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INFECTlON AND IMMUNITY, Sept. 1971, p. 295-306Copyright © 1971 Ameiican Society for Microbiology
United States Standard Diphtheria Toxin for theSchick Text and the Erythema Potency Assay
for the Schick Text DoseMICHAEL F. BARILE, ROBERT W. KOLB, AND MARGARET PITTMAN
Division of Biologics Standards, National Institutes of Health, Bethesda, Maryland 20014
Received for publication 25 May 1971
A diphtheria toxin shown to have high toxicity and avidity and to combine withantitoxin in multiple proportions was selected for the U.S. standard diphtheriatoxin for the Schick test and freeze-dried. Assayed values per vial were 1.09 Lf, 1.09Lr, 1,090 Schick test doses (STD), 33 LD8o, 38 LDw, and 43,000 minimum skin re-active doses. One STD (Lr/1000) is slightly more toxic than one unit of the inter-national standard (Lf/1,000). Experiments showed that the potency assay of Schicktest toxin by guinea pig erythema toxicity, determined relative to the toxicity of thestandard, was highly reproducible and significantly more reproducible than thelethal (minimum lethal dose) test and that the STD, defined as one Lr/1000, wasequivalent to approximately 1/50 minimum lethal dose. The erythema potencyassay was prescribed in the U.S. standards for Schick test toxin effective in 1969.
Revised United States requirements for diph-theria toxin for the Schick test were effective fromJanuary 1969 (19). This paper describes theexperimental work which supported the changefrom the potency requirement that the Schick testdose (STD) shall contain 1/50 minimum lethaldose (MLD) to the requirement that the STDshall be equal in guinea pig intradermal erythema-tous reactivity to that of the STD of the U.S.standard diphtheria toxin for the Schick test. TheSTD is defined as that amount of the standardtoxin which, when mixed with 0.001 unit of theU.S. standard diphtheria antitoxin and injectedintradermally in a guinea pig, will induce anerythematous reaction 10 mm in diameter.The Schick test (16) was developed to determine
if an individual is susceptible or immune todiphtheria, and the dose is defined as 1/50 MLDfor the guinea pig. Because of variation in thesusceptibility of guinea pigs to the lethal activityof diphtheria toxin, the STD was difficult tostandardize (18). In 1925, Glenny (4) proposedthat the STD be standardized as that amount oftoxin which is neutralized by 0.001 unit of a
standard antitoxin. In 1931, the Permanent Com-mission on Biological Standardization of theLeague of Nations (14) accepted the use of twotests, erythema potency and antitoxin-combiningpower, as alternatives to the guinea pig lethal testfor standardization of diphtheria toxin for theSchick test.
In 1957, Gerwing et al. (3) questioned theinclusion of the combining-power test in theroutine assay of the Schick toxin product andrecommended that the STD of the toxin bestandardized to the equivalence of the STD of theinternational Schick toxin (diphtheria) by use ofan animal erythema potency test. The U.S. newlyprescribed potency test (19) is similar to the testdescribed by Gerwing et al. The U.S. specificationfor the parent toxin tends to preclude the need fora combining-power test.
This paper describes the selection of the parenttoxin for the U.S. standard, the studies performedin the development of the dermal erythema po-tency test, the preparation of the standard, theproperties of the standard in comparison with theproperties of the international Schick toxin, andthe relationship of the activity of commercialSchick test toxins assayed by the dermal toxicitytest and the lethal test.
MATERIALS AND METHODS
Animals. Guinea pigs and rabbits were obtainedfrom the Rodent and Rabbit Production Section,Division of Research Services, National Institutesof Health. The guinea pigs weighed 230 to 300 g forlethal tests, 500 to 800 g for skin tests, and 300 to500 g for the L+ test. Rabbits used for skin testsweighed 1,500 to 2,000 g.
Toxins and antitoxin. Three samples of bulk parentdiphtheria toxin were obtained from U.S. manu-
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BARILE, KOLB, AND PITTMAN
facturers. Subsequently, one of these lots was pro-cured for the U.S. standard diphtheria toxin for theSchick test. The international Schick toxin wasstudied for a relationship with the U.S. standard, and18 commercial lots of Schick toxin were assayed todetermine suitability of the erythema assay.The U.S. standard diphtheria antitoxin, filling lots
A15, A16, and A17, was used. The unit of the U.S.standard [antitoxin unit (AU) ] is equivalent to that ofthe international standard [international unit (IU)].
Diluent. Phosphate-buffered saline (PBS; 0.066 M,pH 7.4; reference 17) containingO.2% gelatin (PBSG;reference 13) was used for preparing all dilutions. ThepH and gelatin were critical to prevent deteriorationof the toxin. At pH 7.0, there was about a fourfolddecrease in skin reactivity.MLD. Minimum requirements for diphtheria toxin
for the Schick test (October 15, 1948) defined theMLD as the smallest amount of toxin which, wheninjected subcutaneously into 250 g 4 10% guineapigs, will cause death of all animals injected with atleast 75% of deaths taking place between 72 and 96 hr(2, 5). Experience indicated that the death intervalrequirement of 72 to 96 hr is difficult to meet. Inpractice, a medium death time between 60 and 120 hrwith death of all animals injected was accepted assatisfactory. The lethal action of the standard wastitrated by using nine doses ranging from 0 to Y42vial content and 8 to 20 guinea pigs per dilution in 10experiments. No experiment included the full range ofdoses. Results per dose were combined for estimationsof the lethal dose levels.Minimum skin reactive dose. In our study, the mini-
mum skin reactive dose (MRD) was defined as thesmallest amount of toxin in a volume of 0.1 ml whichinduced an erythematous reaction 10 mm in diameterin guinea pigs or rabbits 40 to 48 hr after intradermalinjection (6, 15). The optimum volume of the injection(0.1 ml) and optimum diluent, PBSG, were determinedin preliminary tests. The nature of the reactions wasobserved up to 100 hr. Injection of Evans Blue dyeused in early tests did not increase the precision ofreading of the reactions and was discontinued.
For each test, eight guinea pigs and eight twofolddilutions of each of two toxins were used. Hair wasremoved from the flanks and back of the animals withan electric clipper followed by application of a depila-tory agent (Nair; Carter Products, Inc., New York,N.Y.). The denuded skin was divided into 16 areas(Fig. 1). The test volume of 0.1 ml was injected byusing a 27-gauge needle and a 1.0-ml tuberculinsyringe. Each of the eight dilutions of each toxin wasinjected into each of the eight guinea pigs in the ran-domized manner as shown in Table 1. Between 40 and48 hr, the reaction induced by each dilution per animalwas recorded as the average of the long and shortdiameters. Then the average diameter of the eightreactions per dilution of toxin was calculated.
Toxin-antitoxin tests: the Lr dose. We determinedthe smallest amounts of toxin (Lr doses) which, whenmixed with 0.1, 0.01, and 0.001 AU of antitoxin, in-duced an erythematous reaction 10 mm in diameter(3, 9). The guinea pigs were prepared as for the MRDtest. For the Lr/10 test, eight small-increment (about
5
Head
9 13
2 6 10 14
3 7 11 15
4 8 12 16
HindFIG. 1. Location of the 16 intradermal sites on a
guinea pig. See Table I for randomization scheme forinjection.
TABLE 1. Randomization scheme for the injectionof eight dilutions of two toxins on each of eight
guinea pigs
Diphtheria Injection sitestoxin
Lot Dilution ia 2 3 4 5 6 7 8
A Al 3 10 14 1 12 7 16 5A2 5 3 12 16 10 1 7 14A3 11 2 6 9 4 15 8 13A4 13 11 4 8 2 9 15 6A5 4 8 15 2 13 6 9 11A6 15 9 11 6 8 13 2 4A7 12 16 7 10 5 14 1 3A8 7 1 3 14 16 5 10 12
B Bi 14 7 1 5 3 16 12 10B2 16 12 5 3 7 10 14 1B3 6 15 9 13 11 8 4 2B4 8 4 13 11 15 2 6 9B5 9 13 2 15 6 4 11 8B6 2 6 8 4 9 11 13 15B7 1 5 10 7 14 12 3 16B8 10 14 16 12 1 3 5 7
a Number of animals.
10%) dilutions of each toxin were mixed, respectively,with a constant amount of standard antitoxin thatprovided 0.1 AU per 0.1-ml test dose. The 16 mixtures(two toxins) were incubated at room temperature(22 i 1 C) for 1 hr and injected intradermally into theeight guinea pigs in the randomized manner, and re-sults were read in the same manner as for the MRDtest. The Lr/100 and Lr/1,000 values were determinedin a similar manner.
The L+ dose. The L+, L+/10, L+/100, and L+/1,000doses of a toxin were determined as the smallestamounts of toxin which, when mixed with 1, 0.1, 0.01,
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U.S. STANDARD DIPHTHERIA TOXIN
TABLE 2. Relative toxicity (MLD anzd MRD)a of toxins A and B
M/RD (pliters)Toxin MLD (pliters)
MRD __uliters_(guinea pig)
Guinea pig Rabbit
A 0.253 3.72 X 104 2.33 X 10-4(3,953)b (2.7 X 106) (4.3 X 106)
B 0.758 10.46 X 10-4 6.18 X 10-4(1,319) (0.96 X 106) (1.6 X 106)
Relative toxicity 3.0 2.8 2.7
a MLD, minimum lethal dose; MRD, minimum skin reactive dose.Values in parentheses denote the number of MLD or MRD doses per milliliter.
TABLE 3. Combining power (Lr and L+ doses) of toxins A anid B
Amt of toxin (juliters)
Diphtheria LLdose +L doseantitoxin unit r dos
Toxin A Toxin B Relative Toxin A Toxin B toxicity
1.0 14.550 37.040 2.50.1 1.250 3.350 2.7 1.790 4.868 2.70.01 0.108 0.289 2.7 0.318 1.050 3.30.001 0.012 0.032 2.7 0.314 1.200 3.8
and 0.001 AU antitoxin, respectively, and injectedsubcutaneously in three or more guinea pigs causeddeath of all animals within 120 hr. For each titration,six small-increment dilutions of each toxin were mixedwith a constant amount of standard antitoxin thatprovided the appropriate unitage per 3-ml test dose.The mixtures were incubated for 1 hr at 22 1 C.
Antitoxin-combining avidity velocity. Antitoxin-combining avidity velocity (10) of the toxins was de-termined at the Lr/100 level. Toxin-antitoxin mix-tures held at 22 :1= 1 C were tested at 0, 0.5, 1, 3, and24 hr to determine that amount of noncombined toxinthat would induce a 10-mm reaction in guinea pigs.
Toxin-combining avidity at different antitoxin con-centrations. Toxin-combining avidity (3, 7) was de-termined with the Lr/10, Lr/100, and L,/1,000 andthe L+, L+/10, L+/100, and L+/1,000 tests.
RESULTSSelection of the parent diphtheria toxin for the
standard. Preliminary comparisons of the threesamples of toxin, by using the guinea pig MLDand the chick mean lethal dose (1, 11) tests,showed that one toxin was much lower in toxicitythan the other two. This toxin was eliminatedfrom further study. The results also pointed upthat the two lethal test systems measured differentdose-response characteristics of the toxins andthat a correlation could not be made in absoluteterms. The impression, not proven, was that the
correlation was poor, and work with the chicktest was discontinued. The other two toxins, codedtoxins A and B, differed about threefold in toxinactivity with toxin A being the more toxic. Asmeasured by the various tests, the relative toxicityvalues did not differ significantly. These valueswere 3.0 in the MLD test and 2.8 (guinea pig) and2.7 (rabbit) in the MRD tests (Table 2); 2.7 in theLr test with 0.1, 0.01, or 0.001 AU; and 2.5, 2.7,3.3, and 3.8 with the L+ test at four antitoxinlevels ranging from 1.0 to 0.001 AU (Table 3).
Figure 2 shows that the two toxins combined inmultiple proportions in the L+ and the Lr tests.In the L+ curves, the deviation from the straightline for toxin A at 0.001 AU and for toxin B at0.01 AU is due to the limit of the sensitivity of thetest for the particular toxin. The avidity of thetwo toxins was comparable as shown by theparallel lines of the Lr and L+ combining valuesat different antitoxin concentrations (Fig. 2) andby the velocity of antitoxin combination at Lr/100which was immediate and stable (Table 4).The results of the above tests indicated that
toxin A had properties recommended for Schicktest toxin (3, 5) and would be suitable for astandard preparation. The results also indicatedthat an erythema potency test for Schick testtoxin would be satisfactory.
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298 BARILE, KOLB, AND PITTMAN
0 l0-lI_ ,L, ASSAY Lr ASSAY
2
I0
z
C I0-
E
< o~~~Toxin A
W lo- 5A Toxin B
1.0 0.1 0.01 0.001 0.1 0.01 0.001
U.S. STANDARD DIPHTHERIA ANTITOXIN(units)
FIG. 2. L+ antd Lr doses of toxins A and B titrated at 10-fold levels of antitoxin with guinwea pigs.
TABLE 4. Avidity of combinationi of toxins A and Bwith anztitoxini at the Lr,'100 level
Toxin in Lr/100 dose (lditers)Incubation Diphtheriatm-h) antitoxintime_(hr) unit Toxin A Toxin B Relative
toxicity
0 0.01 0.105 0.3050 2.90.5 0.01 0.105 0.3025 2.91.0 0.01 0.105 0.3025 2.93.0 0.01 0.1075 0.3025 2.8
24.0 0.01 0.1125 0.3100 2.8
Nature of the skin reaction. The erythematous,edematous, and necrotic manifestations of theskin reactions, within certain dose limits, werelinear (Fig. 3). The erythema and edema reactionsshowed a closer relationship with each otherthan with the necrosis reactions. However, theerythema reactions with average diameters from10 to 20 mm provided a straight line over thewidest range of test doses which was 64-fold andthey remained constant in size from 40 to 72 hr(Fig. 4). Necrosis persisted for a longer period,but the larger doses and the narrower range ofdoses required for linearity made this measure-
ment less desirable than erythema. Erythema andmeasurement between 40 and 48 hr were selectedas criteria for evaluation of reactions.
Skin reactivity of guinea pig and rabbit. Linearand parallel erythema dose responses to toxin Awere obtained in the rabbit as well as in theguinea pig (Fig. 5). A comparison of Fig. 5 withFig. 3 shows the reproducibility of the size of thereactions induced in the guinea pig with a given
amount of toxin. Although the rabbit was some-
what more reactive (Table 2), the guinea pig was
selected as it gave more reliable results. Somerabbits had severe inflammatory reactions to thedepilatory agent and others developed an under-growth of coarse hair which obliterated the re-
actions.Preparation of the U.S. standard diphtheria
toxin for the Schick test. From the data in Tables3 and 4, it was calculated that toxin A contained85.52 Lr per ml. Parent toxin A was diluted in0.066M PBS (pH 7.4) containing 0.1 % humanalbumin to provide, per ml, 1 Lr or 1,000 STD.Each milliliter contained 0.01169 ml of the parenttoxin. Ten thousand glass vials were filled with1.0 ml of the diluted toxin with an accuracy of±0.01 ml, and the contents were freeze-dried.The vacuum in the vials was replaced with argon,and the vials were flame-sealed. The moisturecontent was <1.0%. (In an earlier preparation,nitrogen replaced the vacuum and the moisturecontent was >1.0%. This preparation wasdiscarded.) The drying was performed by a U.S.licensed manufacturer. The preparation satis-factorily passed the general requirements for aproduct for human administration. It was desig-nated lot 2.
Properties of the U.S. standard and comparisonwith the international standard. Assays of the driedU.S. toxin showed that drying had caused no
demonstrable deterioration of the toxicity andcombining power. Table 5 gives the averagevalues per vial which were 1.09 Lf, 1.09 Lr,43,000 MRD, and 38 LD50 (96 hr) or 33 LD80(96 hr). Titrations of the lethal action are givenin Table 8.
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U.S. STANDARD DIPHTHERIA TOXIN
1.46 5.850.73 2.93 11.71
TOXIN CONCENTRATIONS( 107ml.)
FiG. 3. Size of erythematous, edematous, andnecrotic dermal response ofguinea pigs to graded dosesof toxin A at 44 hr after injection.
25
, 20
cncr
W 15ct Ea0=w E< 10wL
5
20 40 60 80TIME FOLLOWING INJECTION
(hours)
100
FIG. 4. Time of appearance and duration of maxi-mum dermal erythema, edema, and necrosis induced bytoxin A.
Table 5 also gives the values (averages) of theinternational standard which were obtained in aWorld Health Organization collaborative study(working document WHO/BS/274, 1954). WHOaccepted this and other working reports andassigned 900 IU per vial (20).
In the preparation of each of the two standards,the aim was to provide 1,000 STD per vial.Based on subsequent assays, the assigned STDvalues were 900 IU for the international standardand 1,090 (Lr/1,000) for the U.S. standard. Therespective Lf contents per vial were 0.9 and 1.09.The 900 IU did not conflict with the British or theCanadian requirements which were in effect atthat time (WHO/BS/274, 1954).
The physical preparation of the U.S. standarddiffered from that of the international standardin that the toxin was less purified, human albuminwas used, and argon replaced the vacuum in thevials, whereas the international preparation con-tained purified toxin and bovine albumin andvials were sealed under vacuum (Table 5).The STD of the U.S. standard is slightly more
reactive than 1 unit of the international standard.With our assayed guinea pig MRD values, theinternational standard had 21 MRD per IU(19,000/900), whereas the U.S. standard had 39MRD per STD (43,000/1,090). In anotherexperiment, each toxin was diluted to provide1 Lr/1,000 per 0.1 ml. Then the MRD values weretitrated simultaneously on guinea pigs by usingserial dilutions. The log doses were plotted againstthe diameter of the reactions. The dilution of theU.S. standard that induced a 10-mm reaction was38, whereas that of the international standard was28. This suggests that the international standardmay contain some toxoid. The presence of toxoidis also suggested by the ratio of 0.9 Lf to 0.74 Lrper vial which was 0.82. With the U.S. standard,the ratio was 1.0. These values indicate that 1 STD(Lr/1,000) of the U.S. standard is slightly moreavid than 1 IU of the international standard.
Stability of the U.S. standard. The stability ofthe toxicity of the standard was determined asprescribed for Schick test toxins (19). Table 6shows that heating of the reconstituted toxin at37 C for 24 hr caused no loss in toxicity. Thetable also shows that the MRD test was repro-ducible within and between laboratories. Thedried standard which has now been stored at
25 k
cnEr 20
c
g0-cjw
w 15o E4(t _w E
< 10
I
cr:w
0.36 1.46 5.85 23.43073 2.93 11.71 46.87
TOXIN CONCENTRATIONS(I07mI )
FiG. 5. Comparison of the dermal erythematousdose response of guinea pigs and rabbits to toxin A.
A ERYTTHEMA* EDEMA* NECROSIS
)_
)F
F-() 0 GUINEA PIGS
0 RABBITS
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TABLE 5. Comparison of the U.S. standard Schick toxin with the international Schick toxin
Property International standard per viala U.S. standard per vialb
Total weight 3.8 mg 5.36 mgAlbumin Bovine, 1.0 mg Human, 1.0 mgNa2HP04 -2H20, 2.37 mg *7H20, 3.57 mgKH2PO4 0.36 mg 0.36 mgGas Vacuum ArgonMoisture < 1 .0%/
Diphtheria toxinTotal solids Purified, 5.0 jig Not purified, 425.0 lAgcTotal N 31.0 1AgcProtein N 0.40 ,gc 0.87 ,ugc
ToxicityLethal dose, guinea pig 20 LD50 (ca. 15 LD75) 38 LD50, 33 LD80dMinimum skin reactive doseRabbit 67,500 54,300eGuinea pig 27,700, l9,Ooob 43,000
Combining powerLf 0.9 1.09fLr guinea pig 0.73, 0.75b 1.09
Schick test dose 900 1,090Lf/l,000 900 1,090Lr/1,000 730, 750b 1,090
a Data from working document WHO/BS/274, 1954, except as indicated otherwise. Values reportedby different participants were averaged. International standard for diphtheria antitoxin was used forcombining-power tests.
bDeterminations made by the authors. U.S. standard diphtheria antitoxin was used for combining-power tests.
c Calculated from assays of parent toxin.d From Table 9 with deaths at 96 hr.eCalculated from Table 2.f Methods: H. R. Dean and R. A. Webb, J. Pathol. Bacteriol., 29:473, 1926. G. Ramon, C. R. Soc.
Biol. 86:661, 711, 813, 1922.
TABLE 6. Heat stability of the U.S. standard diphtheria toxin for Schick test
Avg diameter of erythema reaction (mm)
Laboratory no. I Laboratory no. 2No. of test
Unheateda Heatedb Unheateda Heatedb
1/25c 1/60c 1/25 1/60 1/25 1/60 1/25 1/60
1 12.1 8.7 11.2 8.5 12.2 9.2 12.2 9.02 12.0 9.1 11.8 9.2 12.0 8.8 12.2 8.83 12.0 8.9 10.9 9.4 12.3 9.4 12.3 9.54 11.9 9.1 11.7 9.5 12.3 9.6 12.0 9.1
Avg 12.0 9.0 11.4 9.2 12.2 9.3 12.2 9.1
a Standard reconstituted on day of test.b Vial of standard was reconstituted in 1.0 ml of water and heated at 37 C for 24 hr.c Dilution of 1 :109 dilution of the reconstituted toxin (approximately 1 STD per 0.1-ml injection).
4 C for more than 9 yr has shown no change intoxicity (Table 7).Potency assay of diphtheria toxin for the Schick
test. The suitability of the erythema potencyassay was shown in experiments that provided
information on reproducibility of assay results,relationship between toxicity of the STD asmeasured by the former MLD test and the newerythema toxicity test reative to the standard, andthe greater precision of the latter test.
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U.S. STANDARD DIPHTHERIA TOXIN
TABLE 7. Stability of U.S. standardtoxini for Schick test during 9 yea)
Year
196319641965196619671968196919701971
Totals andmean values
Individualrange
No. oftests
6117
11
74632
57
Dilution oi
1/8
14.4b, C
13.814.414.413.614.214.014.414.2
14.2
12.9-15.6
a New vial of standard toxin was u
of the 57 potency assays. STD, SchicbMean erythema reaction of potenc:
formed per year.cValues are expressed as millimete
In the experiment on reproducibilicate potency tests of a commercial 14
toxin were performed independentlytwo laboratories with different vistandard for each test. The toxin in orstandard was diluted 1:109 to provid0.1 ml and further diluted 1:25 anclot under test was also diluted 1:2'Each dilution in duplicate was injdermally into each of four guinea pigdescribed random scheme. The diamerythema induced by each dilutionat 44 hr. Then the average reactionand ratio of the reactions of the tesstandard at each dilution were calcul8 shows that there was good repwithin and between laboratories anthere was consistency in the toxic contvials of the standard. The diameterstions induced by the two dilutions v
greater than 11 and 8 mm, respectivel,was at the lower limit of the linear d(curve (Fig. 3 and 5). Dilutions of 1 :8the STD were used in the tests repor10 and are currently used for routassays of Schick test toxin.
Lethal reactivity of the standard. Tethe results of the titrations of the leper vial of the standard with 295 guinanimals were observed for 10 days.toxin dilution were accumulated at>5 to 10 days, and survivals at 1(recorded. Considerable variation in
diphtheria occurred within a single dose and was not neces-rs of use sarily dose-related. The range for a single dose was
,f1 STDa as wide as 18 hr to survival at 10 days. Overall,1STD5 however, the death time was dose-related between
1/40 the 1/30 to 1/39 doses. With the accumulateddeaths and survivals at 96 hr, it was calculated by
10 6c the Reed-Muench method that a vial contained1015 37.3 LD5o and 34 LD80 . Inspection of the table10.7 shows that at 96 hr 50% of the animals died at10.8 dose 1/38 and 79% died at dose 1/33 (88% died10.4 at the end of 120 hr). In 18 subsequent tests of the10.7 1/33 vial with 144 guinea pigs (Table 10), 79.9%11.1 died by the end of 96 hr (93% died by the end of10.6 120 hr). Based on 1,090 STD per vial, 1 STD10.9 would be equal to 1/33 LD80 and 1/28.7 LD50.
10.7 The MLD could not be determined directly fromthe table, but it was calculated that each vial
9.7-12.2 contained 24.8 MLD. With this value of 24.8MLD and 1,090 STD per vial, there would be 44STD per MLD. In other words, 1 STD would
sed for each contain about 1/50 MLD.Sk test dose. Table 10 compares the lethal and the erythemay assays per- tests of 18 lots of Schick toxin submitted to the
Division of Biologics Standards for release. Thesers. lots had been evaluated by the lethal test by theity, 10 repli- manufacturers. In the lethal test, eight guinea pigsot of Schick each were injected subcutaneously with 5 ml ofin each of each toxin under test (50 STD) and eight other
ials of the guinea pigs were injected with 5 ml of 1/165ie vial of the dilution of the standard. The latter was estimatede 1 STD per to provide 1 MLD. Later, results given in Table1 1:60. The 8 indicated that this dose contained 1 LD80.5 and 1: 60. Variability of death time was observed as pre-ected intra- viously reported. Only with lot I-1 did 75% of thes in the pre- deaths occur between the specified time of 72 andeters of the 96 hr. Differences between lots were not signifi-were read cant. Combined, 85% of the 144 guinea pigs in-
per dilution jected with the 18 toxins died within 96 hr, 96%it toxin and died within 120 hr, and 4% died between 124 andLated. Table 209 hr. With the standard, 80% died within 96 hr,)roducibility 93% died within 120 hr, 5% died between 125.d also that and 156 hr, and 2% survived. The combinedtent between results indicated that 50 STD of the toxins underof the reac- test were near 1 MLD and that lethality wasvere slightly slightly greater than 1 LD80 of the standard. Ity. The latter seems reasonable to assume that 50 STD of theose-response standard would be approximately 1 MLD.and 1:40 of By the erythema test, no significant differencested in Table between the 18 toxins were shown and there wasmne potency consistency between the reactions of the standard
between tests. The ratio of the potency of eachable 9 shows lot to the standard fell well within the acceptedthaI activity range of 0.77 and 1.30. In fact, the smallest andlea pigs. The largest ratios were 0.88 (lot C-1) and 1.06 (lotDeaths per A-1), respectively. The largest difference between3 4 5, and the ratios of the two dilutions of a single lot was0 days were only 0.09 (lot H-3), whereas the difference of thedeath time mean of the lots was 0.02. An analysis of vari-
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U.S. STANDARD DIPHTHERIA TOXIN
TABLE 9. Titration of lethal action of the U.S. slandard Schick toxin with guinea pigs
Total Accumulated deaths Sur- Per cent dead Sur-Portion Dilutiona No. of no. of vival vivalof vial (5 ml) tests guinea (10 - (10
pigs 3as 4 >5 days) 3 4 5 >5 days)days d_ays days days day) days days days
1/30 1/150 4 46 34 41 44 46 0 74 89 96 100 01/33 1/165 5 61 33 48 54 60 1 54 79c 88 98 21/35 1/175 4 53 29 41 47 53 0 55 77 89 100 01/37 1/185 3 35 12 19 27 33 2 34 54 77 94 61/38 1/190 2 20 8 10 14 18 2 40 50 70 90 101/39 1/195 2 20 3 5 7 15 5 15 25 35 75 251/40 1/200 2 20 2 5 9 16 4 10 25 45 80 201/41 1/205 2 20 5 7 10 15 5 25 35 50 75 251/42 1/210 2 20 7 7 9 17 3 35 35 45 85 15
a Vial of standard toxin (1,090 Schick test doses) was reconstituted in 1 ml of water and diluted inphosphate-buffered saline with 0.2% gelatin. Guinea pigs were injected subcutaneously in groups of8 to 20 per dilution.
b Accumulated results from 10 separate experiments.c Numbers in boldface indicate the approximate LD80 and the LD5, doses.
ance showed that the difference in precision of thelethal and erythema assays was highly significant.The ratio of the skin test variances suggested thatone skin test provided as much precision as 50death-time tests.
DISCUSSIONThe method for evaluating the potency of the
STD of diphtheria toxin has seen changes sincethe original definition of 1/50 MLD for the guineapig (16). Nevertheless, the toxic activity of anSTD has remained quite constant worldwidethrough the years. Variation in guinea pig re-sponse prompted the use of an animal erythemaskin test assay (5). Later, Glenny defined theSTD as that amount of toxin neutralized by 0.001unit of diphtheria antitoxin. The PermanentCommission on Biological Standardization of theLeague of Nations (14) accepted both theerythema and the combining-power potency testsand defined the STD as "(a) that amount oftoxin which when diluted 1/25 produces a positiveand when diluted 1/50 produces a negativeintracutaneous reaction in guinea pigs providedthat (b) when the STD is mixed with 1/750thpart or more of an International Unit (IU) ofdiphtheria antitoxin and injected intracutaneouslycauses no local reaction and when mixed with1/1250th part or less of an IU, similarly injected,causes a marked positive Schick reaction." Thelatest international action was to adopt a standardfor the Schick test toxin (20). The IU was definedas the activity of 1 Lf/1,000 of the standard.Latest available information shows that the toxicactivity of the STD in various countries is equalto the combining power of either 1 Lf/1,000 or
1 Lr/1,000. For the potency assay, Great Britain(British Pharmacopoeia, 1958 and 1968, ThePharmaceutical Press, London) has changed fromuse of the combining power test as defined by theLeague of Nations to the erythema test withpotency expressed relative to the internationalstandard. Canada (Food and Drug Act andRegulations with Amendments to 12 May 1970,C.04.140-C.04.147) has retained the two tests,and Japan (8) uses the MLD, the MRD, and theLr/1,000 combining power tests. The U.S. re-tained the potency requirement of 1/50 MLDuntil recently, when the STD of the U.S. standardwas defined as 1 Lr/1,000 and an erythema po-tency requirement relative to the standard wasprescribed.
This paper has shown that the erythema ac-tivities of an STD of the U.S. standard and of 18commercial lots of Schick toxin, assayed by thelethal test, are equivalent. Therefore, the amountof diphtheria toxin in an STD currently standard-ized on the basis of an Lr/l,000 is the same asthat amount of toxin in an STD previously stand-ardized on the basis of 1/50 MLD. The specifica-tion that the potency of 1 STD be equivalent to1 Lr/1,000 of the U.S. standard conforms withthe recommendation of earlier workers (5). TheU.S. STD, however, is slightly more toxic thanthe IU of the international standard for Schicktoxin. The vial of the international standard hasan assigned unitage of 900. From the valuesobtained in a collaborative study (WHO/BS/274,1954), we have estimated that 1 IU has 1 Lf/i,000,0.81 Lr/1,000, and 31 MRD. Our own assayedvalues were 0.83 L1/1,000 and 28 MRD. It is
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hoped that other laboratories will make compara-tive tests of the two standards.Our experimental observations with the
erythema potency assay are in agreement withother investigations (3, 15). The test is accurateand reproducible. The design of the U.S. assay issimilar to the one recommended by Gerwinget al. (3). In contrast to the limitations of thelethal assay (2, 4, 12, 18), which lacks precisiondue to the variability of death-time of guineapigs, the erythema assay provides a sensitivequantitative measurement of toxin. The size of thereaction is directly proportional to the log of thetoxin concentration. Besides being significantlymore precise, the assay is cheaper. It provides forreplicate testing and simultaneous testing ofseveral toxins on the same animals.
ACKNOWLEDGMENTS
We are grateful to C. J. Maloney, Biometrics Section, Divisioniof Biologics Standards, and Claire Cox for the statistical analysesof the data and to Harry T. Aylor, Donald B. Riggs, and MarionW. Grabowski for excellent technical assistance.
LITERATURE CITED
1. Branham, S. E., and M. F. Wormald. 1954. The use of thechick in titration of diphtheria antitoxin. J. Immunol. 72:478-484.
2. Ehrlich, P. 1897. Die Wertbemessung des Diphtherie-heil-serum und theoretische Grundlagen. Klin. Jahrb. 6:299-305.
3. Gerwing, J., D. A. Long, and M. V. Mussett. 1957. The assayof diphtheria toxin. Bull. W.H.O. 17:537-551.
4. Glenny, A. T. 1925. The principles of immunity applied toprotective inoculation against diphtheria. J. Hyg. 24:301-320.
5. Glenny, A. T. 1931. System of bacteriology in relation tomedicine. London Med. Res. Council 6:106.
6. Glenny, A. T., and K. Allen. 1921. The testing of diphtheriatoxin and antitoxin by intracutaneous injection into guineapigs. J. Pathol. Bacteriol. 24:61-63.
7. Glenny, A. T., and M. Barr. 1932. The "dilution ratio" ofdiphtheria antitoxin as a measure of avidity. J. Pathol.Bacteriol. 35:91-96.
8. Japanese Minimum Requirements of Biologic Products. 1968.Schick toxin (diphtheria toxin for diagnosis), p. 203-288.Ministry of Health and Welfare, Tokyo, Japan.
9. Jensen, C. 1933. Die intrakutane Kaninchen methode zurauswertung von Diphtherie-Toxin und Antitoxin. ActaPathol. Microbiol. Scand., Suppl. XIV.
10. Jerne, N. K. 1951. Study of avidity based on rabbit skin re-sponses to diphtheria toxin-antitoxin mixtures. Acta Pathol.Microbiol. Scand. Suppl. 87:3-183.
11. Kolb, R. W., S. E. Branham, and D. B. Riggs. 1955. Compari-son of the guinea pig and chick in evaluation of diphtheriatoxin for the Schick test. Appl. Microbiol. 3:242-247.
12. Miles, A. A. 1951. Biological standards and the measuremnentof therapeutic activity. Brit. Med. Bull. 7:283-291.
13. Moloney, P. J., and E. M. Taylor. 1931. Stabilization of di-luted diphtheria toxin Trans. Roy. Soc. Canada, Sect. 52 5:149-151.
14. Permanent Commission on Biological Standardization of theHealth Organization of the League of Nations. 1931. Re-port C. H. 1056 (1) annex II series L of N. Publications,III Health m, 10.
15. Romer, P. H. 1909. Ueber den Nachweis sehr Kleiner Mengendes Diphtherie-giftes. Z. Immunitaetsforsch. 3:208-216.
16. Schick, B. 1913. Die Diphtherietoxin-Hautreaktion des
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Menschen als Vorprobe der prophylaktischen Diphtherie-heilseruminjection. Muenchen Med. Wchenschr. 60:2608-2610.
17. Seibert, F. B., and E. H. Dufour. 1954. Comparison betweenthe international standard tuberculins. PPD-S and oldtuberculin. Amer. Rev. Tuberc. 69:585-594.
18. Trevan, J. W. 1927. The error of determination of toxicity.Proc. Roy. Soc. Ser. B 101:483-514.
19. U.S. Public Health Service Regulations (42 C.F.R. Part73:2000-73:2006). Revised September 2, 1970.
20. World Health Organization. 1955. Diphtheria toxoid andSchick test toxin. Techn. Rep. Ser. 96:7-8.
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ERRATA
Mechanisms and Genetics of Resistance to SodiumLauryl Sulfate in Strains of Shigella
and Escherichia coliL. M. CORWIN, SARA W. ROTHMAN, ROSALIND KIM, AND L. A. TALEVI
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118
Volume 4, number 3, page 294, column 1, after last line, add:
ACKNOWLEDGMENTSThis investigation was supported by;the U.S. Army Medical Research and Development Com-
mand, Department of the Army, under research contract no. DADA-17-68-M-8146 under the spon-sorship of the Commission on Enteric Infections of the Armed Forces Epidemiological Board.S. W. R. was a predoctoral fellow of the National Institutes of Health (GM42132). R. K. is sup-ported by a fellowship under the National Defense Education Act.
United States Standard Diphtheria Toxin for theSchick Test and the Erythema Potency Assay
for the Schick Test DoseMICHAEL F. BARILE, ROBERT W. KOLB, AND MARGARET PITTMAN
Division of Biologics Standards, National Institutes of Health, Bethesda, Maryland 20014
Volume 4, no. 3, page 295: The title of this paper should be as above instead of "United StatesStandard Diphtheria Toxin for the Schick Text and the Erythema Potency Assay for the SchickText Dose."