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Factors affecting the reproducibility of the serum neutralization test for infectious bursal disease
Journal of Biological Standardization (1982) 10, 221-229
Factors affecting the reproducibility of the serum neutralization test for infectious bursal disease*
C. Nancy Hebm't, Y N. E. Reed, t J. C. Muskettt and Denise H. Thorntont
An experiment is described in which the main factors contributing to the x~ariability of the serum neutralization test for infecgious bur~al disease were studied. Significant differences were found between test operato~ and readers. Antibody levels ofsera from vaccinated SPF chickens and the method ofpreparlng dilutions did not affect the variability of the test, but there was a tendency towards a decrease in precision with increasing virus concentration.
INTRODUCTION T h e s e r u m n e u t r a l i z a t i o n t e s t ( S N T ) is w i d e l y u s e d i n t h e e s t i m a t i o n o f a n t i b o d y l e v e l s . Thig report describes an i n v e s t i g a t i o n to e x a m i n e possibie factors w h i c h m a y cause variabilit~r i n r e s u l t s , suc.h as d i f f e r e n t t e s t o p e r a t o r s a n d t e s t r e a d e r s , v a r y i n g s t r e n g t h o f v i r u s a n d s e r u m a n d t h e m e t h o d u s e d f o r p r e p a r i n g t h e s e r u m d i l u t i o n s , u s i n g as a n example; the neutralization test for antibodies to infectious bursal disease virus (IBDV) of chickens.
MATERIALS
AND
METHODS
Sera preparatio~ Sera were collected f r o m S P F birds i m m u n i z e d w i t h i n a c t i v a t e d I B D V . F r o m these ~Received for publication 23 January 1982. tMinistry o f Agricultur,:. Fisheries and Food, Central Vete~irtary Laboratory. Weybridge, Surrey, LI.K.
221
C. N. HEBERT E T A L ,
sera t h r e e pools w e r e p r e p a r e d , w h i c h w e r e h i g h , m e d i u m a n d low w i t h respect to t h e i r a n t i b o d y levels to I B D V ; these w e r e d e s i g n a t e d A, B a n d C respectively. T h e s e t h r e e s e r u m p o o l s w e r e i n a c t i v a t e d at 5 6 ° C for 30 r a i n before t e s t i n g .
Virus suspension A l i q u o t s o f a c o n c e n t r a t e d v i r u s s u s p e n s i o n were p r e p a r e d , a n d these w e r e s t o r e d at - - 7 0 ° C as a s t o c k v i r u s s u s p e n s i o n . O n each test occasion d i l u t i o n s f r o m a s i n g l e a l i q u o t w e r e p r e p a r e d to g i v e an e s t i m a t e d 2 0 , 2 0 0 a n d 2 0 0 0 T C I D s 0 / 0 - 0 2 5 m l (R, S a n d T respectively). T h e v i r u s was d i l u t e d in g r o w t h m e d i u m .
Cell culture A s u s p e n s i o n o f cells was m a d e f r o m a 2 4 h p r i m a D, c u l t u r e o f c h i c k e m b r y o fibroblasts. T h e cell c o n c e n t r a t i o n was a d j u s t e d to 3"5 × 105 cells m 1 - 1 o f g r o w t h m e d i u m . T h e g r o w t h m e d i u m was Eagles m i n i m u m essential m e d i u m s u p p i e m e n t e d w i t h 5 % (v/v) c a l f s e r u m , 1% o f a 7 - 5 % (w/v) s o d i u m b / c a r b o n a t e s o l u t i o n a n d 0 " 5 % (v/v) a n t i b i o t i c s o l u t i o n c o n t a i n i n g p e n i c i l l i n , s t r e p t o m y c i n a n d a m p h o t e r i c i n .
Micro-neutralization test G r o w t h m e d i u m ( 0 - 0 2 5 m l ) was p l a c e d in all wells o f t h e m i c r o t i t r e plates. I n i t i a l t w o f o l d d i l u t i o n s o f sera were m a d e by e i t h e r u s i n g d i l u t i n g s t i c k s ( m e t h o d x) or m i c r o p i p e t t e s ( m e t h o d y). F r o m t h e s e initial d i l u t i o n s f u r t h e r t w o f o l d serial d i l u t i o n s w e r e p r e p a r e d u s i n g d i l u t i n g sticks. S e r u m c o n t r o l s w e r e p r e p a r e d f r o m t h e i n i t i a l s e r u m d i l u t i o n s . T h e a p p r o p r i a t e v i r u s s u s p e n s i o n ( 0 - 0 2 5 m l ) was d i s p e n s e d i n t o t h e test wells. N o v i r u s w a s a d d e d to t h e s e r u m c o n t r o l wells, a n d s o m e wells w e r e set aside for cell c o n t r o l s w h e r e n o v i r u s or s e r u m was a d d e d . T h e p l a t e s w e r e t h e n i n c u b a t e d at 3 8 ~ 5 ° C f0k- 6 0 m i n t o a l l o w v i r u s n e u t r a l i z a t i o n to t a k e place. A f t e r virus n e u t r a l i z a t i o n 0 - 2 m l o f t h e ceil s u s p e n s i o n was d i s p e n s e d i n t o each o f t h e wells. T h e p l a t e s w e r e i n c u b a t e d at 3 8 - 5 ° C in a n a t m o s p h e r e o f 5 % C O 2 a n d 7 0 % h u m i d i t y for 4 d a y s a n d read u n d e r a n i n v e r t e d m i c r o s c o p e . T h e t i t r e was e x p r e s s e d as t h e !og2 o f t h e reciprocal s e r u m d i l u t i o n w h i c h g a v e 1 0 0 % v i r u s n e u t r a l i z a t i o n (i.e. no c y t o p a t h i c eFFect c o u l d be seen).
Expe~mental design I n o r d e r t h a t as m a n y variables as p o s s i b l e c o u l d be s t u d i e d s i m u l t a n e o u s l y , a factorial d e s i g n w a s u s e d in w h i c h t w o test o p e r a t o r s (P 1 a n d P 2 ) a n d five readers (J, K , L, M a n d N ) p a r t i c i p a t e d . T h e t h r e e sera w i t h h i g h , m e d i u m a n d l o w a n t i b o d y levels t o I B D V , a n d t h e t h r e e c o n c e n t r a t i o n s o f viral s u s p e n s i o n w e r e all t e s t e d by t h e t w o m e t h o d s in t h e m i c r o - n e u t r a i i z a t i o n t e s t , Five separate assays were p e r f o r m e d , b u t n o t all five readers w e r e a b l e t o read t h e t e s t s o n every occasion. R e a d e r J p a ~ i c i p a t e d in all t h e assays, m a k i n g p o s s i b l e a valid b e t w e e n - a s s a y c o m p a r i s o n . All tests w e r e p e r f o r m e d in d u p l i c a t e ° STATISTICAL
ANALYSIS
OF RESULTS
T h e r e c i p r o c a l e n d - p o i n t s e r u m d i l u t i o n e x p r e s s e d as l o g t o t h e base 2 is u s e d as t h e r e s p o n s e i n t h e - S N T . I n ~he m a i n analyses in t h i s s t u d y t h e m e a n l o g 2 t i t r e o f ~ l u p l i c a t e 222
SERUM N E U T R A L I Z A T I O N TEST FOR IBDV TABLE 1
Mean S N T titres (log2) in five assays: variation between different test operators and readers
C o l u m n no.
1
2
3
4
Operator Assay
s.e. mean*
Reader
P1
P2
P1
P2
(,8> 7-8i
(18) 5-,91
(54)
(54)
j
6-50~ 6-00 J (18) 7-67} 5-53 J
6-37
5-90
(36)
(36)
J N
6- 11 5" 19 (I8) 6-31 5-47
5-89
6-60
(18)
(18)
(54)
(54)
j
8-58
9-501
M N
11-06 8- 11 (18) 6-25 6-72 5"75 5-36 (18) 6-72 6-67 5"89
9,25
9-58
(72)
(72)
6-02
5-53
(54)
(54)
6-43
5 -45
K L 2
3
4 J K L N 5 j L M
9-28 9-97 (18) 5-94 6-25 4-78 5- 17 ( i 8) 5-03 5-92 5-42
~ J / I
1 ~
Operator
Reader
0" 184
0-318
0- 184
0.260
0-197
0-34.1
0-089
0- IV9
0-107
0- 185
*) s.e. mean, standard error of a mean of (n) values. Number of tests included in each mean: Columns 1 and 2, three sera X three virus--concentration X twomed~(~ds; Columns 3 and 4, three sera × three virus concentrations × two methods × two, three or four readers. Number of readings (n)in brackets (means of duplicates). tests was t a k e n as t h e r e s p o n s e , s i n c e t h e v a r i a t i o n b e t w e e n d u p l i c a t e s reflected o n l y t h e errors o f l a b o r a t o r y t e c h n i q u e , a n d t h e r e f o r e w o u l d n o t c o n s t i t u t e a v a l i d e r r o r t e r m for d e t e r m i n i n g t h e s i g n i f i c a n c e o f d i f f e r e n c e s i n t h e factors i n v o l v e d . A s e p a r a t e analysis o f t h e d i f f e r e n c e b e t w e e n d u p l i c a t e s w a s c a r r i e d OUt. W h e r e o n l y o n e t e s t was p e r f o r m e d i n s t e a d Of t w o , t h e d u p l i c a t e t i t r e w a s e s t i m a t e d as t h e v a l u e m o s t c o m m o n l y g i v e n by readers for t h e first t i t r e . M i s s i n g v a l u e s o c c u r r e d i n f r e q u e n t l y (see T a b l e 4 ) a n d w e r e g e n e r a l l y a s s o c i a t e d w i t h t h e h i g h e s t v i r u s c o n c e n t r a t i o n (T). A l l e s t i m a t e d m e a n s w e r e included inthe statistical analyses. A n analysis o f v a r i a n c e was c a r r i e d o u t for each assay, t a k i n g i n t o a c c o u n t t h e f o u r factors, o p e r a t o r / r e a d e r , s e r u m level, v i r u s c o n c e n t r a t i o n a n d d i l u t i o n m e t h o d ~ M e a n l o g 2 t i t r e s for t h e t w o o p e r a t o r s a n d five r e a d e r s o v e r a l l tests p e r f o r m e d w i t h i n e a c h assay are g i v e n in T a b l e I. T h e s e m ~ are c o m p a r a b l e o w i n g t 0 t h e b a l a n c e d d e s i g n o f t h e e x p e r i m e n t , T h e diffe .fence b e t w e e n tes t o p e r a t o r s P 1 a n d P 2 w a s h i g h l y s i g n i f i c a n t in A s s a y 2 ( P < 0 , 0 1 ) a n d in A s s a y s 4 a n d ~5 0"001)~ T h e r e w e r e h i g h l y s i g n i f i c a n t 223
2.42 41'2 1.64 24,7
3"40 34'2 1'48 28'8 1'15 31"3
Mean s . d . c . v .
Assay 4
Mean s . d . c . v .
Assay 5
(36) 13"69 9"61 4-92 (54) 9'68 9'14
1"53 15"8 1"36 14"9
1"57 11"4 1"61 18"8 1"10 22"3
(48) 7"47 5'65 4'19 (72) 6'28 5'26
1"32 2l'1 1"05 20'0
1"42 19"0 1'31 23'3 0'88 20'9
(36) 8'86 5"28 3'68 (54) 6-31 5"56
0"89 14'1 ['10 19"7
1"32 14'9 1"04 19'8 0'92 25'0
(36) (48) (36) 1'1'76 i.44 12.2 6'96 I'33 19'1 8.14 1!25 15'4 9'38 1'81 19'3 6"28 1'17 I8'7 5'69 1'04 18'3 7'1l 0'91 12"8 4"06 0'70 17'2 3'99 0'69 17"2
Mean s.d.c.v.
Assay 3
Mean, Overall mean for all tests; s.d., residual standard deviation; ¢.v., coefficient of variation (%). Number oftests included in mean:sera, two operators × threevirusconcentration X two methods X. readers;virusconcentration, two operator~ X three sera X;:twomethods X ~ readers;method, two operators X three sera X three virus concentration X n readers. Number of readings n in brackets (means ot duplicates).
1'43 23'4 1.89 30'9
2.20 25'6 1'79 28'9 1"34 37"1
(24) 9'94 5"12 3"67 (36) 5,88 6'.61
(36) (24) 8'00 2'00 25'0 6"98 2.64 37"8 6'15 1"08 17'5 7"50 2"00 26,7 4'25 1'12 26"4 4"25 1.20 28'3
Mean s . d . c . v .
Mean s . d . c . v .
A B C Virus concentration (36) 8'60 R S 6"20 3"60 T (54) Method 6'I3 X 6'14 V
Sera
Assay 2
Variability of mean log2 titres within sera, virus concentration and dilution method Assay 1
T^oL• 2.
1,O
7.2 5.2 3"2 0.55 5'7 4.7 0'45
5'5 6'5
4.9 5'9 4.8 0'55
P2
8.8 6.3 2.8
8.I 5.4 4'5
P1
1
7.1 5'8 3'6 0'45
P2
6'3 6'3
5'3 5.6 0'37
8.8 9,3 6.2 4.6 3"9 2"5 0.45
8.1 6.8 4.0
P1
2
3
P2
8.4 8.2 8.8 8.l 0.48
1 3 . 0 12.1 8"3 8.2 4.4 4.0 0.59
1 0 . 9 10.0 8.8 7.6 6.1 6.8 0'59
P1
Assay
P2
7.6 5.8 0'25
9.0 6'3 4.8 0.31
8.3 7.6 4.2 0"31
6'5 6.0
8.1 6'3 4'3
6.8 6.8 5.2
P1
'4
MeanSNT titres (log2) in fiveassaysassessedbya single test reader
" Standard error of a mean of n readings. Number of readings, n; in brackets (means of duplicates).
Serum (6) A B C : s.e. mean* Virus concentration (6) R $ T s.e. mean Dilution method (9) x y s,e. mean
oiseratot:
T~LE ~.
9'2 6'9 3'8 0'32
P2
6.6 6'3
7,7 5"6 0'26
8.6 t0.1 5'4 5'9 3"7 4.0 0.32
8.0 5.6 4.1
P1
5
C. N . H E B E R T E T A L .
differences between readers in all assays. Operator/reader variations w i t h i n assays can, however, be ignored when considering overall mean values for sera, virus concentration a n d test methods (Table 2). In all assays the mean log2 titre of the three sera and the three virus concentrations showed, as anticipated, very h i g h l y significant differences. Means for the two methods of dilution did not differ significantly in the first assay; in assays 2 and 3 the mean difference was significant a t the 5% level, and in assays 4 and 5 it was h i g h l y significant (P < 0-001). Inassays 3, 4 and 5 the mean for me~hod x was rather higher than t h a t for m e t h o d y, but as indicated by the standard deviation, t h e r e was little difference in the precisio n of the two methods. There was also no indication that the S N T is either more or less reproducible with decreasing strength of serum, as shown by the lack of a n y consistent trend in the coefficient of variation (Table 2). There was, however, in three of the five assays a decrease in precision w i t h increasing virus concentration.
Variation between assays. Table 3 shows the overall mean log 2 titres for sera, virus concentration and dilution m e t h o d in tests performed b y both operators, b u t read by a s i n g l e reader, J , in the five separate assays. The level of response in Assay 3 was higher than that i n all other ass'ays, the differences in most instances being statistically significant, sometimes very h i g h l y so. T h e only exception was in tests performed by operator P2, using the highest virus concentration, T. Here the mean log2 titre in Assay 4 was significantly higher than that in Assay ! (P < 0-05) and Assay 2 (P < 0-01L This was the only significant variation between corresponding m e a n Iog2 titres in Assays I, 2 , 4 and 5. T h e mean log2 tixre for Serum A in the first assay i n rests'performed b y P 2 was abnormal, very low readings for tests using dilution m e t h o d y being obtained b y reader J , in contrast t o the other two readers. I n Assay 2. the mean 1092 titre for serum B was higher than that for Serum A, b u t not significantly so.
Differences between duplicate titrations A separate evaluation was carried our on the difference in log2 titre of duplicate tests, to assess the frequency w i t h which identical readings occurred, and where these differed b y o n e or more twofold dilution steps (Table 4 ) . : ~ 4 h e r e the variation was three d i l u t i o n steps or g r e a t e r , this was analysed further tt, determine if there was any association w i t h s t r e n g t h o f s e r u m , d i l u t l o n method, virus concentration oi operator (Table 5):~-There was a h i g h l y significant differencebetWeen Virus Concentrations, greatei variability occurring with lower concentrations, and seven out of the eight differences Of six steps or!over occurring w i t h Concentration R , while similar results were f o u n d w i t h s t r e n g t h of serum -and~dilution method, where the differences approached statistical Significance. T a b l e 6 demonstrateslthe effec t of regarding one of the three sera (A) as a standard and expressing th e r e s u l t s o f ~ e CorreSpond!fig ~itrations ~ i t h SewnB a n d C as ,tide difference from those for Serum :A: ,Only~the readings:by-re'adei J were used i n this inst/mce~ A c 0 m ~ s o n :With: the SignJificanc.e!evels~found in t h e main~analySis shows tha:t there i s a reduction in the Variability, ~particulariFin the differences between Sera A~a n d B, : w h e ~ e~te:variation betwcen:asSa~;s is significank,0nlyat~the5%)l~vel;~:and Xhe~'0ther: m a i n S a t e : n ° t sthtiStically~:iignificant~: A l t h o u g h h i g h | y i . i i g n ! f i c a n t A ! f f e r e n c e s (P, ~ i 0-001) i~cUr~-~ l i ~ i ~ y s and b e ~ n i y i ~ . IConcentratio~ :iintheci c 0 m ~ i s O n of S¢~:~A~,hnd c,:~i~fle ~/arlabiliqr iSC0//siderably.less ;than in!~ii:~m/iinanal~mis: 226
S E R U M N E U T R A L I Z A T I O N TEST FOR I B D V
TABLE 4.
Difference in log2 .titre of duplicate tests in five assays. Percentage of pairs
Lo&2 difference
No.
Percentage
0 1 2 3 4 5 ~6
182 181 92 40 8 10 9 18 522
34-9 34-7 17-6 7-7 1-5 1-9 1-7
M/V Total
M/V, Missing value inserted for one duplicate--result excluded from totals. TABLE-5. Frequency.of.high differences between duplicates Serum N o . of dilution steps 3
4 5 ~6 Total X2 (d~f.)
DISCUSSION
Method
Virus
A
B
C
x
y
R
S
14 4:
13 1 2 1 3
23 3
16 5
17, 12 2 6
Operator T
Pl
P2
lO
22 3 5
17
0 1
6 2 1 6 3 4 4: 4 5 3 0 7 1 7 ,1 0 4 4 29 19 16 39 25 30 2 3 11 - 3 4 30 4-93(2). ~3-00(I) 9'82(2) 0-1.6(1) P - ~ 0 - 1 0 P<~0~10 P < ~ 0 , 0 1 NS
AND CONCLUSIONS
T h e variability o f t i t r e s i n the serum neutralization test in the estimation of antibodies to infectious bursal disease, has for some t i m e given concern, a n d in the present study 5 % ' of readings of titres in duplicate, tests, wtiere all possible e~l~erimental variables were kept~¢onstant, differed b y f o u r t w o f o l d dilution stepS or more, ithat is,, b y a factor0f 16 o r g r e a t e r , O n the other hand, nearly 7 0 % o f titres differed by a f a c t o r o f only two, ~or were: identical. I n 1970,~ Ha!l acad,Felker defined t h e ~ p r o d u c i b i l i t y o f s e r u m t!tres as ~the percen, t a g e / o f d U p l i c a r e : ~ y g l t h a t yield the same titres;6r t i t ~ d i f f e r i n g by: only[:bne d i l u t i o n ,., ~ W ~ : ~ ::D u r h a m (( i 980),: S ~ i n g a;' d i l u t i 0 n : i n t e ~ - . o f [ t~o'~: in \:~this definiti0n ~:P u t forward ~a:~m~/themati~ ~form u ! a f o r a: quantitat!vel measu~e[of~pr0~[ dugibilibz w h i c h i ~ o u l d b e " ~ f o r aigaricry 0f.ierol6gi [cal)t~ks, ~In~ t h e : i X a m P l ~ icited theY:f0~nd h~ reprMuci.bility [ Of around 80[~ ;i!An 6 ~ e ~ ! :asgessment~ 0 f the~rePr~ug~ i b i l i ~ 0 f t h e S N T f m m t h e data u s ~ i/i the present study g i v e s ~ a ~ m e W h / / r ! ! 0 @ i f i g ~ " 227
(A-B)
, . ,
Serum .
.
1 2 3 4 5 1 2 3 4 5
.
.
.
.
P1 P2
Pt P2
.
3"52 3'22
1,72 1.17
Operator (30)
x y
x y
. . . .
3"17 3"57
1,18 1.70
Method (30)~
g S T
S T
R
4'42 3"32 2'35
1.68 1:48 1.18
(20)
Virus
Assays Operators Methods Virus concentration
Assays Operators Methods Virus concentration
P < 0.001 NS NS P < 0'001
P < 0'05 NS NS NS
Significanceof difference between
Number of tests included in mean: assay, two operators × two methods × three Virus concentration; operators, fiveassays x two methods × threevirus concentration; method, fi;tcassays × t'~o ~1~ratorsX three virus concentration; virusconcentration, five assays X two operators X two methods. Number Of pairs of readings in brackets (means of duplicates).
4.42
0"83 :1'29 2'29 0,42 2,38 1'71 3'79 4.04 2.88
.
Assay (12)
T^BL~ 6, Meandlfference in SNT titres (logz) in fiveassays assessed by a single test reader when the results for sera B and C are subtracted from those for serum A
SERUM I~IEUTRALIZATION TEST FOR IBDV
b e c a u s e o f t h e n u m b e r o f d l f f e r e n t variables i n t r o d u c e d w h i c h affect t h e test. A m o r e realistic a p p r o a c h in t h e p r e s e n t s t u d y is to c o n s i d e r o n e o f t h e t h r e e sera as a s t a n d a r d a n d to express t h e ' r e s u l t s for t h e o t h e r t w o sera in ,terms o f t h i s , tfius eliminating all e x t r a n e o u s variables. A l t h o u g h v a r i a t i o n s in i n d i v i d u a l tests are s o m e t i m e s large, t h e overall e f f e c t i n r e m o v i n g o p e r a t o r a n d d i l u t i o n m e t h o d differences is very m a r k e d . Assay-to-assay v a r i a t i o n is also r e d u c e d , a l t h o u g h v i r u s c o n c e n t r a t i o n still a p p e a r s to have s o m e effect o n t h e test. W h e n a n u m b e r o f r e p l i c a t e s are u s e d in a d e s i g n o f t h e p r e s e n t t y p e in w h i c h overall m e a n s f o r t h e d i f f e r e n t factors c a n be e s t i m a t e d , m e a n l o g 2 t i t r e s f o r a n y o n e s e r u m s t r e n g t h , v i r u s c o n c e n t r a t i o n , d i l u t i o n m e t h o d o r test o p e r a t o r r e m a i n r e a s o n a b l y c o n s t a n t , a n d t h e a g r e e m e n t b e t w e e n f o u r o f t h e five assays When assessed b y a s i n g l e r e a d e r w a s s u r p r i s i n g l y g o o d . P e r h a p s t h e m o s t variable f a c t o r to b e e x p e c t e d is t h a t o f t h e d i f f e r e n c e b e t w e e n t e s t readers, s i n c e a d e c i s i o n o n t h e e n d - p o i n t i s clearly a s u b j e c t i v e a s s e s s m e n t . Parallel tests by" d i f f e r e n t o p e r a t o r s are r e a s o n a b l y c o n s i s t e n t , at least w h e n b a s e d o n m e a n logx t i t r e s o f a n u m b e r o.freplicates, b u t it w o u l d a p p e a r t h a t t h e use o f m i c r o - p i p e t t e s for t h e i n i t i a l s e r u m d i l u t i o n g i v e s b e t t e r ' r e p r o d u c i b i l i t y " t h a n t h a t o f t h e faster b u t less p r e c i s e m e t h o d o f u s i n g d i l u t e r s , p a r t i c u l a r l y in r e l a t i o n to virus concentration and test operators and readers. A low virus concentration gives l e s s v a r i a b l e r e s u l t s t h a n a h i g h ° h e , w h e n based o n t h e m e a n o f d u p l i c a t e t i t r a t i o n s , b u t t h e v a r i a t i o n b e t w e e n d u p l i c a t e s t e n d s to be g r e a t e r a t l o w v i r u s c o n c e n t r a t i o n s .
A cknowledgemewts W e would like to thank Miss C. Evans, Mr G. W o o d and M r R. Nicholas for collaborating in the neutralization test, and Mrs I . W h i t e for :assistance with statistical analysis.
REFERENCES Hall, E- .(2." & Felker,, M . B . (1970). Reproducibility in t h e serological h b o r a t o r y . Health Laboratory Science 7 , 63-~68. W o o d , R . J . & D u r h a m , T. M. (1980). Reproducibility of serological titres.Jourrzal of Clinical Mio'obiology 11, 541--545.
229
Factors affecting the reproducibility of the serum neutralization test for infectious bursal disease*
C. Nancy Hebm't, Y N. E. Reed, t J. C. Muskettt and Denise H. Thorntont
An experiment is described in which the main factors contributing to the x~ariability of the serum neutralization test for infecgious bur~al disease were studied. Significant differences were found between test operato~ and readers. Antibody levels ofsera from vaccinated SPF chickens and the method ofpreparlng dilutions did not affect the variability of the test, but there was a tendency towards a decrease in precision with increasing virus concentration.
INTRODUCTION T h e s e r u m n e u t r a l i z a t i o n t e s t ( S N T ) is w i d e l y u s e d i n t h e e s t i m a t i o n o f a n t i b o d y l e v e l s . Thig report describes an i n v e s t i g a t i o n to e x a m i n e possibie factors w h i c h m a y cause variabilit~r i n r e s u l t s , suc.h as d i f f e r e n t t e s t o p e r a t o r s a n d t e s t r e a d e r s , v a r y i n g s t r e n g t h o f v i r u s a n d s e r u m a n d t h e m e t h o d u s e d f o r p r e p a r i n g t h e s e r u m d i l u t i o n s , u s i n g as a n example; the neutralization test for antibodies to infectious bursal disease virus (IBDV) of chickens.
MATERIALS
AND
METHODS
Sera preparatio~ Sera were collected f r o m S P F birds i m m u n i z e d w i t h i n a c t i v a t e d I B D V . F r o m these ~Received for publication 23 January 1982. tMinistry o f Agricultur,:. Fisheries and Food, Central Vete~irtary Laboratory. Weybridge, Surrey, LI.K.
221
C. N. HEBERT E T A L ,
sera t h r e e pools w e r e p r e p a r e d , w h i c h w e r e h i g h , m e d i u m a n d low w i t h respect to t h e i r a n t i b o d y levels to I B D V ; these w e r e d e s i g n a t e d A, B a n d C respectively. T h e s e t h r e e s e r u m p o o l s w e r e i n a c t i v a t e d at 5 6 ° C for 30 r a i n before t e s t i n g .
Virus suspension A l i q u o t s o f a c o n c e n t r a t e d v i r u s s u s p e n s i o n were p r e p a r e d , a n d these w e r e s t o r e d at - - 7 0 ° C as a s t o c k v i r u s s u s p e n s i o n . O n each test occasion d i l u t i o n s f r o m a s i n g l e a l i q u o t w e r e p r e p a r e d to g i v e an e s t i m a t e d 2 0 , 2 0 0 a n d 2 0 0 0 T C I D s 0 / 0 - 0 2 5 m l (R, S a n d T respectively). T h e v i r u s was d i l u t e d in g r o w t h m e d i u m .
Cell culture A s u s p e n s i o n o f cells was m a d e f r o m a 2 4 h p r i m a D, c u l t u r e o f c h i c k e m b r y o fibroblasts. T h e cell c o n c e n t r a t i o n was a d j u s t e d to 3"5 × 105 cells m 1 - 1 o f g r o w t h m e d i u m . T h e g r o w t h m e d i u m was Eagles m i n i m u m essential m e d i u m s u p p i e m e n t e d w i t h 5 % (v/v) c a l f s e r u m , 1% o f a 7 - 5 % (w/v) s o d i u m b / c a r b o n a t e s o l u t i o n a n d 0 " 5 % (v/v) a n t i b i o t i c s o l u t i o n c o n t a i n i n g p e n i c i l l i n , s t r e p t o m y c i n a n d a m p h o t e r i c i n .
Micro-neutralization test G r o w t h m e d i u m ( 0 - 0 2 5 m l ) was p l a c e d in all wells o f t h e m i c r o t i t r e plates. I n i t i a l t w o f o l d d i l u t i o n s o f sera were m a d e by e i t h e r u s i n g d i l u t i n g s t i c k s ( m e t h o d x) or m i c r o p i p e t t e s ( m e t h o d y). F r o m t h e s e initial d i l u t i o n s f u r t h e r t w o f o l d serial d i l u t i o n s w e r e p r e p a r e d u s i n g d i l u t i n g sticks. S e r u m c o n t r o l s w e r e p r e p a r e d f r o m t h e i n i t i a l s e r u m d i l u t i o n s . T h e a p p r o p r i a t e v i r u s s u s p e n s i o n ( 0 - 0 2 5 m l ) was d i s p e n s e d i n t o t h e test wells. N o v i r u s w a s a d d e d to t h e s e r u m c o n t r o l wells, a n d s o m e wells w e r e set aside for cell c o n t r o l s w h e r e n o v i r u s or s e r u m was a d d e d . T h e p l a t e s w e r e t h e n i n c u b a t e d at 3 8 ~ 5 ° C f0k- 6 0 m i n t o a l l o w v i r u s n e u t r a l i z a t i o n to t a k e place. A f t e r virus n e u t r a l i z a t i o n 0 - 2 m l o f t h e ceil s u s p e n s i o n was d i s p e n s e d i n t o each o f t h e wells. T h e p l a t e s w e r e i n c u b a t e d at 3 8 - 5 ° C in a n a t m o s p h e r e o f 5 % C O 2 a n d 7 0 % h u m i d i t y for 4 d a y s a n d read u n d e r a n i n v e r t e d m i c r o s c o p e . T h e t i t r e was e x p r e s s e d as t h e !og2 o f t h e reciprocal s e r u m d i l u t i o n w h i c h g a v e 1 0 0 % v i r u s n e u t r a l i z a t i o n (i.e. no c y t o p a t h i c eFFect c o u l d be seen).
Expe~mental design I n o r d e r t h a t as m a n y variables as p o s s i b l e c o u l d be s t u d i e d s i m u l t a n e o u s l y , a factorial d e s i g n w a s u s e d in w h i c h t w o test o p e r a t o r s (P 1 a n d P 2 ) a n d five readers (J, K , L, M a n d N ) p a r t i c i p a t e d . T h e t h r e e sera w i t h h i g h , m e d i u m a n d l o w a n t i b o d y levels t o I B D V , a n d t h e t h r e e c o n c e n t r a t i o n s o f viral s u s p e n s i o n w e r e all t e s t e d by t h e t w o m e t h o d s in t h e m i c r o - n e u t r a i i z a t i o n t e s t , Five separate assays were p e r f o r m e d , b u t n o t all five readers w e r e a b l e t o read t h e t e s t s o n every occasion. R e a d e r J p a ~ i c i p a t e d in all t h e assays, m a k i n g p o s s i b l e a valid b e t w e e n - a s s a y c o m p a r i s o n . All tests w e r e p e r f o r m e d in d u p l i c a t e ° STATISTICAL
ANALYSIS
OF RESULTS
T h e r e c i p r o c a l e n d - p o i n t s e r u m d i l u t i o n e x p r e s s e d as l o g t o t h e base 2 is u s e d as t h e r e s p o n s e i n t h e - S N T . I n ~he m a i n analyses in t h i s s t u d y t h e m e a n l o g 2 t i t r e o f ~ l u p l i c a t e 222
SERUM N E U T R A L I Z A T I O N TEST FOR IBDV TABLE 1
Mean S N T titres (log2) in five assays: variation between different test operators and readers
C o l u m n no.
1
2
3
4
Operator Assay
s.e. mean*
Reader
P1
P2
P1
P2
(,8> 7-8i
(18) 5-,91
(54)
(54)
j
6-50~ 6-00 J (18) 7-67} 5-53 J
6-37
5-90
(36)
(36)
J N
6- 11 5" 19 (I8) 6-31 5-47
5-89
6-60
(18)
(18)
(54)
(54)
j
8-58
9-501
M N
11-06 8- 11 (18) 6-25 6-72 5"75 5-36 (18) 6-72 6-67 5"89
9,25
9-58
(72)
(72)
6-02
5-53
(54)
(54)
6-43
5 -45
K L 2
3
4 J K L N 5 j L M
9-28 9-97 (18) 5-94 6-25 4-78 5- 17 ( i 8) 5-03 5-92 5-42
~ J / I
1 ~
Operator
Reader
0" 184
0-318
0- 184
0.260
0-197
0-34.1
0-089
0- IV9
0-107
0- 185
*) s.e. mean, standard error of a mean of (n) values. Number of tests included in each mean: Columns 1 and 2, three sera X three virus--concentration X twomed~(~ds; Columns 3 and 4, three sera × three virus concentrations × two methods × two, three or four readers. Number of readings (n)in brackets (means of duplicates). tests was t a k e n as t h e r e s p o n s e , s i n c e t h e v a r i a t i o n b e t w e e n d u p l i c a t e s reflected o n l y t h e errors o f l a b o r a t o r y t e c h n i q u e , a n d t h e r e f o r e w o u l d n o t c o n s t i t u t e a v a l i d e r r o r t e r m for d e t e r m i n i n g t h e s i g n i f i c a n c e o f d i f f e r e n c e s i n t h e factors i n v o l v e d . A s e p a r a t e analysis o f t h e d i f f e r e n c e b e t w e e n d u p l i c a t e s w a s c a r r i e d OUt. W h e r e o n l y o n e t e s t was p e r f o r m e d i n s t e a d Of t w o , t h e d u p l i c a t e t i t r e w a s e s t i m a t e d as t h e v a l u e m o s t c o m m o n l y g i v e n by readers for t h e first t i t r e . M i s s i n g v a l u e s o c c u r r e d i n f r e q u e n t l y (see T a b l e 4 ) a n d w e r e g e n e r a l l y a s s o c i a t e d w i t h t h e h i g h e s t v i r u s c o n c e n t r a t i o n (T). A l l e s t i m a t e d m e a n s w e r e included inthe statistical analyses. A n analysis o f v a r i a n c e was c a r r i e d o u t for each assay, t a k i n g i n t o a c c o u n t t h e f o u r factors, o p e r a t o r / r e a d e r , s e r u m level, v i r u s c o n c e n t r a t i o n a n d d i l u t i o n m e t h o d ~ M e a n l o g 2 t i t r e s for t h e t w o o p e r a t o r s a n d five r e a d e r s o v e r a l l tests p e r f o r m e d w i t h i n e a c h assay are g i v e n in T a b l e I. T h e s e m ~ are c o m p a r a b l e o w i n g t 0 t h e b a l a n c e d d e s i g n o f t h e e x p e r i m e n t , T h e diffe .fence b e t w e e n tes t o p e r a t o r s P 1 a n d P 2 w a s h i g h l y s i g n i f i c a n t in A s s a y 2 ( P < 0 , 0 1 ) a n d in A s s a y s 4 a n d ~5 0"001)~ T h e r e w e r e h i g h l y s i g n i f i c a n t 223
2.42 41'2 1.64 24,7
3"40 34'2 1'48 28'8 1'15 31"3
Mean s . d . c . v .
Assay 4
Mean s . d . c . v .
Assay 5
(36) 13"69 9"61 4-92 (54) 9'68 9'14
1"53 15"8 1"36 14"9
1"57 11"4 1"61 18"8 1"10 22"3
(48) 7"47 5'65 4'19 (72) 6'28 5'26
1"32 2l'1 1"05 20'0
1"42 19"0 1'31 23'3 0'88 20'9
(36) 8'86 5"28 3'68 (54) 6-31 5"56
0"89 14'1 ['10 19"7
1"32 14'9 1"04 19'8 0'92 25'0
(36) (48) (36) 1'1'76 i.44 12.2 6'96 I'33 19'1 8.14 1!25 15'4 9'38 1'81 19'3 6"28 1'17 I8'7 5'69 1'04 18'3 7'1l 0'91 12"8 4"06 0'70 17'2 3'99 0'69 17"2
Mean s.d.c.v.
Assay 3
Mean, Overall mean for all tests; s.d., residual standard deviation; ¢.v., coefficient of variation (%). Number oftests included in mean:sera, two operators × threevirusconcentration X two methods X. readers;virusconcentration, two operator~ X three sera X;:twomethods X ~ readers;method, two operators X three sera X three virus concentration X n readers. Number of readings n in brackets (means ot duplicates).
1'43 23'4 1.89 30'9
2.20 25'6 1'79 28'9 1"34 37"1
(24) 9'94 5"12 3"67 (36) 5,88 6'.61
(36) (24) 8'00 2'00 25'0 6"98 2.64 37"8 6'15 1"08 17'5 7"50 2"00 26,7 4'25 1'12 26"4 4"25 1.20 28'3
Mean s . d . c . v .
Mean s . d . c . v .
A B C Virus concentration (36) 8'60 R S 6"20 3"60 T (54) Method 6'I3 X 6'14 V
Sera
Assay 2
Variability of mean log2 titres within sera, virus concentration and dilution method Assay 1
T^oL• 2.
1,O
7.2 5.2 3"2 0.55 5'7 4.7 0'45
5'5 6'5
4.9 5'9 4.8 0'55
P2
8.8 6.3 2.8
8.I 5.4 4'5
P1
1
7.1 5'8 3'6 0'45
P2
6'3 6'3
5'3 5.6 0'37
8.8 9,3 6.2 4.6 3"9 2"5 0.45
8.1 6.8 4.0
P1
2
3
P2
8.4 8.2 8.8 8.l 0.48
1 3 . 0 12.1 8"3 8.2 4.4 4.0 0.59
1 0 . 9 10.0 8.8 7.6 6.1 6.8 0'59
P1
Assay
P2
7.6 5.8 0'25
9.0 6'3 4.8 0.31
8.3 7.6 4.2 0"31
6'5 6.0
8.1 6'3 4'3
6.8 6.8 5.2
P1
'4
MeanSNT titres (log2) in fiveassaysassessedbya single test reader
" Standard error of a mean of n readings. Number of readings, n; in brackets (means of duplicates).
Serum (6) A B C : s.e. mean* Virus concentration (6) R $ T s.e. mean Dilution method (9) x y s,e. mean
oiseratot:
T~LE ~.
9'2 6'9 3'8 0'32
P2
6.6 6'3
7,7 5"6 0'26
8.6 t0.1 5'4 5'9 3"7 4.0 0.32
8.0 5.6 4.1
P1
5
C. N . H E B E R T E T A L .
differences between readers in all assays. Operator/reader variations w i t h i n assays can, however, be ignored when considering overall mean values for sera, virus concentration a n d test methods (Table 2). In all assays the mean log2 titre of the three sera and the three virus concentrations showed, as anticipated, very h i g h l y significant differences. Means for the two methods of dilution did not differ significantly in the first assay; in assays 2 and 3 the mean difference was significant a t the 5% level, and in assays 4 and 5 it was h i g h l y significant (P < 0-001). Inassays 3, 4 and 5 the mean for me~hod x was rather higher than t h a t for m e t h o d y, but as indicated by the standard deviation, t h e r e was little difference in the precisio n of the two methods. There was also no indication that the S N T is either more or less reproducible with decreasing strength of serum, as shown by the lack of a n y consistent trend in the coefficient of variation (Table 2). There was, however, in three of the five assays a decrease in precision w i t h increasing virus concentration.
Variation between assays. Table 3 shows the overall mean log 2 titres for sera, virus concentration and dilution m e t h o d in tests performed b y both operators, b u t read by a s i n g l e reader, J , in the five separate assays. The level of response in Assay 3 was higher than that i n all other ass'ays, the differences in most instances being statistically significant, sometimes very h i g h l y so. T h e only exception was in tests performed by operator P2, using the highest virus concentration, T. Here the mean log2 titre in Assay 4 was significantly higher than that in Assay ! (P < 0-05) and Assay 2 (P < 0-01L This was the only significant variation between corresponding m e a n Iog2 titres in Assays I, 2 , 4 and 5. T h e mean log2 tixre for Serum A in the first assay i n rests'performed b y P 2 was abnormal, very low readings for tests using dilution m e t h o d y being obtained b y reader J , in contrast t o the other two readers. I n Assay 2. the mean 1092 titre for serum B was higher than that for Serum A, b u t not significantly so.
Differences between duplicate titrations A separate evaluation was carried our on the difference in log2 titre of duplicate tests, to assess the frequency w i t h which identical readings occurred, and where these differed b y o n e or more twofold dilution steps (Table 4 ) . : ~ 4 h e r e the variation was three d i l u t i o n steps or g r e a t e r , this was analysed further tt, determine if there was any association w i t h s t r e n g t h o f s e r u m , d i l u t l o n method, virus concentration oi operator (Table 5):~-There was a h i g h l y significant differencebetWeen Virus Concentrations, greatei variability occurring with lower concentrations, and seven out of the eight differences Of six steps or!over occurring w i t h Concentration R , while similar results were f o u n d w i t h s t r e n g t h of serum -and~dilution method, where the differences approached statistical Significance. T a b l e 6 demonstrateslthe effec t of regarding one of the three sera (A) as a standard and expressing th e r e s u l t s o f ~ e CorreSpond!fig ~itrations ~ i t h SewnB a n d C as ,tide difference from those for Serum :A: ,Only~the readings:by-re'adei J were used i n this inst/mce~ A c 0 m ~ s o n :With: the SignJificanc.e!evels~found in t h e main~analySis shows tha:t there i s a reduction in the Variability, ~particulariFin the differences between Sera A~a n d B, : w h e ~ e~te:variation betwcen:asSa~;s is significank,0nlyat~the5%)l~vel;~:and Xhe~'0ther: m a i n S a t e : n ° t sthtiStically~:iignificant~: A l t h o u g h h i g h | y i . i i g n ! f i c a n t A ! f f e r e n c e s (P, ~ i 0-001) i~cUr~-~ l i ~ i ~ y s and b e ~ n i y i ~ . IConcentratio~ :iintheci c 0 m ~ i s O n of S¢~:~A~,hnd c,:~i~fle ~/arlabiliqr iSC0//siderably.less ;than in!~ii:~m/iinanal~mis: 226
S E R U M N E U T R A L I Z A T I O N TEST FOR I B D V
TABLE 4.
Difference in log2 .titre of duplicate tests in five assays. Percentage of pairs
Lo&2 difference
No.
Percentage
0 1 2 3 4 5 ~6
182 181 92 40 8 10 9 18 522
34-9 34-7 17-6 7-7 1-5 1-9 1-7
M/V Total
M/V, Missing value inserted for one duplicate--result excluded from totals. TABLE-5. Frequency.of.high differences between duplicates Serum N o . of dilution steps 3
4 5 ~6 Total X2 (d~f.)
DISCUSSION
Method
Virus
A
B
C
x
y
R
S
14 4:
13 1 2 1 3
23 3
16 5
17, 12 2 6
Operator T
Pl
P2
lO
22 3 5
17
0 1
6 2 1 6 3 4 4: 4 5 3 0 7 1 7 ,1 0 4 4 29 19 16 39 25 30 2 3 11 - 3 4 30 4-93(2). ~3-00(I) 9'82(2) 0-1.6(1) P - ~ 0 - 1 0 P<~0~10 P < ~ 0 , 0 1 NS
AND CONCLUSIONS
T h e variability o f t i t r e s i n the serum neutralization test in the estimation of antibodies to infectious bursal disease, has for some t i m e given concern, a n d in the present study 5 % ' of readings of titres in duplicate, tests, wtiere all possible e~l~erimental variables were kept~¢onstant, differed b y f o u r t w o f o l d dilution stepS or more, ithat is,, b y a factor0f 16 o r g r e a t e r , O n the other hand, nearly 7 0 % o f titres differed by a f a c t o r o f only two, ~or were: identical. I n 1970,~ Ha!l acad,Felker defined t h e ~ p r o d u c i b i l i t y o f s e r u m t!tres as ~the percen, t a g e / o f d U p l i c a r e : ~ y g l t h a t yield the same titres;6r t i t ~ d i f f e r i n g by: only[:bne d i l u t i o n ,., ~ W ~ : ~ ::D u r h a m (( i 980),: S ~ i n g a;' d i l u t i 0 n : i n t e ~ - . o f [ t~o'~: in \:~this definiti0n ~:P u t forward ~a:~m~/themati~ ~form u ! a f o r a: quantitat!vel measu~e[of~pr0~[ dugibilibz w h i c h i ~ o u l d b e " ~ f o r aigaricry 0f.ierol6gi [cal)t~ks, ~In~ t h e : i X a m P l ~ icited theY:f0~nd h~ reprMuci.bility [ Of around 80[~ ;i!An 6 ~ e ~ ! :asgessment~ 0 f the~rePr~ug~ i b i l i ~ 0 f t h e S N T f m m t h e data u s ~ i/i the present study g i v e s ~ a ~ m e W h / / r ! ! 0 @ i f i g ~ " 227
(A-B)
, . ,
Serum .
.
1 2 3 4 5 1 2 3 4 5
.
.
.
.
P1 P2
Pt P2
.
3"52 3'22
1,72 1.17
Operator (30)
x y
x y
. . . .
3"17 3"57
1,18 1.70
Method (30)~
g S T
S T
R
4'42 3"32 2'35
1.68 1:48 1.18
(20)
Virus
Assays Operators Methods Virus concentration
Assays Operators Methods Virus concentration
P < 0.001 NS NS P < 0'001
P < 0'05 NS NS NS
Significanceof difference between
Number of tests included in mean: assay, two operators × two methods × three Virus concentration; operators, fiveassays x two methods × threevirus concentration; method, fi;tcassays × t'~o ~1~ratorsX three virus concentration; virusconcentration, five assays X two operators X two methods. Number Of pairs of readings in brackets (means of duplicates).
4.42
0"83 :1'29 2'29 0,42 2,38 1'71 3'79 4.04 2.88
.
Assay (12)
T^BL~ 6, Meandlfference in SNT titres (logz) in fiveassays assessed by a single test reader when the results for sera B and C are subtracted from those for serum A
SERUM I~IEUTRALIZATION TEST FOR IBDV
b e c a u s e o f t h e n u m b e r o f d l f f e r e n t variables i n t r o d u c e d w h i c h affect t h e test. A m o r e realistic a p p r o a c h in t h e p r e s e n t s t u d y is to c o n s i d e r o n e o f t h e t h r e e sera as a s t a n d a r d a n d to express t h e ' r e s u l t s for t h e o t h e r t w o sera in ,terms o f t h i s , tfius eliminating all e x t r a n e o u s variables. A l t h o u g h v a r i a t i o n s in i n d i v i d u a l tests are s o m e t i m e s large, t h e overall e f f e c t i n r e m o v i n g o p e r a t o r a n d d i l u t i o n m e t h o d differences is very m a r k e d . Assay-to-assay v a r i a t i o n is also r e d u c e d , a l t h o u g h v i r u s c o n c e n t r a t i o n still a p p e a r s to have s o m e effect o n t h e test. W h e n a n u m b e r o f r e p l i c a t e s are u s e d in a d e s i g n o f t h e p r e s e n t t y p e in w h i c h overall m e a n s f o r t h e d i f f e r e n t factors c a n be e s t i m a t e d , m e a n l o g 2 t i t r e s f o r a n y o n e s e r u m s t r e n g t h , v i r u s c o n c e n t r a t i o n , d i l u t i o n m e t h o d o r test o p e r a t o r r e m a i n r e a s o n a b l y c o n s t a n t , a n d t h e a g r e e m e n t b e t w e e n f o u r o f t h e five assays When assessed b y a s i n g l e r e a d e r w a s s u r p r i s i n g l y g o o d . P e r h a p s t h e m o s t variable f a c t o r to b e e x p e c t e d is t h a t o f t h e d i f f e r e n c e b e t w e e n t e s t readers, s i n c e a d e c i s i o n o n t h e e n d - p o i n t i s clearly a s u b j e c t i v e a s s e s s m e n t . Parallel tests by" d i f f e r e n t o p e r a t o r s are r e a s o n a b l y c o n s i s t e n t , at least w h e n b a s e d o n m e a n logx t i t r e s o f a n u m b e r o.freplicates, b u t it w o u l d a p p e a r t h a t t h e use o f m i c r o - p i p e t t e s for t h e i n i t i a l s e r u m d i l u t i o n g i v e s b e t t e r ' r e p r o d u c i b i l i t y " t h a n t h a t o f t h e faster b u t less p r e c i s e m e t h o d o f u s i n g d i l u t e r s , p a r t i c u l a r l y in r e l a t i o n to virus concentration and test operators and readers. A low virus concentration gives l e s s v a r i a b l e r e s u l t s t h a n a h i g h ° h e , w h e n based o n t h e m e a n o f d u p l i c a t e t i t r a t i o n s , b u t t h e v a r i a t i o n b e t w e e n d u p l i c a t e s t e n d s to be g r e a t e r a t l o w v i r u s c o n c e n t r a t i o n s .
A cknowledgemewts W e would like to thank Miss C. Evans, Mr G. W o o d and M r R. Nicholas for collaborating in the neutralization test, and Mrs I . W h i t e for :assistance with statistical analysis.
REFERENCES Hall, E- .(2." & Felker,, M . B . (1970). Reproducibility in t h e serological h b o r a t o r y . Health Laboratory Science 7 , 63-~68. W o o d , R . J . & D u r h a m , T. M. (1980). Reproducibility of serological titres.Jourrzal of Clinical Mio'obiology 11, 541--545.
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