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The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test
Journal of Biological Standardization (1985) 13, 15 I - I 5 7
The titration of tetanus antitoxin V. E f f e c t o f f o r m a l i z a t i o n m e t h o d for t h e fixation of sheep erythrocytes on the indirect haemagglutination test*
R. K. Gupta, t S. C. Maheshwarit and H. Singht
T w o methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination ( I H A ) test have been compared. The cells fixed with 3% formaldehyde at 4 - 8 ° C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed w i t h 3 ~ formaldehyde at 4 - 8 ° C for 24 h and subsequently treated with 409~ formaldehyde at 4--8°C for a further 24 h (formaldehyde (1I) fixed cells). The correlation between the toxin neutralization (TIM) and I H A titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between T N and I H A titres after treatment o f the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the m i n i m u m protective level of tetanus antitoxin in the sera.
INTRODUCTION The indirect haemagglutination (IHA) test has been widely used throughout the world for detecting and quantitating tetanus antibodies in human and animal sera. Mostly fixed erythrocytes are used in preference to unfixed cells for this test. It has been reported that erythrocytes preserved with g ataraldehyde or formaldehyde show a l t e r e d s e n s i t i v i t y in c o m p a r i s o n w i t h f r e s h , u n f i x e d c e l l s , r e s u l t i n g i n g r e a t d i s c r e p a n c i e s f r o m t h e t i t r e s o b t a i n e d w i t h t h e t o x i n n e u t r a l i z a t i o n ( T N ) t e s t . * L a v e r g n e et al. 2 found that fresh red cells gave IHA reactions which differed from those obtained with o Received for publication 9 J u l y 1984. t Division of Biological Standardization & Quality Control. Central Research Institute, Kasauli (Himachal Pradesh), India. 0092-- 1157185/020151 + 0 7 $ 0 3 . 0 0 / 0
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formalized cells. In o u r previous reports, we found that the sheep e r y t h r o c y t e s fixed with f o r m a l d e h y d e , g l u t a r a l d e h y d e and pyruvic a l d e h y d e had altered sensitivity c o m p a r e d w i t h the unfixed cells, and the correlation b e t w e e n T N and I H A test using cells fixed w i t h different fixative agents varied greatly. 3"'t T h e r e are a n u m b e r o f m e t h o d s for the fixation o f s h e d p e r y t h r o c y t e s w i t h f o r m a l d e h y d e . 1,5-~ In o u r previous studies 3"4 the m e t h o d o f Daniel et al., 6 for fixation o f sheep e r y t h r o c y t e s w i t h f o r m a l d e h y d e , was used. Since the fixation a g e n t and t h e m e t h o d o f fixation o f the cells has a g r e a t effect on the I H A test, we felt the need to s t u d y the effect o f the fixation p r o c e d u r e w i t h f o r m a l d e h y d e on the sensitivity o f the I H A test and t h e correlation b e t w e e n the T N and I H A tests using cells fixed w i t h f o r m a l d e h y d e by two m e t h o d s . MATERIALS
AND
METHODS
Tetanus toxoid Purified t e t a n u s toxoid at 5400 Lf/ml o f p u r i t y 2500 Lf/mg o f protein n i t r o g e n was k i n d l y supplied by D r P. G u p t a , D e p u t y Director o f this Institute. Tetanus toxin Purified freeze d r i e d tetanus toxin (85 Lf/ampoule) was k i n d l y supplied by D r J . G. K r e e f t e n b e r g , Rijks I n s t i t u u t Voor d e V o l k s g e z o n d h e i d , Bilthoven, T h e N e t h e r l a n d s . Standard "antitoxin T h e N a t i o n a l Reference S t a n d a r d for tetanus antitoxi_n ( l 0 I U / m l ) calibrated against the I n t e r n a t i o n a l S t a n d a r d for tetanus a n t i t o x i n was,used for t i t r a t i n g the preparations o f sensitized sheep e r y t h r o c y t e s and for standardization-of tetanus toxin and g u i n e a pig sera. Antisera T h e sera were taken from g u i n e a - p i g s i m m u n i z e d w i t h two doses o f adsorbed tetanus toxoid as described in t h e British P h a r m a c o p o e i a , s T h e sera were stored at - - 2 0 ° C until tested. For t h e I H A tests t h e s e r u m samples were heat inactivated at 56°C for 30 m i n and absorbed w i t h washed p a c k e d sheep e r y t h r o c y t e s at room t e m p e r a t u r e for 30 m i n . T h e dissociation o f I g M m o l e c u l e s into m o n o m e r s w i t h 2 - m e r c a p t o e t h a n o l (2-ME) was effected by t h e methOd described in o u r earlier report. * Sheep erythrocytes O n e v o l u m e o f sheep bloOd was collected in 1-2 v o l u m e s o f Alsever's s o l u t i o n and stored at q-4°C u n t i l n e e d e d biat never for m o r e than two weeks, T h e e r y t h r o c y t e s were w a s h e d t h r e e times" in n o r m a l saline ( 0 " 8 5 % W/v) and c e n t r i f u g e d at 2 0 0 0 r rain - l for seven rain. T h e packed cells were suspended in 3% solution o f f o r m a l d e h y d e in p h o s p h a t e buffered saline (PBS), p H 7-2 to m a k e a 10% cell suspension. Normal rabbit serum ( N R S ) Blood was d r a w n f r o m t h e hearts o f n o r m a l h e a l t h y rabbits. S e r u m was separated and inactivated at 56°C for 30 rain. It was t h e n absorbe~ w i t h washed packed sheep e r y t h r o c y t e s at r o o m t e m p e r a t u r e for 30 rain a n d stored at - 2 0 ° c in small aliquots until r e q u i r e d . 152
TITRATION OF TETANUS A N T I T O X I N ~¢
Fixation of sheep erythrocytes Sheep e r y t h r o c y t e s were fixed w i t h f o r m a l d e h y d e by the following t w o m e t h o d s . ( I ) T h e freshly washed and p a c k e d sheep e r y t h r o c y t e s were m i x e d w i t h 3 % cold f o r m a l d e h y d e in PBS p H 7-2 to p r o v i d e a 10% cell suspension. This suspension was g e n t l y stirred at + 4 ° C for 24 h on a m a g n e t i c stirrer w i t h o u t f r o t h i n g . T h e suspension was d i s t r i b u t e d in t w o parts. Cells o f t h e one p a r t were filtered t h r o u g h four layers o f gauze and w a s h e d six to seven t i m e s w i t h 8 - 1 0 v o l u m e s o f n o r m a l saline. A 10% suspension was m a d e in n o r m a l saline and was stored a t + 4 ° C . T h e cells had a b r o w n i s h coIour. This suspension was labelled as f o r m a l d e h y d e (I) fixed cells. (2) T o the second part, 2 v o l u m e s o f cold 4 0 % f o r m a l d e h y d e was a d d e d and t h e s t i r r i n g was c o n t i n u e d for a n o t h e r 24 h. T h e cells were t h e n filtered t h r o u g h four layers o f g a u z e a n d washed six to seven t i m e s w i t h 8 - 1 0 v o l u m e s o f n o r m a l saline. A 10% suspension was m a d e in n o r m a l saline a n d was s t o r e d at + 4 ° C . T h i s suspension was labelled as f o r m a l d e h y d e (II) fixed cells. T h e cells had a d a r k b r o w n colour. This m e t h o d is essentially the s a m e as t h a t described by Daniel et al. 6
Sensitization of fixed sheep ,~ythrocytes with tetanus toxoid T h e cells w e r e t r e a t e d w i t h various c o n c e n t r a t i o n s o f tannic acid r a n g i n g f r o m I/10 0 0 0 to 1/80 0 0 0 in twofold d i l u t i o n s by the m e t h o d previously reported. 3 T a n n e d sheep e r y t h r o c y t e s w e r e sensitized w i t h t e t a n u s toxoid at p H 6"4 and 7"2 using 25, 50, 100 and 2 0 0 Lf/ml o f t e t a n u s toxoid for 30, 60 a n d 120 rain at 37°C a n d 56°C. T h e m e t h o d o f sensitization has b e e n described elsewhere. 3 Finally t h e sensitized cells were s u s p e n d e d in PBS p H 7-2 w i t h 1 in 10 0 0 0 t h i o m e r s a l and 0 - 5 % N R S in various c o n c e n t r a t i o n s b u t usually at a c o n c e n t r a t i o n o f 0 - 5 % w h i c h was found m o s t suitable.
Indirect haemagglutination test T h e I H A test was p e r f o r m e d as described earlier. 3 A f t e r o b t a i n i n g the o p t i m a l c o n d i t i o n s for s e n s i t i z i n g f o r m a l d e h y d e (I a n d II) fixed sheep e r y t h r o c y t e s , a large batch o f sensitized cells was p r e p a r e d u n d e r o p t i m a l c o n d i t i o n s using b o t h f o r m a l d e h y d e (I a n d II) fixed cells. T h e n all the 77 g u i n e a - p i g sera were t i t r a t e d by t h e I H A before and after t h e t r e a t m e n t o f the sera w i t h 2 - M E u s i n g b o t h types o f cell. T h e titrations o f t h e g u i n e a - p i g sera w e r e d o n e as described elsewhere. 4
Titration of sera by T N T h e T N test was p e r f o r m e d by t h e lethal e n d p o i n t m e t h o d using 1 7 - 2 0 g m i c e o f t h e L A C A strain o f e i t h e r sex. T h e sera w e r e t i t r a t e d at t h e L + / 1 0 0 , L + / 1 0 0 0 a n d L + / 5 0 0 0 doses o f t e t a n u s toxin. T h e toxin dose level used for t i t r a t i n g u n k n o w n sera a n d the r a n g e o f s e r u m d i l u t i o n s tested d e p e n d e d u p o n the t e t a n u s a n t i t o x i n c o n t e n t o f t h e sera as o b t a i n e d by t h e I H A m e t h o d . U s u a l l y an e n d p o i n t for a test s e r u m was o b t a i n e d by t e s t i n g in t h e r a n g e o f t w o to e i g h t t i m e s lower t h a n t h a t d e t e c t e d by I H A . RESULTS
Formaldehyde (!) fixed sheep erythrocytes Sensitization of sheep erythrocytes. Sheep cells w h e n t a n n e d w i t h 1/I 0 0 0 0 , 1/20 0 0 0 , 1/40 0 0 0 a n d 1/80 0 0 0 d i l u t i o n s o f t a n n i c acid s h o w e d differences o f not m o r e t h a n t w o fold in titres w i t h t h e N a t i o n a l R e f e r e n c e 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 (0- I I U / m l ) . T h e titres a n d scores w e r e h i g h e s t at t h e 1/40 0 0 0 c o n c e n t r a t i o n o f t a n n i c acid. 153
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TABLE 1.
T h e effect o f the c o n c e n t r a t i o n o f tetanus toxoid used for sensitization o f f o r m a l d e h y d e (I) fixed cells
C o n c e n t r a t i o n o f tetanus toxoid used for sensitization (Lf/ml)
T i t r e s and scores* with the N a t i o n a l Reference Standard for tetanus a n t i t o x i n (0" 1 I U / m l )
25 50 100 200
16 32 16 4
(18) (21) (17) (7)
* Scores are included in parentheses and represent the sum of the values of the agglutination reaction in each well graded from 0--4.
Table 1 shows the effect of the concentration of sensitizing tetanus toxoid on a n t i t o x i n t i t r e s . T h e o p t i m a l c o n c e n t r a t i o n o f t e t a n u s t o x o i d f o r s e n s i t i z a t i o n was 5 0 L f / m l . T h e m a x i m u m a n t i t o x i n t i t r e w a s o b t a i n e d w h e n t h e periocl o f e x p o s u r e o f t a n n e d e r y t h r o c y t e s to s e n s i t i z i n g t e t a n u s t o x o i d was 1 h. T h e r e was no increase in t h e t i t r e w h e n t h e p e r i o d w a s i n c r e a s e d t o 2 h. T h e p H o f e x p o s u r e o f t a n n e d e r y t h r o c y t e s to sensitizing tetanus toxoid did not have any marked effect on the titres but the score was m a x i m u m a t p H 7 - 2 . T h e cells s e n s i t i z e d a t 5 6 ° C g a v e t h e h i g h e s t t i t r e w i t h m a x i m u m s c o r e . T h e t i t r e w a s r e d u c e d t o o n e - q u a r t e r w h e n t h e cells w e r e s e n s i t i z e d a t 370(2. Importance of the concentration of sensitized erythrocytes. T a b l e 2 s h o w s t h e e f f e c t o f t h e c o n c e n t r a t i o n o f sheep e r y t h r o c y t e s o n titres a n d scores. As t h e c o n c e n t r a t i o n o f t h e cells d e c r e a s e d , t h e s e n s i t i v i t y i n c r e a s e d , b u t t h e H A e n d p o i n t w a s d i f f i c u l t t o r e a d a t t h e l o w e r c o n c e n t r a t i o n . A c o n c e n t r a t i o n o f 0 - 5 % o f cells was f o u n d s u i t a b l e as a compromise between sensitivity and readability. Sensitivity of l H A . A m i n i m u m l e v e l o f 0 - 0 0 3 0 I U / m l o f t e t a n u s a n t i t o x i n was d e t e c t e d u s i n g t h e s e cells s e n s i t i z e d u n d e r o p t i m a l c o n d i t i o n s . Stability of sensitized sheep ceils. T a b l e 3 s h o w s t h e loss o f s e n s i t i v i t y o f s e n s i t i z e d
TABLE 2.
T h e effect o f the c o n c e n t r a t i o n o f sensitized sheep e r y t h r o cytes using f o r m a l d e h y d e (I) fixed cells
Concentration of sensitized cells (%)
T i t r e s and scores* with t h e N a t i o n a l Reference Standard for tetanus antitoxin (0-1 I U / m l )
0- 1 0-25 0-5
2 5 6 (32) 64 (26) 32 (22)
1-0
16 ( 1 6 )
* Scores are indicated in parentheses and represent the sum of the values of the agglutination reaction in each well from O-4.
154
TITRATION TABLE 3.
OF TETANUS ANTITOXIN
V
Loss o f s e n s i t i v i t y o f f o r m a l d e h y d e (I) fixed cells w h e n s t o r e d a t 4 - 8 ° C for n i n e m o n t h s
Storage period at 4-8°C (months)
Titres and scores ~ w i t h the National Reference Standard for t e t a n u s a n t i t o x i n (0- 1 I U / m l )
0 3 6 9
32 ( 2 2 ) 8 (I3) 4(9) 4(8)
* Scores are indicated in parentheses and represent the sum of the values of the a g g l u t i n a t i o n reaction in each well from 0--4.
s h e e p cells w h e n s t o r e d a t 4~.8°C for n i n e m o n t h s . T h e r e was a f o u r f o l d to e i g h t f o l d d e c r e a s e in t h e t i t r e w h e n t h e cells w e r e s t o r e d a t 4 - 8 ° C for t h r e e to n i n e m o n t h s . Correlation between T N a n d I l i a titres. Figure I shows the correlation between titres o f i n d i v i d u a l g u i n e a - p i g sera o b t a i n e d b y T N a n d t h e I H A t e s t before a n d a f t e r t r e a t m e n t o f t h e sera w i t h 2 - M E . T h e I H A t i t r e s before 2 - M E t r e a t m e n t o f t h e sera w e r e a b o u t t w o to six t i m e s h i g h e r t h a n t h e T N t i t r e s . T h e c o r r e l a t i o n coefficient (r) b e t w e e n T N a n d I H A t i t r e s b e f o r e 2 - M E t r e a t m e n t was 0 - 9 7 w i t h t h e r e g r e s s i o n e q u a t i o n y -- 0" 1 8 6 8 x + 0 " 5 9 5 4 . N o s e r u m s a m p l e s h o w e d a l o w e r t i t r e w i t h I H A
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Fig. I. Scatter d i a g r a m for titres of individual g u i n e a - p i g sera obtained by toxin neutralization ( T N ) and indirect h a e m a g g l u t i n a t i o n ( I H A ) tests using formaldehyde (1) fixed sheep erythrocyres before and after the treatment o f the sera with 2-mercaptoethanol (2-ME). A, I H A Titres before 2-ME treatment of the sera; m I H A ritres after 2-ME t r e a t m e n t of the sera. The figures adjacent to some of t h e points indicate a n u m b e r of,sera with coincident titres.
155
R. K. G U P T ~ ET AL. t h a n w i t h T N . T h e / H A titres after 2 - M E t r e a t m e n t o f the sera were closer co "the T N titres. S o m e o f the sera had s l i g h t l y lower titres by I H A t h a n by T N after 2 - M E t r e a t m e n t . T h e correlation coefficient b e t w e e n T N and I H A titres after 2 - M E t r e a t m e n t was 0"95 a n d t h e regression e q u a t i o n was y ----- 0 - 5 1 8 7 x -F 1"0633. Analysis of variance between T N and IHA titres. T h e analysis o f variance b e t w e e n titres o f g u i n e a - p i g sera o b t a i n e d by T N and I H A using f o r m a l d e h y d e (I) fixed cells before 2 - M E t r e a t m e n t o f t h e sera s h o w e d t h e value o f F h i g h l y significant a t 5 % level. A f t e r 2 - M E t r e a t m e n t o f t h e sera there was no statistically significant difference b e t w e e n T N a n d I H A titres.
Formaldehyde ( H ) fixed sheep erythrocytes Ali the results w i t h f o r m a l d e h y d e (II) fixed cells have been described in our previous reports 3"4 u n d e r the h e a d i n g o f f o r m a l d e h y d e fixed sheep e r y t h r o c y t e s .
DISCUSSION T h e reliability o f the I H A test d e p e n d s u p o n various factors involving the sensitization and fixation o f s h e e p e r y t h r o c y t e s w i t h different fixation agents. In our p r e v i o u s s t u d y , 3 it was r e p o r t e d t h a t t h e g l u t a r a l d e h y d e and f o r m a l d e h y d e fixed cells were t h e m o s t sensitive for t h e e s t i m a t i o n Of t e t a n u s a n t i t o x i n . In this report it was found t h a t the sensitivity o f f o r m a l d e h y d e (I) fixed cells was very low c o m p a r e d w i t h t h e sensitivity o f f o r m a l d e h y d e (II) fixed cells. O t h e r workers have also reported low sensitivity o f I H A test w h e n t h e cells w e r e fixed w i t h 3% f o r m a l d e h y d e , v'9 T h e o p t i m a l c o n d i t i o n s for sensitization o f f o r m a l d e h y d e (1) fixed ceils w e r e t h e s a m e as for g l u t a r a l d e h y d e , f o r m a l d e h y d e (II) and p y r u v i c a l d e h y d e fixed cells. 3 T h e c o n c e n t r a t i o n o f sensitized s h e e p e r y t h r o c y t e s h a d g r e a t effect on the titre (Table 2). These findings c o n f i r m the earlier reports. 3.1o. ~, T h e r e was a b o u t four t i m e s loss in the sensitivity o f f o r m a l d e h y d e (I) fixed cells w h e n these were stored at 4 - 8 ° C for three m o n t h s . T h o u g h these findings are similar to o u r earlier report, 3 the f o r m a l d e h y d e (I) fixed cells were n o t sufficiently sensitive after three m o n t h s to d e t e c t even the m i n i m u m protective level o f t e t a n u s a n t i t o x i n (0"01 I U / m l ) in the sera. These cells were used w i t h o u t a n y loss o f sensitivity u p to t w o m o n t h s having an a d v a n t a g e over unfixed cells w h i c h could be used for o n l y seven to ten days after sensitization. 3 T h e correlation b e t w e e n T N a n d I H A titres was b e t t e r w h e n f o r m a l d e h y d e (I) fixed cells were used t h a n w h e n f o r m a l d e h y d e (II) fixed cells were used. "*T h e correlation a n d regression coefficients b e t w e e n T N and I H A titres using f o r m a l d e h y d e (I) fixed cells were s i m i l a r to those o f g l u t a r a l d e h y d e fixed and unfixed cells. 4 T h e differences b e t w e e n T N a n d I H A titres before 2 - M E t r e a t m e n t were statistically significant. These results were s i m i l a r to those r e p o r t e d earlier. "~ T h e I H A titres w i t h f o r m a l d e h y d e (I) fixed Cells were h i g h e r t h a n t h e T N titres c o n f i r m i n g earlier findings. "!"1u'12-15 T h e r e was no statistically significant difference b e t w e e n T N and I H A titres after 2 - M E t r e a t m e n t o f the sera u s i n g f o r m a l d e h y d e (I) fixed cells. T h e s e findings differ from those o b t a i n e d w i t h f o r m a l d e h y d e (lI) fixed ceils and are similar to g l u t a r a l d e h y d e f i x e d and unfixed cells. 4 F r o m this s t u d y it is c o n c l u d e d t h a t not only the a g e n t for fixation o f s h e e p e r y t h r o c y t e s b u t also t h e fixation p r o c e d u r e affect t h e I H A test considerably. T h e f o r m a l d e h y d e (I) fixed cells a l t h o u g h less sensitive, can be used w i t h reliability for t h e t i t r a t i o n o f t e t a n u s a n t i t o x i n in t h e sera. 156
T I T R A T I O N OF T E T A N U S A N T I T O X I N V
Acknowledgements T h e authors are grateful to Dr S. N . Saxena, Director, Central Research Institute, Kasauli for the keen interest in this study. REFERENCES 1. Nyerges G , Lutter J . Tiae influence o f the method of preservation o f erythrocytes on the correlation b e t w e e n tetanus antitoxin values as measured by passive haemagglutination and by seroneutralization tests in mice. J Biol Stand 1980; 8 : 3 1 1 - 3 1 5 . 2. Lavergne M, Mangalo R, Raynaud M. Influence de l'avidit6 des anticorps antidiphth6riques de cheval sur le titre de la reaction d'h6magglutination passive. Ann l'lnst Pasteur 1965; 1 0 9 : 9 4 - 1 1 9 . 3. G u p t a R K , Maheshwari SC, Singh H . The titration of tetanus antitoxin. I. Factors affecting the sensitivity o f the indirect haemagglutination test. J Biol Stand 1984; 12: 11-17. 4. G u p t a R K , Maheshwari SC, Singh H . T h e titration o f tetanus antitoxi'fi II. A comparative evaluation o f the indirect haemagglutination and toxin neutralization tests. J Biol Stand 1984; 12: 1 3 7 - 1 4 3 . 5- Csizmas L. Preparation o f formalinized erythrocytes. Proc Soc Exp Biol Med 1960; 103: 157-160. 6. Daniel T M , W e y z a n d JG/vt. Stavitsky AB. Micro m e t h o d s for the s t u d y o f proteins and antibodies IV. Factors involved in the preparation and use o f stable preparation o f formalinised, tannic acid treated protein sensitized erythrocytes for detection of antigen and antibody. J I m m u n o l 1963; 90: 7 4 1 - 7 5 0 . 7. Pirzurra L, Bistoni F, Pitzurra M, Bastianini L, Per/to S, Vacchiarelli P, Marconi P. Comparison o f passive haemagglutination assay for tetanus antibodies w i t h enzyme-linked i m m u n o s o r b e n t and counterimmunoelectrophoresis methods. In: Proceedings o f the Sixth International Conference on Tet~ nus, Lyon, 3 - 5 D e c e m b e r 1981. Lyon: Fondation Marcel Merieux; 1982: 1 3 9 - 1 4 5 . 8. British Pharmacopoeia. Adsorbed T e t a n u s Toxoid. London: H M S O , 1973: 464. 9- Galazka A, Abgarowicz A. Assays o f diphtheria and tetanus antibodies by the passive haemagglutirration m e t h o d . Epidemiol Rev 1967; ~XXI: 2 3 7 - 2 5 2 . 10. Peel M M . M e a s u r e m e n t o f tetanus antitoxin I. Indirect haemagglutination. J Biol Stand 1980; 8: 1 7 7 - 1 8 9 . 11. Stavitsky AB. H a e m a g g l u t i n a t i o n with tannic acid treated (tanned) erythrocytes. In: CA Williams, M W Chase, eds. Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y , Vol. IV. N e w York: Academic Prress, I 9 7 7 : 3 0 - - 4 1 . 12. Tasman A, Van R a m s h o r t J D , S m i t h L. Determination of diphtheria and tetanus antitoxin with the aid o f haemagglutination. Antonie van Leewenhoek 1960; 2 6 : 4 1 3 - 4 2 9 . 13. Surjan M, N y e r g e s G. H a e m a g g l u t i n a t i o n procedure for the assay o f tetanus antitoxin o f children's sera. Z l m m u n Forsch 1962; 124: 3 9 0 - 4 0 0 . 14. Chatterjee SC. A comparative study o f the haemagglutination and bioassay procedures for the assay o f guinea pigs anti-diphtheria and anti-tetanus sera. l n d i a n J M e d Res 1964; 52: 1241-1249. 15. G u p t a R K , Maheshwari SC, Singh H . T h e titration o f tetanus antitoxin III. A comparative evaluation of.indirect haemagglutination and toxin neutralization titres o f h u m a n sera. J Biol Stand 1984; 12: 1 4 5 - 1 4 9 .
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The titration of tetanus antitoxin V. E f f e c t o f f o r m a l i z a t i o n m e t h o d for t h e fixation of sheep erythrocytes on the indirect haemagglutination test*
R. K. Gupta, t S. C. Maheshwarit and H. Singht
T w o methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination ( I H A ) test have been compared. The cells fixed with 3% formaldehyde at 4 - 8 ° C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed w i t h 3 ~ formaldehyde at 4 - 8 ° C for 24 h and subsequently treated with 409~ formaldehyde at 4--8°C for a further 24 h (formaldehyde (1I) fixed cells). The correlation between the toxin neutralization (TIM) and I H A titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between T N and I H A titres after treatment o f the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the m i n i m u m protective level of tetanus antitoxin in the sera.
INTRODUCTION The indirect haemagglutination (IHA) test has been widely used throughout the world for detecting and quantitating tetanus antibodies in human and animal sera. Mostly fixed erythrocytes are used in preference to unfixed cells for this test. It has been reported that erythrocytes preserved with g ataraldehyde or formaldehyde show a l t e r e d s e n s i t i v i t y in c o m p a r i s o n w i t h f r e s h , u n f i x e d c e l l s , r e s u l t i n g i n g r e a t d i s c r e p a n c i e s f r o m t h e t i t r e s o b t a i n e d w i t h t h e t o x i n n e u t r a l i z a t i o n ( T N ) t e s t . * L a v e r g n e et al. 2 found that fresh red cells gave IHA reactions which differed from those obtained with o Received for publication 9 J u l y 1984. t Division of Biological Standardization & Quality Control. Central Research Institute, Kasauli (Himachal Pradesh), India. 0092-- 1157185/020151 + 0 7 $ 0 3 . 0 0 / 0
~ ) ! 9 8 5 T h e International Association of Biological Standardization
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formalized cells. In o u r previous reports, we found that the sheep e r y t h r o c y t e s fixed with f o r m a l d e h y d e , g l u t a r a l d e h y d e and pyruvic a l d e h y d e had altered sensitivity c o m p a r e d w i t h the unfixed cells, and the correlation b e t w e e n T N and I H A test using cells fixed w i t h different fixative agents varied greatly. 3"'t T h e r e are a n u m b e r o f m e t h o d s for the fixation o f s h e d p e r y t h r o c y t e s w i t h f o r m a l d e h y d e . 1,5-~ In o u r previous studies 3"4 the m e t h o d o f Daniel et al., 6 for fixation o f sheep e r y t h r o c y t e s w i t h f o r m a l d e h y d e , was used. Since the fixation a g e n t and t h e m e t h o d o f fixation o f the cells has a g r e a t effect on the I H A test, we felt the need to s t u d y the effect o f the fixation p r o c e d u r e w i t h f o r m a l d e h y d e on the sensitivity o f the I H A test and t h e correlation b e t w e e n the T N and I H A tests using cells fixed w i t h f o r m a l d e h y d e by two m e t h o d s . MATERIALS
AND
METHODS
Tetanus toxoid Purified t e t a n u s toxoid at 5400 Lf/ml o f p u r i t y 2500 Lf/mg o f protein n i t r o g e n was k i n d l y supplied by D r P. G u p t a , D e p u t y Director o f this Institute. Tetanus toxin Purified freeze d r i e d tetanus toxin (85 Lf/ampoule) was k i n d l y supplied by D r J . G. K r e e f t e n b e r g , Rijks I n s t i t u u t Voor d e V o l k s g e z o n d h e i d , Bilthoven, T h e N e t h e r l a n d s . Standard "antitoxin T h e N a t i o n a l Reference S t a n d a r d for tetanus antitoxi_n ( l 0 I U / m l ) calibrated against the I n t e r n a t i o n a l S t a n d a r d for tetanus a n t i t o x i n was,used for t i t r a t i n g the preparations o f sensitized sheep e r y t h r o c y t e s and for standardization-of tetanus toxin and g u i n e a pig sera. Antisera T h e sera were taken from g u i n e a - p i g s i m m u n i z e d w i t h two doses o f adsorbed tetanus toxoid as described in t h e British P h a r m a c o p o e i a , s T h e sera were stored at - - 2 0 ° C until tested. For t h e I H A tests t h e s e r u m samples were heat inactivated at 56°C for 30 m i n and absorbed w i t h washed p a c k e d sheep e r y t h r o c y t e s at room t e m p e r a t u r e for 30 m i n . T h e dissociation o f I g M m o l e c u l e s into m o n o m e r s w i t h 2 - m e r c a p t o e t h a n o l (2-ME) was effected by t h e methOd described in o u r earlier report. * Sheep erythrocytes O n e v o l u m e o f sheep bloOd was collected in 1-2 v o l u m e s o f Alsever's s o l u t i o n and stored at q-4°C u n t i l n e e d e d biat never for m o r e than two weeks, T h e e r y t h r o c y t e s were w a s h e d t h r e e times" in n o r m a l saline ( 0 " 8 5 % W/v) and c e n t r i f u g e d at 2 0 0 0 r rain - l for seven rain. T h e packed cells were suspended in 3% solution o f f o r m a l d e h y d e in p h o s p h a t e buffered saline (PBS), p H 7-2 to m a k e a 10% cell suspension. Normal rabbit serum ( N R S ) Blood was d r a w n f r o m t h e hearts o f n o r m a l h e a l t h y rabbits. S e r u m was separated and inactivated at 56°C for 30 rain. It was t h e n absorbe~ w i t h washed packed sheep e r y t h r o c y t e s at r o o m t e m p e r a t u r e for 30 rain a n d stored at - 2 0 ° c in small aliquots until r e q u i r e d . 152
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Fixation of sheep erythrocytes Sheep e r y t h r o c y t e s were fixed w i t h f o r m a l d e h y d e by the following t w o m e t h o d s . ( I ) T h e freshly washed and p a c k e d sheep e r y t h r o c y t e s were m i x e d w i t h 3 % cold f o r m a l d e h y d e in PBS p H 7-2 to p r o v i d e a 10% cell suspension. This suspension was g e n t l y stirred at + 4 ° C for 24 h on a m a g n e t i c stirrer w i t h o u t f r o t h i n g . T h e suspension was d i s t r i b u t e d in t w o parts. Cells o f t h e one p a r t were filtered t h r o u g h four layers o f gauze and w a s h e d six to seven t i m e s w i t h 8 - 1 0 v o l u m e s o f n o r m a l saline. A 10% suspension was m a d e in n o r m a l saline and was stored a t + 4 ° C . T h e cells had a b r o w n i s h coIour. This suspension was labelled as f o r m a l d e h y d e (I) fixed cells. (2) T o the second part, 2 v o l u m e s o f cold 4 0 % f o r m a l d e h y d e was a d d e d and t h e s t i r r i n g was c o n t i n u e d for a n o t h e r 24 h. T h e cells were t h e n filtered t h r o u g h four layers o f g a u z e a n d washed six to seven t i m e s w i t h 8 - 1 0 v o l u m e s o f n o r m a l saline. A 10% suspension was m a d e in n o r m a l saline a n d was s t o r e d at + 4 ° C . T h i s suspension was labelled as f o r m a l d e h y d e (II) fixed cells. T h e cells had a d a r k b r o w n colour. This m e t h o d is essentially the s a m e as t h a t described by Daniel et al. 6
Sensitization of fixed sheep ,~ythrocytes with tetanus toxoid T h e cells w e r e t r e a t e d w i t h various c o n c e n t r a t i o n s o f tannic acid r a n g i n g f r o m I/10 0 0 0 to 1/80 0 0 0 in twofold d i l u t i o n s by the m e t h o d previously reported. 3 T a n n e d sheep e r y t h r o c y t e s w e r e sensitized w i t h t e t a n u s toxoid at p H 6"4 and 7"2 using 25, 50, 100 and 2 0 0 Lf/ml o f t e t a n u s toxoid for 30, 60 a n d 120 rain at 37°C a n d 56°C. T h e m e t h o d o f sensitization has b e e n described elsewhere. 3 Finally t h e sensitized cells were s u s p e n d e d in PBS p H 7-2 w i t h 1 in 10 0 0 0 t h i o m e r s a l and 0 - 5 % N R S in various c o n c e n t r a t i o n s b u t usually at a c o n c e n t r a t i o n o f 0 - 5 % w h i c h was found m o s t suitable.
Indirect haemagglutination test T h e I H A test was p e r f o r m e d as described earlier. 3 A f t e r o b t a i n i n g the o p t i m a l c o n d i t i o n s for s e n s i t i z i n g f o r m a l d e h y d e (I a n d II) fixed sheep e r y t h r o c y t e s , a large batch o f sensitized cells was p r e p a r e d u n d e r o p t i m a l c o n d i t i o n s using b o t h f o r m a l d e h y d e (I a n d II) fixed cells. T h e n all the 77 g u i n e a - p i g sera were t i t r a t e d by t h e I H A before and after t h e t r e a t m e n t o f the sera w i t h 2 - M E u s i n g b o t h types o f cell. T h e titrations o f t h e g u i n e a - p i g sera w e r e d o n e as described elsewhere. 4
Titration of sera by T N T h e T N test was p e r f o r m e d by t h e lethal e n d p o i n t m e t h o d using 1 7 - 2 0 g m i c e o f t h e L A C A strain o f e i t h e r sex. T h e sera w e r e t i t r a t e d at t h e L + / 1 0 0 , L + / 1 0 0 0 a n d L + / 5 0 0 0 doses o f t e t a n u s toxin. T h e toxin dose level used for t i t r a t i n g u n k n o w n sera a n d the r a n g e o f s e r u m d i l u t i o n s tested d e p e n d e d u p o n the t e t a n u s a n t i t o x i n c o n t e n t o f t h e sera as o b t a i n e d by t h e I H A m e t h o d . U s u a l l y an e n d p o i n t for a test s e r u m was o b t a i n e d by t e s t i n g in t h e r a n g e o f t w o to e i g h t t i m e s lower t h a n t h a t d e t e c t e d by I H A . RESULTS
Formaldehyde (!) fixed sheep erythrocytes Sensitization of sheep erythrocytes. Sheep cells w h e n t a n n e d w i t h 1/I 0 0 0 0 , 1/20 0 0 0 , 1/40 0 0 0 a n d 1/80 0 0 0 d i l u t i o n s o f t a n n i c acid s h o w e d differences o f not m o r e t h a n t w o fold in titres w i t h t h e N a t i o n a l R e f e r e n c e 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 (0- I I U / m l ) . T h e titres a n d scores w e r e h i g h e s t at t h e 1/40 0 0 0 c o n c e n t r a t i o n o f t a n n i c acid. 153
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TABLE 1.
T h e effect o f the c o n c e n t r a t i o n o f tetanus toxoid used for sensitization o f f o r m a l d e h y d e (I) fixed cells
C o n c e n t r a t i o n o f tetanus toxoid used for sensitization (Lf/ml)
T i t r e s and scores* with the N a t i o n a l Reference Standard for tetanus a n t i t o x i n (0" 1 I U / m l )
25 50 100 200
16 32 16 4
(18) (21) (17) (7)
* Scores are included in parentheses and represent the sum of the values of the agglutination reaction in each well graded from 0--4.
Table 1 shows the effect of the concentration of sensitizing tetanus toxoid on a n t i t o x i n t i t r e s . T h e o p t i m a l c o n c e n t r a t i o n o f t e t a n u s t o x o i d f o r s e n s i t i z a t i o n was 5 0 L f / m l . T h e m a x i m u m a n t i t o x i n t i t r e w a s o b t a i n e d w h e n t h e periocl o f e x p o s u r e o f t a n n e d e r y t h r o c y t e s to s e n s i t i z i n g t e t a n u s t o x o i d was 1 h. T h e r e was no increase in t h e t i t r e w h e n t h e p e r i o d w a s i n c r e a s e d t o 2 h. T h e p H o f e x p o s u r e o f t a n n e d e r y t h r o c y t e s to sensitizing tetanus toxoid did not have any marked effect on the titres but the score was m a x i m u m a t p H 7 - 2 . T h e cells s e n s i t i z e d a t 5 6 ° C g a v e t h e h i g h e s t t i t r e w i t h m a x i m u m s c o r e . T h e t i t r e w a s r e d u c e d t o o n e - q u a r t e r w h e n t h e cells w e r e s e n s i t i z e d a t 370(2. Importance of the concentration of sensitized erythrocytes. T a b l e 2 s h o w s t h e e f f e c t o f t h e c o n c e n t r a t i o n o f sheep e r y t h r o c y t e s o n titres a n d scores. As t h e c o n c e n t r a t i o n o f t h e cells d e c r e a s e d , t h e s e n s i t i v i t y i n c r e a s e d , b u t t h e H A e n d p o i n t w a s d i f f i c u l t t o r e a d a t t h e l o w e r c o n c e n t r a t i o n . A c o n c e n t r a t i o n o f 0 - 5 % o f cells was f o u n d s u i t a b l e as a compromise between sensitivity and readability. Sensitivity of l H A . A m i n i m u m l e v e l o f 0 - 0 0 3 0 I U / m l o f t e t a n u s a n t i t o x i n was d e t e c t e d u s i n g t h e s e cells s e n s i t i z e d u n d e r o p t i m a l c o n d i t i o n s . Stability of sensitized sheep ceils. T a b l e 3 s h o w s t h e loss o f s e n s i t i v i t y o f s e n s i t i z e d
TABLE 2.
T h e effect o f the c o n c e n t r a t i o n o f sensitized sheep e r y t h r o cytes using f o r m a l d e h y d e (I) fixed cells
Concentration of sensitized cells (%)
T i t r e s and scores* with t h e N a t i o n a l Reference Standard for tetanus antitoxin (0-1 I U / m l )
0- 1 0-25 0-5
2 5 6 (32) 64 (26) 32 (22)
1-0
16 ( 1 6 )
* Scores are indicated in parentheses and represent the sum of the values of the agglutination reaction in each well from O-4.
154
TITRATION TABLE 3.
OF TETANUS ANTITOXIN
V
Loss o f s e n s i t i v i t y o f f o r m a l d e h y d e (I) fixed cells w h e n s t o r e d a t 4 - 8 ° C for n i n e m o n t h s
Storage period at 4-8°C (months)
Titres and scores ~ w i t h the National Reference Standard for t e t a n u s a n t i t o x i n (0- 1 I U / m l )
0 3 6 9
32 ( 2 2 ) 8 (I3) 4(9) 4(8)
* Scores are indicated in parentheses and represent the sum of the values of the a g g l u t i n a t i o n reaction in each well from 0--4.
s h e e p cells w h e n s t o r e d a t 4~.8°C for n i n e m o n t h s . T h e r e was a f o u r f o l d to e i g h t f o l d d e c r e a s e in t h e t i t r e w h e n t h e cells w e r e s t o r e d a t 4 - 8 ° C for t h r e e to n i n e m o n t h s . Correlation between T N a n d I l i a titres. Figure I shows the correlation between titres o f i n d i v i d u a l g u i n e a - p i g sera o b t a i n e d b y T N a n d t h e I H A t e s t before a n d a f t e r t r e a t m e n t o f t h e sera w i t h 2 - M E . T h e I H A t i t r e s before 2 - M E t r e a t m e n t o f t h e sera w e r e a b o u t t w o to six t i m e s h i g h e r t h a n t h e T N t i t r e s . T h e c o r r e l a t i o n coefficient (r) b e t w e e n T N a n d I H A t i t r e s b e f o r e 2 - M E t r e a t m e n t was 0 - 9 7 w i t h t h e r e g r e s s i o n e q u a t i o n y -- 0" 1 8 6 8 x + 0 " 5 9 5 4 . N o s e r u m s a m p l e s h o w e d a l o w e r t i t r e w i t h I H A
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Fig. I. Scatter d i a g r a m for titres of individual g u i n e a - p i g sera obtained by toxin neutralization ( T N ) and indirect h a e m a g g l u t i n a t i o n ( I H A ) tests using formaldehyde (1) fixed sheep erythrocyres before and after the treatment o f the sera with 2-mercaptoethanol (2-ME). A, I H A Titres before 2-ME treatment of the sera; m I H A ritres after 2-ME t r e a t m e n t of the sera. The figures adjacent to some of t h e points indicate a n u m b e r of,sera with coincident titres.
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R. K. G U P T ~ ET AL. t h a n w i t h T N . T h e / H A titres after 2 - M E t r e a t m e n t o f the sera were closer co "the T N titres. S o m e o f the sera had s l i g h t l y lower titres by I H A t h a n by T N after 2 - M E t r e a t m e n t . T h e correlation coefficient b e t w e e n T N and I H A titres after 2 - M E t r e a t m e n t was 0"95 a n d t h e regression e q u a t i o n was y ----- 0 - 5 1 8 7 x -F 1"0633. Analysis of variance between T N and IHA titres. T h e analysis o f variance b e t w e e n titres o f g u i n e a - p i g sera o b t a i n e d by T N and I H A using f o r m a l d e h y d e (I) fixed cells before 2 - M E t r e a t m e n t o f t h e sera s h o w e d t h e value o f F h i g h l y significant a t 5 % level. A f t e r 2 - M E t r e a t m e n t o f t h e sera there was no statistically significant difference b e t w e e n T N a n d I H A titres.
Formaldehyde ( H ) fixed sheep erythrocytes Ali the results w i t h f o r m a l d e h y d e (II) fixed cells have been described in our previous reports 3"4 u n d e r the h e a d i n g o f f o r m a l d e h y d e fixed sheep e r y t h r o c y t e s .
DISCUSSION T h e reliability o f the I H A test d e p e n d s u p o n various factors involving the sensitization and fixation o f s h e e p e r y t h r o c y t e s w i t h different fixation agents. In our p r e v i o u s s t u d y , 3 it was r e p o r t e d t h a t t h e g l u t a r a l d e h y d e and f o r m a l d e h y d e fixed cells were t h e m o s t sensitive for t h e e s t i m a t i o n Of t e t a n u s a n t i t o x i n . In this report it was found t h a t the sensitivity o f f o r m a l d e h y d e (I) fixed cells was very low c o m p a r e d w i t h t h e sensitivity o f f o r m a l d e h y d e (II) fixed cells. O t h e r workers have also reported low sensitivity o f I H A test w h e n t h e cells w e r e fixed w i t h 3% f o r m a l d e h y d e , v'9 T h e o p t i m a l c o n d i t i o n s for sensitization o f f o r m a l d e h y d e (1) fixed ceils w e r e t h e s a m e as for g l u t a r a l d e h y d e , f o r m a l d e h y d e (II) and p y r u v i c a l d e h y d e fixed cells. 3 T h e c o n c e n t r a t i o n o f sensitized s h e e p e r y t h r o c y t e s h a d g r e a t effect on the titre (Table 2). These findings c o n f i r m the earlier reports. 3.1o. ~, T h e r e was a b o u t four t i m e s loss in the sensitivity o f f o r m a l d e h y d e (I) fixed cells w h e n these were stored at 4 - 8 ° C for three m o n t h s . T h o u g h these findings are similar to o u r earlier report, 3 the f o r m a l d e h y d e (I) fixed cells were n o t sufficiently sensitive after three m o n t h s to d e t e c t even the m i n i m u m protective level o f t e t a n u s a n t i t o x i n (0"01 I U / m l ) in the sera. These cells were used w i t h o u t a n y loss o f sensitivity u p to t w o m o n t h s having an a d v a n t a g e over unfixed cells w h i c h could be used for o n l y seven to ten days after sensitization. 3 T h e correlation b e t w e e n T N a n d I H A titres was b e t t e r w h e n f o r m a l d e h y d e (I) fixed cells were used t h a n w h e n f o r m a l d e h y d e (II) fixed cells were used. "*T h e correlation a n d regression coefficients b e t w e e n T N and I H A titres using f o r m a l d e h y d e (I) fixed cells were s i m i l a r to those o f g l u t a r a l d e h y d e fixed and unfixed cells. 4 T h e differences b e t w e e n T N a n d I H A titres before 2 - M E t r e a t m e n t were statistically significant. These results were s i m i l a r to those r e p o r t e d earlier. "~ T h e I H A titres w i t h f o r m a l d e h y d e (I) fixed Cells were h i g h e r t h a n t h e T N titres c o n f i r m i n g earlier findings. "!"1u'12-15 T h e r e was no statistically significant difference b e t w e e n T N and I H A titres after 2 - M E t r e a t m e n t o f the sera u s i n g f o r m a l d e h y d e (I) fixed cells. T h e s e findings differ from those o b t a i n e d w i t h f o r m a l d e h y d e (lI) fixed ceils and are similar to g l u t a r a l d e h y d e f i x e d and unfixed cells. 4 F r o m this s t u d y it is c o n c l u d e d t h a t not only the a g e n t for fixation o f s h e e p e r y t h r o c y t e s b u t also t h e fixation p r o c e d u r e affect t h e I H A test considerably. T h e f o r m a l d e h y d e (I) fixed cells a l t h o u g h less sensitive, can be used w i t h reliability for t h e t i t r a t i o n o f t e t a n u s a n t i t o x i n in t h e sera. 156
T I T R A T I O N OF T E T A N U S A N T I T O X I N V
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