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An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines
Btolog~cals (1991) 19, 281-286
An Alternative to the Toxin Neutralization Assay in Mice for the Potency Testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon Components of Multivalent Sheep Vaccines K. R. Wood
Hoechst Animal Health, Walton Manor, Walton, Milton Keynes, Bucks, MK7 7AJ, U.K.
Abstract. Potency testing of vetennary vaccines containing clostnd~al antigens currently requires the vaccination of laboratory rabbits followed by the determination of spec#ic antitoxin concentrahon ~n the rabbtt sera by toxin neutrahzatlon test in mice ELISAs are descnbed as an alternatwe method to toxin neutrahzation for the determination of Clostnd~um tetanl, Clostndlum septlcum, Clostndlum novyl Type B and Clostndlum perfnngens Type D epsllon anhtoxlns The assays were found to be rapid, spec#~c and economical and showed good correlation wtth the toxin neutrahzatJon test
Introduction
Materials and methods
The potency of the Clostrzdium tetani, Cl. novyi Type B, Cl. sept~cum and Cl. perfringens Type D epsilon components of sheep vaccines is currently tested by their ability to stimulate an antibody response in rabbits. This is measured ~n wvo using toxin neutralization tests in mice. 1~ Whilst these tests are known to be sensitive and reliable, they are slow (3-4 days) and can be somewhat imprecise as often only a ranged result is obtained. They are also relatively expensive and require the use of large numbers of mice, the effects on which may vary from paralysis to death due to non-neutrahzed toxin. Alternative assays would therefore be of great benefit and result in a substantial reduction in the numbers of mice used in the assessment of vaccine potency. Several alternative methods have previously been proposed including haemagglutination 5 and ELISA 6 for tetanus antitoxin tltratmn, in Vero cells for Cl. septicum antitoxin 7 and monoclonal antibody based competition ELISA for Cl. perfringens epsilon antitoxin, s By far the most work has been done on replacement by ELISA but this has been found by some workers to give poor correlation at low levels of detection, 9-H e.g in the t~tration of h u m a n sera. However, it was thought that ELISAs could be a satisfactory method in clostndlal potency tests in rabbits where the cut-off level is relatively high.
Punhcatlon of CI perfnngens epsllon prototoxln
1045-1056/91/040281+06 $03 00/0
Cl. perfringens Type D was grown using the normal HUK production method and the cells removed by filtration. The culture filtrate was mixed with an equal volume of saturated ammonium sulphate solution, pH 7.1, by gradual addition with stirring. The mixture was stirred for 1 h at room temperature and then overnight at 4°C before centrifugation at 4000 g for 20 min at 4°C. The precipitate was redissolved in and dlalysed against 10 1 of distilled water for 48 h at 4°C and then freeze-dried. This material was then dissolved in 0.005 M sodium phosphate buffer, pH 7.3, and applied to a 1 ml Mono Q fast protein liquid chromatography (FPLC) ion exchange column (Pharmacia). Elution was performed using a gradient of increasing sodium chloride concentration to a maximum of 1 M and the eluate was monitored for protein peaks by measuring absorbance at 280 nm. Fractions were treated with 0.04% (w/v) trypsin (Difco) for 30 min at 37°C and assayed for toxic activity by intravenous injection of 0.5 ml into mice which were monitored for deaths occurring within 24 h. A peak containing toxic fractions was identified and fractions from this peak were frozen immediately after elution in subsequent FPLC runs. They were later pooled, concentrated against polyethylene gly© 1991 The International Association of Biological Standardization
282
K . R . Wood
col 6000 (Ubichem) and either dialysed against distilled water for 72 h or desalted using a disposable desalting column (Bio-Rad) prior to freeze-drying. Presence of any alpha toxin contaminatmn was tested for by comparing the effects on Nagler egg-yolk agar (Dlfco) with those of known positive alpha toxin: alpha toxin produced egg-yolk turbidity within 24 h at 37°C. The purified prototoxin, crude ammonium sulphate precipitate and molecular weight markers (Sigma) were subjected to polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) and the purity and molecular weight of the purified prototoxin were assessed. Ouchterlony gel diffusion was performed on a 1 mg/ml solution of purified prototoxin which was applied to the central well of a 1~ agarose gel with various clostndial antitoxin solutions in the surrounding wells. ELISA methods CI tetanl antitoxin ELISA. M129B micro-ELiSA plates (Dynatech, Billingshurst, U.K.) were coated with 5.9 pg/ml (as protein) toxin (Calbiochem, Nottingham, U.K.) in 0.01 M phosphate-buffered saline (PBS), pH 7.2 and incubated at 37°C overnight. Some 0.25 ml 0.1% (w/v) bovine serum albumin in PBS (BSA, Sigma Chemical Company, Poole, U.K.) was then added and the plates were incubated at 37°C for 1 h prior to additmn of standard and test rabbit sera at appropriate dilutions in PBS containing 0.05% Tween 20 (PBST): standard serum was a house-standard rabbit serum containing 50 IU/ml tetanus antitoxin (by toxin neutralization assay) which was diluted within the range 1/10 000 and 1/320 000; test rabbit sera (pools from ten rabbits) were routinely diluted to 1/5000. The plates were again incubated at 37°C for 1 h before the addition of a 1/2000 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate (Sigma, Poole, U.K.) m PBST and further incubation at 37°C for 1 h. Tetramethyl benzldme (0.042 mM) (ICN Biomedicals, High Wycombe, U.K.) in 0.1 M sodium acetate-otric acid buffer, pH 6.0, containing 8-8 mM hydrogen peroxide was then added as the substrate and the colour allowed to develop for 15 min at room temperature, the reaction then being terminated by the addition of 50 pl 1 M sulphuric acid. All volumes were 200/zl per well unless stated and the plates were washed three times with 0 35 ml PBST prmr to the addition of each new reagent. All standard and test samples were added to the plate in duplicate and the absorbance was measured at 450 nm using a Titertek Multiscan
s p e c t r o p h o t o m e t e r (Flow L a b o r a t o r i e s , High Wycombe, U.K.) relative to a blank row of wells which had all reagents added except the test or standard solutions, these being replaced by PBST diluent at the appropriate stage. The tetanus antitoxin concentration of the test samples was determined by comparison of absorbance with those of the standard curve which was originally constructed manually but later calculated by means of computer-generated standard curve using a four-parameter logistic curvefit (Titersoft, Flow Laboratories, High Wycombe, U.K.). CI novyl antitoxin ELISA. The methodology was essentially the same as for Cl. tetant with the following differences: the coating antigen was a 1 /zg/ml solution ofCl. novyz Type B toxin (Wellcome Research Laboratories) which was adsorbed overmght at 4°C; the standard serum was a house-standard rabbit serum containing 20 IU/ml Cl. novyt antitoxin (assayed by toxin neutralization) diluted to between 1/2000 and 1/32 000. Test rabbit sera were routinely diluted to 1/2000. The conjugate was as used in the Cl. tetant ELISA but diluted to 1/1000. Standard and test sera were routinely apphed to the plate as four replicates per solution. CI septlcum anhtoxln ELISA. The methodology was the same as for Cl. tzovyt except that the plates were coated with a 2 pg/m] soluUon of Cl. septicum toxin (Wellcome Research Laboratories) in 0.01 M sodium carbonate-biocarbonate buffer (pH 9.6) overmght at 4°C. The standard serum was a house standard rabbit serum containing 9 IU/m] Cl. septicum antitoxin (assayed by toxin neutralization) diluted to between 1/2000 and 1/32 000. CI perfnngens epsllon anhtoxln ELISA. The methodology was similar to t h a t of the other assays for volumes and incubation times and temperatures but differed m respect of the following: Linbro microELISA plates (Flow Laboratories) were coated with a 2 pg/ml solution of purified CI perfringens epsilon prototoxin in 0.01 M PBS (pH 7.2) overnight at 4°C. No BSA blocking stage was used. The standard serum was a house-standard rabb,t serum containing 20 IU/ml Cl. perfringens epsllon antitoxin (assayed by toxin neutralization) diluted to between 1/200 and 1/6400 whilst test rabbit sera were routinely diluted to 1/500. Both were diluted in duplicate and each of these was applied to the plate in duplicate, i.e. four wells per solution.
An alternative to the mouse toxin neutralization assay
Preparation of house-standard rabblt sera CI tetan,. A r a b b i t was inoculated s u b c u t a n e o u s l y with two 1 ml doses of c o n c e n t r a t e d t e t a n u s toxoid ( B e h r i n g w e r k e , M a r b u r g , G e r m a n y - - 1 5 0 IU/ml adsorbed on to a l u m i n i u m hydroxide) with an interval of 28 days. After a f u r t h e r seven days c o n c e n t r a t e d toxoid (Hoechst U.K. Ltd, Milton Keynes, U.K.) from a p r o d u c t i o n b a t c h was diluted to 1/10 in P B S and was injected i n t r a v e n o u s l y with doses i n c r e a s i n g from 0.5 to 2.0 ml in 0.5 ml i n c r e m e n t s at 3 day intervals. T h e r a b b i t was t h e n bled and the s e r u m r e m o v e d for s t a n d a r d i z a t i o n b:y toxin n e u t r a l i z a t i o n a s s a y and t h e n stored in small aliquots at - 2 0 ° C .
283
the ELISAs by t e s t i n g rabbit sera k n o w n to be negative for the antitoxin u n d e r test b u t pomt~ve for all the o t h e r c o m p o n e n t s p r e s e n t in H o e c h s t U. K. multiv a l e n t vaccines.
Correlation studies S e r a from rabbits vaccinated with various Hoechst U.K. vaccines (see Table 1) were assayed for Cl. tetam, Cl. novyi, Cl. sept~cum and Cl. perfringens epsilon a n t i t o x i n by both E L I S A and toxin neutralization. The n u m b e r of batches t e s t e d was 50, 50, 80 and 55, respectively. E a c h s e r u m sample was pooled from the i n d i w d u a l sera of 10 vaccinated rabbits.
CI now,. A m o n o v a l e n t vaccine was p r e p a r e d by a d s o r b i n g Cl. novyi toxoid (Hoechst U.K. Ltd, Milton Keynes, U.K.) from a production b a t c h on a l u m i n i u m hydroxide. A r a b b i t was injected i n t r a v e n o u s l y with 4 ml of the vaccine followed by a second 4 ml dose 4 weeks later. T h e r a b b i t was bled a f t e r a f u r t h e r 2 weeks and the s e r u m was r e m o v e d for s t a n d a r d i z a tion by toxin n e u t r a l i z a t i o n a s s a y before storage in small a h q u o t s at - 2 0 ° C .
Results
CI perfnngens epsllon prototoxln punhcat~on T h e e l u a t e from the F P L C column was m o n i t o r e d for absorbance at 280 n m and prototoxin was found to be p r e s e n t in the first peak. When subjected to S D S - P A G E the purified sample produced only one band. It was e s t i m a t e d by comparison with known _ m a r k e r s (Fig. 1) t h a t the molecular weight was 37 500 D - a figure which c o m p a r e s well with those previously obtained, z2-~3Only one precipitin line was visible on O u c h t e r l o n y gel diffusion (Fig. 2), between the purified prototoxin and National S t a n d a r d Cl. perfringens Type D antitoxin. No reaction was seen with a n y of the o t h e r clostndial antitoxins tested.
CI sept,cum and CI perfnngens epsllon. Standard s e r u m was p r e p a r e d in the same m a n n e r as for Cl. novyi s t a n d a r d s e r u m , except t h a t 2 ml doses were injected in the case ofCl. perfrmgens epsilon vaccine.
Assay variation In each of the a s s a y s a r a b b i t s e r u m pool was t e s t e d n i n e or 10 times in the same a s s a y and b e t w e e n 10 and 15 t i m e s m s e p a r a t e a s s a y s in o r d e r to establish the i n t r a - and i n t e r - a s s a y variation, respectively.
House-standard rabbit sera T h e s e were s t a n d a r d i z e d by toxin n e u t r a h z a t i o n as follows: Cl. tetant, 50 IU/ml; Cl. novyi, 20 IU/ml; Cl. septtcum 9 IU/ml; Cl. perfrtngens epsilon, 20 IU/ml.
Cross-reactlwty studies C r o s s - r e a c t i w t y studies were p e r f o r m e d for each of
T a b l e 1. S u m m a r y of the composition of H o e c h s t U.K. vaccines used in the s t u d y Antigenic c o m p o n e n t s Vaccine
Clostridmm Cl. Cl. tetam n o v y ~ sept,cum
Heptavac P Heptavac Ovivac P Ovivac Lambivac Pulpy kidney and t e t a n u s Braxy blackleg
+ + + + + +
+ + -
+ + + + -
-
-
+
Cl. perfringens B C D
Cl. chauvoei
Pasteurella haemolyttca
+ +
+ +
+ +
+ +
+
_
_
+
+
+
_
_
+
+
--
+
+
+
--
_
+
-
284
K.R. Wood
n
1
3
2
Figure 1. SDS-PAGE profile showing molecular weight markers (Lane 1), crude ammonium sulphate preclpate (Lane 2) and the purffied sample from the first peak of FPLC eluate (Lane 3) The gel was stained with 0.25% Coomassie Bnlhant Blue R250.
Assay vanatlon
Correlation studies
The variation within and between ELISAs is summarized in Table 2.
In the toxin neutralization assay in mice the result is often presented as a range (e.g. 4-6 IU/ml antitoxin). This is because the assay is set up using defined dilutions of test sera which, when reacted with a known concentration of standard toxin, will, after injection, either protect a pair of mice or not. In the above example, if the mice survive at the 6 IU/ml dilution but die at the 4 IU/ml dilution then the serum is said to contain 4-6 IU/ml. If, however, both mice survive at the 6 IU/ml dilution and only one of a pair
Cross-react~wty studies When rabbit sera known to be negatwe for the antitoxin under test were assayed in the ELISAs, little or no colour development occurred, any absorbance always being well below those of the highest dilution of the standard curve (data not shown) showing good specfficity for each of the assays.
Table 2. Inter- and intra-assay variation in the ELISAs
Replicates
Mean concentration (IU/ml)
Coefficient of SD (IU/ml) variation (%)
Cl. tetani
Intra-assay Inter-assay
10 10
6.1 7.7
0.39 0.61
6.4 7.9
Cl. novyi
Intra-assay Inter-assay
10 10
6-6 6-6
0.34 0.38
5.2 5.7
Cl. septicum
Intra-assay Inter-assay
10 15
3.8 3.7
0.15 0.33
4.0 8.9
Cl. perfringens epsilon
Intra-assay Inter-assay
9 10
5.8 5-5
0.36 0.28
6.22 5.14
An alternative to the mouse toxin neutralization assay
285
T a b l e 3. S u m m a r y of correlation coefficients for results by ELISA and toxin neutralization
Cl. Cl. Cl. Cl. 5
"
~
4
Figure 2. Ouchterlony gel diffusion. Central well; I mg/ml purified prototoxin; peripheral wells: 1-National Standard Cl. perfnngens Type D antitoxin, 100 IU/ml; 2-National Standard Cl. perfr~ngens Type A antitoxin, 200 IU/ml; 3-Cl perfringens beta antitoxin (rabbit serum), 200 IU/ml; 4National Standard Cl. sept~cum antitoxin, 100 IU/ml; 5National Standard Cl. novyi Type B antitoxin, 110 IU/ml; 6-Cl. tetan~ antitoxin (Behring), 100 IU/ml. The gel was stained with 0.25% Coomassie Brilliant Blue R250.
20
E
r2
0.94 0.93 0-84 0.93
0-88 0-86 0.71 0-87
survive at the 4 IU/ml dilution the serum is t h e n said to contain 4 IU/ml antitoxin. As the data for m a n y of the batches of sera by toxin neutralization test was in this form, in order to make statistical comparisons the m e a n of any ranged results was used. Correlation data are s h o w n in Table 3. Correlation was significant for all the assays at -<0.001. Regression plots between results produced by toxin neutralization and ELISA are s h o w n in Fig. 3 along with the equations of regression. In each case, the gradient and intercept were not significantly different from 1.0 and zero, respectively, after analysis by Student's t-test (p = 0-05).
A _J E
a)
septicum tetani novyi perfringens eps~lon
r
16 14
(b)
12 I0
- . . - .
o
8
o o/ 5 E x
°.~ 7e 0
./
Oleo
•o
_c F-
I
I
5
I0 ELISA
j 15
20
2
I 4
J__ 6 ELISA
(IU/rnL)
I 8 (IU/mL)
I I0
I 12
14
30 20
(c)
A
-~
(d) 25
E 15
20
8
8 o I0
ado
o
o
II
J
c_
0
J 5
I I0
ELISA ( l U / m L )
I 15
20
0
5
I I0
I 15
I 20
I 25
30
ELLS& ( l U / m t )
Figure 3. Regression plots showing the relationship between results obtained by ELISA and by toxin neutralization. (a) Cl. tetanL antitoxin y = 1.07x + 0.1. (b) Cl. novy~ antitoxin, y = 1.10x - 0.05. (c) Cl. septicum antitoxin, y = 0.95x + 0.38. (d) Cl. perfnngens epsilon antitoxin, y = 1.00x + 0.70.
286
K.R. Wood
Discussion
References
C o r r e l a t i o n b e t w e e n r e s u l t s o b t a i n e d by E L I S A a n d T N w a s g e n e r a l l y good for all four a s s a y s . T h e criterion for the clostridial c o m p o n e n t s p a s s i n g p o t e n c y is t h a t a c e r t a i n c o n c e n t r a t i o n of specific a n t i b o d y is achieved in the r a b b i t s e r a (Cl. tetani 2.5 IU/ml; Cl. septzcum 2.5 IU/ml; Cl. novyi 3.5 IU/ml; Cl. perfringens epsilon 5.0 IU/ml. 1-4 F o r all t h e a s s a y s , b o t h E L I S A a n d T N p r o d u c e d r e s u l t s which would h a v e p a s s e d a n d failed the s a m e vaccine b a t c h e s . C r i t i c i s m s of E L I S A s as r e p l a c e m e n t s for t h e T N a s s a y h a v e m a i n l y s t e m m e d f r o m w o r k done on t h e t i t r a t i o n of h u m a n sera, p a r t i c u l a r l y for Cl. tetani a n t i t o x i n w h e r e poor correlation a t low s e r u m titres h a s b e e n a problem. 9-n On the o t h e r h a n d , good corr e l a t i o n h a s also b e e n r e p o r t e d with t e t a n u s a n t i t o x i n in h u m a n s e r a by E L I S A a n d T N over a wide r a n g e of titres.12 It is also recognized by the a u t h o r t h a t unlike t h e T N assay, the E L I S A s do not n e c e s s a r i l y m e a s u r e only protective antitoxin, but the correlation o b s e r v e d s u g g e s t s t h a t s i m i l a r entities are b e i n g measured: it m a y h a v e b e e n e x p e c t e d t h a t t h e E L I S A , if m e a s u r i n g b o t h protective a n d non-protective antibodies, would h a v e p r o d u c e d c o n s i s t e n t l y h i g h e r r e s u l t s t h a n t h a t of the T N assay, b u t this w a s not the case. T h e benefits of r e p l a c e m e n t of t h e s e T N a s s a y s are significant a n d include a reduction in the use of subs t a n t i a l n u m b e r s of mice which is of both ethical a n d economic i m p o r t a n c e a n d a reduction in the t i m e t a k e n to a c h m v e a potency r e s u l t f r o m 3 - 4 d a y s to a p p r o x i m a t e l y 3 h All four a s s a y s h a v e now b e e n accepted as b a t c h r e l e a s e potency t e s t s for v e t e r i n a r y vaccines by the U.K. R e g u l a t o r y a u t h o r i t i e s a n d h a v e b e e n successfully i n t r o d u c e d into the H o e c h s t A n i m a l H e a l t h Q u a l i t y A s s u r a n c e L a b o r a t o r i e s as a l t e r n a tives to t h e toxin n e u t r a l i z a t i o n assay.
1. Biological assay of Clostrtdtum tetant antitoxin. British Pharmacopoeia Veterinary, London: HMSO, 1985, A l l 6 2. Biological assay of Clostrtdtum novyt alpha antitoxin. British Pharmacopoeia Vetennary, London: HMSO, 1985, All3. 3. Biological assay of Clostrtdtum septicum antitoxin. British Pharmacopoeia Vetennary, London: HMSO, 1985, All5. 4 Biological assay of Clostrtdzumperfrtngens antitoxins. British Pharmacopoeia Veterinary, London: HMSO, 1985, All4. 5. Gupta RK, Maheshwari SC, Singh H. The titration of tetanus antitoxin V. Effect of formahsation method for the fixation of sheep erythrocytes on the indirect haemaglutmatlon test. J Biol Stand 1985; 13:151-157. 6. German-Fattal M, Bizzml B, German A. Immunity to tetanus: tetanus antitoxin and anti-BIIb m human sera J Blol Stand 1987, 15: 223-230. 7. Knight PA, Tflleray JH, Quemlnet J. In vttro tests for the measurement of veterinary clostrldlal toxins, toxoids and antisera. 1. titration ofClostrtdtum septtcum toxins and antitoxins m cell culture. Biologicals 1990; 18: 181-189. 8 Sojka MG, White VJ, Thorns CJ, Roeder PL. The detection of Clostrldtum perfrtngens epsilon antitoxin in rabbit serum by monoclonal antibody based competition ELISA J Blol Stand 1989; 17. 117-124 9. Hendmksen CFM, v d Gun JW, Nagel J, Kreeftenberg JG. The toxin binding inhibition test as a reliable tn wtro alternative to the toxin neutralisation test m mice for the estlmatmn of tetanus antitoxin in human sera. J Biol Stand 1988; 16 287-297. 10 Gentlh G, Plni C, Collotti C. The determination of the potency of human tetanus lmmunoglobuhns by an enzyme-linked immunosorbent assay. J Blol Stand 1984; 12: 167-173. 11. Hagenaars AM, van Delft RW, Nagel J. Comparison of ELISA and toxin neutrahsatmn for the determination of tetanus antlbodms. J Immunoassay 1984; 5 1-11. 12 Habeeb AFSA. Studies on epsflon prototoxin of Clostridtum perfrtngens Type D. Physmcochemical and chemical propertms of epsilon prototoxin. Biochim Biophys Acta 1975; 412.62-69. 13 Worthington RW, Mulders MSG. Physical changes in the epsflon prototoxm molecule of Clostrtdtum perfrtngens during enzymatic actwatlon. Infect Immun 1977, 18:549-551 14. Melwlle-Sm~th ME, Seagroatt VA, Watkins JT. Comparison of enzyme-hnked lmmunosorbent assay (ELISA) with the toxin neutralisation test m mine as a method for the estimation of tetanus antitoxin m human sera. J Biol Stand 1983; 11: 137-144.
Acknowledgement T h e a u t h o r would like to e x p r e s s t h a n k s to M r N o r m a n B a k e r for e n t h u s i a s t i c s u p p o r t a n d encoura g e m e n t at inception a n d t h r o u g h o u t the project.
Received for pubhcation 19 January 1991; accepted 31 May 1991.
An Alternative to the Toxin Neutralization Assay in Mice for the Potency Testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon Components of Multivalent Sheep Vaccines K. R. Wood
Hoechst Animal Health, Walton Manor, Walton, Milton Keynes, Bucks, MK7 7AJ, U.K.
Abstract. Potency testing of vetennary vaccines containing clostnd~al antigens currently requires the vaccination of laboratory rabbits followed by the determination of spec#ic antitoxin concentrahon ~n the rabbtt sera by toxin neutrahzatlon test in mice ELISAs are descnbed as an alternatwe method to toxin neutrahzation for the determination of Clostnd~um tetanl, Clostndlum septlcum, Clostndlum novyl Type B and Clostndlum perfnngens Type D epsllon anhtoxlns The assays were found to be rapid, spec#~c and economical and showed good correlation wtth the toxin neutrahzatJon test
Introduction
Materials and methods
The potency of the Clostrzdium tetani, Cl. novyi Type B, Cl. sept~cum and Cl. perfringens Type D epsilon components of sheep vaccines is currently tested by their ability to stimulate an antibody response in rabbits. This is measured ~n wvo using toxin neutralization tests in mice. 1~ Whilst these tests are known to be sensitive and reliable, they are slow (3-4 days) and can be somewhat imprecise as often only a ranged result is obtained. They are also relatively expensive and require the use of large numbers of mice, the effects on which may vary from paralysis to death due to non-neutrahzed toxin. Alternative assays would therefore be of great benefit and result in a substantial reduction in the numbers of mice used in the assessment of vaccine potency. Several alternative methods have previously been proposed including haemagglutination 5 and ELISA 6 for tetanus antitoxin tltratmn, in Vero cells for Cl. septicum antitoxin 7 and monoclonal antibody based competition ELISA for Cl. perfringens epsilon antitoxin, s By far the most work has been done on replacement by ELISA but this has been found by some workers to give poor correlation at low levels of detection, 9-H e.g in the t~tration of h u m a n sera. However, it was thought that ELISAs could be a satisfactory method in clostndlal potency tests in rabbits where the cut-off level is relatively high.
Punhcatlon of CI perfnngens epsllon prototoxln
1045-1056/91/040281+06 $03 00/0
Cl. perfringens Type D was grown using the normal HUK production method and the cells removed by filtration. The culture filtrate was mixed with an equal volume of saturated ammonium sulphate solution, pH 7.1, by gradual addition with stirring. The mixture was stirred for 1 h at room temperature and then overnight at 4°C before centrifugation at 4000 g for 20 min at 4°C. The precipitate was redissolved in and dlalysed against 10 1 of distilled water for 48 h at 4°C and then freeze-dried. This material was then dissolved in 0.005 M sodium phosphate buffer, pH 7.3, and applied to a 1 ml Mono Q fast protein liquid chromatography (FPLC) ion exchange column (Pharmacia). Elution was performed using a gradient of increasing sodium chloride concentration to a maximum of 1 M and the eluate was monitored for protein peaks by measuring absorbance at 280 nm. Fractions were treated with 0.04% (w/v) trypsin (Difco) for 30 min at 37°C and assayed for toxic activity by intravenous injection of 0.5 ml into mice which were monitored for deaths occurring within 24 h. A peak containing toxic fractions was identified and fractions from this peak were frozen immediately after elution in subsequent FPLC runs. They were later pooled, concentrated against polyethylene gly© 1991 The International Association of Biological Standardization
282
K . R . Wood
col 6000 (Ubichem) and either dialysed against distilled water for 72 h or desalted using a disposable desalting column (Bio-Rad) prior to freeze-drying. Presence of any alpha toxin contaminatmn was tested for by comparing the effects on Nagler egg-yolk agar (Dlfco) with those of known positive alpha toxin: alpha toxin produced egg-yolk turbidity within 24 h at 37°C. The purified prototoxin, crude ammonium sulphate precipitate and molecular weight markers (Sigma) were subjected to polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) and the purity and molecular weight of the purified prototoxin were assessed. Ouchterlony gel diffusion was performed on a 1 mg/ml solution of purified prototoxin which was applied to the central well of a 1~ agarose gel with various clostndial antitoxin solutions in the surrounding wells. ELISA methods CI tetanl antitoxin ELISA. M129B micro-ELiSA plates (Dynatech, Billingshurst, U.K.) were coated with 5.9 pg/ml (as protein) toxin (Calbiochem, Nottingham, U.K.) in 0.01 M phosphate-buffered saline (PBS), pH 7.2 and incubated at 37°C overnight. Some 0.25 ml 0.1% (w/v) bovine serum albumin in PBS (BSA, Sigma Chemical Company, Poole, U.K.) was then added and the plates were incubated at 37°C for 1 h prior to additmn of standard and test rabbit sera at appropriate dilutions in PBS containing 0.05% Tween 20 (PBST): standard serum was a house-standard rabbit serum containing 50 IU/ml tetanus antitoxin (by toxin neutralization assay) which was diluted within the range 1/10 000 and 1/320 000; test rabbit sera (pools from ten rabbits) were routinely diluted to 1/5000. The plates were again incubated at 37°C for 1 h before the addition of a 1/2000 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate (Sigma, Poole, U.K.) m PBST and further incubation at 37°C for 1 h. Tetramethyl benzldme (0.042 mM) (ICN Biomedicals, High Wycombe, U.K.) in 0.1 M sodium acetate-otric acid buffer, pH 6.0, containing 8-8 mM hydrogen peroxide was then added as the substrate and the colour allowed to develop for 15 min at room temperature, the reaction then being terminated by the addition of 50 pl 1 M sulphuric acid. All volumes were 200/zl per well unless stated and the plates were washed three times with 0 35 ml PBST prmr to the addition of each new reagent. All standard and test samples were added to the plate in duplicate and the absorbance was measured at 450 nm using a Titertek Multiscan
s p e c t r o p h o t o m e t e r (Flow L a b o r a t o r i e s , High Wycombe, U.K.) relative to a blank row of wells which had all reagents added except the test or standard solutions, these being replaced by PBST diluent at the appropriate stage. The tetanus antitoxin concentration of the test samples was determined by comparison of absorbance with those of the standard curve which was originally constructed manually but later calculated by means of computer-generated standard curve using a four-parameter logistic curvefit (Titersoft, Flow Laboratories, High Wycombe, U.K.). CI novyl antitoxin ELISA. The methodology was essentially the same as for Cl. tetant with the following differences: the coating antigen was a 1 /zg/ml solution ofCl. novyz Type B toxin (Wellcome Research Laboratories) which was adsorbed overmght at 4°C; the standard serum was a house-standard rabbit serum containing 20 IU/ml Cl. novyt antitoxin (assayed by toxin neutralization) diluted to between 1/2000 and 1/32 000. Test rabbit sera were routinely diluted to 1/2000. The conjugate was as used in the Cl. tetant ELISA but diluted to 1/1000. Standard and test sera were routinely apphed to the plate as four replicates per solution. CI septlcum anhtoxln ELISA. The methodology was the same as for Cl. tzovyt except that the plates were coated with a 2 pg/m] soluUon of Cl. septicum toxin (Wellcome Research Laboratories) in 0.01 M sodium carbonate-biocarbonate buffer (pH 9.6) overmght at 4°C. The standard serum was a house standard rabbit serum containing 9 IU/m] Cl. septicum antitoxin (assayed by toxin neutralization) diluted to between 1/2000 and 1/32 000. CI perfnngens epsllon anhtoxln ELISA. The methodology was similar to t h a t of the other assays for volumes and incubation times and temperatures but differed m respect of the following: Linbro microELISA plates (Flow Laboratories) were coated with a 2 pg/ml solution of purified CI perfringens epsilon prototoxin in 0.01 M PBS (pH 7.2) overnight at 4°C. No BSA blocking stage was used. The standard serum was a house-standard rabb,t serum containing 20 IU/ml Cl. perfringens epsllon antitoxin (assayed by toxin neutralization) diluted to between 1/200 and 1/6400 whilst test rabbit sera were routinely diluted to 1/500. Both were diluted in duplicate and each of these was applied to the plate in duplicate, i.e. four wells per solution.
An alternative to the mouse toxin neutralization assay
Preparation of house-standard rabblt sera CI tetan,. A r a b b i t was inoculated s u b c u t a n e o u s l y with two 1 ml doses of c o n c e n t r a t e d t e t a n u s toxoid ( B e h r i n g w e r k e , M a r b u r g , G e r m a n y - - 1 5 0 IU/ml adsorbed on to a l u m i n i u m hydroxide) with an interval of 28 days. After a f u r t h e r seven days c o n c e n t r a t e d toxoid (Hoechst U.K. Ltd, Milton Keynes, U.K.) from a p r o d u c t i o n b a t c h was diluted to 1/10 in P B S and was injected i n t r a v e n o u s l y with doses i n c r e a s i n g from 0.5 to 2.0 ml in 0.5 ml i n c r e m e n t s at 3 day intervals. T h e r a b b i t was t h e n bled and the s e r u m r e m o v e d for s t a n d a r d i z a t i o n b:y toxin n e u t r a l i z a t i o n a s s a y and t h e n stored in small aliquots at - 2 0 ° C .
283
the ELISAs by t e s t i n g rabbit sera k n o w n to be negative for the antitoxin u n d e r test b u t pomt~ve for all the o t h e r c o m p o n e n t s p r e s e n t in H o e c h s t U. K. multiv a l e n t vaccines.
Correlation studies S e r a from rabbits vaccinated with various Hoechst U.K. vaccines (see Table 1) were assayed for Cl. tetam, Cl. novyi, Cl. sept~cum and Cl. perfringens epsilon a n t i t o x i n by both E L I S A and toxin neutralization. The n u m b e r of batches t e s t e d was 50, 50, 80 and 55, respectively. E a c h s e r u m sample was pooled from the i n d i w d u a l sera of 10 vaccinated rabbits.
CI now,. A m o n o v a l e n t vaccine was p r e p a r e d by a d s o r b i n g Cl. novyi toxoid (Hoechst U.K. Ltd, Milton Keynes, U.K.) from a production b a t c h on a l u m i n i u m hydroxide. A r a b b i t was injected i n t r a v e n o u s l y with 4 ml of the vaccine followed by a second 4 ml dose 4 weeks later. T h e r a b b i t was bled a f t e r a f u r t h e r 2 weeks and the s e r u m was r e m o v e d for s t a n d a r d i z a tion by toxin n e u t r a l i z a t i o n a s s a y before storage in small a h q u o t s at - 2 0 ° C .
Results
CI perfnngens epsllon prototoxln punhcat~on T h e e l u a t e from the F P L C column was m o n i t o r e d for absorbance at 280 n m and prototoxin was found to be p r e s e n t in the first peak. When subjected to S D S - P A G E the purified sample produced only one band. It was e s t i m a t e d by comparison with known _ m a r k e r s (Fig. 1) t h a t the molecular weight was 37 500 D - a figure which c o m p a r e s well with those previously obtained, z2-~3Only one precipitin line was visible on O u c h t e r l o n y gel diffusion (Fig. 2), between the purified prototoxin and National S t a n d a r d Cl. perfringens Type D antitoxin. No reaction was seen with a n y of the o t h e r clostndial antitoxins tested.
CI sept,cum and CI perfnngens epsllon. Standard s e r u m was p r e p a r e d in the same m a n n e r as for Cl. novyi s t a n d a r d s e r u m , except t h a t 2 ml doses were injected in the case ofCl. perfrmgens epsilon vaccine.
Assay variation In each of the a s s a y s a r a b b i t s e r u m pool was t e s t e d n i n e or 10 times in the same a s s a y and b e t w e e n 10 and 15 t i m e s m s e p a r a t e a s s a y s in o r d e r to establish the i n t r a - and i n t e r - a s s a y variation, respectively.
House-standard rabbit sera T h e s e were s t a n d a r d i z e d by toxin n e u t r a h z a t i o n as follows: Cl. tetant, 50 IU/ml; Cl. novyi, 20 IU/ml; Cl. septtcum 9 IU/ml; Cl. perfrtngens epsilon, 20 IU/ml.
Cross-reactlwty studies C r o s s - r e a c t i w t y studies were p e r f o r m e d for each of
T a b l e 1. S u m m a r y of the composition of H o e c h s t U.K. vaccines used in the s t u d y Antigenic c o m p o n e n t s Vaccine
Clostridmm Cl. Cl. tetam n o v y ~ sept,cum
Heptavac P Heptavac Ovivac P Ovivac Lambivac Pulpy kidney and t e t a n u s Braxy blackleg
+ + + + + +
+ + -
+ + + + -
-
-
+
Cl. perfringens B C D
Cl. chauvoei
Pasteurella haemolyttca
+ +
+ +
+ +
+ +
+
_
_
+
+
+
_
_
+
+
--
+
+
+
--
_
+
-
284
K.R. Wood
n
1
3
2
Figure 1. SDS-PAGE profile showing molecular weight markers (Lane 1), crude ammonium sulphate preclpate (Lane 2) and the purffied sample from the first peak of FPLC eluate (Lane 3) The gel was stained with 0.25% Coomassie Bnlhant Blue R250.
Assay vanatlon
Correlation studies
The variation within and between ELISAs is summarized in Table 2.
In the toxin neutralization assay in mice the result is often presented as a range (e.g. 4-6 IU/ml antitoxin). This is because the assay is set up using defined dilutions of test sera which, when reacted with a known concentration of standard toxin, will, after injection, either protect a pair of mice or not. In the above example, if the mice survive at the 6 IU/ml dilution but die at the 4 IU/ml dilution then the serum is said to contain 4-6 IU/ml. If, however, both mice survive at the 6 IU/ml dilution and only one of a pair
Cross-react~wty studies When rabbit sera known to be negatwe for the antitoxin under test were assayed in the ELISAs, little or no colour development occurred, any absorbance always being well below those of the highest dilution of the standard curve (data not shown) showing good specfficity for each of the assays.
Table 2. Inter- and intra-assay variation in the ELISAs
Replicates
Mean concentration (IU/ml)
Coefficient of SD (IU/ml) variation (%)
Cl. tetani
Intra-assay Inter-assay
10 10
6.1 7.7
0.39 0.61
6.4 7.9
Cl. novyi
Intra-assay Inter-assay
10 10
6-6 6-6
0.34 0.38
5.2 5.7
Cl. septicum
Intra-assay Inter-assay
10 15
3.8 3.7
0.15 0.33
4.0 8.9
Cl. perfringens epsilon
Intra-assay Inter-assay
9 10
5.8 5-5
0.36 0.28
6.22 5.14
An alternative to the mouse toxin neutralization assay
285
T a b l e 3. S u m m a r y of correlation coefficients for results by ELISA and toxin neutralization
Cl. Cl. Cl. Cl. 5
"
~
4
Figure 2. Ouchterlony gel diffusion. Central well; I mg/ml purified prototoxin; peripheral wells: 1-National Standard Cl. perfnngens Type D antitoxin, 100 IU/ml; 2-National Standard Cl. perfr~ngens Type A antitoxin, 200 IU/ml; 3-Cl perfringens beta antitoxin (rabbit serum), 200 IU/ml; 4National Standard Cl. sept~cum antitoxin, 100 IU/ml; 5National Standard Cl. novyi Type B antitoxin, 110 IU/ml; 6-Cl. tetan~ antitoxin (Behring), 100 IU/ml. The gel was stained with 0.25% Coomassie Brilliant Blue R250.
20
E
r2
0.94 0.93 0-84 0.93
0-88 0-86 0.71 0-87
survive at the 4 IU/ml dilution the serum is t h e n said to contain 4 IU/ml antitoxin. As the data for m a n y of the batches of sera by toxin neutralization test was in this form, in order to make statistical comparisons the m e a n of any ranged results was used. Correlation data are s h o w n in Table 3. Correlation was significant for all the assays at -<0.001. Regression plots between results produced by toxin neutralization and ELISA are s h o w n in Fig. 3 along with the equations of regression. In each case, the gradient and intercept were not significantly different from 1.0 and zero, respectively, after analysis by Student's t-test (p = 0-05).
A _J E
a)
septicum tetani novyi perfringens eps~lon
r
16 14
(b)
12 I0
- . . - .
o
8
o o/ 5 E x
°.~ 7e 0
./
Oleo
•o
_c F-
I
I
5
I0 ELISA
j 15
20
2
I 4
J__ 6 ELISA
(IU/rnL)
I 8 (IU/mL)
I I0
I 12
14
30 20
(c)
A
-~
(d) 25
E 15
20
8
8 o I0
ado
o
o
II
J
c_
0
J 5
I I0
ELISA ( l U / m L )
I 15
20
0
5
I I0
I 15
I 20
I 25
30
ELLS& ( l U / m t )
Figure 3. Regression plots showing the relationship between results obtained by ELISA and by toxin neutralization. (a) Cl. tetanL antitoxin y = 1.07x + 0.1. (b) Cl. novy~ antitoxin, y = 1.10x - 0.05. (c) Cl. septicum antitoxin, y = 0.95x + 0.38. (d) Cl. perfnngens epsilon antitoxin, y = 1.00x + 0.70.
286
K.R. Wood
Discussion
References
C o r r e l a t i o n b e t w e e n r e s u l t s o b t a i n e d by E L I S A a n d T N w a s g e n e r a l l y good for all four a s s a y s . T h e criterion for the clostridial c o m p o n e n t s p a s s i n g p o t e n c y is t h a t a c e r t a i n c o n c e n t r a t i o n of specific a n t i b o d y is achieved in the r a b b i t s e r a (Cl. tetani 2.5 IU/ml; Cl. septzcum 2.5 IU/ml; Cl. novyi 3.5 IU/ml; Cl. perfringens epsilon 5.0 IU/ml. 1-4 F o r all t h e a s s a y s , b o t h E L I S A a n d T N p r o d u c e d r e s u l t s which would h a v e p a s s e d a n d failed the s a m e vaccine b a t c h e s . C r i t i c i s m s of E L I S A s as r e p l a c e m e n t s for t h e T N a s s a y h a v e m a i n l y s t e m m e d f r o m w o r k done on t h e t i t r a t i o n of h u m a n sera, p a r t i c u l a r l y for Cl. tetani a n t i t o x i n w h e r e poor correlation a t low s e r u m titres h a s b e e n a problem. 9-n On the o t h e r h a n d , good corr e l a t i o n h a s also b e e n r e p o r t e d with t e t a n u s a n t i t o x i n in h u m a n s e r a by E L I S A a n d T N over a wide r a n g e of titres.12 It is also recognized by the a u t h o r t h a t unlike t h e T N assay, the E L I S A s do not n e c e s s a r i l y m e a s u r e only protective antitoxin, but the correlation o b s e r v e d s u g g e s t s t h a t s i m i l a r entities are b e i n g measured: it m a y h a v e b e e n e x p e c t e d t h a t t h e E L I S A , if m e a s u r i n g b o t h protective a n d non-protective antibodies, would h a v e p r o d u c e d c o n s i s t e n t l y h i g h e r r e s u l t s t h a n t h a t of the T N assay, b u t this w a s not the case. T h e benefits of r e p l a c e m e n t of t h e s e T N a s s a y s are significant a n d include a reduction in the use of subs t a n t i a l n u m b e r s of mice which is of both ethical a n d economic i m p o r t a n c e a n d a reduction in the t i m e t a k e n to a c h m v e a potency r e s u l t f r o m 3 - 4 d a y s to a p p r o x i m a t e l y 3 h All four a s s a y s h a v e now b e e n accepted as b a t c h r e l e a s e potency t e s t s for v e t e r i n a r y vaccines by the U.K. R e g u l a t o r y a u t h o r i t i e s a n d h a v e b e e n successfully i n t r o d u c e d into the H o e c h s t A n i m a l H e a l t h Q u a l i t y A s s u r a n c e L a b o r a t o r i e s as a l t e r n a tives to t h e toxin n e u t r a l i z a t i o n assay.
1. Biological assay of Clostrtdtum tetant antitoxin. British Pharmacopoeia Veterinary, London: HMSO, 1985, A l l 6 2. Biological assay of Clostrtdtum novyt alpha antitoxin. British Pharmacopoeia Vetennary, London: HMSO, 1985, All3. 3. Biological assay of Clostrtdtum septicum antitoxin. British Pharmacopoeia Vetennary, London: HMSO, 1985, All5. 4 Biological assay of Clostrtdzumperfrtngens antitoxins. British Pharmacopoeia Veterinary, London: HMSO, 1985, All4. 5. Gupta RK, Maheshwari SC, Singh H. The titration of tetanus antitoxin V. Effect of formahsation method for the fixation of sheep erythrocytes on the indirect haemaglutmatlon test. J Biol Stand 1985; 13:151-157. 6. German-Fattal M, Bizzml B, German A. Immunity to tetanus: tetanus antitoxin and anti-BIIb m human sera J Blol Stand 1987, 15: 223-230. 7. Knight PA, Tflleray JH, Quemlnet J. In vttro tests for the measurement of veterinary clostrldlal toxins, toxoids and antisera. 1. titration ofClostrtdtum septtcum toxins and antitoxins m cell culture. Biologicals 1990; 18: 181-189. 8 Sojka MG, White VJ, Thorns CJ, Roeder PL. The detection of Clostrldtum perfrtngens epsilon antitoxin in rabbit serum by monoclonal antibody based competition ELISA J Blol Stand 1989; 17. 117-124 9. Hendmksen CFM, v d Gun JW, Nagel J, Kreeftenberg JG. The toxin binding inhibition test as a reliable tn wtro alternative to the toxin neutralisation test m mice for the estlmatmn of tetanus antitoxin in human sera. J Biol Stand 1988; 16 287-297. 10 Gentlh G, Plni C, Collotti C. The determination of the potency of human tetanus lmmunoglobuhns by an enzyme-linked immunosorbent assay. J Blol Stand 1984; 12: 167-173. 11. Hagenaars AM, van Delft RW, Nagel J. Comparison of ELISA and toxin neutrahsatmn for the determination of tetanus antlbodms. J Immunoassay 1984; 5 1-11. 12 Habeeb AFSA. Studies on epsflon prototoxin of Clostridtum perfrtngens Type D. Physmcochemical and chemical propertms of epsilon prototoxin. Biochim Biophys Acta 1975; 412.62-69. 13 Worthington RW, Mulders MSG. Physical changes in the epsflon prototoxm molecule of Clostrtdtum perfrtngens during enzymatic actwatlon. Infect Immun 1977, 18:549-551 14. Melwlle-Sm~th ME, Seagroatt VA, Watkins JT. Comparison of enzyme-hnked lmmunosorbent assay (ELISA) with the toxin neutralisation test m mine as a method for the estimation of tetanus antitoxin m human sera. J Biol Stand 1983; 11: 137-144.
Acknowledgement T h e a u t h o r would like to e x p r e s s t h a n k s to M r N o r m a n B a k e r for e n t h u s i a s t i c s u p p o r t a n d encoura g e m e n t at inception a n d t h r o u g h o u t the project.
Received for pubhcation 19 January 1991; accepted 31 May 1991.