Bmlog~cals { 1991) 19, 299-310

Evaluation of a Trapping ELISA for the Differentiation of Foot-and-mouth Disease Virus Strains Using Monoclonal Antibodies A. R. Samuel, N. J. Knowles, G. D. Samuel* and J. R. Crowthert

AFRC Institute for Animal Health, Pirbnght Laboratory, Ash Road, P/rbnght, Wok/ng, Surrey GU24 ONE U.K. Abstract. A trappMng enzyme-hnked immunosorbent assay (ELISA) has been evaluated for the differenttat~on of foot-and-mouth dMsease wrus (FMDV) strains using a panel of seven anti-serotype O monoclonal antMbodtes (MAbs) The vanatlon of results within and between tests performed on the same day and on d~fferent days was examined using three strains of FMDV. Cntena for estabhshlng antigenic differences between the straMns as defined by the indwidual MAbs are proposed based on the vain ablhty measured, which can be used as standards by workers performing this test with other MAbs and FMDV strains Introduction The origin of wruses causing outbreaks of foot-andmouth disease (FMD) in countries where vaccination is performed must be identified to ascertain whether the outbreak is caused by the administration of an incompletely inactivated vaccine, laboratory escape or by an introduced strain. Background information on variation of field strains from around the world aids the recogmtion of strains in new environments. Variation in virus during the preparation of vaccines may also be important since some adaptation from stock viruses (field strains) into, for example, suspension cell culture or Frenkel culture, is necessary. Variation is also relevant when considering challenge strains which are adapted to grow in cattle, mice or guinea pigs and which are then used to challenge vaccinated ammals to estimate the vaccine potency. The types of study involved in the examination of variation can be divided into serological and biochemical. The serological studies attempt to measure the antigenicity of viruses by examination of the relative reactions with reference antisera with respective viruses. The main assays have involved virus neutralization, complement fixation and, more recently, various enzyme-linked immunosorbent assays (ELISAs). All such assays involve the interaction of virus and polyclonal antisera and are best * Present address: 13 Ashley Road, Farnborough, Hampshire GU14 7EZ. t To whom correspondence should be addressed. 1045-1056/91/040299+ 12 $03 00/0

assessed where homologous antisera are produced against each isolate examined so t h a t two-way testing comparing two viruses can be obtained. The preparation of antisera is time consuming, suffers from the high animal-to-animal and species-tospecies variation of polyclonal response to the same antigen(s) and is selective in terms of assays performed. For example, complement fixation is dependent on certain isotypes. Results of assays may also suffer from high test-to-test variation, so t h a t several e s t i m a t i o n s are needed to obtain acceptable confidence limits. However, these systems have served well in the evaluation of variation; the main draw-back is the time taken fully to profile viruses where two-way tests are performed, particularly where a large number of strains have to be analysed. Biochemical techniques used to differentiate FMDV have included polyacrylamide gel electrophoresis and electrofocusing of virus proteins and RNase T1 oligonucleotide mapping and nucleotide sequencing of the viral genome. The methods used to identify differences in electrophoretic mobilities (molecular weight and/or charge changes) of the proteins can be useful for identifying differences between strains but are difficult to correlate with antigenic properties. RNase T1 oligonucleotide mapping can be useful for looking at undefined differences or similarities in the nucleic acid, but the method only allows analysis of 5-10% of the genome and interpretation of the maps can be very subjective. Nucleotide sequencing offers a method of examining the viral O 1991 The International Associationof BiologicalStandardization

300

A.R. Samuel et aL

genome in detail and valid comparisons may be made u s i n g only very s h o r t s e q u e n c e s (150-300 nucleotides). ~ Sequencing is preferable to other biochemical and serological methods for use as a tool for epidemiologmal studies, such as tracing the origin of outbreaks. Nucleotlde sequencing of regions import a n t in antigenicity used m conjunctmn with monoclonal antibody (MAb) analysis can also be useful for studying antigemc variation. A number of laboratories throughout the world have prepared MAbs against various serotypes of FMDV. These have been characterized to varying degrees in specific assays, particularly the ELISA. They have also been used to determine the location of distract epitopes on the virus capsid by sequencing virus m u t a n t s which escape neutralization by these MAbs. A method for the use of MAbs m a trapping ELISA to differentiate FMD viruses has already been descmbed 4 and similar methodologies have been in practice m this laboratory for the past three years. ~~ This paper examines the variation in binding of a single panel of MAbs to three FMD type O viruses using a trapping ELISA. The type of evaluation described here is applicable to other MAb panels used for the differentmtion of viruses within other FMDV serotypes.

Materials and methods

Viruses and cell cultures Viruses originally isolated from bovine epithelial samples in primary bovine thyroid (BTY) cells were passaged m BHK-21 cells, the final passage level of virus used in this study is indicated m brackets. Footand-mouth disease virus OlfLausanne/Switzerland/ 65 (BHK3) which was used to produce the MAbs; OJSharqula/Egypt/72 (BHK4) and O/BAR/4/89 (BHK3; an isolate from Bahrain m 1989) were prepared by infecting confluent 175 cm'-' flasks of BHK21 cells overnight at 37°C (25 ml Eagle's minimum essential medmm per flask). The infectious medium was clarified by centrifugation at 2000 g for 10 mm and the supernatant was stored at -20°C after addition of an equal volume of stemle glycerol Monoclonal antibodies A panel of seven virus neutrahzing MAbs produced against the FMDV strata Or/Lausanne/Switzerland/ 65 were described by Brocchi e t a l . 7 and Capuccl e t a l . 8 These have been studied extensively by McCullough e t al.9-r2 and McCahon e t a l . ~,3Monoclonal

antibody mouse ascites preparations were stored at -20°C undiluted in small volumes before use in the profiling assays. Studies using fresh test dilutions made from the stored samples showed t h a t there was no deleterious effect on tltre of any of the MAbs on freeze/thawing. Later practice involved the preparation of large volumes of the MAbs at the test dilution in blocking buffer containing a preservative (0.01% sodium ethylmercurithiosalicylate [ThimerosalJ; Sigma, Poole, U.K.) and storage at 4°C in Micronic tubes (Flow Laboratories, U.K.) to facilitate the efficient dispensing of the multiple panels of MAbs, or m bottles. Polyclonal ant~sera Rabbit anti-FMDV polyclonal antisera were prepared against purified FMDV O~/BFS 1860 by multiple inoculation of animals as described by Roeder & LeBlanc Smith. '4 These antisera are used routinely in the World Reference Laboratory for FMD, for the immunological trapping of viruses in a sandwich ELISA. ~ These antisera have been specificity tested and selected for their broad intratypic reactivity and low intertypic cross-reactivity and therefore should react with all FMD type O viruses, at the concentrations used in the assay. Guinea-pig antisera were prepared in the same way and were used to detect trapped virus. These antisera were mixed with an equal volume of normal bovine serum before titration to adsorb antibodies produced against bovine serum proteins associated with the purified viruses. Rabbit anti-mouse and rabbit anti-guinea-pig immunoglobulins conjugated to horseradish peroxidase (HRPO) were obtained from a commercial source (Dako, U.K.). ELISA method A single dilution of the different virus samples was trapped by the rabbit serum A single dilution of the pretitrated MAbs or polyclonal type specific guineapig serum was added, followed by anti-species conjugate to measure the amount of mouse or guinea-pig antibodies bound to the wells. The same test were set up on three separate days, the plan for the addition of antigens to the plates is shown in Fig. 1. Thus, every plate received the homologous OlfLausanne in row A. Respective plates r e c e w e d the homologous virus O~/Lausanne, O1/Sharqma and O/BAR/4/89 in 6 rows B to H, single plates were set up on the individual days. Other plates received m duplicate rows B and C, OlfLausanne, rows D and E, O1/Sharquia and rows F

Evaluation of a trapping ELISA

Monoelonal Ab 1 2

A B

C D E F G H

3 4

5 6 7 8 9

)4

Polyclonal 10

11

12

Homologous virus Virus A

V~rus B Virus C No v,rus

Figure 1. Plate layout of trapping ELISA showing standardized format for the addition of test wruses and monoclonal antibodies. and G, O/BAPd4/89, three plates of this design were set up on the individual days. This format, using duplicates of test viruses, has been used in routine testing with varaous MAb panels over the past two years. The last row H in all plates received diluent containing no antigen. The monoclonal and guineapig antibodms were added identically to all plates as illustrated in Fig. 1. The data were analysed to determine the variation in individual plates, the variation in tests performed on the same day on different plates and the overall day-to-day test variation. Fifty microlitre volumes were used throughout the assay. Microtitre ELISA plates (Nunc immulon I, Nunc, Roskilde, Denmark) were coated with antiFMDV type O specific rabbit antisera, diluted in coating buffer (0.05 M sodium carbonate, 0.05 M sodium bicarbonate buffer, pH 9.6) and left at 37°C for 1 h. The antisera were pre-tltrated and diluted so t h a t they had the capacity to trap type O FMDV (when added in excess) to give a reading of 1.5-2.0 optical density (OD) units as detected w~th the relevant polyclonal guinea-pig serum. Whole rabbit immune serum was diluted to 1/5000 for use as trapping reagent and there was no need to prepare IgG fractions. Plates were washed by flooding and emptying four times with PBS and then tapped free the residual fluid. The glycerlnated virus samples were added to the plates diluted w~th an equal volume of PBS containing 5% bovine serum albumin (BSA fraction V) and 0.1% Tween 20, to prevent non-specific binding to wells. Plates were covered with hds and then incubated for 1 h at 37°C while being agitated on a rotary shaker (Rotatest, Luckhams Ltd). Plates were then washed as above to remove unreacted virus. Monoclonal antibodies (50 pl), diluted in the same buffer as the virus, were then added. These had been previously titrated in an identical system using

301

O1/Lausanne as the virus. Antibodies were added at half the last dilution (twice the concentration) which gave the maximum plateau height of ELISA activity. Previously titrated polyclonal antibody (guinea-pig) was added in columns 10, 11 and 12. Plates were incubated for 1 h at 37°C and washed as before then 50 pl of a pretitrated optimal dilution of anti-mouse HRPO conjugate was added per well to detect mouse antibody binding and anti-guinea-pig HRPO conjugate diluted in the same blocking buffer for the detection of guinea-pig polyclonal antisera binding. The plates were incubated for 1 h and then washed. Substrate s o l u t i o n (50 pl per well of o r t h o p h e n y l e n e diamine/hydrogen peroxide in citrate/phosphate buffer, pH 5.5) was then added and colour reaction inhibited after 10 min incubation at room temperature by the addition of 50 pl per well of 1.25 M sulphurac acid m distilled water. The OD readings of the plates were measured using a multichannel spect r o p h o t o m e t e r ( M u l t i s k a n Plus Mk II; Flow Laboratorms, U.K.) at a wavelength of 492 nm and the data processed as described in the results. These assays relied upon their being an excess of virus available for trapping by the rabbit antibody. The effect of using more dilute antigens on the ELISA MAb profiles was examined for the three viruses. Plates were set up as above except that different dilutions of the viruses were produced so that the ELISA values obtained with the polyclonal sera represented approximately three quarters, half and a quarter of the maximum value where virus was in excess. Results were obtained to compare the MAb binding under identical conditions described above with the different concentrations of homologous and heterologous isolates. Results

Data were processed rapidly by hnking the ELISA reader to a microcomputer and using appropriate software developed at this Institute. The OD reading obtained in row H was subtracted from each respective test value m that column. The reading in row H determines the background colour obtained for the interaction of trapping antibody, monoclonal or polyclonal antibody and respective conjugate. The resulting values were then expressed as a percentage of the mean polyclonal value obtained for each row, i.e. as a percentage of the mean OD readings of wells 10, 11 and 12 for respective row. This attempts to normalize the respective readings according to the amount of the different wrus samples attached to the wells. These values were then related to those obtained for the homologous virus (O1/Lausanne) by expressing

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A. R, Samuel et al

Table 1. Data processing for comparison of three type O foot-and-mouth disease viruses (a) Actual optical density results Antibodies

Homologous O1/Lausanne O~/Lausanne O~/Sharquia O~/Sharquia O/BAR/4/89 O/BAR/4/89 BGt

B2

C6

C8

C9

D9

A8

G5

1.96 1.89 1.94 1.51 1.54 1.41 1.36 0-03

1.73 1.62 1.60 0.22 0.27 0-14 0.17 0.01

1.41 1.25 1.31 0.71 0-73 0.70 0.67 0.03

1.27 1.39 1.48 0.22 0.19 0.02 0.02 0.02

1.43 1.34 1-38 0.80 0.78 0.62 0.61 0.02

1.27 1.16 1.21 0-63 0.61 0.64 0.64 0.03

2.13 2.09 2.12 1.12 0.97 0.03 0.01 0.00

B*

B

PC

PC

PC

1.94 1.92 1-91 1.63 1.56 1.09 1.01 0.02

1.89 2-31 1 - 6 5 1.94 1 - 8 7 1.95 1.69 1.71 1.60 1.72 1.03 1.24 1.04 1.13 0.03 0.15

PC

PC

1.93 1.90 1.89 1.61 1.55 1.08 0.99

1 - 8 7 2.16 1 - 6 3 1.79 1-84 1.81 1.66 1.56 1.58 1.58 1.01 1.09 1.01 0.99

(b) Results minus optical density value of background control for each column Antibodies

Homologous O~/Lausanne O~/Lausanne O:/Sharqula O:/Sharquia O/BAR/4/89 O/BAR/4/89

B2

C6

C8

C9

D9

A8

G5

1.93 1.86 1.91 1.48 1.51 1.38 1.33

1.72 1.61 1.59 0.21 0.26 0.13 0.17

1.38 1.23 1.28 0.68 0.71 0.67 0-64

1.26 1.38 1.47 0.20 0-18 0.00 0.01

1.41 1.32 1.37 0.78 0.76 0.61 0.61

1.25 1.14 1-18 0.61 0.58 0.61 0.61

2.13 2.09 2.12 1.12 0.97 0.04 0.05

B*

B

PC

(c) Values in (b) expressed as a percentage of the mean optical density of the polyclonal antibody for each row Antibodies

Homologous O~/Lausanne O:/Lausanne O~/Sharquia O~/Sharquia O/BAR/4/89 O/BAR/4/89

B2

C6

C8

C9

D9

A8

G5

97 104 103 91 96 130 133

86 90 86 12 16 12 16

69 69 69 42 45 63 64

63 77 79 12 11 0 0

70 74 73 48 48 57 59

62 64 63 37 37 57 61

107 117 114 69 64 4 2

B*

B

PC

PC

PC 100 100 100 100 100 100 100

(d) Values as percentage of the homologous value for each antibody Antibodies

Homologous O1/Lausanne O:/Lausanne O~/Sharqma O~/Sharqma O/BAR/4/89 O/BAR/4/89

B2

C6

C8

C9

D9

A8

G5

100 107 106 93 98 134 137

100 104 100 13 18 13 18

100 100 100 60 65 91 92

100 122 125 19 17 0 0

100 105 104 68 68 81 84

100 103 101 59 59 91 98

100 109 106 64 59 3 1

B*

B

Wells not used t Background control (no virus added). Note: minor dlscrepancms in OD minus background occur due to rounding-up of values.

PC

PC

PC

Evaluation of a trapping ELISA

t h em as a percentage of the homologous values for the respective MAbs in each column. The homologous virus was always placed in row A, so each plate was controlled by the values obtained in this row. The processing is illustrated in Table 1, which shows the comparison of O J L a u s a n n e , O,/Sharquia and O/BAR/ 4/89 placed as duphcate samples. Table 2 shows the results obtained comparing the viruses as duplicates on three separate plates on three different days. The m e a n value for each MAb is shown as percentage of O~/Lausanne percentage values obtained in row A [processed as in Table l(d)]. Table 3 shows the mean and s t a n d a r d deviation (SD)

303

from the mean and coefficient of variation (CV) (1 × SD expressed as per cent mean) for the data in Table 2. Values are to the nearest integer, although the coefficient of variation is calculated from the actual figures. The variation in the results where plates contained six rows of each of the viruses is shown in Table 4. are expressed as the percentage values of the polyclonal value for each row [processed as shown in Table l(c)]. The results of the above plates expressed as the percentage of the O,/Lausanne sample on row A [processed as in Table l(d)] are shown in Table 5. The data for each plate or day can be analysed by calculating

T a b l e 2. Percentage reactivity values for each virus against the MAb panel for the three tests repeated on three consecutive days O,/Lausanne test plate I MAb B2 C6 C8 C9 D9 A8 G5

II

III

A

B

C

A

B

C

A

B

C

90 103 108 113 100 106 92

95 100 103 161 102 92 96

103 104 104 198 100 101 100

99 105 110 85 104 90 103

93 100 98 94 97 95 100

103 100 116 102 103 104 101

94 89 107 97 85 91 107

97 96 97 104 95 95 94

107 102 100 123 105 102 108

O,/Sharquia test plate I

II

III

MAb

A

B

C

A

B

C

A

B

C

B2 C6 C8 C9 D9 A8 G5

77 13 78 11 70 55 60

97 8 71 16 70 54 43

89 18 84 35 72 69 64

85 17 70 8 59 36 50

96 20 85 18 73 51 56

92 16 76 20 66 51 56

80 15 70 11 55 52 53

85 13 58 12 60 49 40

95 16 62 18 68 59 46

O/BAR/4/89 test plate I MAb B2 C6 C8 C9 D9 A8 G5

II

III

A

B

C

A

B

C

118 14 116 1 82 100 2

139 4 100 9 83 72 1

127 13 126 5 71 107 0

100 9 125 1 55 46 2

108 22 117 4 65 74 1

150 14 143 4 81 70 3

A 97 8 95 2 57 50 2

B

C

107 10 82 4 50 62 4

136 16 92 0 83 95 2

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A.R. Samuel et al

T a b l e 3. Mean, s t a n d a r d d e v m t i o n a n d coefficient of v a r i a t i o n of s t a n d a r d deviation, of t e s t r e s u l t s h o w n in Table 2. T h e p l a t e - t o - p l a t e v a n a t m n on each d a y for each MAb is s h o w n u n d e r I, II a n d III, t h e c o m p a r i s o n of r e s u l t s over t h r e e t e s t s is s h o w n in l a s t column

O~/Lausanne test I MAb B2 C6 C8 C9 D9 A8 G5

II

Mean

SD

CV

96 102 105 157 101 100 96

7 2 6 42 1 7 4

7 2 6 27 1 7 4

Mean 98 101 103 97 101 96 10l

III

SD

CV

Mean

SD

5 3 6 4 4 7 2

5 3 6 5 4 7 2

99 96 101 108 95 96 103

7 7 5 13 10 6 8

All t h r e e t e s t s CV 7 7 5 12 11 6 8

Mean

SD

CV

98 100 103 121 99 97 100

2 3 2 31 6 2 4

2 3 2 26 6 2 4

O1/Sharquia t e s t I MAb B2 C6 C8 C9 D9 A8 G5

Mean 88 13 78 21 71 59 56

II

SD

CV

10 5 7 13 1 8 11

12 38 8 60 1 14 20

Mean 91 18 77 15 66 46 54

III

SD

CV

6 2 8 6 7 9 4

6 11 10 4 11 19 7

Mean 87 15 63 14 61 53 50

All t h r e e t e s t s

SD

CV

8 4 6 4 6 5 9

9 26 10 29 10 10 17

Mean 89 15 73 17 66 53 53

SD

CV

2 2 8 3 5 6 3

2 17 11 22 8 12 6

O/BAR/4/89 t e s t I MAb B2 C6 C8 C9 D9 A8 G5

II

III

All t h r e e t e s t s

Mean

SD

CV

Mean

SD

CV

Mean

SD

CV

Mean

SD

CV

128 10 114 5 82 93 1

11 6 13 4 11 19 1

8 55 12 80 13 20 100

119 15 128 3 67 63 2

27 7 13 2 13 15 1

23 43 10 66 20 29 50

113 11 90 2 63 69 3

20 4 8 2 17 23 1

18 37 7 100 27 33 33

120 12 110 3 70 75 2

7 3 19 2 10 16 1

6 25 18 66 14 21 33

t h e coefficmnt of v a r m t m n a n d its v a r m t i o n P e r c e n t a g e values of 10 or less are excluded f r o m the calculatmn. R e s u l t s are s h o w n in Tables 6 a n d 7. T h u s , the d a t a in Tables 2 a n d 3 i l l u s t r a t e the p l a t e - t o - p l a t e v a r i a t i o n for t e s t s m a d e on the s a m e day a n d the t e m p o r a l v a r i a t i o n on t h r e e s e p a r a t e days, w h e r e t e s t v i r u s e s were a n a l y s e d in duplicate. This is a c o n v e m e n t f o r m a t w h e r e t h r e e s e p a r a t e w r u s e s can be reacted w~th up to nine MAbs in a single plate. T h e r e s u l t s which t r e a t e d O1/Lausanne as a t e s t w r u s are m o s t easily e x a m i n e d s m c e for each MAb a v a l u e of 100% should be o b t a i n e d MAb C9 in

Test I g a v e a s p u r i o u s r e s u l t as c o m p a r e d to all subs e q u e n t a n a l y s e s , due to an e x t r e m e l y low r e s u l t for t h e h o m o l o g o u s v i r u s reaction w i t h C9 on two p l a t e s for t h a t day. T h e m e a n a n d SD for all the t e s t r e s u l t s showed a s i m i l a r v a r m t m n for all t h e MAbs except C9 which w a s a g a i n influenced by t h e high r e s u l t in Test I. T h e i n t r a - t e s t v a r i a t i o n w a s h i g h e r t h a n t h e day-to-day variatmn. The mtra- and inter-test variations for t h e MAbs b i n d i n g to the o t h e r two t e s t v i r u s e s w e r e h i g h e r t h a n for t h e homologous strain. T h e h i g h e s t v a r i a U o n s w e r e o b s e r v e d w h e r e the bindm g w a s g r e a t l y r e d u c e d (low OD readings), e.g.

Evaluation of a trapping ELISA

305

T a b l e 4. Mean, s t a nda r d deviation and coefficient of vamatmn where a single virus was added to all test wells O~/Lausanne test I MAb B2 C6 C8 C9 D9 A8 G5

II

III

Mean

SD

CV

Mean

SD

CV

129 104 62 74 77 57 117

4 5 7 16 9 13 9

3 5 11 22 12 23 8

112 98 62 86 69 45 112

4 10 4 8 7 7 4

3 10 6 9 10 14 3

Mean 110 92 68 91 80 68 112

All three tests

SD

CV

Mean

SD

CV

4 5 8 4 5 2 4

3 5 12 4 6 3 4

117 98 64 84 75 57 114

10 6 3 9 6 12 3

9 6 5 10 8 20 3

O~/Sharqula test I MAb B2 C6 C8 C9 D9 A8 G5

Mean 90 14 51 12 57 37 73

II

SD

CV

9 2 5 1 4 3 6

10 11 10 9 8 10 9

III

All three tests

Mean

SD

CV

Mean

SD

CV

Mean

102 20 47 15 64 39 58

17 5 16 1 17 9 14

16 25 33 7 26 24 20

108 15 53 13 62 38 64

11 1 4 1 4 2 11

10 7 7 8 7 5 17

100 16 50 13 61 38 65

SD

CV

9 3 3 2 4 1 8

9 19 6 12 6 3 12

O/BAR/4/89 test I MAb B2 C6 C8 C9 D9 A8 G5

II

III

Mean

SD

CV

Mean

SD

CV

Mean

108 6 73 1 57 33 1

14 5 8 1 8 3 1

13 75 12 100 13 15 100

181 12 43 2 51 25 1

20 2 9 2 4 2 1

11 14 20 100 9 2 100

126 15 79 5 62 39 3

O~/Sharquia against C6, C9; O/BAR/4/89 against C6, C9 and G5. Higher day-to-day variation was also observed for MAbs C8, D9 and A8. Table 6 emphasises the differences in the variability of results for O~/Sharquia and O/BAR/4/89 by comparing the mean and standard deviations of the mean coefficient of variations for most of the MAb reactions on and between each day. The data in Table 3 showed t h a t there were significant differences (t-test P < 0.05) in the binding of O~/Lausanne and O1/Sharquia with all the MAbs and for O/BAR/4/89 with C6, C9 and G5 MAbs. Table 5 shows the results where the viruses were

All three tests

SD

CV

Mean

SD

CV

5 2 3 1 3 4 2

4 11 3 20 5 10 66

138 8 65 3 57 32 2

38 4 19 2 6 7 1

28 50 30 66 10 21 50

titrated in six rows and examines more closely the influence of any variation over the whole plate for a specific assay. Examination of the O~/Lausanne data shows t h a t there was an increased intra- and intertest variation for each of the MAbs as compared to the data dealing with virus duplicates, indicating t h a t there was a factor involving the sample position on the plate. Again, the variation for the O~/Sharquia and O/BAR/4/89 was higher than for the homologous virus. The results showed significant differences between the binding of O~/Lausanne and O~/Sharquia (t-test P < 0.05) for all the MAbs and with O/BAR/4/89 for C6, C9, A8 and G5. Table 7 examines

306

A.R. Samuel et al

T a b l e 5. M e a n , s t a n d a r d d e v i a t i o n a n d coefficient of v a r i a t i o n of the m e a n p e r c e n t a g e v a l u e s for MAbs e x p r e s s e d as p e r c e n t a g e v a l u e of t h e O ~ / L a u s a n n e r e a c t i o n in row A for t e s t s p e r f o r m e d on t h r e e different d a y s (virus in six positions) O1/Lausanne test I MAb B2 C6 C8 C9 D9 A8 G5

II

Mean

SD

CV

95 99 106 171 81 119 88

3 6 11 30 6 30 7

3 6 9 18 7 25 8

III

Mean

SD

CV

106 119 112 107 121 96 101

3 12 5 12 8 10 4

3 10 4 11 7 10 4

All t h r e e t e s t s

Mean

SD

CV

93 90 86 109 86 94 98

3 4 11 3 1 3 4

3 4 13 3 1 3 4

Mean

SD

CV

98 103 101 129 99 103 96

7 15 14 36 20 14 7

7 15 14 28 20 14 7

O1/Sharquia t e s t I MAb B2 C6 C8 C9 D9 A8 G5

Mean 65 14 72 16 59 76 59

II

SD

CV

6 2 7 1 5 7 5

9 14 10 6 8 10 8

III

Mean

SD

CV

104 22 62 20 73 54 57

17 6 8 2 8 7 10

16 27 13 10 11 13 18

Mean 82 15 70 12 67 59 58

All t h r e e t e s t s

SD

CV

8 1 5 1 5 3 10

10 7 7 8 7 5 18

Mean 84 17 68 16 66 63 63

SD

CV

20 4 5 4 7 11 11

25 24 7 28 10 17 17

O/BAR/4/89 t e s t I MAb B2 C6 C8 C9 D9 A8 G5

II

III

All t h r e e t e s t s

Mean

SD

CV

Mean

SD

CV

Mean

SD

CV

Mean

SD

CV

119 9 123 1 77 66 0

29 6 51 1 11 11 0

24 66 41 100 14 17 0

173 13 71 2 62 57 1

19 2 14 2 5 3 1

11 15 20 100 8 5 100

109 15 94 4 73 54 2

4 2 3 1 4 5 2

4 13 3 25 5 9 100

133 12 96 2 71 59 1

34 3 26 2 8 6 1

26 25 27 100 11 10 100

the v a r i a t i o n of the CV for the MAbs for v a l u e s r e l a t e d to the homologous (O1/Lausanne) p e r c e n t a g e r e a c t i o n (from Table 5) a n d t e s t s a n a l y s i n g the m e a n coefficient of v a r i a t i o n a n d s t a n d a r d d e w a t i o n of the v a l u e s of each v i r u s e x p r e s s e d as a p e r c e n t a g e of its own polyclonal OD [ d a t a t r e a t e d as in Table l(c)] from Table 4. T h e r e is no significant difference b e t w e e n t h e s e r e s u l t s (P > 0.05). T h e CVs w h e r e the m e a n p e r c e n t a g e reaction of the MAb w a s 15% w e r e excluded since t h e s e b i a s e d the v a r i a t i o n d a t a greatly. This w a s m o s t e x a g g e r a t e d w h e r e the m e a n

v a l u e s w e r e f r o m 1-5%, e.g. MAb C9 × O/BAR/4/89 ( m e a n = 5%, SD = 4, CV = 80). Table 8 gives a m e a s u r e of the overall v a r i a t i o n in the row A r e s u l t s r e l a t i n g t h e O 1 / L a u s a n n e r e a c t i o n w i t h the MAbs as a p e r c e n t a g e of t h e polyclonal OD. T h e m e a n coefficient of v a r i a t i o n for all MAbs w a s 12 w i t h a SD of 6, MAb C9 s h o w e d t h e g r e a t e r v a r i a t i o n . Table 9 i l l u s t r a t e s the effect of diluting v i r u s s a m p l e s on t h e t r a p p i n g plates. Generally, t h e r e w a s i n c r e a s e d v a r i a t i o n in the duplicate s a m p l e s as the polyclonal OD r e d u c e d on dilution. C o m p a r i s o n s

Evaluation of a trapping ELISA

307

T a b l e 6. Mean and s t a n d a r d deviations of the mean coefficmnts of variation of data shown in Table 3 for all MAbs (excluding value < 10) I

O/Lausanne* O~/Sharquia O/BAR/4/89

II

III

Total

Mean

SD

Mean

SD

Mean

SD

Mean

SD

4-4 22.0 13.2

2.6 22-0 5.0

4.6 9.7 25-0

1.7 4.8 12.0

8.0 14-4 21.0

2.6 10.0 11.0

3.1 11.1 17.0

46 6.7 7.3

'~Excluding C9 result m I.

T a b l e 7. Mean and s t a nda r d deviation of the mean coefficients of variation of the MAbs (excluding results