The Elisa guidebook

A representative mAb profile from some of the clusters is shown in Fig. 22A¨CD. Such profiles relating binding of mAbs are typically produced. Their evaluation is more difficult. Comparison by eye estimation is not valid; this section has attempted to introduce statistical methods for the easy comparison of data.

11.3.3¡ª Discussion

The method used for the rapid analysis of virus isolates relied on the specificity of mAbs. In this study, a relatively large panel was used, the mAbs of which were prepared against several classical type A viruses representing epidemiologically important groups. Thus, one aspect of the study allows an

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Fig. 20.

Grouping of viruses according to their similarity of binding profiles with mAbs.

examination of the distribution of epitopes identified by the mAbs. The characterization of the specific mAbs in terms of, e.g., reaction with conformational and linear sites, amino acid sequences important to binding, and trypsin sensitivity, allows various antigenic properties to be ascribed to the viruses where binding of antibody is observed. Such properties can be used to directly compare possible biological properties of isolates and thus predict problems in vaccine formulation, identify specific changes to viruses during the manufacture of vaccine and examine viruses and virus proteins produced and expressed using molecular biological techniques. The data collected for the mAbs is included to allow a comprehensive list of properties to be available to other researchers.

The use of a large number of virus isolates also allowed the rapid comparison of all the mAbs. Thus, the variation in antigenic properties of the isolates allowed identification of patterns of reaction of the mAbs, thereby producing groups (clusters) of identically or similarly reactive mAbs and distinguishing them from other clusters. This approach is only possible where a large number

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Fig. 21.

Virus clusters (1¨C16) taken from data in Fig. 21. The closeness of relationships is indicated by boxes.

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Fig. 22.

(A) Profiles showing individual mAb reactions with particular viruses. 1¨C4, members of those cluster groups specified in Fig. 22. (B) Profiles showing individual mAb reactions with particular viruses. 6, 7, 9, and 10: members of those cluster groups specified in Fig. 22. (C) Profiles showing individual mAb reactions with particular viruses. 11¨C14: Members of those cluster groups specified in Fig. 22. (D) Profiles showing individual mAb reactions with particular viruses. 8, 15, and 16: Members of those cluster groups specified in Fig. 22. Scale (y-axis) is from 0¨C120% binding with respect to homologous binding of mAb to parental strain.

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of epidemiologically distinct isolates is available (as is true of the WRL), and a limitation of the variation in antigenic makeup of the isolates would have an effect on the patterns of reaction of the mAbs. Thus, if only two strains were used in this study, it would be likely that only two to three groups of mAbs would be observed. The clusters observed for the mAbs through examination of many isolates are verified with reference to data from this and previous studies on mAb escape mutants as well as reference to the binding characteristics of the mAbs.

This exercise also allows a limited panel to be designated whereby the relationships between isolates can be made with a low number of mAbs, which greatly simplifies assays. Confidence that limited panels reflect true antigenic differences comes only through a thorough examination of mAbs against a large number of viruses. Such an approach is also quite useful to workers in laboratories without access to a large number of isolates because the mAbs they produced can be compared to others by standardization laboratories. The mAbs can be identified as fitting into particular clusters (already defined), and properties common to those clusters can be ascribed.

The method used to examine the virus/mAb binding used mAb in excess (i.e., at a high concentration). This can have a distinct effect on the binding profiles observed. The relative affinity of each of the mAbs depends on the exact differences between the epitopes presented on the heterologous viruses as compared to the homologous virus. The assay used is essentially a binding assay, and small differences in affinity constants among isolates are not reflected by differences in binding in which there is a large excess of antibody molecules. Thus, any differences noted in this study reflect relatively large differences in affinity (a significant difference in epitope). Such differences also fit in with the examination of the virus-neutralizing capacity of the mAb for homologous and heterologous viruses. Not all mAbs that bind 100% to heterologous isolates will neutralize that virus (results not shown). This is a result of the differences in the conditions in the virus neutralization test (VNT). In the VNT, the amount of virus used represents about 100 TCID50 (approx 105 virus particles assuming 1 TCID50 is equivalent to 103 noninfectious particles). In the ELISA, approx 0.05 µg is present to bind with mAb (approximately 8 ¡Á 109 virus particles). The same concentration of mAb is used in both assays; thus, by the Law of Mass Action, the ELISA tends to effect reaction owing to the high concentration of virus (approx ¡Á 80,000 times that in the VNT). Therefore, mAbs with reduced affinity tend to have the reaction driven in the ELISA but not in the VNT.

Differences in affinity for strongly binding mAbs can be assessed easily using competitive assays in which a homologous system involving pretitrated

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virus and homologous mAb (submaximal binding concentration) is challenged by the addition of heterologous virus. Here, the relationship of competition slopes comparing homologous and heterologous viruses reflects the relative affinity of the mAb for the two isolates (data not shown).

12¡ª Conclusion

Figure 23 shows the features of mAbs relevant to the ELISA. Although the mAb is, by definition, a specific reagent with respect to the binding to epitopes, the physical state of the mAb, its density, and the distribution of epitopes all affect assay performance.

Let us review the diagrams in Fig. 23:

1.Diagram 1: This indicates that the orientation of the mAb on the plastic surface can affect subsequent binding to antigens (e.g., in capture ELISA). Since the mAb is a single population of molecules with identical chemical structure, any tendency for such orientations (depending on the exact nature of plastics used and the solutions used to bind the mAb), will be translated to all molecules. This means that a variety of plates and solutions of different ionic strengths and pHs can be used in cases in which an mAb apparently does not perform well as a capture reagent.

2.Diagram 2: This indicates that the density of mAb can affect binding, even when the orientation is correct and Fab molecules are present. Full-dilution ranges of mAbs should be performed to allow an assessment of binding properties, since it is possible that lower concentrations of mAb are better spaced to allow capture. This is also dependent on the nature of the antigen.

3.Diagram 3: This reminds us that the isotype of an mAb can be important. Generally, IgM molecules are poor capture reagents. The use of mAbs of different isotypes can be exploited through the use of specific antimouse isotype reagents.

4.Diagram 4a: When bivalent molecules of mAb are reacting with epitopes on a complex, the spacing of the epitopes has a profound effect on the actual affinity of the mAb. When epitopes are spaced too far to allow bivalent binding, effectively a single Fab interaction takes place. The orientation of the epitopes is also important.

5.Diagram 4b: When small molecules (e.g., polypeptides) are coated, mAb may also have optimal bivalent binding in which the spacing (and presentation/orientation) is optimal.

6.Diagram 4c: Here, the spacing is too large to allow bivalent binding and hence the effective affinity is reduced as compared to diagram 4a.

7.Diagram 4d: The deliberate processing of mAbs to Fab fragments affects the affinity. Here, the spacing of the molecules is not as important as in diagrams 4a and 4b, since the Fab fragments are free to interact in solution.

8.Diagram 4f: This probably reflects the most common situation in which mAbs are used as a relatively impurified mixture of bivalent and monovalent molecules.

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Fig. 23.

Properties of mAbs relevant to performance of ELISA. 1, The orientation of molecules affects capture properties; 2, density of molecules affects performance through interference. The correct spacing of mAbs also is important with reference to epitope spacing and density; 3, the isotype of mAbs can be important; 4a, optimal binding of bivalent mAb needs spacing of epitopes on multivalent antigen target; 4b, spacing of small molecules is important to maximal bivalent binding; 4c, where the distance between FAB fragments is too large, only monovalent binding takes place; 4d¨C4e, Fab fragments are free to bind and the reaction is limited only by concentration of epitopes; 4f, the most common mixture of bivalent and Fab molecules used in assays.

Here, the binding of Fab fragments and bivalent molecules can be regarded as competitive. Assays developed with such reagents may suffer since the distribution of Fab to bivalent molecules is different from batch to batch and owing to physical changes on storage. When a purified product is used, the results may be

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different. This is also relevant when considering mAbs as capture reagents (as in diagram 1).

References

1.Grandic, P. (1994) Monoclonal antibody purification guide. Part 3. American Biotech. Lab. 12(8), 16, 18.

2.Butcher, R. N., Obi T. U., and McCullough, K. C. (1991) Rapid isolation of monoclonal hybridoma cultures by a fusion-cloning method: the requirement for aminopterin. Biologicals 19, 171¨C175.

3.McCullough, K. C., Crowther, J. R., Butcher, R. N., Carpenter, W. C., Brocchi, E., Capucci, L., and De Simone, F. (1986) Immune protection against foot-and-mouth disease virus studied using virus neutralising and nonneutralising concentrations of monoclonal antibodies. Immunology 58, 421¨C429.

4.McCahon, D., Crowther, J. R., Belsham, G. J., Kitson, J. D. A., Duchesne, M., Have, P., Meleon, R. H., Morgan, D. O., and De Simone, F. (1989) Evidence for at least four antigenic sites on type O foot-and-mouth disease virus involved in neutralisation: identification by single and multiple site monoclonal antibody resistant mutants. J. Gen. Virol. 70, 639¨C664.

5.McCullough, K. C., Crowther, J. R., Carpenter, W. C., Brocchi, E., Capucci, L., De Simone, F., Xie, Q., and McCahon, D. (1987) Epitopes on foot-and-mouth disease virus particles. I. Topology. Virology 157, 516¨C525.

6.Nakane, P. K. and Kawaoi, A. (1974) Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22, 1084¨C1091.

7.Samuels, A. R., Knowles, N. J., Samuel, G. D., and Crowther, J. R. (1991) Evaluation of a trapping ELISA for the differentiation of foot-and-mouth disease virus strains using monoclonal antibodies. Biologicals 19, 229¨C310.

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8¡ª

Validation of Diagnostic Tests for Infectious Diseases

1¡ª Validation

Validation involves all processes that determine the performance of an assay to achieve a defined set of objectives. Only when actual data have been obtained can test parameters be assessed and confidence in results be assigned in a statistical sense. Validation is a continuous process, in which increasing knowledge about an assay is gained each time it is run. The continuous process also involves data obtained when the test is performed in hitherto untried scenarios. Since most assays begin in the research arena, the use of validated assays in the form of kits by a wider range of scientists in laboratories varying widely in expertise, equipment, and climatic conditions can cause problems. The objective in validation is to be able to define an assay in terms of statistically quantifiable parameters with measured confidence. The designation of ''validated assay" is only merited when it has been defined in terms of its capacity to classify samples with regard to the presence or absence of a particular analyte. Validation relies on examination of as many factors as possible. At any stage, quantifiable parameters must be defined describing the test and mechanisms to reevaluate be put into place.

A validated assay, therefore, depends on the characteristics of assay design that ensure the results. This leads to a robust assay (not easily affected by physical factors, operators, or geographical location where used or where samples came from). Such assays generate data that can be compared directly irrespective of which laboratory uses it, and to what population of animals it is applied.

In the context of ELISA, the development and validation of an assay is usually made using a limited number of tests, on samples from a selected group(s) of animals or patients, and made over a short time frame. The data define the performance of the assay, and these performance characteristics are published, and possibly certified by governing authorities. In this case, what constitutes a validated assay obviously depends directly on the experiences limited to the

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samples analyzed. The conditions established during this validation phase can also be modified based on experience with the assay's capacity to correctly classify the infection status of animals from various populations over a longer time period. Such a situation is unavoidable since a single laboratory cannot have access to all samples at all times. However, the validation methods made must be clearly described so that at least variations from the accepted criteria can be determined and possibly accounted for.

1.1¡ª

Definition of a Validated Assay

The concept of a validated assay has many shades of meaning among laboratory diagnosticians and veterinary clinicians. For this chapter, a validated assay is described in terms of its use as an assay that provides results that consistently identify animals as being positive or negative for the presence of a specific analyte (antibody or antigen), and by inference accurately predict the infection status of animals with a known (measurable) degree of statistical certainty. The principles underlying the development and maintenance of such a validated assays are examined herein.

1.2¡ª

Components of Assay Validation

The development and validation of an assay is a multicomponent operation consisting of at least three general areas:

1.Feasibility of the method including choice and optimization of reagents and protocols.

2.Determination of the assay's performance characteristics.

3.Continuous monitoring of assay performance during routine use.

The third component may not be immediately considered as part of assay validation, but it is included because a test can be considered valid only when the data generated and their interpretation are, respectively, accurate and meaningful and updated. The development of an indirect ELISA for antibody detection can be used to illustrate points 1¨C3. This is a test format that can be difficult to validate since there is signal amplification owing to both specific and nonspecific components.

1.3¡ª

Feasibility Studies

In our ELISA example, feasibility studies are first made to examine whether the selected reagents have the capacity to distinguish between a range of antibody concentrations and the infectious agent in question, while providing minimal background activity. This can be a rapid process (a few weeks) and uses a minimum of samples. It establishes whether the test is feasible for further examination.