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In vitro tests for the measurement of veterinary clostridial toxins, toxoids and antisera
Bidogicals
W90)
18, 181-189
In Vitro Tests for the Measurement of Veterinary Clostridial Toxiqs, Toxoids and Antisera I. Titration of Clostridium Septicum Toxins and Antitoxins in Cell Culture P. A. Knight, J. H. Tilleray and J. Queminet t Welcome Biotechnology Limited, Langley Court, Beckenham, Kent, BR3 3BS, U.K.
Abstract. The assay of Clostridium septicurn antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in viva and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in viva counterparts, permitting detection of substantially lower titres than is possible using in viva indicators. It is suggested that cell culture indicators may prove useful for the titration of CI septicurn antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations
Introduction The need for a substantial reduction in the use of animals in the development and testing of medicinal products has been identified by many authorities.‘3 Large numbers of laboratory animals are used in the routine potency testing of bacterial vaccines. These potency tests may involve the direct challenge of vaccinated animals with live organisms. Alternatively, they may involve inoculating animals with the vaccine under test to elicit a humoral antibody response, then titrating the antibody by means of a toxin neutralization test. The potency tests for clostridial toxoid vaccines and antitoxin specified by the European Pharmacopoeia fall into the latter cate4*5 Monographs exist for the testing of vaccines WY. against Clostridium novyi, Cl perfringens (beta and epsilon), CL septicurn and CZ tetani and for antitoxins against Cl novyi, Cl perfiingens (beta and epilson) and CL tetuni. All of these tests involve the titration of antitoxin in the sera of vaccinated rabbits using toxin neutralization assays. The indicator system used to determine neutralization is generally lethality, or paralysis in laboratory animals. The replacement of these in vivo indicators would be especially desirable since approximately 50% of the animals used can be 1045-1056/90/030181+9
$03.00/O
expected to die or suffer paralysis as a result of non-neutralized toxin. The use of dermonecrosis as an alternative indicator can reduce the number of animals affected but is not universally accepted for routine testing. The immunogenicity of bacterial vaccines is not simply related to the quantity of antigen present. It is also affected by several other factors that are difflcult to quantify except by means of animal tests.6 Consequently, it remains necessary to inoculate rabbits with vaccines under test to elicit an antitoxin response. However, the use of in vitro assays for the measurement of antitoxin in the sera from vaccinated rabbits may be possible. Alternative assays for antitoxin could involve serological methods to quantify specific antibody, or a toxin neutralisation test using an in vitro indicator system. However, serological methods are not inherently specific. They measure any antibodies directed against the vaccine test antigen irrespective of their protective activity and are therefore capable of producing misleading results. Several methods have been proposed to increase the specificity of serological assays for antitoxin; some success has been claimed for antigen capture assays,7 toxin binding inhibition assays’ and subtractive @ 1990 The International
Association of Biological
Standardization
182
P. A. Knight
tests9 Each of these tests can improve the specificity for antitoxin but is unlikely to equal the assurance of toxin neutralization tests. Cell cultures can be effective indicators of free toxin in toxin neutralization tests. However, this type of assay is restricted to those toxins that are.cytopathic for recognised cell lines. The culture filtrates of CZ septicum, Cl perfringens and Cl novyi are lethal to certain cell cultures. However, the major lethal toxins may not be the only cytopathic entities present in clostridial culture filtrates. It is therefore necessary to demonstrate, as far as possible, that the lethal and cytopathic properties are mediated by the same entity. An effective demonstration of such identity would be seen in the abrogation of both effects by a single monoclonal antibody. In the absence of neutralizing monoclonal antibody, alternative evidence would be necessary to ascribe the various biological activities to a single entity. A combination of gel filtration, ion-exchange chromatography and hydrophobic interaction chromatography would confirm that the entities responsible for each biological activity share similar molecular sizes, charge characteristics and hydrophobicity. The application of cell culture assays in the testing of CZ septicum toxin and antitoxin is discussed below. Cl septicum is an organism of considerable medical and veterinary importance, causing gas gangrene in man and braxy, malignant oedema and blackquarter in farm animals. Immunity to these diseases is principally mediated by antitoxin raised against CZ septicum alpha toxin. Potency tests for antisera involve the titration of antibodies raised against alpha toxin.l” The potency test for vaccines containing CZ septicum alpha toxoid involves the titration of alpha antitoxin in the sera of vaccinated rabbits. In both cases the indicator for toxin neutralization is lethality in the mouse.4 Variations in the susceptibility of mice to CZ septicum alpha toxin dicate that substantial numbers of animals are required to give adequate assurance that the titre of the serum pool from vaccinated rabbits is greater than the required minimum of 2.5 IU/ml. CZ septicum alpha toxin has been shown to be dermonecrotic” as well as lethal and can therefore be titrated in guinea-pig skin in toxin neutralization assays. The guinea-pig skin test allows several titrations to be performed on a single animal, permitting a substantial reduction in the number of animals required for the quality assurance testing of CZ septicum vaccines. It was subsequently adopted as the standard method of titration of septicum antitoxin at the Wellcome Research Laboratories. The dermone-
et
al.
0 E
1 2000 Frr cl 1000 P .r 3 500
00 Mouse
lethal
Figure 1. Cl se&cum antitoxin titres of native and purified equine antisera measured using mouse lethal and guineapig dermonecrotic indicators.
erotic and lethal indicators of neutralisation have shown an excellent degree of correlation in the titration of equine antitoxins (Fig. 1). The cytopathic activity of CE septicurn culture filtrates was first reported by Penso and Vicari.l’ In more recent studies, the filtrates were found to be cytophathic for a number of established cell lines. Furthermore, it was shown that the cytopathic activity could be abrogated by antitoxin. Consequently, the cytopathic activity was used as an indicator for l3 A series of chromatographic toxin neutralization. separations carried out in an attempt to elucidate biochemical similarities between the cytopathic, lethal and dermonecrotic activities of CZ septicurn toxin is also described. The present paper describes and evaluates the use of in uiuo and in vitro indicator systems for the titration of CZ septicum antitoxins generated in routine vaccine potency tests and field trials in farm animals. Materials
and methods
Toxins
Test toxin preparations consisted of toxin which had been purified by ammonium sulphate precipitation, redissolved, dispensed into vials and lyophilized as specified in the European Pharmacopoeia.r” Chromatographic fractions were diluted in cell culture medium for the titration of cytopathic and haemolytic activities and in nutrient broth for dermonecrotic and lethal activities.
Titration
of clostridlum
septicum toxins and antitoxins
Antisera
The antisera studied were pools of rabbit serum prepared in accordance with the provisions of the European Pharmacopoeia test for CZ septicurn vaccine4 and sera from farm animals used in field trials of vaccine. Before titration in cell culture the sera were decomplemented by heating at 56°C for 30 min and sterilized by filtration through a O-22 pm membrane. All sera were tested and found to be free from cytopathic activity before assay. The sera had already been titrated by the routine dermonecrotic test. The reference antitoxin was a solution of Fabs fragment from an equine antitoxin which had been standardised against the British National Standard for Gas Gangrene Antitoxin by the dermonecrotic titration method. Cell culture methods
Cell lines. MDCK cells, an established dog kidney cell line were obtained from Flow Laboratories, Rickmansworth, Herts, U.K. VERO African Green Monkey Kidney Cells were obtained from the same source. Cell culture titrations on rabbit sera were performed in VERO cells maintained in medium 199 or Eagles minimal essential medium, supplemented with 5% (v/v) foetal bovine serum, penicillin (40 000 IU/ml) and streptomycin (20 000 IU/ml). The same media were used as diluents for all reagents used in the test. Titrations were performed in sealable 96-well cell culture titration plates from Alpha Laboratories, Eastleigh, Hants, U.K. The level of test used with rabbit serum pools was chosen to ensure that the concentrations of toxin and antibodies in the reaction mixture were the same as those prescribed in the European Pharmacopoeia (i.e. 0.04 U in 100 ~1 = 0.2 /.L in 500 ~1). Test sera were diluted to expected concentrations of 1.6 U/ml and 1.44 U/ml and volumes of 50, 40, 33, 27, 22, 18, ,15 and 12 ~1 of each dilution were dispensed into a column of eight wells using a Hamilton Microlab M programmable dispenser. Reference antitoxin was diluted to 1.6 U/ml and volumes of 40, 36, 33, 30, 27, 24, 22 and 30 ~1 were dispensed similarly. The total volume in each well was made up to 50 ~1 using medium and 0.04 units equivalent of test toxin in 50 ~1 were subsequently added. Controls consisting of serum only, medium only and a two fold series of toxin dilutions were included. The toxin/antitoxin mixtures were agitated for 30 seconds on a plate
in cell culture
183
shaker and allowed to stand at ambient temperature for 1 h before the addition of 2.5 x lo4 MDCK or VERO cells in 50 ~1. Cells were also added to the controls. After the addition of cells the plate was sealed with film taking care to ensure that each well was gas tight. Results were assessed after 24 h by microscopic examination or by the change of indicator colour’4 at 6 days. The end points selected were O-10% living cells by microscopic examination or an orange colouration in the indicator. Cattle and sheep sera were titrated at a level of L + (cells)/400 in a volume of a 100 ~1 in order to allow titres as low as 0.05 U/ml to be measured. Aliquants of 50 ~1 of sera were serially diluted in cell culture medium in the microtitre wells. 50 ~1 of test toxin solution containing 0.05 units equivalent/ml was added to each well. Controls similar to those already described for the titration of rabbit serum pools were included in each test. 2.5 x lo4 MDCK cells were added to each well in a volume of 50 ~1 after which the culture responses were assessed as above. Dermonecrotic tests
The method of titration using the dermonecrotic indicator was parallel to that already described for the cell culture method. Sera were diluted to approximately 2 U/ml in borate buffered saline and similar series of volumes within the range 0.2 ml to 1 ml were dispensed into tubes. One unit of test toxin in 0.5 ml of nutrient broth was added to each tube. The contents of each tube were mixed by inversion. After 30 minutes 0.2 ml of each mixture was injected intradermall-y into guinea-pig skin. The injection sites were examined for signs ofdermonecrosis after 2 days, the end point being minimal necrosis. Cattle and sheep sera were tested by a capillary method in which equal volumes of a twofold dilution series of toxin were mixed with aliquants of undiluted serum. After 30 min at room temperature 0.2 ml of each dilution was inoculated intradermally into guinea-pig skin. Chromatographic separations
These were performed using a Fast Protein Liquid Chromatography (FPLC) system (Pharmacia Biotechnology Limited, Milton Keynes, U.K.). Ge/ filtration chromatography. Test toxin was reconstituted in distilled water and clarified by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Superose 12 column (10 x 300 mm) equilibrated with phosphate buffered saline pH7.2
184
P. A. Knight
et
a/.
(PBS). The sample was then eluted in PBS at a flow rate of O-5 ml/mm, the absorbance of the eluate at 280 nm monitored and 20 fractions of 2 ml each collected. Anion exchange chromatography. Test toxin was reconstituted in, and dialysed against 0.05 M diethanolamine+HCl buffer, pHQ-0 for c 20 h at 4”C, then clari&d by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Mono Q in diethanolamine column (5 x 50 mm) equilibrated buffer. The material was then eluted with a 20 ml linear gradient of 0 to 0.35 M sodium chloride in diethanolamine buffer, followed by a 2 ml linear gradient from 0.35 M to 1-O M sodium chloride. The flow rate was 1 ml/min throughout. The eluate was monitored at 280 nm and 17 fractions of 2 ml each collected. Hydrophobic interaction chromatography. Test toxin was reconstituted in distilled water and clarified by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Phenyl Superose column with O-1 M sodium phos(5 x 50 mm) equilibrated phate buffer, pH 7-O containing 2 M sodium sulphate. The material was then eluted with a 15 ml linear gradient of decreasing salt concentration to zero. The flow rate was O-5 ml/min throughout. The eluate was monitored at 280 nm and 13 fractions of 2 ml each collected. Estimates of bias Possible bias arising from the use of the cell culture method was investigated by comparing the computed regression of log cell culture titres on log dermonecrotic titres for each of the trial groups using parallel line regression analysis. An estimate of bias ratio was then obtained by similar methods to those used to estimate potency ratio in conventional bio-assays. One of the preparations was arbitrarily selected as a reference and the regression for the sera of each trial group in turn was compared with it to produce an estimate of bias ratio and its 95% fiducial limits. Results Exposure of VERO cells to CZ septicum toxin resulted in rapid cell death. Cultures exposed to toxic mixtures contained only unattached rounded cells [Fig. 2(a)]. Weakly toxic mixtures permitted survival of a few cells which were only partially extended [Fig. 2(b)]. This level was chosen as the titration end point. There
Figure 2. Effect of Cl septicurn toxin on cultures of VERO cells. (a) Toxic mixture; no surviving cells; (b) end-point; less than 10% confluent surviving cells; (c) minimal toxic effect; 20% confluent cells; (d) no toxin; confluent cell growth.
was little evidence of sub lethal cytopathic effects although growth was inhibited by very weakly toxic mixtures [Fig. 2(c)] compared with atoxic mixtures Fig. 2(d). The relative sensitivities of the lethal, dermonecrotic and cytopathic indicators of free toxin are shown in Table 1. The cytopathic indicator system proved to be much more sensitive than the in uiuo systems. Two of the toxins examined contained barely sufficient indicating doses to justify the use of 10% incremental steps in the test series using the lethal and dermoneerotic indicators. Tests were completely justified when the cytopathic indicator was used. Figure 3 shows the characteristic titration curve for standard antitoxin. Clear and consistent end points were discernable even when the incremental step was only 10%.
Tltratlon
Table 1. Relative
of clostrldlum
septlcum toxlns and antltoxlns
sensitivity
of lethal,
dermonecrotic
Indicating
doses per unit equivalent
Lethal
Dermonecrotic
and cytopathic
18!5
In cell culture
indicators
for CZ septicum
alpha
toxin Indicating Toxin preparation AVX1887 AVXl888 AVX1897
117 46 47
158 65 60
7380 2468 3844
fellow
Irange ‘ink 40 36 33 Volume of antitoxin
30 27 solution
Figure 3. Mean percentage f SEM confluence of VERO cells exposed to mixtures containing L + (cells)/25 of CZ septicurn toxin and varying volumes of diluted standard
antitoxin (n = 12).
3
4.
Lethal (L+/5)
Cytopathic
5
6 7 Dermonecrotic
doses in test dose
Dermonecrotic (Lr5)
23.4
31.6 13 12
9.2 9.4
Cytopathic L+(cellY25 296 99
153
The presence of orthocresoYether (OCE) preservative made the first series of sera cytopathic at dilutions of less than 1 in 10. To overcome this problem the level of toxin used in the cell culture assay was lowered sufficiently to just produce cytopathic effects in the presence of O-01 U of antitoxin (L+(cells)/lOO). The correlation between the titres obtained for the OCE-preserved sera in cell culture and by the dermonecrotic test is shown in Fig. 4. Also shown is the correlation obtained using unpreserved sera at the Ph Eur level of test (L+/25). In each case, a close degree of correlation occurred (r = 0.95). The results obtained upon a series of preservativefree sera in a comparison of VERO cell and guinea-pig skin titrations are summarised in Table 2. The mean estimates of antitoxin titres in cell culture were in close agreement with the in uiuo values (r = 0.95). The association between the titres obtained in cell culture and in uiuo titrations of septicum antitoxin for a series of bovine and ovine sera from field trials is shown in Figs 5 and 6 respectively. In each case, a close degree of correlation resulted (r = 0.91 and 0.88 respectively). Table 3 shows the estimates of bias ratio for each of the populations of animal sera obtained from the field trial. In no instance did the bias ratio for any vaccine formulation differ significantly from unity.
titre
4 (U/ml)
5
678
Figure 4. Comparison of estimates of the antitoxin titre of (a) OCE preserved and (b) unpreserved rabbit sera obtained using the guineapig intradermal and cell culture indicators.
188
P. A. Knight
et al.
Table 2. CZ septicum antitoxin titres of sera from rabbits immunised with various clostridial vaccines titrated in guinea-pig skin and VERO cell culture Cell culture
O-25
tests
Vaccine
Titre in G pig skin
Mean titre
cov
NN NN NN NN NN NN NN NN Ref.
6.33 4.43 8.50 7.00 4.40 6.79 4.03 5.93 4.03
6.89 4.28 7.73 6.60 4.41 6.16 3.83 4.69 3.75
11% 10% 14% 18% 8.1% 14.3% 5.1% 4.9% 18.7%
I
8184 8026 7852 8206 8441 8476 8475 8478 vaccine
Dermonecrotic
U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml
3
2 titre
Ratio of titres
4
U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml
0.051 co.05
(U/ml)
Figure 5. Comparison of estimates of Cl septicum antitoxin in bovine sera obtained by titration using the guinea-pig dermonecrotic and MDCK cell culture indicators.
Figures 7, 8 and 9 show the distributions of the lethal, dermonecrotic and cytopathic activities among fractions obtained from Cl septicum test toxin by gel filtration, ion exchange and hydrophobic interaction chromatography. For each biological activity, the titre is expressed as the reciprocal of the dilution at which the effect is extinguished. The distribution of protein indicated by absorbance at 280 nm is also shown. Each method of separation shows that the biological effects are closely associated. There is some evidence that the association between cytopathic and lethal activities may be closer than between dermonecrotic and the other two indicators.
in vitro/in
vivo
1.09 o-97 0.91 0.94 1.00 0.91 o-95 0.79 0.93
’ 0.5 Dermonecrotic
I I.0 titre
I
I
2-o (U/ml)
4-o
8-O
Figure 6. Comparison of estimates of Cl septicum antitoxin in sheep sera obtained by titration based upon the gnineapig dermonecrotic and MDCK cell culture indicators.
Discussion Titrations of septicum antitoxin in rabbit sera using the cell culture assay have been shown to be precise and reproducible within 20%. They have also been found to correlate closely (r = O-95) with titrations carried out using the guinea-pig dermonecrotic method which in turn reflects the mouse lethal test accurately (Fig. 1). These results suggest that the cell culture assay could be used as a replacement for the existing in vivo methods prescribed in the potency tests for vaccines and antitoxins. The results of cell culture titrations of sera from
Tttration of clostrldium
septkum
toxins and antltoxins
187
in cell culture!
Table 3. Bias ratio for cell culture titrations of CZ septicurn antitoxin measured in sera of different inoculated with vaccines of different formulations prepared from different antigen sources Antigen source
Vaccine
Formulation
Species
Regression slope (b)
Ref. 1
1 1
1 2
Sheep Sheep
1-l o-93
2
2
3
Sheep
O-89
3
1
4
Sheep
0.93
4
1
5
Sheep
0.91
5
2
6
Sheep
1.09
6
1
7
Sheep
1.33
1,2&3
Cattle
0438
7
l&2
species
Linearity and parallelism
Bias ratio
95% fid limits
Linear Linear parallel Curved (P = 0.037) parallel Linear parallel Linear parallel Linear parallel Linear parallel Linear parallel
1-o 1.25
(0~9cz.80)
0.82
(O-57-1.15)
l-16
(O-77-1*80)
1.13
(0.77-1.59)
1.15
(0+X3-1.56)
1.00
(O-70-1-29)
O-90
(O-61-1.26)
Mean slope = 1.037.
1000
Cytopathic
-
activity
Lethality
Dermonecrosis
0
8
16
24
I -
32
7. Distribution of biological properties of CZ septzcumculture filtrates following gel filtration chromato-
graphy.
Dermonecrosis
Elutlon
Figure
volume
(ml)
8. Distribution of biological filtrates following chromatography.
septicurn culture
properties of CZ anion exchange
188
P. A. Knight
150
-
Dermonecrosis
loo-
Q
.-._
Protein --._
--.._ --._
A. 4
-------JO 8 I2 Elu tion
Figure
9. Distribution
volume
-. 16
20
24
(~1)
of biological
properties
of Cl
septicum culture filtrates following hydrophobic interaction
chromatography.
cattle and sheep also correlated closely (r = O-91 and 048 respectively) with the results of guinea-pig dermonecrotic titrations. The cell culture tests were performed at a much lower level of test (O-025 U of toxin per ml of mixture) than the intradermal tests (0.25 to 1 U/ml of toxin). The use of a lower level of test can be expected to lead to lower estimates of potency for non-avid sheep and cattle sera when they are compared with highly avid equine antitoxin reference preparations. This effect was reflected in cell culture titres which were generally about one half of those in their in uiuo counterparts. This difficulty may be readily overcome by selection of a laboratory reference antitoxin more representative of the sera to be tested. It is particularly important that the relationship linking titres obtained by an alternative method to titres obtained by the in uiuo method should be consistent for sera raised against different vaccines. If this were not the case, the use of the alternative test would be likely to bias the assessment of vaccines in field trials or in laboratory potency tests. The sheep and cattle sera upon which Figs. 5 and 6 are based were produced in field trials involving seven vaccine formulations using antigen from two distinct sources. The results obtained in the estimation of bias showed that, in each case the 95% fiducial limits of estimate of the bias ratio embraced unity, indicating that use of the cell culture method
et al.
would not biase the results of a comparative field trial for or against any of the formulations (Table 3). By retaining the principle of toxin neutralization, the cell culture method, unlike serological methods is completely specific to neutralizing antibodies. Antibodies directed against impurities in the toxin preparation or antibodies directed against non-neutralizing epitopes on the toxin molecule are not detected. The results of the chromatographic separation studies on CZ septicurn toxin indicate that the molecular species responsible for the dermonecrotic, lethal and cytopathic activities share similar molecular size, charge characteristics and hydrophobicity. This implies that the three biological activities are likely to be shared by the same molecule and supports the evidence of the correlation studies reported. The use of the cell culture indicator offers several advantages over the in uiuo indicators currently used for the titration of septicum antitoxin in sera. Firstly the cell culture assay obviates the use of animals entirely. Very large numbers of animals are used by manufacturers for routine potency testing of clostridial vaccines and antitoxins. The avoidance of the trauma of lethal intoxication and dermonecrosis in such large numbers of animals must be welcome on animal welfare grounds. Secondly the cell cultures assay is more sensitive, creating further advantages. To achieve the 10 indicating doses of toxin necessary to support 10% incremental steps it is necessary that the toxin preparations contain 50 reacting doses per L + dose. It is often difficult to satisfy this requirement and the greater sensitivity of the cell cultures to toxin (2500 CCTDEO per L + (cells)) ensures that even toxins of marginal specific toxicity in the lethal test provide more than 100 indicating doses in the cell culture test. The greater sensitivity of the cell culture method also permits the measurement of lo-fold lower titres than is possible by in uiuo tests, which cannot detect less than 0.5 U/ml of antitoxin. The ability to measure titres down to O-05 U/ml has great potential value for monitoring field trials of vaccine where titres consistently greater than 0.5 U/ml are rarely maintained in all of the animals despite evidence of adequate protection long after the disappearance of titres measurable by in uiuo methods. Thirdly, the cell culture test requires much smaller volumes of serum than are necessary for in uiuo methods of titration. It is concluded that cytopathic indicator systems for toxin neutralisation provide a valid and practicable alternative to the lethal and dermonecrotic indicators currently used for the titration of CZ septicurn antitoxins in animal sera.
Tit&Ion
of clostrldium
septlcum toxins and antttoxins
Acknowledgements The authors extend their thanks and appreciation to Miss G. M. Baxter, Miss W. Wailer and Miss H. Edmonds for their assistance in performance of comparative in viva and in vitro assays.
References 1. Council of Europe European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Specific Purposes. Article 6 in COM (85) Final ISBM: 92-77-10665-4, Commission of the European Community. 2. Russel WMS, Burch RL. The Principle of Humane Experimental Technique. London: Methuen and Co., 1959. 3. Scientific Procedures on Living Animals. Cmnd 8883. London: H.M. Stationery Office, May 1983. 4. European Pharmacopoeia. Clostridium novyi, perfringens and septicum Vaccines for Veterinary Use, 2nd edn, part II-8 numbers 362,363,364. 5. European Pharmacopoeia. Clostridium Novyi Alpha, Perfringens Beta Perfringens Epsilon Antitoxin for Veterinary Use. 2nd edn, part II-8 numbers 339,340, 341. 6. Knight PA. Are Potency Tests for Tetanus Vaccines Really Necessary?Dev Biol Standard 1986; 64: 39-46. 7. Knight PA, Tilleray J, Queminet J. Studies on the correlation of a range of immunoassaysfor diphtheria toxin with the guinea pig intradermal test. Dev Biol Standard 1986; 64: 25-32.
189
in cell culture
8. Hendriksen, CFM, Gun VDJ, Nagel J, and Kreeften-
berg JG. The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralisation test in mice for the estimation of tetanus antitoxin in human sera. J Biol Stand 1988; 16: 287-97. 9. Simonsen 0, Schou C, Heron I. Modification of the ELISA for the routine determination of antitoxic immunity to tetanus. J Biol Stand 1987; 15: 143-157. British Pharmacopoeia (Veterinary 1989) Biological assay of clostridium septicum antitoxin Appendix XIVB All5 HMSO London. 10. British Pharmacopoeia Biological Assay for Gas Gangrene Antitoxin (Septicurn) 1988; II App. 14B5 A155. 11. Glenny AT, LlewellynJones M, Mason JH. The intracutaneous method of testing the toxin and antitoxins of the ‘Gas Gangrene’ organisms. J Path Bact 1931; 34: 201-211. 12. PensoG, Vicari G. Studio dei fenomeni immunitari per mezzo delle colture di tessato-V. Sull’azione citopatogena della tossine di Clostridium septicurn. Rend Institute Superior di Santa 1959; 22: 135. 13. Knight PA, Burnett C, Whitaker AM, Queminet J. The titration of clostridial toxoids and antisera in cell culture. Dev Biol Standard 1986; 64: 129-136. 14. Shand FL. A modified colour test technique for the estimation of polio virus neutralising antibody. J Med Lab Technol 1900; 18: 7.5.
Received for publication 6 November accepted 8 May 1990.
1989;
W90)
18, 181-189
In Vitro Tests for the Measurement of Veterinary Clostridial Toxiqs, Toxoids and Antisera I. Titration of Clostridium Septicum Toxins and Antitoxins in Cell Culture P. A. Knight, J. H. Tilleray and J. Queminet t Welcome Biotechnology Limited, Langley Court, Beckenham, Kent, BR3 3BS, U.K.
Abstract. The assay of Clostridium septicurn antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in viva and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in viva counterparts, permitting detection of substantially lower titres than is possible using in viva indicators. It is suggested that cell culture indicators may prove useful for the titration of CI septicurn antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations
Introduction The need for a substantial reduction in the use of animals in the development and testing of medicinal products has been identified by many authorities.‘3 Large numbers of laboratory animals are used in the routine potency testing of bacterial vaccines. These potency tests may involve the direct challenge of vaccinated animals with live organisms. Alternatively, they may involve inoculating animals with the vaccine under test to elicit a humoral antibody response, then titrating the antibody by means of a toxin neutralization test. The potency tests for clostridial toxoid vaccines and antitoxin specified by the European Pharmacopoeia fall into the latter cate4*5 Monographs exist for the testing of vaccines WY. against Clostridium novyi, Cl perfringens (beta and epsilon), CL septicurn and CZ tetani and for antitoxins against Cl novyi, Cl perfiingens (beta and epilson) and CL tetuni. All of these tests involve the titration of antitoxin in the sera of vaccinated rabbits using toxin neutralization assays. The indicator system used to determine neutralization is generally lethality, or paralysis in laboratory animals. The replacement of these in vivo indicators would be especially desirable since approximately 50% of the animals used can be 1045-1056/90/030181+9
$03.00/O
expected to die or suffer paralysis as a result of non-neutralized toxin. The use of dermonecrosis as an alternative indicator can reduce the number of animals affected but is not universally accepted for routine testing. The immunogenicity of bacterial vaccines is not simply related to the quantity of antigen present. It is also affected by several other factors that are difflcult to quantify except by means of animal tests.6 Consequently, it remains necessary to inoculate rabbits with vaccines under test to elicit an antitoxin response. However, the use of in vitro assays for the measurement of antitoxin in the sera from vaccinated rabbits may be possible. Alternative assays for antitoxin could involve serological methods to quantify specific antibody, or a toxin neutralisation test using an in vitro indicator system. However, serological methods are not inherently specific. They measure any antibodies directed against the vaccine test antigen irrespective of their protective activity and are therefore capable of producing misleading results. Several methods have been proposed to increase the specificity of serological assays for antitoxin; some success has been claimed for antigen capture assays,7 toxin binding inhibition assays’ and subtractive @ 1990 The International
Association of Biological
Standardization
182
P. A. Knight
tests9 Each of these tests can improve the specificity for antitoxin but is unlikely to equal the assurance of toxin neutralization tests. Cell cultures can be effective indicators of free toxin in toxin neutralization tests. However, this type of assay is restricted to those toxins that are.cytopathic for recognised cell lines. The culture filtrates of CZ septicum, Cl perfringens and Cl novyi are lethal to certain cell cultures. However, the major lethal toxins may not be the only cytopathic entities present in clostridial culture filtrates. It is therefore necessary to demonstrate, as far as possible, that the lethal and cytopathic properties are mediated by the same entity. An effective demonstration of such identity would be seen in the abrogation of both effects by a single monoclonal antibody. In the absence of neutralizing monoclonal antibody, alternative evidence would be necessary to ascribe the various biological activities to a single entity. A combination of gel filtration, ion-exchange chromatography and hydrophobic interaction chromatography would confirm that the entities responsible for each biological activity share similar molecular sizes, charge characteristics and hydrophobicity. The application of cell culture assays in the testing of CZ septicum toxin and antitoxin is discussed below. Cl septicum is an organism of considerable medical and veterinary importance, causing gas gangrene in man and braxy, malignant oedema and blackquarter in farm animals. Immunity to these diseases is principally mediated by antitoxin raised against CZ septicum alpha toxin. Potency tests for antisera involve the titration of antibodies raised against alpha toxin.l” The potency test for vaccines containing CZ septicum alpha toxoid involves the titration of alpha antitoxin in the sera of vaccinated rabbits. In both cases the indicator for toxin neutralization is lethality in the mouse.4 Variations in the susceptibility of mice to CZ septicum alpha toxin dicate that substantial numbers of animals are required to give adequate assurance that the titre of the serum pool from vaccinated rabbits is greater than the required minimum of 2.5 IU/ml. CZ septicum alpha toxin has been shown to be dermonecrotic” as well as lethal and can therefore be titrated in guinea-pig skin in toxin neutralization assays. The guinea-pig skin test allows several titrations to be performed on a single animal, permitting a substantial reduction in the number of animals required for the quality assurance testing of CZ septicum vaccines. It was subsequently adopted as the standard method of titration of septicum antitoxin at the Wellcome Research Laboratories. The dermone-
et
al.
0 E
1 2000 Frr cl 1000 P .r 3 500
00 Mouse
lethal
Figure 1. Cl se&cum antitoxin titres of native and purified equine antisera measured using mouse lethal and guineapig dermonecrotic indicators.
erotic and lethal indicators of neutralisation have shown an excellent degree of correlation in the titration of equine antitoxins (Fig. 1). The cytopathic activity of CE septicurn culture filtrates was first reported by Penso and Vicari.l’ In more recent studies, the filtrates were found to be cytophathic for a number of established cell lines. Furthermore, it was shown that the cytopathic activity could be abrogated by antitoxin. Consequently, the cytopathic activity was used as an indicator for l3 A series of chromatographic toxin neutralization. separations carried out in an attempt to elucidate biochemical similarities between the cytopathic, lethal and dermonecrotic activities of CZ septicurn toxin is also described. The present paper describes and evaluates the use of in uiuo and in vitro indicator systems for the titration of CZ septicum antitoxins generated in routine vaccine potency tests and field trials in farm animals. Materials
and methods
Toxins
Test toxin preparations consisted of toxin which had been purified by ammonium sulphate precipitation, redissolved, dispensed into vials and lyophilized as specified in the European Pharmacopoeia.r” Chromatographic fractions were diluted in cell culture medium for the titration of cytopathic and haemolytic activities and in nutrient broth for dermonecrotic and lethal activities.
Titration
of clostridlum
septicum toxins and antitoxins
Antisera
The antisera studied were pools of rabbit serum prepared in accordance with the provisions of the European Pharmacopoeia test for CZ septicurn vaccine4 and sera from farm animals used in field trials of vaccine. Before titration in cell culture the sera were decomplemented by heating at 56°C for 30 min and sterilized by filtration through a O-22 pm membrane. All sera were tested and found to be free from cytopathic activity before assay. The sera had already been titrated by the routine dermonecrotic test. The reference antitoxin was a solution of Fabs fragment from an equine antitoxin which had been standardised against the British National Standard for Gas Gangrene Antitoxin by the dermonecrotic titration method. Cell culture methods
Cell lines. MDCK cells, an established dog kidney cell line were obtained from Flow Laboratories, Rickmansworth, Herts, U.K. VERO African Green Monkey Kidney Cells were obtained from the same source. Cell culture titrations on rabbit sera were performed in VERO cells maintained in medium 199 or Eagles minimal essential medium, supplemented with 5% (v/v) foetal bovine serum, penicillin (40 000 IU/ml) and streptomycin (20 000 IU/ml). The same media were used as diluents for all reagents used in the test. Titrations were performed in sealable 96-well cell culture titration plates from Alpha Laboratories, Eastleigh, Hants, U.K. The level of test used with rabbit serum pools was chosen to ensure that the concentrations of toxin and antibodies in the reaction mixture were the same as those prescribed in the European Pharmacopoeia (i.e. 0.04 U in 100 ~1 = 0.2 /.L in 500 ~1). Test sera were diluted to expected concentrations of 1.6 U/ml and 1.44 U/ml and volumes of 50, 40, 33, 27, 22, 18, ,15 and 12 ~1 of each dilution were dispensed into a column of eight wells using a Hamilton Microlab M programmable dispenser. Reference antitoxin was diluted to 1.6 U/ml and volumes of 40, 36, 33, 30, 27, 24, 22 and 30 ~1 were dispensed similarly. The total volume in each well was made up to 50 ~1 using medium and 0.04 units equivalent of test toxin in 50 ~1 were subsequently added. Controls consisting of serum only, medium only and a two fold series of toxin dilutions were included. The toxin/antitoxin mixtures were agitated for 30 seconds on a plate
in cell culture
183
shaker and allowed to stand at ambient temperature for 1 h before the addition of 2.5 x lo4 MDCK or VERO cells in 50 ~1. Cells were also added to the controls. After the addition of cells the plate was sealed with film taking care to ensure that each well was gas tight. Results were assessed after 24 h by microscopic examination or by the change of indicator colour’4 at 6 days. The end points selected were O-10% living cells by microscopic examination or an orange colouration in the indicator. Cattle and sheep sera were titrated at a level of L + (cells)/400 in a volume of a 100 ~1 in order to allow titres as low as 0.05 U/ml to be measured. Aliquants of 50 ~1 of sera were serially diluted in cell culture medium in the microtitre wells. 50 ~1 of test toxin solution containing 0.05 units equivalent/ml was added to each well. Controls similar to those already described for the titration of rabbit serum pools were included in each test. 2.5 x lo4 MDCK cells were added to each well in a volume of 50 ~1 after which the culture responses were assessed as above. Dermonecrotic tests
The method of titration using the dermonecrotic indicator was parallel to that already described for the cell culture method. Sera were diluted to approximately 2 U/ml in borate buffered saline and similar series of volumes within the range 0.2 ml to 1 ml were dispensed into tubes. One unit of test toxin in 0.5 ml of nutrient broth was added to each tube. The contents of each tube were mixed by inversion. After 30 minutes 0.2 ml of each mixture was injected intradermall-y into guinea-pig skin. The injection sites were examined for signs ofdermonecrosis after 2 days, the end point being minimal necrosis. Cattle and sheep sera were tested by a capillary method in which equal volumes of a twofold dilution series of toxin were mixed with aliquants of undiluted serum. After 30 min at room temperature 0.2 ml of each dilution was inoculated intradermally into guinea-pig skin. Chromatographic separations
These were performed using a Fast Protein Liquid Chromatography (FPLC) system (Pharmacia Biotechnology Limited, Milton Keynes, U.K.). Ge/ filtration chromatography. Test toxin was reconstituted in distilled water and clarified by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Superose 12 column (10 x 300 mm) equilibrated with phosphate buffered saline pH7.2
184
P. A. Knight
et
a/.
(PBS). The sample was then eluted in PBS at a flow rate of O-5 ml/mm, the absorbance of the eluate at 280 nm monitored and 20 fractions of 2 ml each collected. Anion exchange chromatography. Test toxin was reconstituted in, and dialysed against 0.05 M diethanolamine+HCl buffer, pHQ-0 for c 20 h at 4”C, then clari&d by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Mono Q in diethanolamine column (5 x 50 mm) equilibrated buffer. The material was then eluted with a 20 ml linear gradient of 0 to 0.35 M sodium chloride in diethanolamine buffer, followed by a 2 ml linear gradient from 0.35 M to 1-O M sodium chloride. The flow rate was 1 ml/min throughout. The eluate was monitored at 280 nm and 17 fractions of 2 ml each collected. Hydrophobic interaction chromatography. Test toxin was reconstituted in distilled water and clarified by filtration through a 0.22 pm filter. A sample (500 ~1) was loaded onto a Phenyl Superose column with O-1 M sodium phos(5 x 50 mm) equilibrated phate buffer, pH 7-O containing 2 M sodium sulphate. The material was then eluted with a 15 ml linear gradient of decreasing salt concentration to zero. The flow rate was O-5 ml/min throughout. The eluate was monitored at 280 nm and 13 fractions of 2 ml each collected. Estimates of bias Possible bias arising from the use of the cell culture method was investigated by comparing the computed regression of log cell culture titres on log dermonecrotic titres for each of the trial groups using parallel line regression analysis. An estimate of bias ratio was then obtained by similar methods to those used to estimate potency ratio in conventional bio-assays. One of the preparations was arbitrarily selected as a reference and the regression for the sera of each trial group in turn was compared with it to produce an estimate of bias ratio and its 95% fiducial limits. Results Exposure of VERO cells to CZ septicum toxin resulted in rapid cell death. Cultures exposed to toxic mixtures contained only unattached rounded cells [Fig. 2(a)]. Weakly toxic mixtures permitted survival of a few cells which were only partially extended [Fig. 2(b)]. This level was chosen as the titration end point. There
Figure 2. Effect of Cl septicurn toxin on cultures of VERO cells. (a) Toxic mixture; no surviving cells; (b) end-point; less than 10% confluent surviving cells; (c) minimal toxic effect; 20% confluent cells; (d) no toxin; confluent cell growth.
was little evidence of sub lethal cytopathic effects although growth was inhibited by very weakly toxic mixtures [Fig. 2(c)] compared with atoxic mixtures Fig. 2(d). The relative sensitivities of the lethal, dermonecrotic and cytopathic indicators of free toxin are shown in Table 1. The cytopathic indicator system proved to be much more sensitive than the in uiuo systems. Two of the toxins examined contained barely sufficient indicating doses to justify the use of 10% incremental steps in the test series using the lethal and dermoneerotic indicators. Tests were completely justified when the cytopathic indicator was used. Figure 3 shows the characteristic titration curve for standard antitoxin. Clear and consistent end points were discernable even when the incremental step was only 10%.
Tltratlon
Table 1. Relative
of clostrldlum
septlcum toxlns and antltoxlns
sensitivity
of lethal,
dermonecrotic
Indicating
doses per unit equivalent
Lethal
Dermonecrotic
and cytopathic
18!5
In cell culture
indicators
for CZ septicum
alpha
toxin Indicating Toxin preparation AVX1887 AVXl888 AVX1897
117 46 47
158 65 60
7380 2468 3844
fellow
Irange ‘ink 40 36 33 Volume of antitoxin
30 27 solution
Figure 3. Mean percentage f SEM confluence of VERO cells exposed to mixtures containing L + (cells)/25 of CZ septicurn toxin and varying volumes of diluted standard
antitoxin (n = 12).
3
4.
Lethal (L+/5)
Cytopathic
5
6 7 Dermonecrotic
doses in test dose
Dermonecrotic (Lr5)
23.4
31.6 13 12
9.2 9.4
Cytopathic L+(cellY25 296 99
153
The presence of orthocresoYether (OCE) preservative made the first series of sera cytopathic at dilutions of less than 1 in 10. To overcome this problem the level of toxin used in the cell culture assay was lowered sufficiently to just produce cytopathic effects in the presence of O-01 U of antitoxin (L+(cells)/lOO). The correlation between the titres obtained for the OCE-preserved sera in cell culture and by the dermonecrotic test is shown in Fig. 4. Also shown is the correlation obtained using unpreserved sera at the Ph Eur level of test (L+/25). In each case, a close degree of correlation occurred (r = 0.95). The results obtained upon a series of preservativefree sera in a comparison of VERO cell and guinea-pig skin titrations are summarised in Table 2. The mean estimates of antitoxin titres in cell culture were in close agreement with the in uiuo values (r = 0.95). The association between the titres obtained in cell culture and in uiuo titrations of septicum antitoxin for a series of bovine and ovine sera from field trials is shown in Figs 5 and 6 respectively. In each case, a close degree of correlation resulted (r = 0.91 and 0.88 respectively). Table 3 shows the estimates of bias ratio for each of the populations of animal sera obtained from the field trial. In no instance did the bias ratio for any vaccine formulation differ significantly from unity.
titre
4 (U/ml)
5
678
Figure 4. Comparison of estimates of the antitoxin titre of (a) OCE preserved and (b) unpreserved rabbit sera obtained using the guineapig intradermal and cell culture indicators.
188
P. A. Knight
et al.
Table 2. CZ septicum antitoxin titres of sera from rabbits immunised with various clostridial vaccines titrated in guinea-pig skin and VERO cell culture Cell culture
O-25
tests
Vaccine
Titre in G pig skin
Mean titre
cov
NN NN NN NN NN NN NN NN Ref.
6.33 4.43 8.50 7.00 4.40 6.79 4.03 5.93 4.03
6.89 4.28 7.73 6.60 4.41 6.16 3.83 4.69 3.75
11% 10% 14% 18% 8.1% 14.3% 5.1% 4.9% 18.7%
I
8184 8026 7852 8206 8441 8476 8475 8478 vaccine
Dermonecrotic
U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml
3
2 titre
Ratio of titres
4
U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml U/ml
0.051 co.05
(U/ml)
Figure 5. Comparison of estimates of Cl septicum antitoxin in bovine sera obtained by titration using the guinea-pig dermonecrotic and MDCK cell culture indicators.
Figures 7, 8 and 9 show the distributions of the lethal, dermonecrotic and cytopathic activities among fractions obtained from Cl septicum test toxin by gel filtration, ion exchange and hydrophobic interaction chromatography. For each biological activity, the titre is expressed as the reciprocal of the dilution at which the effect is extinguished. The distribution of protein indicated by absorbance at 280 nm is also shown. Each method of separation shows that the biological effects are closely associated. There is some evidence that the association between cytopathic and lethal activities may be closer than between dermonecrotic and the other two indicators.
in vitro/in
vivo
1.09 o-97 0.91 0.94 1.00 0.91 o-95 0.79 0.93
’ 0.5 Dermonecrotic
I I.0 titre
I
I
2-o (U/ml)
4-o
8-O
Figure 6. Comparison of estimates of Cl septicum antitoxin in sheep sera obtained by titration based upon the gnineapig dermonecrotic and MDCK cell culture indicators.
Discussion Titrations of septicum antitoxin in rabbit sera using the cell culture assay have been shown to be precise and reproducible within 20%. They have also been found to correlate closely (r = O-95) with titrations carried out using the guinea-pig dermonecrotic method which in turn reflects the mouse lethal test accurately (Fig. 1). These results suggest that the cell culture assay could be used as a replacement for the existing in vivo methods prescribed in the potency tests for vaccines and antitoxins. The results of cell culture titrations of sera from
Tttration of clostrldium
septkum
toxins and antltoxins
187
in cell culture!
Table 3. Bias ratio for cell culture titrations of CZ septicurn antitoxin measured in sera of different inoculated with vaccines of different formulations prepared from different antigen sources Antigen source
Vaccine
Formulation
Species
Regression slope (b)
Ref. 1
1 1
1 2
Sheep Sheep
1-l o-93
2
2
3
Sheep
O-89
3
1
4
Sheep
0.93
4
1
5
Sheep
0.91
5
2
6
Sheep
1.09
6
1
7
Sheep
1.33
1,2&3
Cattle
0438
7
l&2
species
Linearity and parallelism
Bias ratio
95% fid limits
Linear Linear parallel Curved (P = 0.037) parallel Linear parallel Linear parallel Linear parallel Linear parallel Linear parallel
1-o 1.25
(0~9cz.80)
0.82
(O-57-1.15)
l-16
(O-77-1*80)
1.13
(0.77-1.59)
1.15
(0+X3-1.56)
1.00
(O-70-1-29)
O-90
(O-61-1.26)
Mean slope = 1.037.
1000
Cytopathic
-
activity
Lethality
Dermonecrosis
0
8
16
24
I -
32
7. Distribution of biological properties of CZ septzcumculture filtrates following gel filtration chromato-
graphy.
Dermonecrosis
Elutlon
Figure
volume
(ml)
8. Distribution of biological filtrates following chromatography.
septicurn culture
properties of CZ anion exchange
188
P. A. Knight
150
-
Dermonecrosis
loo-
Q
.-._
Protein --._
--.._ --._
A. 4
-------JO 8 I2 Elu tion
Figure
9. Distribution
volume
-. 16
20
24
(~1)
of biological
properties
of Cl
septicum culture filtrates following hydrophobic interaction
chromatography.
cattle and sheep also correlated closely (r = O-91 and 048 respectively) with the results of guinea-pig dermonecrotic titrations. The cell culture tests were performed at a much lower level of test (O-025 U of toxin per ml of mixture) than the intradermal tests (0.25 to 1 U/ml of toxin). The use of a lower level of test can be expected to lead to lower estimates of potency for non-avid sheep and cattle sera when they are compared with highly avid equine antitoxin reference preparations. This effect was reflected in cell culture titres which were generally about one half of those in their in uiuo counterparts. This difficulty may be readily overcome by selection of a laboratory reference antitoxin more representative of the sera to be tested. It is particularly important that the relationship linking titres obtained by an alternative method to titres obtained by the in uiuo method should be consistent for sera raised against different vaccines. If this were not the case, the use of the alternative test would be likely to bias the assessment of vaccines in field trials or in laboratory potency tests. The sheep and cattle sera upon which Figs. 5 and 6 are based were produced in field trials involving seven vaccine formulations using antigen from two distinct sources. The results obtained in the estimation of bias showed that, in each case the 95% fiducial limits of estimate of the bias ratio embraced unity, indicating that use of the cell culture method
et al.
would not biase the results of a comparative field trial for or against any of the formulations (Table 3). By retaining the principle of toxin neutralization, the cell culture method, unlike serological methods is completely specific to neutralizing antibodies. Antibodies directed against impurities in the toxin preparation or antibodies directed against non-neutralizing epitopes on the toxin molecule are not detected. The results of the chromatographic separation studies on CZ septicurn toxin indicate that the molecular species responsible for the dermonecrotic, lethal and cytopathic activities share similar molecular size, charge characteristics and hydrophobicity. This implies that the three biological activities are likely to be shared by the same molecule and supports the evidence of the correlation studies reported. The use of the cell culture indicator offers several advantages over the in uiuo indicators currently used for the titration of septicum antitoxin in sera. Firstly the cell culture assay obviates the use of animals entirely. Very large numbers of animals are used by manufacturers for routine potency testing of clostridial vaccines and antitoxins. The avoidance of the trauma of lethal intoxication and dermonecrosis in such large numbers of animals must be welcome on animal welfare grounds. Secondly the cell cultures assay is more sensitive, creating further advantages. To achieve the 10 indicating doses of toxin necessary to support 10% incremental steps it is necessary that the toxin preparations contain 50 reacting doses per L + dose. It is often difficult to satisfy this requirement and the greater sensitivity of the cell cultures to toxin (2500 CCTDEO per L + (cells)) ensures that even toxins of marginal specific toxicity in the lethal test provide more than 100 indicating doses in the cell culture test. The greater sensitivity of the cell culture method also permits the measurement of lo-fold lower titres than is possible by in uiuo tests, which cannot detect less than 0.5 U/ml of antitoxin. The ability to measure titres down to O-05 U/ml has great potential value for monitoring field trials of vaccine where titres consistently greater than 0.5 U/ml are rarely maintained in all of the animals despite evidence of adequate protection long after the disappearance of titres measurable by in uiuo methods. Thirdly, the cell culture test requires much smaller volumes of serum than are necessary for in uiuo methods of titration. It is concluded that cytopathic indicator systems for toxin neutralisation provide a valid and practicable alternative to the lethal and dermonecrotic indicators currently used for the titration of CZ septicurn antitoxins in animal sera.
Tit&Ion
of clostrldium
septlcum toxins and antttoxins
Acknowledgements The authors extend their thanks and appreciation to Miss G. M. Baxter, Miss W. Wailer and Miss H. Edmonds for their assistance in performance of comparative in viva and in vitro assays.
References 1. Council of Europe European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Specific Purposes. Article 6 in COM (85) Final ISBM: 92-77-10665-4, Commission of the European Community. 2. Russel WMS, Burch RL. The Principle of Humane Experimental Technique. London: Methuen and Co., 1959. 3. Scientific Procedures on Living Animals. Cmnd 8883. London: H.M. Stationery Office, May 1983. 4. European Pharmacopoeia. Clostridium novyi, perfringens and septicum Vaccines for Veterinary Use, 2nd edn, part II-8 numbers 362,363,364. 5. European Pharmacopoeia. Clostridium Novyi Alpha, Perfringens Beta Perfringens Epsilon Antitoxin for Veterinary Use. 2nd edn, part II-8 numbers 339,340, 341. 6. Knight PA. Are Potency Tests for Tetanus Vaccines Really Necessary?Dev Biol Standard 1986; 64: 39-46. 7. Knight PA, Tilleray J, Queminet J. Studies on the correlation of a range of immunoassaysfor diphtheria toxin with the guinea pig intradermal test. Dev Biol Standard 1986; 64: 25-32.
189
in cell culture
8. Hendriksen, CFM, Gun VDJ, Nagel J, and Kreeften-
berg JG. The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralisation test in mice for the estimation of tetanus antitoxin in human sera. J Biol Stand 1988; 16: 287-97. 9. Simonsen 0, Schou C, Heron I. Modification of the ELISA for the routine determination of antitoxic immunity to tetanus. J Biol Stand 1987; 15: 143-157. British Pharmacopoeia (Veterinary 1989) Biological assay of clostridium septicum antitoxin Appendix XIVB All5 HMSO London. 10. British Pharmacopoeia Biological Assay for Gas Gangrene Antitoxin (Septicurn) 1988; II App. 14B5 A155. 11. Glenny AT, LlewellynJones M, Mason JH. The intracutaneous method of testing the toxin and antitoxins of the ‘Gas Gangrene’ organisms. J Path Bact 1931; 34: 201-211. 12. PensoG, Vicari G. Studio dei fenomeni immunitari per mezzo delle colture di tessato-V. Sull’azione citopatogena della tossine di Clostridium septicurn. Rend Institute Superior di Santa 1959; 22: 135. 13. Knight PA, Burnett C, Whitaker AM, Queminet J. The titration of clostridial toxoids and antisera in cell culture. Dev Biol Standard 1986; 64: 129-136. 14. Shand FL. A modified colour test technique for the estimation of polio virus neutralising antibody. J Med Lab Technol 1900; 18: 7.5.
Received for publication 6 November accepted 8 May 1990.
1989;