The Elisa guidebook

2. Box 2: The Cm OD values are decreasing, possibly indicating that the antigen or mAb reactivity, or both, is being lost. The reduction in OD will be reflected by a relative increase in competing ability shown by the increase in PI% values. The results of PI% SDC will be most likely reflected as in box 2C, in which both

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Fig. 25.

Summary of most likely relationship of trends in OD SDC Cm and PI% SDC C++ and C+.

controls increase in PI%. Although, since the C++ contains an excess of antibodies, a situation as in box 2G is also possible.

3. Box 3: The Cm controls increase in OD value in time. This would be unusual. However, here the increase in reaction could reduce both the C++ and C+ control sera's ability to compete as in box 3B. Again, the excess antibodies in the C++ might maintain the C++ PI% against that of the C+ as in box 2G.

An overall summary of the most likely scenario for the SDC OD and PI% relationship is shown in Fig. 25.

7.3.1¡ª Irregular Plots

The same criteria for evaluating and comparing the Cm OD data and the C++ and C+ PI% data can be used to evaluate any time points plotted. This will establish any effects of the increase or decrease in Cm OD at any time on the expected PI% values.

When irregular results are obtained with high interest variation, one can note which of the results (at a given time) are affected most. Examination of the operator, methods, actual plate data, and day-to-day variables can be associated with good or bad results. Reference can be made to the individual plate data. Measures to solve the problems could then be used and results obtained after corrections compared.

7.4¡ª

Sources of Variation

As already stated, the kit is a complex association of reagents and equipment in the hands of different operators. The sources of variation thus come

7.4.3¡ª Reagents

The protocols given in the kit's manual should be strictly adhered to. Failure to maintain required minimal temperatures or to alter dilutions can severely harm the assay parameters. Such harm can be assessed with reference to the test performance before and after an identified abuse of reagent. However, such abuses are either not identified or not reported for other reasons. Note should be taken of likely susceptibilities of reagents to various physical conditions (see Table 9).

7.4.4¡ª

Water Quality

Water quality is consistently the chief source of problems in cases in which results (OD values) are generally much lower than expected. Attempts to obtain water from other sources should be made. The reasons for the alteration in color are not clear, and it is better to obtain water from other sources rather than waste time in curing an internal problem.

7.4.5¡ª

Poor Washing

Poor washing applies to washing tips and glassware and plasticware. It is better not to reuse anything that has had contact with enzyme. When tips are reused, they must be acid washed and then very thoroughly rinsed in distilled water. The pH of the water should be checked because it can often be very

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acidic (poor plant-producing water). Poorly rinsed vessels in which original dilutions of reagents are made also adds to variation because activities can be reduced or totally destroyed in a highly alkaline or acidic pH range.

7.4.6¡ª Mathematics

Errors in diluting reagents are a major problem. Calculations should always be checked and all bottles and so forth clearly labeled with the name of the reagent and dilution. The fundamental principles involved in good performance of ELISA should be learned through in-house training, external training, and reading of the literature. There is no substitute for training, which leads to improvements in practical skills and a thorough understanding of what one is doing.

7.5¡ª

More Problem Solving

Most problems are not really taken care of until it is too late. Operators must examine results constantly. The object of this section is to detail what should be done and when actions should be taken. The very reason for ICQ charts is to examine all results at all times. Actions are dictated by the results. Figure 26 highlights measures to be taken when certain observations are made.

7.5.1¡ª Stage 1:

Running the Kit for the First Time

In stage 1, a single vial for conjugate, Ag, and control C++ and C+ as well as mAb is selected. The test should be made exactly as described in the kit's manual. Results should be read and processed. The correct value for Cm OD, and allowable limits, are shown in the manual. The expected mean OD is 0.7, with the variation given at 1, 2, and 3 SDs from this mean expected value. The initial running of the reagents should give this mean, or a mean within the 2 ¡Á SD range recommended. Be concerned in cases in which the mean is outside the 2 ¡Á SD limits set, or when the mean is within the limits but when one end of the error bars plotted is inside the upper or lower mean or ¨C3 ¡Á SD limits. Usually the OD readings are low when there is a problem.

7.5.1.1¡ª Situation A

In situation A, the results are good. The mean is between the 2 ¡Á SD limits. The extremes of the error bars are also within the 2 ¡Á SD limits. The reagents are performing well and testing can begin. The PI% values for C++ and C+ should be examined to check that they are within prescribed limits.

7.5.1.2¡ª Situation B

In situation B, the means are wither low or high. The usual situation is that they are too low. Even when the means are within the 2 ¡Á SD limits, note that

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Fig. 26.

Scheme for evaluating kits.

we should be cautious when the error bars are in the 2 to 3 ¡Á SD range. Reasons for low color may be that the reagent was not made up properly, either by incorrect dilution or losing material on opening the vial (freeze-dried material can be lost); that the reagents were damaged transport; or that the laboratory has poor water. A poor result means that stage 2 must be run.

7.5.2¡ª Stage 2

In case there was a general poor dilution or problem of loss, it is good practice to select new vials of all reagents, and retest. The initial vials can be stored. Be extra cautious in diluting the new vial reagents accurately. Also be careful

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all material is solubilized and that none is lost when the vials are opened, or left on caps on dilution.

7.5.2.1¡ª Situation C

If the results are good, then this points to the errors in stage 1 being dilution and making up reagents. Proceed with the testing.

7.5.2.2¡ª Situation D

In cases in which there is no improvement, then some retitration and examination of the water in the laboratory is necessary in stage 3.

7.5.3¡ª Stage 3

7.5.3.1¡ª

Simple First Checks

1.Are you using the correct filter? This should be 492 nm. If you have relatively strong color but the machine is reading only 0.1¨C0.4, then check the filter.

2.Make sure the substrate (H2O2) and chromophore ortho-phenylenediamine are made up properly. Check the viability by dipping the pipet tip into antimouse conjugate and putting tip into freshly made up substrate/chromophore. This should turn dark brown very quickly.

3.Double-check all dilutions you are making from vials.

4.Check the pH of the substrate buffer after the addition of chromophore!

7.5.3.2¡ª

Obtaining Another Source(S) of Distilled Water and Repeating Stage 1

Water can have a limiting effect on color development. If replacement of water allows results as in Subheading 7.5.3.1. then proceed with a secured source of water.

7.5.3.3¡ª

Retitrate the Antigen

Often there can be some lowering of antigen activity owing to denaturation (also on continued testing) and through aggregation. A simple examination of antigen can be made by keeping all other reactants constant.

1.Dilute antigen 1/25, 1/50, 1/100, and 1/200, as indicated in table 10.

2.Coat the plates under standard conditions.

3.Wash.

4.Add mAb at the recommended dilution. Add C++ and C+ for reference. Cc is included in column 5.

5.Incubate.

6.Wash

7.Add conjugate.

8.Incubate.

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Table 10

Possible Results from Titrations (a)

9.Add substrate/chromophore.

10.Stop and read at 492 nm.

Some possible results can be examined.

7.5.3.3.1¡ª Result (a)

Table 10 presents possible results from titrations (a). If there is an increase of OD at 1/25 and/or 1/50, this indicates that there is a loss in activity of the antigen so that recommended dilution is not suitable.

Action:

1.Use the antigen at a retitrated dilution that gives the mean OD for Cm within recommended limits. Check that the PI% values for C++ and C+ are within limits. In the example, a dilution of 1/50 is better.

2.Obtain a new batch of antigen.

Different results may be found as shown in result (b).

7.5.3.3.2¡ª Result (b)

Table 11 presents possible results from titrations. Here, increasing the antigen concentration has no effect on increasing the OD.

Action: Retitrate the antigen, mAb, and conjugate.

7.5.4¡ª

Retitration of Antigen, Conjugate and Mab

The antigen (Ag), mAb, and conjugate can be titrated on one plate (see Fig. 27). The antigen is diluted beginning at 10 times the recommended concentration into wells in columns 1 and 7. The antigen is then diluted in a twofold range over four wells (columns 1¨C4 and 7¨C10. Columns 5, 6, 11, and 12 receive only buffer. After incubation and washing, the mAb is added and then diluted beginning at 10 times the recommended dilution in rows A and E. The mAb is diluted in blocking buffer in a twofold range from A1¨C5, A7¨C11 to D1¨C5,

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Table 11

Possible Results from Titrations (b)

Fig. 27.

Scheme for titration of all reagents.

D7¨C11; and E1¨C5, E7¨C11 to H1¨C5, H7¨C11, respectively. Columns 6 and 12 receive only blocking buffer. After incubation and washing, the conjugate is added in all wells of the four quarters of the plate indicated at four different dilutions representing 8, 4, 2, and 1 times the recommended dilutions. After incubation and washing, substrate/chromophore is added and the late stopped at the recommended time. The color developed is then read at 492 nm. Table 12 presents combinations of probable results. We have already assessed that under the standardized conditions, the antigen appears weak, so we are investigating mainly the probable situations in columns 5¨C8 in Table 12. However,

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Table 12

Possible Combinations of Resultsa

aM, mAb; Ag, antigen, C, conjugate.

the titration recommended will investigate all the scenarios. Sketch graphs of the plates as they might appear in the various scenarios are given below in Figs. 28 (1¨C3) and 29 (4¨C6). The situations 7 and 8 are not illustrated since there is no color obtained and in this situation (which would be very unusual), new kit regents should be tested and the materials returned to source. From the pattern obtained it should be apparent where the problem is in the reagents.

7.6¡ª

Some Points to Help Testing

The following tips will help in testing:

1.Check the antigen since it is the factor that most often causes problems. Make sure that none is lost on opening a vial (also true for all other reagents), and that all is resuspended. Check that no reagent escapes or is stuck to lids.

2.Check the pH of all buffers and if possible make up reagents. The wrong pH for substrate/chromophore will affect color development.

3.Practice good pipeting technique at all times. There can be large errors when pipeting small volumes. This is particularly true when tests are first run by inexperienced operators. The variation in testing should go down on practice. This will be seen also with reference to data plotted on charts. The target maximum error between replicates is 10¨C15%. If there are many duplicates for controls and test sera with error >10%, then tighten up your technique for reagent transfer. Slow down the testing (transfer).

4.Examine data obtained with respect to actual OD values generated (unprocessed). Ignore the computer-generated processed data while examining OD values. Assess each plate by eye with respect to controls. Reject those in which Cm values are obviously too small. Reassess the processed plate data with respect to all results for all plates in a test. This can also be done when data are plotted on charts. Do you agree with the action recorded by software-processed data?

5.What are the results from sera tested? Are you getting clear positive sera (high PI% competition of 80¨C100%), even when the Cm OD is low or out of limits? In this case, results may be acceptable, e.g., when in sero monitoring. However, attempt to increase Cm OD. Are you getting clear negatives (competition PI% values of¨C20 to 20%) even when the OD for Cm is low? Again, results may be acceptable. How many sera are in the problem area (e.g., 40¨C55% PI) from

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Fig. 28.

Situations 1¨C3 on titration of reagents.

the samples tested. All? A few? If there are many, then the whole system should be retitrated.

6. Plot all processed data as they are processed on to the IQC charts. The continuous examination and transparent recording of data is essential.

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