The Elisa guidebook

14.Small-volume bottles.

15.Multichannel spectrophotometer.

16.Clock.

17.Graph paper.

5.1.5¡ª Methods

Since you are now familiar with the indirect assay, the steps in the optimization of the capture ELISA should be straightforward. The first essential step is to determine the amount of capture antibody to be attached to the wells. We have two situations in the laboratory depending on the availability of specific reagents. We can use capture antibody as an IgG preparation, or if sufficiently

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high-titer serum is available against the antigen, as whole serum. The easiest way to avoid serum effects is to prepare the IgG. Salt fractionation is usually adequate and does not affect antibody activity. Care must be taken to assess the effect of chemical preparation of IgG from mAbs.

5.1.5.1¡ª

Use of IG Preparations

The advantage of using IG preparations is that the weight of Ig can be calculated, so that a defined quantity of reagent may be added to the plate. In general, a maximum amount of protein will attach to the wells. Because we know that a maximum possible binding of subsequently added antigen may be expected, we may add the Ig at "saturating" level. Thus, a good estimation of the activity of a capture antibody (the particular dilution/concentration to be used) can be assessed. As an example, if capture antibody is added at 5 µg/mL in 50-µL amounts, this represents the saturating amount of antibody protein that will attach to the wells. The ultimate activity will depend on the concentration of the specific Ig (against the Ag) in the capture antibody and the spacing of the capture molecules.

Some assays perform better at lower than saturating levels of capture antibody so that a titration is needed. Generally, the amount of specific antibodies in a serum as a percentage of the total protein is about 1¨C5%. The preparation of Ig eliminates a large percentage of the serum proteins not involved in the assay (e.g., serum albumins). Therefore, the activity of the Ig protein (relative increase in the IgG fraction that will attach to each well) is effectively increased. In other words, there is a greater proportion of IgG sticking to the wells to act as trapping antibody if Ig preparations are used.

5.1.5.2¡ª

Use of Whole Serum

Dilutions of untreated serum can be used. However, as already indicated above, the proportion of specific IgG is low, and other serum proteins attach in a competitive manner. One cannot assume that putting on a low dilution of serum will give a good level of capture antibody. The most usual event is that a bell-shaped curve of capture ability is obtained, with little activity at high concentrations of serum and a rise in activity as the serum is diluted. In general, serum has to be diluted to around 1/500 to 1/2000. Thus, we must have fairly high titers to be able to use whole serum. Figure 24 demonstrates the activity of whole and IgG capture antibodies as they are diluted down, to illustrate the bell-shaped curve.

5.1.5.3¡ª

Titration of Capture Antibody Using IGG

1. Dilute the sheep anti-guinea pig IgG preparation to 5 µg/mL in carbonate buffer. Add 50 µL to each well on the plate except column 12.

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Fig. 24.

Comparison of capture of IgG using whole serum or IgG as capture antibody. C, IgG; +, serum.

2.Add adsorption buffer alone to row 12. Incubate at 37¡ãC for 2 h or overnight if more convenient (remember to put lids on the plates).

3.Wash the plates. (From now on we are performing a procedure similar to the indirect ELISA.)

4.Place the microtiter plate with well A1 at the top left-hand corner. Add 50 µL of blocking buffer to each well.

5.Make a dilution range of guinea pig IgG (the antigen of interest) from 5 µg/mL from column 1 (8 wells) to column

11in blocking buffer. Thus, add 50 µL of guinea pig IgG at 10 µg/mL to the first row 1 using a multichannel pipet. Mix and double dilute across the plate (you should be competent at this now). Remember to discard the last 50 µL in the tips so that each well should only contain 50 µL of fluid (check!).

6.Incubate the plates at room temperature or at 37¡ãC for 1 h.

7.Wash the plates.

8.Add 50 µL of blocking buffer to each well.

9.Dilute rabbit anti-guinea pig serum to 1/100 in blocking buffer (make up 1.0 mL: add 10 µL of undiluted serum to 1.0 mL of buffer). Mix. Add 50 µL of the dilution to row A using a single-channel pipet. Dilute across rows A¨CH using a multichannel pipet. We now have a twofold dilution range from 1/200 (row A) to 1/25,600 (row H).

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

11.Wash the plate.

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Table 7

CBT of Guinea Pig IgG vs Rabbit Anti-Guinea Pig IgG¡ª

Constant Capture Antibody, Constant Conjugate

	1	2	3	4	5	6	7	8	9	10	11	12
A	1.67	1.67	1.68	1.65	1.54	1.34	1.09	0.89	0.67	0.54	0.34	0.23
B	1.68	1.68	1.65	1.56	1.51	1.31	1.04	0.84	0.59	0.51	0.32	0.17
C	1.56	1.54	1.52	1.43	1.34	1.23	0.99	0.76	0.52	0.43	0.23	0.09
D	1.12	1.09	1.00	0.94	0.87	0.78	0.67	0.56	0.45	0.34	0.19	0.08
E	1.00	0.97	0.89	0.78	0.67	0.56	0.43	0.34	0.23	0.21	0.17	0.08
F	0.78	0.74	0.71	0.56	0.51	0.43	0.32	0.21	0.19	0.14	0.09	0.10
G	0.54	0.51	0.51	0.42	0.36	0.32	0.21	0.16	0.14	0.09	0.08	0.09
H	0.34	0.34	0.32	0.21	0.18	0.15	0.16	0.09	0.08	0.07	0.08	0.09

Guinea pig IgG diluted 1¨C11; anti-guinea pig IgG diluted A¨CH.

12.Make up an optimum dilution of sheep anti-rabbit conjugate. Add 50 µL to each well using multichannel pipets.

13.Incubate for the standard time as used in the optimization of conjugate (1 h at 37¡ãC or room temperature).

14.Wash the plate.

15.Add substrate and stop the color development at 10 min.

5.1.5.3.1¡ª Data

Essentially we have made a CBT of the antigen against the detecting antibody (as in the indirect assay). Thus, we have assumed that the capture antibody, put on the plate as an IgG, is at maximal reactivity. The results are therefore similar to those obtained in the indirect assay, and can be treated similarly. Each row A¨CH had an identical dilution series of the antigen (guinea pig IgG) being captured by the same amount of antibody, thus the same amount of guinea pig IgG should be present attached via antibody to the wells 1¨C11. The rabbit antibody against the antigen has been titrated at different dilutions so that we can examine which dilution shows the best detection of IgG in rows A¨CH. The use of 5 µg/mL of capture IgG was taken as that which from experience saturates the plastic sites available on the plate wells. Once the antigen and detecting serum optima have been established using this level of capture IgG, they can be altered to examine the effect on the assay. Table 7 gives the spectrophotometric plate readings. A representation of the plate is also shown in Fig. 25.

5.1.5.3.2¡ª Plots of Data

Figure 26 shows the data plotting the OD results obtained at the different antigen dilutions for each dilution of rabbit anti-guinea pig serum.

5.1.5.3.3¡ª Assessment of Data

Column 12 contained no antigen (guinea pig IgG), and therefore examination of the color here gives a measure of the bind-

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Fig. 25.

Diagrammatic representation of the plate showing results of CBT.

ing of the detection system to the plate or capture antibody. Thus, rows A and B show higher levels of color than the other rows. The value around 0.09 appears to be the plate background expected in the presence of the same dilution of conjugate. Thus, the end point detection of IgG is affected in rows A and B. Examination of the plateau heights indicates that the trapping system is saturated in columns 1¨C4, since we obtain similar OD values; as an example, we have around 1.67 for the first four wells using the 1/200 detecting antibody, although the actual plateau height value reduces on dilution of the detecting rabbit anti-guinea pig serum. Examination of Fig. 26, which relates the curves obtained for the detection of trapped Ig for different dilutions of the rabbit anti-guinea pig Ig, easily shows that the last dilution giving an optimal titration is in row C. After this dilution, the effect of is to reduce more markedly the OD in the plateau region (where the trapped Ig is in excess) and also to affect the sensitivity of detection of the Ig at higher dilutions, as indicated by a reduction in the end points where the test background is the same as the plate background.

5.1.5.4¡ª

Retitration of Capture IGG

The optimal dilutions chosen will depend on how the test is to be used. If an antigen is to be detected, then we might require high detection limits in the system, so that we can use a dilution of detecting antiserum to maximize this.

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Fig. 26.

Titration of guinea pig IgG using constant capture conditions. Each line represents titration of the same dilution range of IgG using a different concentration of rabbit anti-guinea pig IgG. The conjugate dilution is constant. Data are from Table 7.

We will see later that capture assays are used in competitive situations in which the amount of antigen to be captured needs to be reduced so that a variation in reagent concentrations for that application may be necessary.

The established optima for the antigen and detecting serum can be reassessed using lower concentratons of capture IgG. Thus, a full CBT can be performed using 2.5, 1.25, and 0.625 µg/mL of the capture IgG. However, a simpler procedure is to coat plates with a dilution range of the capture IgG and use constant antigen, detecting antiserum and pre-titrated conjugate dilutions. Table 8 gives the results of a typical titration of this sort. Here plates were coated with capture anti-IgG at 5 µg/mL, in a twofold range from row A to H, only columns 1 to 4; thus, quadruplicate samples were being examined. After incubation and washing, antigen (guinea pig IgG) at 0.625 µg/mL was added in blocking buffer. Following incubation and washing, the detecting antibody (rabbit anti-guinea pig IgG) was added at 1/400 diluted in blocking buffer. After incubation and washing, the anti-rabbit conjugate was added at the dilution used to optimize the reagents.

Table 8 shows that the capture IgG produces similar results at 5 and 2.5 µg/mL; thus, the latter dilution can be used in an assay to capture antigen.

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Table 8

Titration of Capture IgG Against Optimal Antigen,

Detecting Antibody, and Conjugate

Capture IgG
concentration
(µg/mL)		1	2	3	4	Mean
5.0	A	1.50	1.48	1.49	1.51	1.50
2.5	B	1.49	1.47	1.51	1.46	1.48
1.25	C	1.25	1.21	1.24	1.27	1.24
0.63	D	0.95	0.94	0.96	0.93	0.95
0.32	E	0.67	0.69	0.69	0.66	0.68
0.16	F	0.36	0.37	0.40	0.37	0.38
0.08	G	0.14	0.12	0.15	0.12	0.13
0.08	H	0.05	0.04	0.04	0.03	0.04

Lower concentrations produce lower OD values, indicating that not all the available antigen is being captured. The reduced ability to bind antigen (when in excess) is accompanied by a decreased ability to detect small amounts of antigen (the minimum detection limit is reduced).

Similar titrations of the other reagents can be made in which only one is diluted and the others are kept constant. Thus, in the previous case we know we have three conditions optimized under experimental conditions with control sera and antigen. The capture IgG can be used at 2.5 µg/mL, the antigen can be used at 0.625 µg/mL, and the rabbit detector at 1/400, with the anti-rabbit conjugate at a constant dilution as assessed originally against the relevant IgG attached to a microplate.

We may wish to reassess the conjugate dilution under standardized conditions. Thus, using the capture IgG, antigen, and detecting antiserum optima just given, replicate wells can be used to titrate different dilutions of conjugate. An example is given in Table 9. Here, a dilution of 2¨C400 of conjugate gives similar results. Effectively, a dilution of 1/800 gives "optimal" results (OD value around 1.45), for an assay.

5.1.5.5¡ª

Titration of Capture Antibody When Used as Whole Serum

As already stated, whole serum can be used to coat plates and act as a capture reagent. However, this is not recommended because we cannot measure the protein Ig because it is contaminated with "blocking" serum proteins that compete for plastic binding sites preferentially over the IgG. The simplest method is to perform a CBT relating dilutions of capture serum to dilutions of detecting antibody and keep the antigen constant.

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Table 9

Assessment of Constant Capture System with Different Dilutions of Conjugate

Conjugate
dilution		1	2	3	4	Mean
200	A	1.95	1.87	1.87	1.95	1.92
400	B	1.84	1.82	1.84	1.82	1.83
800	C	1.45	1.41	1.44	1.47	1.44
1600	D	0.95	0.94	0.96	0.93	0.95
3200	E	0.77	0.79	0.79	0.76	0.78
6400	F	0.36	0.37	0.40	0.37	0.38
12,800	G	0.15	0.14	0.15	0.14	0.15
None	H	0.05	0.04	0.04	0.03	0.04

The diagram below illustrates this:

5.1.5.5.1¡ª Reaction Scheme

I-Ab	= dilution range of trapping antibody
Ag	= constant dilution of antigen (high concentration)
AB	= dilution range of detecting antibody
Anti-AB*E	= conjugated anti species antibody
S	= substrate/chromophore
READ	= OD

This assay is not described in detail. However, a description of the test is given with relevant points highlighted. You should now have enough experience to be able to set up the exact practical details with help from the exercise titrating IgG as capture antibody.

5.1.5.6¡ª Method

1.Dilute the serum containing capture antibody on plates in carbonate/bicarbonate buffer (begin at 1/100, twofold dilutions). Incubate and then wash the plates.

2.Add constant (excess) antigen diluted in blocking buffer. (It is difficult to specify here what excess might be for specific systems, for example, an undiluted tissue culture sample containing virus might be expected to have a high concentration of antigen.) Incubate for 1 h at standard conditions.

3. Wash and add dilutions of detecting antibodies in blocking buffer to obtain a CBT (dilute in opposite direction to the capture serum). Incubate and then wash the plates.

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Table 10

Dilutions of Capture Serum 1/100 to 1/51,200

	1	2	3	4	5	6	7	8	9	10	11	12
A	0.67	0.96	1.34	1.35	1.32	1.11	0.98	0.76	0.56	0.45	0.23	0.12
B	0.66	0.99	1.42	1.37	1.29	1.09	0.89	0.75	0.54	0.36	0.22	0.11
C	0.65	0.98	1.36	1.34	1.15	0.99	0.87	0.72	0.52	0.31	0.17	0.09
D	0.56	0.88	1.23	1.19	1.01	0.88	0.74	0.65	0.43	0.26	0.14	0.09
E	0.45	0.67	1.00	1.09	0.98	0.78	0.56	0.45	0.34	0.21	0.12	0.07
F	0.23	0.43	0.78	0.76	0.56	0.45	0.40	0.33	0.23	0.16	0.12	0.08
G	0.15	0.23	0.34	0.35	0.21	0.15	0.16	0.09	0.08	0.07	0.09	0.09
H	0.15	0.19	0.18	0.17	0.16	0.10	0.09	0.08	0.09	0.07	0.07	0.09

Detecting serum deluted A¨CH 1/200 2 fold.

4.Add optimal conjugate, incubate, and wash.

5.Add substrate and then stop at 10 min.

5.1.5.6.1¡ª Data

Table 10 presents typical results. Data are plotted in Fig. 27. Rows A¨CH contain dilution ranges of the capture serum 1/100 to 1/51,200 and column 1 = 1/100, and column 12 = 1/51,200. Row A received the detecting antiserum at 1/200, row B at 1/400 and so on to row H at 1/12,800.

5.1.5.6.2¡ª Conclusion

1.The optimal dilution of capture serum is around row 5 (last row showing maximum OD).

2.The optimal dilution of detecting second antibody is around row C¨CD (last showing maximal titration curve of antigen).

3.Bell-shaped curves are obtained where low dilutions of capture serum give low OD values (columns 1 and 2).

5.1.5.7¡ª

Notes on Use of Capture ELISAs to Detect Antigens

Once the optimal conditions have been established, the capture assays can be used in several ways.

5.1.5.7.1¡ª

Diagnosis of Specific Disease Agents

Here, a sample possibly containing antigen is added to a capture system (microtiter plate wells coated with an antiserum against a specific disease). Any bound antigen is then detected by another antibody from a different species. Such assays are important in serotyping in which the second antibody

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Fig. 27.

Graph of data in Table 10. Columns 1¨C12 contain dilutions of capture antibody on wells. Rows A¨CH have different dilutions of detecting antibody.

may further ''divide" the disease agent into a serological grouping, such as is used routinely to serotype FMDV into one of seven distinct serotypes. The use of capture antibody means that relatively crude or contaminated samples can be used. Antigens may be quantified with reference to a standard antigen titration on the same plate.

Single dilutions of material containing the Ag can then be titrated in the same system and the developing OD read against the standard titration. Again, the use of the capture antibody ensures an efficient and proportional uptake of the antigen on to the plate, which is unaffected by contaminating proteins.

5.2¡ª

Use of Capture ELISA to Detect and Titrate Antibodies

Essentially, the same parameters have to be standardized as for capture ELISA for antigen detection. However, the test is used to measure antibodies against a fixed amount of antigen captured on the plate. Thus, we must optimize the system to have the correct amount of capture antibody and antigen necessary to bind any test or control antisera. The test offers the ability to specifically capture antigen using a solid-phase antibody. Thus, relatively crude

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preparations can be used in cases in which the required antigen concentration may be low. Care must be taken to avoid reactions of the conjugate with components of the assay.

5.2.1¡ª

Learning Principles

1.Optimizing of capture antibodies.

2.Optimizing of detecting antibody.

5.2.2¡ª

Reaction Scheme

I	= microplate
ABX	= trapping antibody (species X)
Ag	= antigen
AbY	= test or control sera (species Y)
Anti-Ab*E	= antispecies Y antibody conjugated with enzyme
S	= substrate/color detection system
W	= washing step
+	= addition and incubation of reagents

5.2.3¡ª

Materials and Methods

1.Capture antibody (ABX): sheep anti-guinea pig Ig at 5 mg/mL in PBS.

2.Ag: guinea pig Ig at 1 mg/mL.

3.Test antisera (AbY: three rabbit anti-guinea pig Ig sera; also seronegative (Ab) rabbit sera.

4.Anti-AbY*E: rabbit antigoat Ig conjugated to HRP.

5.Microplates.

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

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

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

9.Solution of OPD in citrate buffer.