 1/50 in blocking buffer (PBS containing 1% BSA and 0.05% Tween20). Make up 1 mL; therefore, add 20 µL to 1 mL of buffer.

8.Add 50 µL of blocking buffer to all wells using a multichannel pipet.

9.Add 50 µL of the 1/50 anti-guinea pig serum to each well of row A. Mix and transfer 50 µL to row B, mix and transfer 50 µL to row C, and repeat this procedure to row H. We now have a twofold dilution series of antibody the opposite way to the IgG antigen.

10.Incubate the plate at room temperature or 37¡ãC for 1 h.

11.Wash and blot the plate.

12.Make up the antispecies conjugate (kept at ¨C20¡ãC) to the optimal dilution found in the direct ELISA (or as instructed) in the blocking buffer. Make up enough for all the wells of the plate plus 0.5 mL (approx 5.5 mL). This might appear wasteful but is convenient practice since it allows for minor errors in pipeting and avoids the need to make up a small volume of conjugate when one runs out on the last row (i.e., when the exact volume to fill the plate wells is made up). Add 50 µL of the dilution to each well using the multichannel pipet and a clean trough.

13.Incubate at room temperature or 37¡ãC for 1 h.

14.Wash and blot the wells.

15.Thaw out the OPD (10 mL). Add 5 µL of H2O2 immediately before use. Mix well. Add 50 µL of this to each well, using a multichannel pipet and clean the troughs (make sure that the trough is not contaminated with conjugate from the previous addition to the plate).

16.Incubate for 10 min (note color changes).

17.Stop any color development by adding 50 µL of 1.0 M sulfuric acid to each well.

18.Read the plate by eye and with a multichannel spectrophotometer after titration of antigen (guinea pig IgG) and antibody (anti-guinea pig serum) as described above.

2.6¡ª Results

Table 3 presents the microplate reader results. Note that these produce a similar picture to the direct ELISA results. Also, you should have observed that there was a similar development of color throughout the 10-min incubation after addition of the substrate solution. Figure 7 shows the data graphically. Plots relating the concentration (or dilution) of the IgG (Ag) to the OD for all the different dilutions of rabbit anti-IgG are shown.

Plot the data relating the IgG concentration on the plate plotted as a log10 twofold series (micrograms/milliliter per well, or dilution if the actual concen-

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Table 3

Plate Data from CBT of Conjugate and Antigen

Rows A and B indicate that antibodies are in excess, and we have some problems of nonspecific attachment to the plate without antigen having been adsorbed (well 12). Note that in these rows, the plateau regions extend to well 4. Thus, no more antigen (IgG) is able to absorb to the plate above the concentration in well 4. Rows C and D give optimal titrations of the IgG in that maximum values do not exceed 1.6 OD, and high end point titers are obtained. Below these dilutions, sensitivity for the detection of IgG is lost. Thus, to detect the antigen optimally, and to use a single dilution of antiserum under the conditions of the ELISA described, use a dilution of about 1/400 to 1/800.

The optimum dilution of antigen that might be used as a single dilution to detect and maybe quantify antibodies is best assessed as the dilution (or concentration) that shows good binding across the whole range of antiserum dilutions. The best way to illustrate this is to draw a graph of the plate data, but this time, plot the dilutions of serum gainst the OD for the various antigen concentrations (or dilutions). This was done in Fig. 7.

At the first four concentrations (dilutions) of antigen (IgG), there is little difference in the end-point detection for the dilutions of antiserum. are reduced. After this, the OD readings and the end point detections are reduced. At the extreme, in column 10, barely any antibody is detectable, even where the serum is most concentrated. The higher values in rows A¨CC correspond to the nonspecific binding to the wells seen in row 12. Thus, the dilution of antigen found in columns 3 and 4 is optimal to detect antibodies.

3¡ª

Use of Indirect ELISA to Titrate Antibodies

The optimized reagents in Subheading 2.4. can be exploited to measure antibodies directed against the guinea pig IgG target.

3.1¡ª

Learning Principles

1. Titrating antibodies from positive sera using full-dilution ranges.

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2.Establishing ELISA-negative antibody levels for control of nonimmune sera.

3.Duplicating samples tested.

3.2¡ª

Reaction Scheme

In this exercise, the Ag and Anti-Ab*E are used at optimal dilution. The test or standard Abs are added as dilution ranges.

3.3¡ª

Basis of Assay

We are now able to titrate antibodies, since we know the antigen optimum and the conjugate optimum dilutions for our given system. Thus, if sera are reacted with the antigen on the plate, and if they contain antibodies against the guinea pig IgG, they will be picked up by the subsequent addition of the conjugate. The seropositive serum titration curves may then be compared with each other and to the seronegative curves to establish antibody titers and examine the result of nonspecific reactions at the various dilutions of the negative sera, within the system.

3.4¡ª

Materials and Reagents

1.Ag: guinea pig IgG, 1 mg/mL.

2.Ab: three rabbit serum samples after injection with guinea sera test bled at different times following inoculation with guinea pig IgG and three rabbit sera from antibody-negative animals (prebleeds).

3.Anti-Ab*E: sheep anti-rabbit serum linked to HRP.

4.Microplates.

5.Multichannel and single-channel pipets.

6.10and 1-mL pipets.

7.Carbonate/bicarbonate buffer, pH 9.6, 0.05 M.

8.PBS containing 1% BSA and 0.05% Tween-20.

9.OPD solution.

10.Hydrogen peroxide.

11.Washing solution (PBS).

12.1 M sulfuric acid in water.

13.Paper towels.

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14.Small-volume bottles/microdilution system.

15.Multichannel spectrophotometer.

16.Clock.

17.Graph paper.

3.5¡ª

Titration of the Antigen Dilution or Concentration for Use in Measuring Antibodies

Titration is performed as described earlier for the indirect assay (see Subheadings 2.5. and 2.6.), in which we also titrated the optimum dilution of conjugate. Now we are concerned with the titration of antibodies against guinea pig IgG in rabbit sera. Therefore, we make the CBT of guinea pig IgG against the positive rabbit antiserum and use a constant dilution of anti-rabbit conjugate.

Note that while is setting up an indirect ELISA, a positive serum against the particular antigen being detected is needed. Such sera are often available as determined from other serological assays, from systems whereby specific antibodies are expected (e.g., from experimentally infected or vaccinated animals or from animals during the course of an outbreak). The exact conditions of the ELISA may therefore have to be altered during the developmental stages when many sera have been examined as compared to the originally used positive serum. For now, the original ''optimal" conditions are determined using a defined (experimentally derived) positive serum.

3.6¡ª

Titration of Different Sera

1.Dilute guinea pig IgG (Ag) to optimum concentration in carbonate/bicarbonate buffer, pH 9.5, 0.05 M (as determined in Subheading 2.1.).

2.Add 50 µL to each well of the plate using a multichannel pipet.

3.Incubate at 37¡ãC for 2 h.

4.Wash and blot the plate.

5.Add 50 µL of blocking buffer to all the wells using a multichannel pipet and trough.

6.Take the six sera supplied. Label the three positive sera 1, 2, and 3. Label the three negative sera 4, 5, and 6. Dilute each one to 1/20 in blocking buffer in small bottles, and make up a final volume of 0.5 mL of each (25 µL +

475µL of blocking buffer).

7.Turn the plate with 50 µL of blocking buffer per well so that well H1 is on the left-hand top corner (see Fig. 8). Add 50 µL of serum 1 dilution to wells H1 and H2, add 50 µL of serum 2 dilution to wells H3 and H4, and add 50 µL of serum 3 dilution to wells H5 and H6. Repeat the process adding sera 4, 5, and 6 to wells H7 and H8, H9 and H10, and H11 and H12. We now have each of the sera diluted effectively to 1/40 in 100 µL of blocking buffer in the left-hand extreme row (H) of the plate (see Fig. 9).

8.Use the multichannel pipet with 12 tips attached to mix, and the dilute the sera across wells G, F, E, D, C, B, and A, transferring 50 µL of each dilution. We now

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Fig. 8.

Orientation of plate for dilutions of sera.

have a twofold dilution range of the sera, in duplicate, that is, there are two dilution series of each of the sera (see

Fig. 9).

9.Incubate at 37¡ãC or room temperature for 1 h (the exact conditions you used in the indirect CBT are best).

10.Wash and blot the plate.

11.Add 50 µL of anti-rabbit conjugate per well (diluted in blocking buffer).

12.Incubate at 37¡ãC (or room temperature) for 1 h (conditions as for 1-h incubation in CBT).

13.Wash and blot the plate.

14.Add substrate and chromophore (50 µL).

15.Stop color development after 10 min.

16.Read the plate in a multichannel spectrophotometer. Remember to watch the plate as the color develops and make relevant notes.

3.7¡ª

Explanation of Data

Figure 10 gives typical results from this assay, and Table 4 presents the OD readings.