The Elisa guidebook

Page 108

Table 16

BRs for Data in Table 15a

	1	2	3	4	5	6	7	8	9	10	11	12
A	5	5	5	5	5	5	5	4	3	2	1	1
B	10	10	10	10	9	7	6	5	3	3	2	1
C	10	10	10	10	9	7	6	5	3	3	2	1
D	38	38	38	38	36	26	22	18	12	10	6	1
E	36	36	36	34	30	26	22	16	10	6	4	1
F	16	14	12	10	8	6	4	4	1	1	1	1
G	10	10	10	8	6	4	2	1	1	1	1	1
H	4	2	2	1	1	1	1	1	1	1	1	1

aData are rounded up to one decimal place.

D (1/400). The dilution in row E (1/800) gives quite similar results and could be used to detect the antigen when the availability of the conjugate is a strong consideration.

2.2.4¡ª

Further Refinement

Stages (i) and (ii) enable a good estimate of the concentrations of each reactant to be made. The idealized example is when the tests work well. Even here we may require further CBTs to establish more precise conditions; for example, a CBT of dilutions of capture antibody against constant antigen and dilutions of conjugate could be examined. The initial CBTs also give an opportunity to set up limited studies on field samples. Thus, if you wish to titrate antigen in samples, you could coat plates with antibody, add serial dilutions of test antigens and then detect these with the conjugate. This would investigate how proper field samples behave in a test and possibly give clues as to the need to modify conditions.

The titration of all three reactants also allows them to be used in similar assays with other reagents. Thus, we may wish to examine another antigen in the assay. The capture serum and detecting conjugate can be used at the dilutions found from CBTs, but the new antigen titrated. Similarly, other capture antibody preparations can be used in tests involving the antigen and conjugate used at the optimal dilutions, as found by CBTs.

As for the description of the direct ELISA, the purpose for which the assay is being developed should always be the strongest factor in test reagent optimization. Ultimately, the test will have to be proved to perform on particular samples and under specific conditions, and validation of ELISAs must meet such conditions.

Page 109

Table 17

CBT, Low OD Data, Stage (i) Direct Sandwich ELISAa

	1	2	3	4	5	6	7	8	9	10	11	12
A	0.4	0.4	0.4	0.4	0.4	0.3	0.3	0.2	0.2	0.1	0.1	0.1
B	0.4	0.4	0.4	0.4	0.4	0.3	0.3	0.2	0.2	0.1	0.1	0.1
C	0.4	0.4	0.4	0.4	0.4	0.3	0.3	0.2	0.2	0.1	0.1	0.1
D	0.4	0.3	0.4	0.4	0.4	0.4	0.4	0.4	0.3	0.3	0.2	0.1
E	0.4	0.4	0.4	0.3	0.3	0.2	0.2	0.2	0.1	0.1	0.1	0.1
F	0.4	0.4	0.3	0.2	0.2	0.2	0.1	0.1	0.1	0.1	0.1	0.1
G	0.3	0.2	0.2	0.2	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
H	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1

aCapture serum diluted is in columns 1¨C11, antigen diluted is in rows A¨CH, and the constant was conjugate.

2.2.5¡ª

Bad Results

The idealized example is typical of good results in which all reactants perform well, i.e., can be used at high dilution and give OD values that are relatively high. When one or more of the reactants is at a low concentration or has poor binding characteristics in the assay, the CBT soon indicates where the problems reside.

Although the number of examples cannot be exhaustive, demonstration of a few bad results is probably far more informative than giving the ideal situation.

2.2.5.1¡ª

Low Color Generally over Plate

Table 17 gives the results of a similar CBT for stage (i) of the direct sandwich ELISA (results shown in

Table 13).

Generally there is low color. There is a plateau corresponding to a maximum value of 0.4 OD units, from column 1 to 5 (highest concentrations of capture antibody), which indicates that the antigen is being captured. There are several reasons for the low OD value in this region:

1.The capture antibodies specific for the antigen are at a low concentration with respect to other serum proteins or do not bind as well as other proteins. Thus, the amount of antigen captured is limited. In this situation, there is no observed increase in OD on increasing the capture antibody concentration.

2.The amount of antigen available for capture is low. This is unlikely since dilution of antigen (from A to B to C, and so on) does not decrease the OD observed in columns 1¨C4, indicating that there is an excess of antigen to row F (after which we observe a reduction in OD).

3.The activity of the conjugate is low.

Page 110

The CBT can be repeated using increased staring concentrations of the titrated components. Thus, we could begin the capture antibody concentration at 10X that used in the first CBT. It is unlikely, however, that this will increase the OD values since we did observe that there was an extensive (columns 1¨C5) plateau maximum indicating that there was maximal activity being measured that did not alter on dilution of the antigen.

In cases in which there is an observed increase in OD on increasing capture antibody, the CBT can be reassessed and a stage (ii) CBT can be performed. When there is no increase in OD values with increased concentrations of antigen or conjugate, the strongest candidate for replacement is the capture antibody. When this antibody is the same as that used for conjugation, both should be replaced.

2.2.5.2¡ª Extremes of Color

When there is a very high color in the majority of the wells, the CBT must be repeated with lower concentrations of reactants. Table 18 gives data from such a plate. The reagent responsible for the high readings may be directly identified from the CBT. Close examination of background values is also necessary because the results may be owing to a very high nonspecific binding of one of the reactants.

The data in Table 18 shows a high background for the conjugate in the absence of capture serum (column 12). Row H indicates that there is no color obtained in which there is antigen and capture antibody in the absence of conjugate. Thus, there is unwanted nonspecific color through attachment of the enzyme conjugate to the wells. This could indicate that the conjugate is being used at far too high a concentration, so that the blocking buffer conditions are not preventing nonspecific adsorption of the enzyme-labeled proteins. There is a titration of antigen on diluting the capture serum, indicated in the backgrounds in column 12; for example, in row G, which has a background of 0.9, a plateau of approx 2.0 is observed with an OD above background observed in column 11 and a reduction in color gradually from columns 3 and 4 to column 11.

A further CBT can be made using the conjugate beginning with that used in row F in the first attempt. Table 19 gives idealized results. Here, the background is eliminated by row D (results are the same in D12, E12, F12, and G12). The effect of diluting the capture antibody is to titrate the antigen after an initial plateau (region of capture antibody/antigen excess). The conjugate dilution up to row D is not suitable owing to the high backgrounds obtained. Thus, a dilution of conjugate at about that in rows D and E can be assessed in the second stage of the CBT, in which the capture antibody and antigen can be varied.

																		Page 111
Table 18
High Color in CBTa
		1		2		3		4		5		6	7	8	9	10	11	12
A	2	* 5	2	* 6	2	* 5	2	* 7	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.6	1.6
B	2	* 8	2	* 6	2	* 5	2	* 8	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.6	1.6
C	2	* 7	2	* 5	2	* 4	2	* 6	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.6	1.6
D	2	* 6	2	* 7	2	* 4	2	* 6	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.5	1.4
E	2	* 2	2	* 3	2	* 3	2	* 6	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.5	1.3
F	2	* 1	2	* 2	2	* 2	2	* 6	2	* 7	2	* 1	2.0	1.9	1.7	1.6	1.5	1.0
G	2.0		2.0		1.9		1.9		1.8		1.7		1.4	1.3	1.2	1.6	1.3	0.9
H	0.1		0.1		0.1		0.1		0.1		0.1		0.1	0.1	0.1	0.1	0.1	0.1
aCapture antibody titrated is in columns 1¨C11, and constant antigen A12 to H12, conjugate diluted is in rows
A¨CG. *Readings are out of accurate range for reader.
Table 19
Repeat of CBT in Which High Color Was Obtained Using More Dilute Conjugatea
		1		2		3		4		5		6	7	8	9	10	11	12
A	2	* 1	2 * 2		2 * 2		2 * 6		2 * 7		2 * 1		2.0	1.9	1.7	1.6	1.5	1.0
B	2.0		2.0		1.9		1.9		1.8		1.7		1.4	1.3	1.2	1.6	1.3	0.9
C	1.9		1.9		1.9		1.9		1.8		1.6		1.3	1.1	0.9	0.5	0.3	0.3
D	1.8		1.8		1.8		1.8		1.8		1.5		1.1	0.8	0.6	0.4	0.2	0.1
E	1.6		1.6		1.6		1.6		1.6		1.3		1.1	0.7	0.5	0.3	0.2	0.1
F	1.2		1.1		1.1		1.1		1.1		1.0		0.9	0.6	0.4	0.2	0.1	0.1
G	0.9		0.9		0.8		0.8		0.7		0.6		0.5	0.4	0.3	0.2	0.1	0.1
H	0.1		0.1		0.1		0.1		0.1		0.1		0.1	0.1	0.1	0.1	0.1	0.1

aCapture antibody titrated is in columns 1¨C11, constant antigen is in A12¨CH12, and conjugate diluted is in rows A¨CG (beginning with dilution used in row F in the initial CBT).

2.2.5.3¡ª

Very Weak Reactions

In cases in which little color is observed, we run into more difficulties because there is no obvious indicator of whether one or all the reagents are not functioning.

A special case is when there is no color development even after a significant time of incubation of the substrate/chromophore. Then the most likely culprit is the operator who forgets to add substrate to the reaction mixture. This can be tested by dipping a microtip into a conjugate and putting the tip into the remaining substrate/chromophore solution. This should show a rapid color

Page 112

change; if not, the operator should repeat the test making sure that the proper substrate/chromophore mixture is correct.

When there is color, the CBT can be repeated. Should very low color then be obtained, the initial CBT should be repeated beginning with much higher concentrations of the two reagents being titrated. This can be a relatively futile operation since we know the dilutions of reactants and have usually added these at a high concentration in the initial CBT. Obviously, the third reactant (conjugate) can be critical, so a higher concentration can be added. Once again, when conjugates are not reacting at dilutions from 1/100 and below, there is little practical value in pursuing their use.

Thus, low color can result from an error such as failing to add proper reagents, making a mistake in the original dilution, or having all the reactants of inadequate strength.

It is easier to assess the reason for low color when there is an indication that one of the reagents is active. This is illustrated in Table 20, in which there is some color development in one area of the plate associated with column 1 and rows A¨CE. Since we have a constant amount of antigen and 1.5 OD units in column 1 is observed, this indicates that both the antigen and conjugate can function although the activity of the conjugate is also rapidly diminished after row C.

The fault here lies with the capture antibody, whose ability to capture rapidly dilutes out by column 3. The CBT could be repeated with higher concentrations in column 1. As already indicated, since the capture antibodies are present as a small component of the total serum proteins, and its capture activity resides in the ability of these specific antibodies to bind to the wells, it may be impossible to achieve a better capture reagent with this serum.

2.2.6¡ª

CBT for Other Systems

The last examples are meant to indicate the first developmental steps in analyzing the suitability of available reagents. In all cases, there is always going to be the need to make adjustments to allow establishment of defined test protocols. The CBT allows only a rough estimate of activities.

More complicated systems (e.g., those relying on four reactants) rely on establishing rough parameters for two of the reagents and examining the affect of diluting the other two. Such assays can be helped greatly through the developmental work with other ELISA systems using the same reagents. Examples are given next.

2.2.6.1¡ª

Developing an Indirect Sandwich ELISA

We already have titrated capture antibodies for use in a direct sandwich ELISA. Thus, the effective concentrations of capture antibodies, antigen, and conjugate are known. Now we may wish to develop an indirect sandwich

Page 113

Table 20

Results Where One or More Reagents Are Weaka

	1	2	3	4	5	6	7	8	9	10	11	12
A	1.5	0.5	0.3	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
B	1.5	0.4	0.2	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
C	1.2	0.3	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
D	0.8	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
E	0.4	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
F	0.2	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
G	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
H	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1

aCapture antibody titrated is in columns 1¨C11, constant antigen is in A12¨CH12, and conjugate diluted is in rows A¨CG.

ELISA replacing the labeled antibodies directly prepared against the target antigen by a nonlabeled detecting serum and an antispecies conjugate. A good reason to do this would be to allow the use of many different animal sera for examination. A good starting point would be to perform a CBT using constant capture antibodies and antigen and titration of the detecting serum and conjugate. The species of the detecting serum would have to be different from that of the capture antibodies since we are adopting an antispecies conjugate (the antispecies conjugate has to be tested for non-ELISA activity against the coating antibodies). Conditions for the optimal coating and antigen concentrations can be used initially. If the test is successful, adjustments can be made by altering any one of the reactant's concentrations.

The establishment of the concentrations of reagents in tests other than those finally used is not uncommon. In fact, pretitration in other systems could be used as a deliberate tool. In the majority of cases, laboratories are working with a limited range of antigens and antibodies, and their exploitation in different systems often results from the need to improve methods defined by a specific task at hand. The reagent link extends to developments in monoclonal antibodies (mAbs) in which polyclonal antibodies may by used at some stage to help production of a more specific or sensitive test. As an example, an indirect sandwich ELISA based on polyclonal sera is available, and we are investigating the use of a detecting mAb (possibly to see whether we can increase specificity for detecting captured antigen). Here, the initial CBT should involve constant capture antibodies and antigen, and titrate the mAb and antimouse conjugate. We know for certain (based on original polyclonal-based ELISA) that we can provide enough captured antigenic target for the mAb. Similarly, the activity of the mAb as a capture reagent can be assessed using the constant components of antigen, polyclonal detecting serum, and conjugate.

Page 115

5¡ª

Theoretical Considerations

This chapter examines the aspects of using ELISA to solve problems, definitions of terms met in serology, antibody structure and the production of antibodies in animals, units, dilutions, and molarities. Antibodies¡ªA Laboratory Manual (1) is an excellent manual of techniques relevant to ELISA and all scientists involved in experimental work involving antibodies should have this manual. The manuals given in refs. 2 and 3 also provide extensive relevant practical information.

1¡ª

Setting up and Use of ELISA

The main aim in the development or use of established ELISAs is to measure some reactant. The need to measure a substance is the major reason for the assay. ELISAs can be used in pure and applied fields of science, but the chief reason they are worth developing is their high sample-handling capacity, ideal analytical sensitivity, and ease of performance. Another factor is the ease of reading, so that time is not wasted when a test has gone wrong. Thus, ELISAs can be assessed by eye before machine reading; for example, time is not wasted in reading 1000 sample points before this insight is obtained (as in radioimmunoassay). Care must be taken not to discard successful tests merely because ELISAs are in fashion. The relationship between ELISA results and other test system results must be established so that a large amount of comparative work using ELISA and one or more assays might be involved in setting up the ELISA as a standard assay.

2¡ª

What Is Known Already

A body of knowledge is often available in the scientific literature on any problem faced by an investigator. Therefore, a survey is necessary. This would mainly involve the detailing of work concerning the biological agent (antigen) being examined and any work involved with its relationship to defined hosts in experimental (laboratory animals) and field studies. This

Page 116

knowledge can be divided into two categories: the biochemical/molecular biological aspects of the agent, and the immunological aspects (serology and immunology per se).

The literature may deal with the exact agent the investigator wishes to study or a similar agent and also reveal whether ELISAs have been performed. Obviously the two aspects of biochemistry and serology are related via the host. The main task at the beginning of any study is to define the aims properly.

Scientists should also examine published work with care since often assays have been poorly devised or have been validated with little data. Scientists should also seek advice from others in related fields and examine whether reagents have already been produced that might be applicable to their own problems. A particular area is the use of monoclonal antibodies (mAbs).

An excellent catalog of immunological reagents is available, Linscott's that lists and updates reagents and suppliers of thousands of polyclonal and monoclonal reagents of direct relevance to ELISA. Much information is also available in catalogs of commercial firms that often have detailed technical descriptions of the use of their products.

3¡ª

Complexity of Problems

Complexity of problems is manifested throughout the interrelationship between the agent and host. The concept of the relationship between antigenicity /immunogenicity and protection should be examined in this light. Thus, the major dogmas of antibodies and antigens must be examined. This book does not investigate the theories surrounding immunology, and textbooks should be consulted for more details. This chapter highlights relevant knowledge to allow more information to be sought. The following definitions are helpful.

3.1¡ª Antigenicity

Antigenicity is the ability of proteins and carbohydrates to elicit the formation of antibodies, which, by definition, bind specifically to the antigens used for injection into animals. Antibodies may be produced as a consequence of replication of an agent or by injection of inactivated whole or parts of that agent.

This can further be refined so that defined peptides or polypeptides are used. The antigens used to elicit antibodies can, in turn, be used in tests such as the ELISA.

3.2¡ª Immunogenicity

Immunogenicity is a measure of the effect of binding of antibodies elicited by any substance. More specifically, the effect is one of producing some degree of immunity against the disease agent. Generally, such measurements are made in vitro or in animal systems other than those being examined in the field.

Page 117

3.3¡ª Protection

Production of antibodies and demonstration of immunogenic responses does not necessarily mean that animals will be protected against challenge of the disease agent. The relationship of immunoassay results to protection is never straightforward because of the many other factors involved in immunological response, e.g., cellular immunity.

4¡ª

Antigenic Considerations

Inherent to the understanding of what is being measured in any ELISA is the definition of terms involving antigens and antibodies and an understanding of the implications of the size, number of possible antigenic sites (epitopes), distribution of epitopes (distance between them), variability of epitopes, effect on variation in epitopes on different assay systems, and so forth. As the size (molecular weight) of disease agents increases, complexity increases.

4.1¡ª

Size Considerations

Immunoassays must be developed with as much knowledge as possible of all previous studies. The complexity of agents generally increases with their size. On theoretical grounds, the relationship of size to possible complexity can be demonstrated by examining spherical agents of different diameters, beginning at 25 nm (e.g., the size of a foot-and-mouth disease virus [FMDV] particle).

One can calculate that the area bound by a Fab molecule (single arm combining site of antibody without Fc) is approx 20 nm2. This can represent an antigenic site (epitope). Thus, the number of possible sites on the virus is the surface area of the virion divided by the surface area of the combining site. The surface area of a sphere is 4 ¡Á π ¡Á r2; therefore, for FMDV 4 ¡Á 3.14 ¡Á 12.52 = 12.56 ¡Á 156.25 = 1962 nm2. By dividing this by 20, the maximum number of Fab sites possible is 98.

If the diameter of the agent is increased by twofold to 50 nm, using the same calculation the number of sites is 392. Another twofold increase in diameter gives 1468. These are small agents. If the calculations are done for agents increasing in diameter by 10-fold steps, for Fab and whole IgG (binding bivalently effective area 60 nm2), the data would be as given in Table 1.

The numbers in Table 1 illustrate that the surface area increases as a square function of the diameter. Such a calculation is based on the facts that the whole surface is antigenic (rarely true) and that the molecules bind maximally. However, experimentally, relative figures close to the theoretical are obtained. This has implications in immunoassays since one can calculate how much antibody is needed to saturate any agent, or measure the level of antibody attachment as a function of available surface.