The Elisa guidebook

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Fig. 15.

Outline of antigen(s) detected in ELISA.

(A) Relative sizes of whole antibody molecules, virus (146S), and subunits (12S); (B) profile of virus and subunits; (C) 12S subunit with internal and external (as exposed on 146S) epitopes; (D) capture of 12S by mAbs directed against external epitopes with orientation of internal epitopes to

detecting antibodies; (E) polyclonal serum containing antibodies against both internal and external epitopes orientate 12S in both ways.

ELISA has been used successfully as an analytical method for the estimation of total degradation of the 146S using a defined polyclonal system in the analysis of the pH stability of 146S and 75S particles.

All the mAbs examined were capable of detecting 146S and 12S when used as detecting reagents, as shown in Fig. 9. The reduction in plateau height was again observed. However, mAbs used in combination did not detect 12S

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even at high concentration, but were capable of specifically measuring 146S. Figure 12 illustrates this and also shows that mAbs B2 and D9 did not detect TTV or DNV, although both these mAbs have been shown to react with continuous epitopes (linear determinants) on the VP1 loop of type O FMDV in the indirect ELISA. However, the sandwich conditions presumably inhibit any second antibody binding since denaturation and disruption of the virus produces small polypeptides and peptides containing the linear epitopes that bind exclusively to the capture antibodies.

The use of such virus-neutralizing mAbs reacting with linear epitopes on VP1 on the outside of the capsid that are sensitive to proteolytic cleavage allows not only the quantification of virus but also a qualitative assessment of the antigen. This is vital in preparing vaccines that show poor immunogenicity when cleavage of VP1 has occurred. mAbs that react with similar epitopes have been characterized, so it is envisaged that there will be little difficulty in identifying reagents suitable for this assay for the assessment of vaccines. The prerequisite is that the mAb can bind to the vaccine strain of interest.

Titration of 146S in the presence of large excesses of 12S could interfere with the specific quantification of 146S. This could occur since the capture mAbs bind 12S, which may affect the capture potential for 146S. The results in Figs. 12 and 13 confirm that this was not a problem. There was a slight increase in the expected OD values for the 146S , when 12S was added at 100 and 50 times the weight of 146S, particularly using the C8 and C9 systems.

Such ratios are not expected to be present in infectious tissue culture samples prepared during the manufacture of vaccine.

The use of standard curves for calculation of virus weight should be successful if precautions are taken to avoid thermal and chemical effects on standard preparations. Thus, once a purified virus has been assessed spectrophotometrically and stored in small aliquots at ¨C70¡ãC or in liquid nitrogen, and when used as single batches, it should be possible to standardize assays precisely. This was shown by titration of the same virus on 10 different days where the best range for the estimation for virus was when OD values were from approx 1.2 to 0.4, corresponding to 0.5 and 0.03 µg/mL of virus/mL. The data in Tables 18 and 19 indicate that reproducibility of the assay is acceptable for the purpose of assessing of 146S in vaccines.

11¡ª

Use of mAbs to Examine Antigenic Variation in Type A FMDV

11.1¡ª Background

Any mAbs produced against members of serotype A FMDVs can be been used to examine antigenic differences. The mAbs used in this study were obtained from various laboratories in Europe and South America. A microtiter plate sandwich ELISA was used to measure the binding of the mAbs with virus

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field isolates, vaccine strains, and mAb escape mutants relative to binding with homologous virus. Different amounts of serological and biochemical data were available as to the characterization of the mAbs, particularly for the identification of the critical amino acid sequences bound by individual mAbs. The use of a relatively high number of viruses allowed mAbs of similar reactivities to be compared and grouped using multivariate statistics. The antigenic relationships between the viruses were also evaluated in the same way, and the relevance of results to the epidemiology of strains was examined. The study has allowed the frequency of different epitopes on type A viruses to be examined and the consideration of the epidemiological significance of the findings. Recommendations are made for the use of a limited number of mAbs to act as a standard panel to allow rapid antigenic analysis of isolates.

mAbs against most serotypes of FMDVs have been prepared and characterized in many laboratories worldwide. Such reagents offer the potential for rapid antigenic characterization of virus isolates in simple binding assays such as ELISA. Antigenic variation of FMD viruses is important in many areas involving the control of diseases, such as assessing field strains for their potential threat to animals vaccinated with vaccine strains, comparing vaccine strains among producers, monitoring the vaccine strain throughout production, examination of challenge strains used to evaluate vaccine efficacy which are produced by passage in animals, and examining of persistent viruses (carrier state).

This study shows how mAbs produced against serotypes A5, A10, A22, and A24 viruses, from different laboratories, can be used to group viruses according to their similar properties; examine the distribution and variation of the epitopes; recommend an assay for the rapid comparison of epitopes on type A viruses; and to define a limited panel of type A mAbs that might be useful in comparing type A field, vaccine, and challenge strains.

11.2¡ª

Materials and Methods

11.2.1¡ª Viruses

The viruses were obtained from the World Reference Laboratory (WRL), at Pirbright, UK. Certain isolates were selected as representatives of vaccine strains. The isolates were amplified by growth in tissue culture usually through bovine thyroid (BTY) primary cells, then passaged in continuous monolayer cultures of baby hamster kidney (BHK-21). Some of the viruses also were passaged in continuous renal swine cells (RS). The passage history of most the isolates is indicated by the number following the cell line. Most of the samples for use in the ELISA were obtained by a further passage of seed stock virus in BHK cells, but the last manipulation of the virus is shown by the last cell line indicated. When monolayers were totally disrupted, the mixture was cen-

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trifuged (2000g for 10 min) to remove debris, and the supernatant was stored at ¨C70 or ¨C20¡ãC after the addition of an equal volume of sterile glycerol.

11.2.2¡ª Antisera

Rabbit and guinea pig polyclonal antisera against type A5, A22, and A24 viruses were prepared as described in ref. 2. These sera, used as capture antibodies and detecting antibodies, respectively, in ELISAs were produced after multivaccination of animals with purified inactivated virus, containing antibodies with a wide spectrum of activity against all FMDVs components.

11.2.3¡ª

Monoclonal Antibodies

The mAbs used in this study came from the IAH, Pirbright, UK, and from various laboratories in Europe and South America. These were obtained as ascites fluids or tissue culture preparations.

11.2.4¡ª

Data Obtained for mAbs

Different methods were used to characterize the various mAbs, including use of various ELISAs with various antigenic preparations of the viruses. The data were also used to evaluate the findings of the sandwich ELISA.

11.3¡ª

Sandwich ELISA to Compare Binding of mAbs to FMDVs: Antigenic Profiling

The sandwich ELISA was conducted as described in ref. 7. Briefly, microtiter plate wells were coated with a pooled mixture of rabbit antibodies produced against type-purified isolates characterizing A24, A22, and A5 FMD virus subtypes. Such a mixture has been shown to be effective in capturing most of the type A FMDV isolates examined in the WRL at Pirbright. The rabbit antiserum was added in 50-µL vol to the wells and diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6. Plates were incubated overnight at 4¡ãC or 1 h at 37¡ãC. Plates were then washed with PBS, and the various virus preparations were added in duplicate in 50-µL vol, (as shown in Fig. 16) diluted in 50 µL of blocking buffer (PBS containing 3% bovine serum albumin, 0.1% Tween-20, and 5% nonimmune [normal] bovine serum). The homologous virus was always included on row A of each plate to demonstrate the maximal interaction of the mAbs so that relative assessments of binding could be examined, as discussed next.

Plates were washed and mAbs were added as shown in Fig. 17. The mAbs were diluted in blocking buffer as described for the virus dilution. The dilution of mAb used was determined from the studies on mAb binding to homologous virus using indirect and sandwich ELISAs; an excess of mAb was always used. The last two columns of the plates received guinea pig serotype-specific serum

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Fig. 16.

Sandwich ELISA for mAb profiling. All wells of the plates are coated with polyclonal anti-FMDV-type specific serum. A mixture of rabbit antibodies against A5, A22, and A24 serotypes is used. After incubation and washing, a single dilution of different virus suspensions is added (in blocking buffer) as shown, in duplicate rows for test samples (B, C; D, E; and F, G). Row A contains virus responsible for eliciting mAbs used, and row H receives only blocking buffer.

at a pretitrated dilution in blocking buffer. This serum was broadly reactive (produced in a way similar to the rabbit capture antibodies), and has been tested to react with all viruses within a serotype. The values in these columns served to estimate the amount of each virus captured. The last row received no virus but the respective column mAb. This acted as the background control for any mAb interaction. Plates were incubated at 37¡ãC for 1 h while being rotated. Plates were washed, and then each well that had mAb added received antimouse IgG enzyme conjugate at a pretitrated optimal dilution in blocking buffer. The last two columns received anti¨Cguinea pig conjugate as described for the virus titrations. The plates were incubated at 37¡ãC for 1 h with rotation and then the OPD substrate solution was added. The color development was stopped after 10 min and the color quantified by a multichannel spectrophotometer.

The dilution of each virus was usually determined by previous titration in a sandwich ELISA in which plates were coated and viruses diluted in triplicate as twofold series, in blocking buffer. After incubation with rotation, the plates were washed and a pretitrated antiserotype-specific guinea pig serum was added diluted in blocking buffer. The plate was then incubated as for the virus stage and washed, and then each well received a dilution (in blocking buffer) of anti-guinea pig HRP conjugate. After incubation as before, H2O2/OPD substrate was added. Color development was stopped at 10 min. The plates were read in a multichannel spectrophotometer (492 nm). The developed color

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Fig. 17.

Addition of mAbs to profiling plates. mAbs 1¨C10 are added in columns 1¨C10 at a single dilution in blocking buffer. Polyclonal anti¨Ctype A serum is added in columns 11 and 12. Plates are incubated and washed, after which antimouse enzyme conjugate is added to columns 1¨C10 and anti¨Cguinea pig enzyme conjugate to columns 11 and 12. After incubation and washing, substrate chromophore is added and color development stopped. The OD values

are read and the relative binding of mAbs to viruses is determined with reference to the reactions of the mAbs with the homologous virus. The OD values in row H are subtracted from each OD value in the respective column. The OD values for each duplicate mAb reaction are then averaged. This mean OD value is then expressed as a percentage of the mean value obtained for the virus/ guinea pig binding for each respective virus. Finally, this percentage value is expressed as a percentage of the value obtained for the homologous virus. This, the relative weights of each virus are taken into consideration by reference to the examination of the guinea pig polyclonal readings, and results are therefore comparing the relative binding of the same mAbs to different viruses as compared to the homologous virus.

was related to the dilution, and the dilution giving 1.2 to 1.5 OD was used in the antigenic profiling ELISA described in Subheading 11.3.1. The plates were incubated for 1 h at 37¡ãC while being rotated at approximately three revolutions per second. In practice, it was found that most tissue culture preparations contained high levels of virus and that dilutions of 1/3 to 1/16 could be used to provide excess virus for the trapping rabbit serum. Therefore, the amount of rabbit serum was limiting in this step.

11.3.1¡ª Processing of Data

Processing of the data is described in ref. 7. Briefly, the OD values in the last row (mAb negative antigen control) were subtracted from each column.

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The OD values for each duplicate were then averaged. This mean value was then expressed as a percentage of the mean value obtained for the virus/guinea pig binding for each respective virus. Finally, this percentage value was expressed as a percentage of the value obtained for the homologous virus. Thus, the relative weights of each virus were taken into consideration by reference to the examination of the guinea pig polyclonal readings. Results are therefore comparing the relative binding of the same mAbs to different viruses as compared to the homologous virus. This is illustrated in Tables 20 with simplified results.

The criteria for assessing the results and statistical considerations were examined in ref. 7. The data were analyzed with a computer-based package using multivariate statistics to perform hierarchical analysis (block method, complete linkage). The mAbs were then assessed according to their reactions with the viruses, and the viruses were related according to their reactions with the mAbs. Cluster analyses were made grouping the reactions of the mAbs and viruses, respectively. The reactions were related to obtain dendrograms of the mAb and the virus groups.

11.3.2¡ª Results

All the mAbs were examined initially for their binding characteristics against 20 selected field strains, using the antigenic profiling sandwich ELISA. From all data, 30 of the mAbs were selected for use in larger-scale antigenic profiling studies, in which a higher number of virus isolates was examined. The conclusive final relationships for the mAbs and the antigenic relationships of the viruses were based on the use of these 30 mAbs and 60 viruses. The data in the dendrograms in Fig. 18 show the cluster analysis for the mAbs. Ten clusters were selected as being distinct; these are indicated in Fig. 19 (in which the origin of the mAbs is also indicated). That the mAbs in these clusters reacted with different epitopes was further confirmed through the examination of other available data. In Fig. 19, mAb L13 is boxed in because it reacts with a conformational epitope whereas the other mAbs bind to a linear epitope.

The relationship of the viruses as elucidated from the binding pattern with the mAbs is shown as a dendrogram in Fig. 20. The distance at which the viruses within a cluster are assessed as being very similar but different to another cluster has been put at 5.0. This is demonstrated by the line drawn across the dendrogram in Fig. 20. This value is based on assessing the relevance of the observed clusters to existing epidemiological knowledge of virus isolates. Sixteen clusters are produced, as shown in Fig. 21. The virus profiling data showed two major clusters separating A22-like viruses from the A24-like and A5/A10-like viruses, the latter two clusters were more closely related. The first cluster group indicates closely related South American A24 Cruzerio iso-

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Table 20

Stylized Data and Methods for Calculation

			(i) Untreated sylized OD data (duplicates made the same)
	1	2	3	4	5	6	7	8	9	10	11	12
A	1.1	1.5	1.3	1.6	1.0	0.8	0.9	1.1	1.2	1.3	1.6	1.6
B	0.1	1.4	1.3	1.1	0.1	0.1	0.7	1.1	0.1	0.1	1.5	1.5
C	0.1	1.4	1.3	1.1	0.1	0.1	0.7	1.1	0.1	0.1	1.5	1.5
D	1.1	1.4	1.2	0.1	0.1	0.1	0.1	1.2	1.0	1.0	1.5	1.5
E	1.1	1.4	1.2	0.1	0.1	0.1	0.1	1.2	1.0	1.0	1.5	1.5
F	0.5	1.1	1.3	0.1	0.5	0.8	0.9	1.1	0.1	0.1	1.2	1.2
G	0.5	1.1	1.3	0.1	0.5	0.8	0.9	1.1	0.1	0.1	1.2	1.2
H	0.1	0.2	0.3	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.2	0.2
				(ii) OD minus row H value in each column
	1	2	3	4	5	6	7	8	9	10	11/12
A	1.0	1.3	1.0	1.5	0.9	0.7	0.8	1.0	1.1	1.2	1.4
B	0	1.2	1.0	1.0	0	0.1	0.6	1.0	0	0	1.3
C	0	1.2	1.0	1.0	0	0.1	0.6	1.0	0	0	1.3
D	1.0	1.2	0.9	0	0	0.1	0	1.1	0.9	0.9	1.3
E	1.0	1.2	0.9	0	0	0.1	0	1.1	0.9	0.9	1.3
F	0.4	0.9	1.0	0	0.4	0.7	0.8	1.0	0	0	1.0
G	0.4	0.9	1.0	0	0.4	0.7	0.8	1.0	0	0	1.0
				(iii) Means of OD values after subtraction
	1	2	3	4	5	6	7	8	9	10	11/12
A	1.0	1.3	1.0	1.5	0.9	0.7	0.8	1.0	1.1	1.2	1.4
B/C	0	1.2	1.0	1.0	0	0.1	0.6	1.0	0	0	1.3
D/E	1.0	1.2	0.9	0	0	0.1	0	1.1	0.9	0.9	1.3
F/G	0.4	0.9	1.0	0	0.4	0.7	0.8	1.0	0	0	1.0
		(iv) Percentage value of OD mAb/OD value polyclonal, for that virus in 11/12
	1	2	3	4	5	6	7	8	9	10	11/12
A	71	93	71	107	64	50	57	71	79	86	100
B/C	0	92	77	77	0	8	46	77	0	0	100
D/E	77	92	70	0	0	8	0	84	70	70	100
F/G	40	90	100	0	40	70	80	100	0	0	100
		(v) mAb percentage in each column as percentage of homologous virus value

	1	2	3	4	5	6	7	8	9	10
A	100	100	100	100	100	100	100	100	100	100
B/C	0	99	108	72	0	16	80	108	0	0
D/E	108	99	99	0	0	16	0	118	89	81
F/G	56	97	140	0	61	140	158	140	0	0

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Fig. 18.

Grouping of mAbs according to reaction patterns against viruses. Analysis was by multivariate statistics.

lates. The profiling confirms the identity of two vaccine and challenge strains produced after passage in cattle and indicates their similarity to the A24 Cruz reference strain. Viruses from Peru in 1971 and 1972 appear to be similar to each other and to the reference strain. The second cluster within the A24-like viruses comprises two virus isolates from Brazil in 1979 and A24 Venceslau in 1970. Both these clusters have a similar relationship to clusters 3 and 4. These clusters contain South American viruses expected to be A24-like (A Col/Sab/85 and A Col/Boy/89); however, viruses of African origin (A Egypt 1/77, A Cam 5/75, and A Sau 1/76) are also included. Cluster 5 has more similarity to the A5/A10 viruses and again contains both South American (A Bra/68, A27 Col/67) and African (A Libya 3/79, A Alg 5/75) viruses. This cluster is similar to clusters 6 and 7, which contain early European (A5 West/51, A Greece 1/76), Middle Eastern (A Sau 23/68) and African (A Ken 1/76) viruses. Cluster 8 is more distinct than cluster 1¨C7 and comprises the A10 Holl/42, A10 Arg/61 (known to be related), and A5 Spa/73 virus. In turn, this group is strongly related to A5 viruses from Italy and France.

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Fig. 19.

Grouping of mAbs according to reaction patterns against viruses. Analysis was by multivariate statistics. There was clustering of mAbs with similar reactivities.

Data were examined in light of the origin of mAb and known reactions from other tests. Serotypes used to generate mAbs are shown as A5, A22, A24, and A10. C, mAbs binding to conformation-dependent epitopes; L, mAbs binding to conformation-independent epitopes (linear); CTERM, mAbs demonstrated to react with the C-terminus of structural protein VP1.