The Elisa guidebook

competing Ig was reduced the effect was also reduced. Plate 2 results show that there is a low color representing the background.

To calculate the degree of competition, a calculation of the range of values with 100% and 0% competition is needed. The 100% competition values for both dilutions of conjugate used can be taken from mean values on plate 2. We have 1/x = 0.07; and 1/y = 0/05.

The 0% values for each of the conjugate dilutions and each of the IgG coating conditions are read for the respective concentrations in column A of plate 1. These are contained within the line drawn on the results of plate 1.

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Table 4

Mean Values in Competition Assay Minus the Background Values for Relevant Conjugate Dilution Used

These results show the color obtained for a particular coating concentration of Ig with the conjugates at different dilutions.

2.4.4¡ª Calculations

The mean value of the conjugate control for both the dilutions of conjugate obtained in plate 2 is subtracted from the values obtained in which that dilution of conjugate was used. This is shown in Table 4.

The values for the reaction between the conjugate and various concentrations of coating Ig without competition (0% competition) are shown in the gray boxes. This value is now used in the following formula to calculate the effect of adding the competing Ig:

As an example: taking the first line of data we have the following values:

Value

0.24 100 ¨C [(0.24/1.75) ¡Á 100] = 100 ¨C (0.14 ¡Á 100) = 100 ¨C 14 = 86% 0.29 100 ¨C [(0.29/1.75) ¡Á 100] = 100 ¨C (0.17 ¡Á 100) = 100 ¨C 17 = 83% 0.45 100 ¨C [(0.45/1.75) ¡Á 100] = 100 ¨C (0.26 ¡Á 100) = 100 ¨C 26 = 74% 0.92 100 ¨C [(1.18/1.75) ¡Á 100] = 100 ¨C (0.67 ¡Á 100) = 100 ¨C 67 = 33% 1.27 100 ¨C [(1.39/1.75) ¡Á 100] = 100 ¨C (0.79 ¡Á 100) = 100 ¨C 79 = 21% 1.47 100 ¨C [(1.72/1.75) ¡Á 100] = 100 ¨C (0.98 ¡Á 100) = 100 ¨C 98 = 2% 1.74 100 ¨C [(1.74/1.75) ¡Á 100] = 100 ¨C (0.99 ¡Á 100) = 100 ¨C 99 = 1%

For the second row or results we have a different value for the % competition OD: 1.5 as follows:

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Value

0.20100 ¨C [(0.20/1.45) ¡Á 100] = 100 ¨C (0.13 ¡Á 100) = 100 ¨C 13 = 87%

0.29100 ¨C [(0.29/1.45) ¡Á 100] = 100 ¨C (0.19 ¡Á 100) = 100 ¨C 19 = 81%

0.44100 ¨C [(0.66/1.45) ¡Á 100] = 100 ¨C (0.46 ¡Á 100) = 100 ¨C 46 = 54%

0.84100 ¨C [(1.05/1.45) ¡Á 100] = 100 ¨C (0.72 ¡Á 100) = 100 ¨C 72 = 28%

1.08100 ¨C [(1.21/1.45) ¡Á 100] = 100 ¨C (0.83 ¡Á 100) = 100 ¨C 83 = 17%

1.21100 ¨C [(1.38/1.45) ¡Á 100] = 100 ¨C (0.95 ¡Á 100) = 100 ¨C 95 = 5%

1.40100 ¨C [(1.40/1.45) ¡Á 100] = 100 ¨C (0.97 ¡Á 100) = 100 ¨C 97 = 3%

Similar calculations can be made on all the other results. These are shown in Table 5, which lists all the data.

From these data we can determine optimal coating and conjugate dilutions to be used to test Ig in samples. Although it looks complicated, the whole procedure is accomplished in 2 d and is usually straightforward. Again, the calculations, seem problematic, but careful consideration will indicate that they are relatively simple.

In practice, examination of the data in the tabular form should be enough to set up optimal conditions. The optimal conditions can be assessed best if data are plotted. Figure 2 illustrates features of the competition curves. All the curves illustrate that there is competition for the expected binding of the conjugates. The best combination of reagents to analyze samples is obtained by considering the effective analytical sensitivity of each combination. The estimate of sensitivity is made by examining the weight of competing Ig at the 50% competition point. Thus, a line drawn across the 50% point is shown in Fig. 2. The weight of Ig standard needed to give 50% inhibition of the conjugate binding for each combination can be measured as shown in Fig. 3, by plotting the perpendicular to the x-axis where the curve crosses the 50% competition line. Ideally, the weight of competing Ig at the 50% competitive point should be the same as that added to coat the wells, assuming that all the Ig added was bound to the wells.

Note that it is much easier to plot the competition data on semilog graph paper. The competition is plotted on an arithmetic scale and related to log10 of the competing Ig concentration. In this way, the values for the various

concentrations can be directly read from the scale. This approach also simplifies all other data plotting in which the x-axis is a scale of activity. Figure 4 presents the same data plotted in this way. The values for the combination are shown in Table 6.

2.4.5¡ª Conclusion

All conditions show that the conjugate was competed for on the addition of the known standard concentrations of Ig. A standard curve relating added Ig concentration to competition is obtained. The choice of best conditions is a

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Table 5

Percentage Competition Values

Fig. 2.

The six lines show the competition data for the various combinations of coating concentrations and conjugate dilution. Lines 1 and 2 show data for 1.5 µg/mL, 3 and 4 for 0.75 µg/mL, and 5 and 6 for 0.375 µg/mL coating. The conjugate dilution for 1, 3, and 5 was 1/x;

for 2, 4, and 6, the conjugate dilution was 2/y. The 50% competition point is indicated by the dashed line.

balance between required sensitivity and likely accuracy of the test. Screening for secretion of mouse Ig does not require high precision, the results are relative within the standard system used with reference to the conjugate. The sen-

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Fig. 3.

The six lines show the competition data for the various combinations of coating concentrations and conjugate dilution. The competing Ig at 50% is indicated for each of the combinations 1¨C6.

sitivity is lowest when there are high levels of antigen Ig coating and higher levels of conjugate. In this case, there is more conjugate and antigenic target to be competed for. Reference to the OD values obtained for the 0% control indicates that this is rather high (OPD substrate system). The intermediate values of coating plates result in good titrations over the range of Ig added with OD values for 0% competition in a good area for this substrate. The sensitivity is increased here from about 1 µg/mL in 50 µL to about 0.4 µg/mL. The curves are also extended more into the area below which 50% competition is achieved. The third coating concentration gives a slightly higher sensitivity, but the OD values for control 0% competition can be regarded as too low. The variation in results increases with reaction in the colored product. An optimal dilution of coating Ig at about 0.75 µg/mL with a conjugate dilution equivalent to 1/y appears suitable for screening.

2.5¡ª

Use of Competition Assay to Assess Samples

The conditions established can now be used routinely for screening unknown samples for the presence of mouse Igs. The conditions for coating, washing,

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Fig. 4.

Competition data plotted on long-semilog scale. The weight of competing Ig can be read directly from the x-axis. Combinations 1¨C6 are shown.

buffers, incubation, and so forth, are those described previsouly. A suitable template for addition of samples and controls (known Ig standards) should be developed. An example is shown in Fig. 5.

1.Wells should be coated with Ig at 0.75 µg/mL (50 µL per well). After incubation and washing, samples from mAb hybridoma studies can be added. A template as shown in Fig. 5 is suitable.

2.Add 25 µL of blocking buffer to each well except the control wells (see below). Add 25 µL of each mAb sample. Test mAb in duplicate if possible.

3.Add a dilution range of standard Ig preparation from 5 µg/mL by seven wells as in stage 2 (duplicate row).

4.Set up 0% controls (wells with blocking buffer added to Ig-coated wells).

5.Set up 100% competition controls.

6.Add conjugate to relevant wells (50 µL at 1/y) found in first stages.

7.Incubate, wash, add substrate, develop color, and stop.

8.Read the plates.

Typical results are shown in Table 7. Figure 5 shows the template used. Duplicate samples of mAb are titrated vertically.

For accurate treatment of data, the means of the results are calculated. The mean of the 100% competition data (A11 and 12 and B1 and 12) is subtracted

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Table 7

Examination of Samples

from all means. The percentage competition is then calculated for the data using the same formula as in

Subheading 2.4.4.

The standard curve can be plotted on semilog paper. The percentage values for the samples can be used to read off the relative activity as compared to the standard curve. This is illustrated in Fig. 6, in which only three samples are shown being read from the standard curve. When the sample values are equal to 100%, the effective concentration in the samples cannot be calculated. When they are lower then effectively the concentration of Ig can be assessed. Examination of the stopped plate by eye is useful and may be enough to indicate which wells or samples contain Ig. Thus, assessment as to wells which have no or very little color is enough to indicate Ig secretion. Similarly where wells have color equivalent to that in the 0% control wells then they can be deemed ''negative" with respect to Ig secretion to the limits of sensitivity as defined by the standard curve. The standard curve should also be examined in a by eye assessment and should indicate a gradual increase in color as expected by a decreasing level of competition.

Table 8 shows the data in Table 7 and represents mean of replicates minus the mean value for the 100% competition controls, and the % competition values. The mean of replicates of 100% competition data is 0.05

3¡ª

Screening of mAbs for Specific Activity

A reminder is needed that the purpose of producing mAbs is to solve a particular problem. The screening of mAbs must reflect this purpose as early as possible. Reference to Table 1 indicates when specific screening is best attempted. ELISAs measuring binding of fusion products to antigens are ideal for testing a high number of clones in a 96-well format. The screening does require some pretesting of reagents to allow confidence that a system will

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Fig. 6.

Competition data for standards plotted on log-semilog scale. The weight of competing IgG in samples can be read directly from the x-axis. Three examples of test samples are shown, as indicated in the text. The estimated micrograms/milliliter values are shown for each sample below the x-axis.

detect mAb if produced. The usual source of developmental reagents comes from collecting serum from mice primed and vaccinated in the same way (or the actual mice used) as was used to prepare the mAbs. The polyclonal serum should contain antibodies that bind to a selected target antigen(s). This and preimmune serum can be used for developing an ELISA system for screening purposes.

A review of what is available in terms of likelihood, specificity, physical parameters, and availability is needed in order to assess the most suitable test. This also infers that there may be other reagents already exploited in ELISAs (e.g., polyclonal capture antibodies) that can be used. Table 9 shows some considerations. The development of assays to detect bound mouse antibodies is no different from that for other assays. We need to be certain that a test will measure this binding and be reasonably assured that the test has good analytical sensitivity (can detect low levels of antibody).