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A comparison of enzyme immunoassay and bioassay for the quantitative determination of antibodies to tetanus toxin
Journal of Biological Staudardi:atitaz ( ! 983 ) ! !, ! 2 3 - i 28
A comparison of enzyme immLinoassay a n d b i o a s s a y for t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f a n t i b o d i e s to t e t a n u s toxin*
.J. c . Cox, t R. R. Premier,'~ ~/. Finger~4 and .J. G. R. Hurrellt
Antibodies ¢o tetanus toxin were induced in sheep by hyperimmunizar~on over 24 g'ecks. BIc-eds taken at weeks 4 . 8 . 2 0 and tO were assayed for antibody titre by tx)th an enzyme immt, noass.iy (EIA) using a newly-described urease enzyme/substrate system and by bio.Lssay in mice. There g'as a very good correlation between the two assay systems and. x~-ith the exccptiLm of tht- wct-k -1 bleeds, the relationship was the same at all stages of h H x ' r i m m u n i z a t i o n re/~.irdlcss of tttre. adjuvant, or whether toxin or toxoid was used as i m m u n o g e n or for coatin.t: the plates. T h e results c-stablish that the EIA can replace the bioassay for the determination of tetanus ~lnt itoxin in ovine sera.
INTRODUCTION An enzyme immunoassay (EIA) procedure for the estimation of antibodies to tetanus toxin was first reported by Stiffler-Rosenberg & Fey I and has subsequently tbund application in various epidemiological s t u d i e s . 2"3 I n t h e d e t e r m i n a t i o n of the potency o f t e t a n u s a n t i t o x i n , t h e b i o l o g i c a l a s s a y is s t i l l s p e c i f i e d i n t h e B r i t i s h P h a r m a c o p o e i a (1980). 4 This test has a number of shortcomings. Most importantly, it is l a b o u r intensive, takes 4 days to provide a result and large numbers of mice are requited to °Received for publication 17 September 1982. t l m m u n o c h e m i s t r y R. & D . . C o m m o n w e a l t h Serum Laboratories. Parkville. Victoria, 3052, Australia. *Biostatistical Unit, C o m m o n w e a l t h Serum Laboratories. Parkville, Victoria. 3l)52. Australia. 0092-115718i/02Ot2~+06 SO3.00/O
( ~ 1983 The International Association o) Biologit'al Standardization 123
J. c . c o x
ET AL.
o b t a i n p r e c i s i o n . F o r t h e s e reasons it is d e s i r a b l e t o use an in vitro test p r o c e d u r e for all d e t e r m i n a t i o n s o f t e t a n u s a n t i t o x i n t i t r e p r o v i d e d t h a t a reliable c o r r e l a t i o n can be e s t a b l i s h e d b e t w e e n t h e results o f t h e in vitro test a n d t h o s e o f t h e bioassay. In t h i s s t u d y , 2 0 2 s e r u m s a m p l e s w e r e assayed by b o t h E I A a n d bioassay a n d t h e results c o m p a r e d . A g o o d c o r r e l a t i o n was o b s e r v e d u n d e r all c i r c u m s t a n c e s . MATERIALS
AND
METHODS
Production of antisera Sera w e r e available f r o m s h e e p b e i n g u s e d in a s t u d y o f a d j u v a n t s , d o s i n g p r o c e d u r e s a n d o t h e r p a r a m e t e r s i n v o l v e d in t h e p r o d u c t i o n o f i m m u n e g l o b u l i n to t e t a n u s t o x i n . Five g r o u p s each o f t e n y o u n g ( a p p r o x i m a t e l y 6 m o n t h s old) h e a l t h y s h e e p were d o s e d at 6 - w e e k intervals w i t h 1 0 0 0 Lf o f t e t a n u s t o x o i d f o r m u l a t e d as b e l o w a n d a d m i n i s t e r e d s u b c u t a n e o u s l y in a final v o l u m e o f 1 m l .
Group A. T e t a n u s t o x o i d ( p u r i t y 2 7 0 0 L f m g - 1 p r o t e i n IM) purified a c c o r d i n g to t h e p r o c e d u r e o f O g n y a n o v a ~ was a d s o r b e d t o a l h y d r o g e l a n d e m u l s i f i e d in oil (25 p a r t s Bayol F, t w o p a r t s Arlacel 80). T h e a d j u v a n t was essentially t h e s a m e as t h a t d e s c r i b e d b y W e l l s , G i l m o u r , B u r r e l l s & T h o m p s o n . c' Group B. Dose as for G r o u p A b u t , in a d d i t i o n t w o 1-1 b l e e d s w e r e t a k e n 2 w e e k s a p a r t , every" 6 w e e k s . Group C. P u r i f i e d t e t a n u s t o x o i d e m u l s i f i e d in an e q u a l v o l u m e o f F r e u n d s C o m p l e t e A d j u v a n t . A f t e r t h e first t w o doses, F r e u n d s I n c o m p l e t e A d j u v a n t was used. Group D. P u r i f i e d t e t a n u s t o x o i d a d s o r b e d to a l h y d r o g e l . Group E. U n p u r i f i e d t e t a n u s t o x o i d a d s o r b e d to a l h y d r o g e l . A s i x t h g r o u p o f t e n y o u n g , h e a l t h y s h e e p , G r o u p F, was s e n s i t i z e d w i t h 1-0 m l o f t e t a n u s t o x o i d v a c c i n e (CSL AustraJia). T h r e e m o n t h s later t h e s h e e p received 15 L f o f u n p u r i f i e d t e t a n u s t o x i n f o l l o w e d by I 1 s u b c u t a n e o u s doses o f u n p u r i f i e d t e t a n u s t o x i n a d s o r b e d to p o t a s s i u m a l u m . T h r e e doses w e e k -1 w e r e g i v e n , t h e t o x i n doses i n c r e a s i n g f r o m 1-5 to 4 5 0 L f o v e r t h e i m m u n i z a t i o n course. A s e c o n d c o u r s e o f six doses r a n g i n g f r o m 3 7 - 5 t o 4 5 0 L f o f t o x i n was g i v e n 6 w e e k s later. S h e e p in g r o u p s A to E w e r e b l e d ( t e n t o 2 0 m l ) at intervals o f 2 w e e k s a n d s h e e p in g r o u p F w e r e bled t h r e e t i m e s at intervals o f 2 w e e k s after each d o s e course. B l o o d was a l l o w e d to clot n a t u r a l l y a n d t h e s e r u m r e m o v e d a n d s t o r e d a t - - 2 0 ° C .
EIA T e t a n u s t o x o i d , p u r i f i e d a c c o r d i n g t o t h e p r o c e d u r e o f O g n y a n o v a 5 to a p u r i t y o f 2 7 0 0 L f m g " l o f p r o t e i n N was d i l u t e d t o 1 0 L f m l " l in 0- 1 M s o d i u m c a r b o n a t e buffer p H 9 " 6 a n d 9 6 - w e l l d i s p o s a b l e p o l y v i n y l c h l o r i d e m i c r o t i t r e p l a t e s ( C o o k e ) were c o a t e d by i n c u b a t i o n o f 0 - 1 m l o f a n t i g e n at 3 7 ° C for 2 h. T h e c o n t e n t s were a s p i r a t e d a n d t h e p l a t e s w a s h e d t h r e e t i m e s in wash b u f f e r ~ 0 - 0 1 M p h o s p h a t e b u f f e r e d saline, p H 7 - 2 (PBS) c o n t a i n i n g 0 - 0 5 % ( v / v ) T w e e n 2 0 . Plates w e r e s t o r e d at 4°C. T e t a n u s t o x i n , p u r i f i e d a c c o r d i n g to t h e p r o c e d u r e o f O g n y a n o v a s to a p u r i t y o f 1 4 0 0 L f r a g - ) o f p r o t e i n N was also c o a t e d o n to p l a t e s as d e s c r i b e d above. 124
COMPARISON
OF EIA AND
BIOASSAY FOR TETANUS
T e s t sera w e r e d i l u t e d a c c u r a t e l y 1 in 1000 in d i l u t i n g b u f f e r - - w a s h buffer c o n t a i n i n g 0 " 5 % (w/v) b o v i n e s e r u m a l b u m i n - - a n d 0- 1 m l o f s e r u m d i l u t i o n was a d d e d t o i n d i v i d u a l wells. Plates w e r e sealed a n d i n c u b a t e d at 37°C for 30 rain, w a s h e d t h r e e t i m e s w i t h wash buffer a n d t h e n 0- 1 m l urease-labelled rabbit I g G a n t i - s h e e p I g G 7 d i l u t e d to 5 ~ g o f I g G m l - l in d i l u t i n g buffer was a d d e d to each ~,ell. Plates w e r e i n c u b a t e d a n d washed as before, t h e n w a s h e d t h r e e t i m e s in 0 - 1 4 5 M NaCI. Urease s u b s t r a t e s o l u t i o n 7 0- I m l , was a d d e d t o each well a n d the reaction allowed to p r o c e e d at r o o m t e m p e r a t u r e . T h e c o l o u r c h a n g e d f r o m yellow to p u r p l e at a rate w h i c h was p r o p o r t i o n a l to t h e a m o u n t o f specific a n t i b o d y w i t h i n t h e well. Results were read by eye w h e n the h i g h a n d low t i t r e s t a n d a r d sheep sera, i n c l u d e d on every p l a t e , had r e a c h e d t h e i r assigned titres (usually after 20 rain). T h e h i g h s t a n d a r d s e r u m had a log2 E I A t i t r e o f 11-5 ± 0 - 5 , t h e low s t a n d a r d a Iog2 EIA titre o f 3"5 ± 0"5. In t h e e v e n t t h a t t h e s e t w o sera did not reach t h e i r assigned titre at the s a m e t i m e , all assays on t h a t plate w o u l d have b e e n d i s r e g a r d e d b u t this c o n t i n g e n c y d i d not arise.
B ioassay All d i l u t i o n s were p e r f o r m e d in t h e d i l u t i n g buffer used for the EIA. T h e assay s y s t e m used t h e L + / 1 0 toxin dose. T h i s is essentially as described in the British P h a r m a c o p o e i a (1980) a e x c e p t t h a t a lethal r a t h e r t h a n a paralytic e n d p o i n t was used. Sera w e r e screened a t fivefold d i l u t i o n s a r o u n d t h e e x p e c t e d e n d p o i n t a n d t h e n t i t r a t e d at six levels w i t h i n t h e a p p r o p r i a t e range. A s u b s i d i a r y s t a n d a r d s e r u m o f 35 I n t e r n a tional U n i t s m l - i was assayed on all occasions. This s e r u m had been s t a n d a r d i z e d a g a i n s t t h e Second I n t e r n a t i o n a l S t a n d a r d for T e t a n u s A n t i t o x i n at t h e L ÷ / 1 0 level. It h a d been p r e p a r e d by a 1 in 2 0 0 d i l u t i o n in 50% glycerol saline o f a b a t c h o f e q u i n e tetanus antitoxin concentrate. Biostatislica/ procedt, res T h e basic m o d e l used was: log2 (EIA titre) = tog2 (bioassay) + c o n s t a n t (k). T h i s m o d e l was used to s i m p l i f y relationships and was justified on the basis o f very g o o d correlations b e i n g o b t a i n e d for all g r o u p s a n d t i m e s o f b l e e d i n g , t o g e t h e r w i t h actuaI slopes (b) o f b e t w e e n 0 - 9 2 and 1-06 b e i n g o b t a i n e d for t h e alternative: log2 (EIA titre) = b log2 (bioassay) + a. T h e c o n s t a n t s k i for each t i m e / g r o u p were t h e n c o m p a r e d u s i n g s t a n d a r d t-tests and t h e f u r t h e r h y p o t h e s i s t h a t kT(the m e a n log ratio for t i m e s / g r o u p s ) was a c o n s t a n t for all t i m e s (after e l i m i n a t i o n o f 4 w e e k g r o u p s ) was tested. RESULTS
Comparison of toxin a n d toxoid coatings F i f t y - e i g h t h i g h t i t r e sera w e r e ,assayed by EIA in d u p l i c a t e u s i n g b o t h toxin a n d toxoid c o a t e d t o t h e wells. T h e sera w e r e t h e 8 and 20 w e e k bleeds from g r o u p s A , B and C a n d h a d a r a n g e o f log2 titres f r o m 8"5 to 14-0. T h e average t i t r e for each d u p l i c a t e u s i n g t o x i n - c o a t e d wells was s u b t r a c t e d f r o m t h e average t i t r e o b t a i n e d for t h a t s e r u m u s i n g t o x o i d - c o a t e d wells a n d t h e m e a n d i f f e r e n c e t h e n c a l c u l a t e d . T h e m e a n difference 125
./. C. c o x
ETAL.
o f 0 " 0 5 2 h a d 9 5 9 ~ c o n f i d e n c e l i m i t s o f 0 - 2 0 t o - - 0 - 10 s h o w i n g t h a t t h e u s e o f t o x i n o r t o x o i d m a d e n o s i g n i f i c a n t d i f f e r e n c e t o t h e E I A assay r e s u l t .
Correlation of EIA and bioassay A comparison of the means of the results of the bioassay and of the means of the r e s u l t s o f t h e E I A o f a t o t a l o f 2 0 2 sera f r o m t h e six g r o u p s o f s h e e p is s h o w n in T a b l e 1, a n d t h e a r i t h m e t i c ratios b e t w e e n e a c h p a i r o f m e a n s a r e g i v e n in" T a b l e 2. T h e geometric mean of the ratios at week 4 was compared with the geometric means of the ratios at each of the other bleed times and found to be very highly significantly different
TABLE I. T h e g e o m e t r i c mean (log2) titres o f six groups o f sheep (202 sera) i m m u n i z e d with tetanus t o , old and/or tetanus toxin as obtained by EIA and bioassay.
Sheep A B C D E F
Assay method
Week 4 bleed
Bioassay EIA Bioassay EIA Bioassay EIA Bioassay EIA Bioassay EIA
6-47 10-7 6- 33 10.4 5"59 9" 50 0"24 3"65 -- I "81 2 "05
Bioassay EIA
0"34 4" 15
Week 8 bleed
W e e k 20 bleed
W e e k 30 bleed
8- 33 11-75 8-02 10-85 7"69 10-85 2-04 4-70 0- 17 2"90
9" 07 11-60 8-92 11.90 9"48 12" 50 3"94 6"30 0"20 3"80
7-86 10-70 8-08 11-50 7"90 10-80
6"34 9-40
5" 34 8- lO
TABLE 2. T h e arithmetic ratios b e t w e e n the mean tetanus antitoxin titres o f the sera from six groups o f sheep as obtained by EIA and b y bioassay.
Group A B C D E F Geometric mean ratio (weeks) 126
Week 4 bleed
Week 8 bleed
Week 20 bleed
W e e k 30 bleed
G e o m e t r i c mean ratio (groups)
18-8 16-8 15-0 10-6 14-5 14-0
10-7 7- I 8"9 6"3 6.6
5"8 7-9 8-1 5" 1 12- 1 8"3
7-2 10-7 7-5
6-8
7-6 8-4 8-2 5"7 9"0 7-5
14r7
7"8
7"6
7"9
7"7
COMPARISON OF EIA AND BIOASSAY FOR TETANUS f r o m tTae o t h e r t h r e e m e a n s in a t w o - r a i l e d test (P < 0 - 0 0 1 for weeks 8, 20 a n d 30). T h e o r e t i c a l g r o u n d s for t h e exclusion o f w e e k 4 f r o m the overall m e a h will be discussed. T h e overall m e a n r a t i o for all w e e k s o t h e r t h a n w e e k 4 was 7-7 w i t h 9 5 % c o n f i d e n c e l i m i t s o f 4-8 to 12-4. All 15 ratios fell w i t h i n this range. S i m i l a r l y , t h e g e o m e t r i c m e a n s f o r t h e six g r o u p s (after exclusion o f t h e w e e k 4 bleeds) w e r e calculted. All m e a n s w e r e w i t h i n t h e 9 5 % l i m i t s o f 5-6 to 10-5. DISCUSSION In t h e results r e p o r t e d here, a d i r e c t r e l a t i o n s h i p has been s h o w n b e t w e e n the results o f an EIA a n d those o f a bioassay, i.e. E I A r e s u l t = c o n s t a n t × bioassay result. T h e value o f this c o n s t a n t was f o u n d to be i n d e p e n d e n t o f t i t r e , i m m u n i z a t i o n course, a d j u v a n t a n d t h e use o f toxin or toxoid e i t h e r as i m m u n o g e n or for c o a t i n g the EIA plates. T h e value was affected b y t i m e o f bleed. T h u s at w e e k 4, a m e a n ratio of 14-7 was observed whereas at w e e k s 8, 2 0 a n d 30, t h e ratio was effectively c o n s t a n t w i t h a m e a n o f 7-7. T h e p r o b a b l e e x p l a n a t i o n o f this o b s e r v a t i o n is t h e p r e s e n c e o f I g M a n t i - t e t a n u s toxoid in t h e early ( w e e k 4) bleeds. T h e urease c o n j u g a t e used was p r i m a r i l y d i r e c t e d against t h e Fc p o r t i o n o f s h e e p I g G , a l t h o u g h c r o s s - r e a c t i v i t y w i t h l i g h t chains had not been r e m o v e d b y a d s o r p t i o n . T h e p r e s e n c e o f significant levels o f I g M w o u l d be e x p e c t e d in t h e w e e k 4 bleeds b u t not in t h e w e e k 8 or s u b s e q u e n t bleeds and t h e l i g h t chains o f b o u n d I g M w o u l d have b e e n r e c o g n i z e d b y t h e c o n j u g a t e . I t has been r e p o r t e d t h a t l g M has n o in vivo t o x i n n e u t r a l i z i n g c a p a c i t y , s and therefore it w o u l d be e x p e c t e d t h a t the E I A w o u l d o v e r e s t i m a t e t h e a m o u n t o f in vivo t o x i n - n e u t r a l i z i n g a n t i b o d y . T h e c o r r e l a t i o n b e t w e e n t o x i n a n d t o x o i d - c o a t e d plates in the EIA was i n v e s t i g a t e d because, i f t h e r e w e r e a n y differences, the m e a s u r e m e n t o f t o x i n - b o u n d a n t i b o d y w o u l d parallel m o r e closely a n t i b o d y m e a s u r e d by the bioassay. It was also possible t h a t assay s e n s i t i v i t y m i g h t have been i m p r o v e d . T h e results s h o w e d that tile t w o c o a t i n g s p r o v i d e d i n d i s t i n g u i s h a b l e results a n d h e n c e toxoid was used t h r o u g h o u t the E I A bioassay c o r r e l a t i o n . I n c o n c l u s i o n , t h e s e results s h o w t h a t an E I A can be used in place o f a bioassay for t h e e s t i m a t i o n o f a n t i b o d i e s to t e t a n u s toxin. W h e n p e r f o r m e d a c c o r d i n g to t h e p r o c e d u r e s d e s c r i b e d here, E I A t i t r e = 8 × bioassay titre. A ck~owledgements The authors thank Mirella Barbaro and Anne Stacpoole for tochnical assistance, Dr C. E. Liefman and Mr H. M. Chandler for advice and co-operation and the Director of CSL fi~r supporting this investigation. REFERENCES 1. Stiffler-Rosenberg G , Fey H. Messung yon tetanus-antitoxin mit diem enzyme-linked immunosorbent assay (ELISA). Schweiz Med Wochenschr 1977; 107:1101-110-1. 2. Viljanen MK, Nieminen S. I m m u n i t y to tetanus in Finland. ScandJ Infect Dis 1980; 12: 211-213. 3. Layton GT, A micro-enzyme-linked immunosorbent assay (ELISA) and radio immunosorbent technique (RIST) for the detection of i m m u n i t y to clinical tetanus. Med Lab Sci 19~10; 37: 323--329. 4. British Pharmacopoeia. Biological assay o f tetanus antitoxin, p A130 London: Her Majesty's Stationer,/Office, 1980. 127
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ET AL.
5. Ognyanova O. Purification of tetanus toxin and toxoid by ion exchange chromatography. J H y g Epidemiol Microbiol lmmunol 1974; 18: 3 4 2 - 3 4 6 . 6. Wells P W , G i l m o u r NJL, Burreils C, Thomson D A . A serological comparison of Paste#rella haemolytica vaccines containig different adjuvants. Res V e t Sci 1979; 27: 2 4 8 - 2 5 0 . 7. Chandler H M , Cox J C , Healey K. MacGregor A, Premie~ R R . t'lurreil J G R . An investigation of the use ofurease-antibody conjugates in enzyme immunoassays. J l m m u n o Methods 1982; 53: 1 8 7 - I 9 4 . 8. Edsall G. Problems in the immunology and control of tetanus. Med J Aust 1976; 2: 216--220.
12~
A comparison of enzyme immLinoassay a n d b i o a s s a y for t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f a n t i b o d i e s to t e t a n u s toxin*
.J. c . Cox, t R. R. Premier,'~ ~/. Finger~4 and .J. G. R. Hurrellt
Antibodies ¢o tetanus toxin were induced in sheep by hyperimmunizar~on over 24 g'ecks. BIc-eds taken at weeks 4 . 8 . 2 0 and tO were assayed for antibody titre by tx)th an enzyme immt, noass.iy (EIA) using a newly-described urease enzyme/substrate system and by bio.Lssay in mice. There g'as a very good correlation between the two assay systems and. x~-ith the exccptiLm of tht- wct-k -1 bleeds, the relationship was the same at all stages of h H x ' r i m m u n i z a t i o n re/~.irdlcss of tttre. adjuvant, or whether toxin or toxoid was used as i m m u n o g e n or for coatin.t: the plates. T h e results c-stablish that the EIA can replace the bioassay for the determination of tetanus ~lnt itoxin in ovine sera.
INTRODUCTION An enzyme immunoassay (EIA) procedure for the estimation of antibodies to tetanus toxin was first reported by Stiffler-Rosenberg & Fey I and has subsequently tbund application in various epidemiological s t u d i e s . 2"3 I n t h e d e t e r m i n a t i o n of the potency o f t e t a n u s a n t i t o x i n , t h e b i o l o g i c a l a s s a y is s t i l l s p e c i f i e d i n t h e B r i t i s h P h a r m a c o p o e i a (1980). 4 This test has a number of shortcomings. Most importantly, it is l a b o u r intensive, takes 4 days to provide a result and large numbers of mice are requited to °Received for publication 17 September 1982. t l m m u n o c h e m i s t r y R. & D . . C o m m o n w e a l t h Serum Laboratories. Parkville. Victoria, 3052, Australia. *Biostatistical Unit, C o m m o n w e a l t h Serum Laboratories. Parkville, Victoria. 3l)52. Australia. 0092-115718i/02Ot2~+06 SO3.00/O
( ~ 1983 The International Association o) Biologit'al Standardization 123
J. c . c o x
ET AL.
o b t a i n p r e c i s i o n . F o r t h e s e reasons it is d e s i r a b l e t o use an in vitro test p r o c e d u r e for all d e t e r m i n a t i o n s o f t e t a n u s a n t i t o x i n t i t r e p r o v i d e d t h a t a reliable c o r r e l a t i o n can be e s t a b l i s h e d b e t w e e n t h e results o f t h e in vitro test a n d t h o s e o f t h e bioassay. In t h i s s t u d y , 2 0 2 s e r u m s a m p l e s w e r e assayed by b o t h E I A a n d bioassay a n d t h e results c o m p a r e d . A g o o d c o r r e l a t i o n was o b s e r v e d u n d e r all c i r c u m s t a n c e s . MATERIALS
AND
METHODS
Production of antisera Sera w e r e available f r o m s h e e p b e i n g u s e d in a s t u d y o f a d j u v a n t s , d o s i n g p r o c e d u r e s a n d o t h e r p a r a m e t e r s i n v o l v e d in t h e p r o d u c t i o n o f i m m u n e g l o b u l i n to t e t a n u s t o x i n . Five g r o u p s each o f t e n y o u n g ( a p p r o x i m a t e l y 6 m o n t h s old) h e a l t h y s h e e p were d o s e d at 6 - w e e k intervals w i t h 1 0 0 0 Lf o f t e t a n u s t o x o i d f o r m u l a t e d as b e l o w a n d a d m i n i s t e r e d s u b c u t a n e o u s l y in a final v o l u m e o f 1 m l .
Group A. T e t a n u s t o x o i d ( p u r i t y 2 7 0 0 L f m g - 1 p r o t e i n IM) purified a c c o r d i n g to t h e p r o c e d u r e o f O g n y a n o v a ~ was a d s o r b e d t o a l h y d r o g e l a n d e m u l s i f i e d in oil (25 p a r t s Bayol F, t w o p a r t s Arlacel 80). T h e a d j u v a n t was essentially t h e s a m e as t h a t d e s c r i b e d b y W e l l s , G i l m o u r , B u r r e l l s & T h o m p s o n . c' Group B. Dose as for G r o u p A b u t , in a d d i t i o n t w o 1-1 b l e e d s w e r e t a k e n 2 w e e k s a p a r t , every" 6 w e e k s . Group C. P u r i f i e d t e t a n u s t o x o i d e m u l s i f i e d in an e q u a l v o l u m e o f F r e u n d s C o m p l e t e A d j u v a n t . A f t e r t h e first t w o doses, F r e u n d s I n c o m p l e t e A d j u v a n t was used. Group D. P u r i f i e d t e t a n u s t o x o i d a d s o r b e d to a l h y d r o g e l . Group E. U n p u r i f i e d t e t a n u s t o x o i d a d s o r b e d to a l h y d r o g e l . A s i x t h g r o u p o f t e n y o u n g , h e a l t h y s h e e p , G r o u p F, was s e n s i t i z e d w i t h 1-0 m l o f t e t a n u s t o x o i d v a c c i n e (CSL AustraJia). T h r e e m o n t h s later t h e s h e e p received 15 L f o f u n p u r i f i e d t e t a n u s t o x i n f o l l o w e d by I 1 s u b c u t a n e o u s doses o f u n p u r i f i e d t e t a n u s t o x i n a d s o r b e d to p o t a s s i u m a l u m . T h r e e doses w e e k -1 w e r e g i v e n , t h e t o x i n doses i n c r e a s i n g f r o m 1-5 to 4 5 0 L f o v e r t h e i m m u n i z a t i o n course. A s e c o n d c o u r s e o f six doses r a n g i n g f r o m 3 7 - 5 t o 4 5 0 L f o f t o x i n was g i v e n 6 w e e k s later. S h e e p in g r o u p s A to E w e r e b l e d ( t e n t o 2 0 m l ) at intervals o f 2 w e e k s a n d s h e e p in g r o u p F w e r e bled t h r e e t i m e s at intervals o f 2 w e e k s after each d o s e course. B l o o d was a l l o w e d to clot n a t u r a l l y a n d t h e s e r u m r e m o v e d a n d s t o r e d a t - - 2 0 ° C .
EIA T e t a n u s t o x o i d , p u r i f i e d a c c o r d i n g t o t h e p r o c e d u r e o f O g n y a n o v a 5 to a p u r i t y o f 2 7 0 0 L f m g " l o f p r o t e i n N was d i l u t e d t o 1 0 L f m l " l in 0- 1 M s o d i u m c a r b o n a t e buffer p H 9 " 6 a n d 9 6 - w e l l d i s p o s a b l e p o l y v i n y l c h l o r i d e m i c r o t i t r e p l a t e s ( C o o k e ) were c o a t e d by i n c u b a t i o n o f 0 - 1 m l o f a n t i g e n at 3 7 ° C for 2 h. T h e c o n t e n t s were a s p i r a t e d a n d t h e p l a t e s w a s h e d t h r e e t i m e s in wash b u f f e r ~ 0 - 0 1 M p h o s p h a t e b u f f e r e d saline, p H 7 - 2 (PBS) c o n t a i n i n g 0 - 0 5 % ( v / v ) T w e e n 2 0 . Plates w e r e s t o r e d at 4°C. T e t a n u s t o x i n , p u r i f i e d a c c o r d i n g to t h e p r o c e d u r e o f O g n y a n o v a s to a p u r i t y o f 1 4 0 0 L f r a g - ) o f p r o t e i n N was also c o a t e d o n to p l a t e s as d e s c r i b e d above. 124
COMPARISON
OF EIA AND
BIOASSAY FOR TETANUS
T e s t sera w e r e d i l u t e d a c c u r a t e l y 1 in 1000 in d i l u t i n g b u f f e r - - w a s h buffer c o n t a i n i n g 0 " 5 % (w/v) b o v i n e s e r u m a l b u m i n - - a n d 0- 1 m l o f s e r u m d i l u t i o n was a d d e d t o i n d i v i d u a l wells. Plates w e r e sealed a n d i n c u b a t e d at 37°C for 30 rain, w a s h e d t h r e e t i m e s w i t h wash buffer a n d t h e n 0- 1 m l urease-labelled rabbit I g G a n t i - s h e e p I g G 7 d i l u t e d to 5 ~ g o f I g G m l - l in d i l u t i n g buffer was a d d e d to each ~,ell. Plates w e r e i n c u b a t e d a n d washed as before, t h e n w a s h e d t h r e e t i m e s in 0 - 1 4 5 M NaCI. Urease s u b s t r a t e s o l u t i o n 7 0- I m l , was a d d e d t o each well a n d the reaction allowed to p r o c e e d at r o o m t e m p e r a t u r e . T h e c o l o u r c h a n g e d f r o m yellow to p u r p l e at a rate w h i c h was p r o p o r t i o n a l to t h e a m o u n t o f specific a n t i b o d y w i t h i n t h e well. Results were read by eye w h e n the h i g h a n d low t i t r e s t a n d a r d sheep sera, i n c l u d e d on every p l a t e , had r e a c h e d t h e i r assigned titres (usually after 20 rain). T h e h i g h s t a n d a r d s e r u m had a log2 E I A t i t r e o f 11-5 ± 0 - 5 , t h e low s t a n d a r d a Iog2 EIA titre o f 3"5 ± 0"5. In t h e e v e n t t h a t t h e s e t w o sera did not reach t h e i r assigned titre at the s a m e t i m e , all assays on t h a t plate w o u l d have b e e n d i s r e g a r d e d b u t this c o n t i n g e n c y d i d not arise.
B ioassay All d i l u t i o n s were p e r f o r m e d in t h e d i l u t i n g buffer used for the EIA. T h e assay s y s t e m used t h e L + / 1 0 toxin dose. T h i s is essentially as described in the British P h a r m a c o p o e i a (1980) a e x c e p t t h a t a lethal r a t h e r t h a n a paralytic e n d p o i n t was used. Sera w e r e screened a t fivefold d i l u t i o n s a r o u n d t h e e x p e c t e d e n d p o i n t a n d t h e n t i t r a t e d at six levels w i t h i n t h e a p p r o p r i a t e range. A s u b s i d i a r y s t a n d a r d s e r u m o f 35 I n t e r n a tional U n i t s m l - i was assayed on all occasions. This s e r u m had been s t a n d a r d i z e d a g a i n s t t h e Second I n t e r n a t i o n a l S t a n d a r d for T e t a n u s A n t i t o x i n at t h e L ÷ / 1 0 level. It h a d been p r e p a r e d by a 1 in 2 0 0 d i l u t i o n in 50% glycerol saline o f a b a t c h o f e q u i n e tetanus antitoxin concentrate. Biostatislica/ procedt, res T h e basic m o d e l used was: log2 (EIA titre) = tog2 (bioassay) + c o n s t a n t (k). T h i s m o d e l was used to s i m p l i f y relationships and was justified on the basis o f very g o o d correlations b e i n g o b t a i n e d for all g r o u p s a n d t i m e s o f b l e e d i n g , t o g e t h e r w i t h actuaI slopes (b) o f b e t w e e n 0 - 9 2 and 1-06 b e i n g o b t a i n e d for t h e alternative: log2 (EIA titre) = b log2 (bioassay) + a. T h e c o n s t a n t s k i for each t i m e / g r o u p were t h e n c o m p a r e d u s i n g s t a n d a r d t-tests and t h e f u r t h e r h y p o t h e s i s t h a t kT(the m e a n log ratio for t i m e s / g r o u p s ) was a c o n s t a n t for all t i m e s (after e l i m i n a t i o n o f 4 w e e k g r o u p s ) was tested. RESULTS
Comparison of toxin a n d toxoid coatings F i f t y - e i g h t h i g h t i t r e sera w e r e ,assayed by EIA in d u p l i c a t e u s i n g b o t h toxin a n d toxoid c o a t e d t o t h e wells. T h e sera w e r e t h e 8 and 20 w e e k bleeds from g r o u p s A , B and C a n d h a d a r a n g e o f log2 titres f r o m 8"5 to 14-0. T h e average t i t r e for each d u p l i c a t e u s i n g t o x i n - c o a t e d wells was s u b t r a c t e d f r o m t h e average t i t r e o b t a i n e d for t h a t s e r u m u s i n g t o x o i d - c o a t e d wells a n d t h e m e a n d i f f e r e n c e t h e n c a l c u l a t e d . T h e m e a n difference 125
./. C. c o x
ETAL.
o f 0 " 0 5 2 h a d 9 5 9 ~ c o n f i d e n c e l i m i t s o f 0 - 2 0 t o - - 0 - 10 s h o w i n g t h a t t h e u s e o f t o x i n o r t o x o i d m a d e n o s i g n i f i c a n t d i f f e r e n c e t o t h e E I A assay r e s u l t .
Correlation of EIA and bioassay A comparison of the means of the results of the bioassay and of the means of the r e s u l t s o f t h e E I A o f a t o t a l o f 2 0 2 sera f r o m t h e six g r o u p s o f s h e e p is s h o w n in T a b l e 1, a n d t h e a r i t h m e t i c ratios b e t w e e n e a c h p a i r o f m e a n s a r e g i v e n in" T a b l e 2. T h e geometric mean of the ratios at week 4 was compared with the geometric means of the ratios at each of the other bleed times and found to be very highly significantly different
TABLE I. T h e g e o m e t r i c mean (log2) titres o f six groups o f sheep (202 sera) i m m u n i z e d with tetanus t o , old and/or tetanus toxin as obtained by EIA and bioassay.
Sheep A B C D E F
Assay method
Week 4 bleed
Bioassay EIA Bioassay EIA Bioassay EIA Bioassay EIA Bioassay EIA
6-47 10-7 6- 33 10.4 5"59 9" 50 0"24 3"65 -- I "81 2 "05
Bioassay EIA
0"34 4" 15
Week 8 bleed
W e e k 20 bleed
W e e k 30 bleed
8- 33 11-75 8-02 10-85 7"69 10-85 2-04 4-70 0- 17 2"90
9" 07 11-60 8-92 11.90 9"48 12" 50 3"94 6"30 0"20 3"80
7-86 10-70 8-08 11-50 7"90 10-80
6"34 9-40
5" 34 8- lO
TABLE 2. T h e arithmetic ratios b e t w e e n the mean tetanus antitoxin titres o f the sera from six groups o f sheep as obtained by EIA and b y bioassay.
Group A B C D E F Geometric mean ratio (weeks) 126
Week 4 bleed
Week 8 bleed
Week 20 bleed
W e e k 30 bleed
G e o m e t r i c mean ratio (groups)
18-8 16-8 15-0 10-6 14-5 14-0
10-7 7- I 8"9 6"3 6.6
5"8 7-9 8-1 5" 1 12- 1 8"3
7-2 10-7 7-5
6-8
7-6 8-4 8-2 5"7 9"0 7-5
14r7
7"8
7"6
7"9
7"7
COMPARISON OF EIA AND BIOASSAY FOR TETANUS f r o m tTae o t h e r t h r e e m e a n s in a t w o - r a i l e d test (P < 0 - 0 0 1 for weeks 8, 20 a n d 30). T h e o r e t i c a l g r o u n d s for t h e exclusion o f w e e k 4 f r o m the overall m e a h will be discussed. T h e overall m e a n r a t i o for all w e e k s o t h e r t h a n w e e k 4 was 7-7 w i t h 9 5 % c o n f i d e n c e l i m i t s o f 4-8 to 12-4. All 15 ratios fell w i t h i n this range. S i m i l a r l y , t h e g e o m e t r i c m e a n s f o r t h e six g r o u p s (after exclusion o f t h e w e e k 4 bleeds) w e r e calculted. All m e a n s w e r e w i t h i n t h e 9 5 % l i m i t s o f 5-6 to 10-5. DISCUSSION In t h e results r e p o r t e d here, a d i r e c t r e l a t i o n s h i p has been s h o w n b e t w e e n the results o f an EIA a n d those o f a bioassay, i.e. E I A r e s u l t = c o n s t a n t × bioassay result. T h e value o f this c o n s t a n t was f o u n d to be i n d e p e n d e n t o f t i t r e , i m m u n i z a t i o n course, a d j u v a n t a n d t h e use o f toxin or toxoid e i t h e r as i m m u n o g e n or for c o a t i n g the EIA plates. T h e value was affected b y t i m e o f bleed. T h u s at w e e k 4, a m e a n ratio of 14-7 was observed whereas at w e e k s 8, 2 0 a n d 30, t h e ratio was effectively c o n s t a n t w i t h a m e a n o f 7-7. T h e p r o b a b l e e x p l a n a t i o n o f this o b s e r v a t i o n is t h e p r e s e n c e o f I g M a n t i - t e t a n u s toxoid in t h e early ( w e e k 4) bleeds. T h e urease c o n j u g a t e used was p r i m a r i l y d i r e c t e d against t h e Fc p o r t i o n o f s h e e p I g G , a l t h o u g h c r o s s - r e a c t i v i t y w i t h l i g h t chains had not been r e m o v e d b y a d s o r p t i o n . T h e p r e s e n c e o f significant levels o f I g M w o u l d be e x p e c t e d in t h e w e e k 4 bleeds b u t not in t h e w e e k 8 or s u b s e q u e n t bleeds and t h e l i g h t chains o f b o u n d I g M w o u l d have b e e n r e c o g n i z e d b y t h e c o n j u g a t e . I t has been r e p o r t e d t h a t l g M has n o in vivo t o x i n n e u t r a l i z i n g c a p a c i t y , s and therefore it w o u l d be e x p e c t e d t h a t the E I A w o u l d o v e r e s t i m a t e t h e a m o u n t o f in vivo t o x i n - n e u t r a l i z i n g a n t i b o d y . T h e c o r r e l a t i o n b e t w e e n t o x i n a n d t o x o i d - c o a t e d plates in the EIA was i n v e s t i g a t e d because, i f t h e r e w e r e a n y differences, the m e a s u r e m e n t o f t o x i n - b o u n d a n t i b o d y w o u l d parallel m o r e closely a n t i b o d y m e a s u r e d by the bioassay. It was also possible t h a t assay s e n s i t i v i t y m i g h t have been i m p r o v e d . T h e results s h o w e d that tile t w o c o a t i n g s p r o v i d e d i n d i s t i n g u i s h a b l e results a n d h e n c e toxoid was used t h r o u g h o u t the E I A bioassay c o r r e l a t i o n . I n c o n c l u s i o n , t h e s e results s h o w t h a t an E I A can be used in place o f a bioassay for t h e e s t i m a t i o n o f a n t i b o d i e s to t e t a n u s toxin. W h e n p e r f o r m e d a c c o r d i n g to t h e p r o c e d u r e s d e s c r i b e d here, E I A t i t r e = 8 × bioassay titre. A ck~owledgements The authors thank Mirella Barbaro and Anne Stacpoole for tochnical assistance, Dr C. E. Liefman and Mr H. M. Chandler for advice and co-operation and the Director of CSL fi~r supporting this investigation. REFERENCES 1. Stiffler-Rosenberg G , Fey H. Messung yon tetanus-antitoxin mit diem enzyme-linked immunosorbent assay (ELISA). Schweiz Med Wochenschr 1977; 107:1101-110-1. 2. Viljanen MK, Nieminen S. I m m u n i t y to tetanus in Finland. ScandJ Infect Dis 1980; 12: 211-213. 3. Layton GT, A micro-enzyme-linked immunosorbent assay (ELISA) and radio immunosorbent technique (RIST) for the detection of i m m u n i t y to clinical tetanus. Med Lab Sci 19~10; 37: 323--329. 4. British Pharmacopoeia. Biological assay o f tetanus antitoxin, p A130 London: Her Majesty's Stationer,/Office, 1980. 127
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5. Ognyanova O. Purification of tetanus toxin and toxoid by ion exchange chromatography. J H y g Epidemiol Microbiol lmmunol 1974; 18: 3 4 2 - 3 4 6 . 6. Wells P W , G i l m o u r NJL, Burreils C, Thomson D A . A serological comparison of Paste#rella haemolytica vaccines containig different adjuvants. Res V e t Sci 1979; 27: 2 4 8 - 2 5 0 . 7. Chandler H M , Cox J C , Healey K. MacGregor A, Premie~ R R . t'lurreil J G R . An investigation of the use ofurease-antibody conjugates in enzyme immunoassays. J l m m u n o Methods 1982; 53: 1 8 7 - I 9 4 . 8. Edsall G. Problems in the immunology and control of tetanus. Med J Aust 1976; 2: 216--220.
12~