Biologicals (1990) 18, 11-17

Quantitative Estimation of Diphtheria and Tetanus

Toxoids. 4. Toxoids as International Reference Materials
Defining Lf-units for Diphtheria and Tetanus Toxoids7

Jsrn Lyng*
* WHO International Laboratory for Biological Standards, State Serum Institute, DK-2300
Copenhagen S, Denmark

Abstract. The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in
some countries also to follow immunization of horses for production of antitoxins, has hitherto been
defined by means of antitoxin preparations.
A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an
international collaborative study for their suitability to serve as reference reagents in the flocculation
tests and for defining the Lf-units.
It was shown that flocculation tests using the reference toxoids are very reproducible and reliable
and the WHO Expert Committee on Biological Standardization established:
-the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation
Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and
-the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation
Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.

Introduction containing the “prkcipitb indicateur” of Ramon, con-

The first potency units defined for diphtheria and tains toxin and antitoxin in the proportion of 1 Lf of
tetanus preparations were the antitoxin units. As a toxin to 1 unit of antitoxin’.
matter of fact the whole field of international stand- It should be clear from the above that whereas the
ardization of biological therapeutics by means of antitoxin unit is a basic unit defined by means of a
International Units defined with the help of Interna- standard preparation, namely as the amount of anti-
tional Standard Preparations dates back to when toxin contained in a certain amount of the standard
Paul Ehrlich in 1898 defined a unit for diphtheria preparation, the Lf-unit was originally a relative
antitoxoin by means of a dried preparation of unit, related to the unit for antitoxin. During the
serum from horses immunized against diphtheria years this relationship has become weaker and
toxoid.’ weaker. The Lf-unit has become an independent
When Ramon, 25 years later, developed the first in unit.
vitro titration for diphtheria and tetanus toxins and Today the Lf-unit for tetanus toxin (and toxoid) and
antitoxins,2934 he measured toxin relative to antitoxin the International Unit for Tetanus Antitoxin are still
(with known potency in antitoxin units defined by internationally defined by one and the same interna-
Erhlich’s standard serum). tional standard preparation but the two units are no
In 1924 Glenny and Oke115 introduced a term for a longer related. The International Standard for Teta-
new unit expressing the amount of toxin. This new nus Antitoxin defines the IU for Tetanus Antitoxin by
unit was given the name ‘limit of flocculation’ (Lf) and defining the potency of the total contents of an
was defined as: ‘amount of toxin equivalent to one ampoule of the standard preparation as 1400 IU. The
unit of standard antitoxin as established by floccu- Lf-unit, however, is defined by stating that the total
lation . . . . The “indicating mixture”, or the mixture contents of an ampoule of the same preparation is
equal to 1000 Lf-equivalents.6T7,8
t A list of the participating laboratories is found in When the second International Standard for Teta-
Appendix 1. nus Antitoxin (TE) was discussed by the WHO Expert
1045-1056/90/010011+07 $03.00/O @ 1990 The International Association of Biological Standardization
12 J. Lyng

Committee on Biological Standardization,7 the Com- hagen the solution was distributed into ampoules and
mittee also discussed a suggestion that the Lf-unit of freeze dried; each ampoule contained approximately
tetanus toxoids could be defined not by a relationship 900 Lf (see Results). The potency per ampoule in IU of
to the antitoxin unit but directly by means of a Diphtheria Toxoid? Plain or Adsorbed has not been
reference toxoid. In doing this the Lf-unit would be estimated.
made a basic unit just like the normal IUs. Diphtheria toxoid DA is a liquid toxoid, lot H, from
For diphtheria the situation is very similar, the Copenhagen. It has a purity of 2000 Lf/mg protein
only difference being that the Lf-unit for diphtheria is nitrogen.
defined by means of a separate antitoxin preparation, Diphtheria toxoid DC is a dilution (1 + 1) of DA.
the International Reference Preparation for Diph- Diphtheria toxoid DBis a liquid toxoid kindly sup-
theria Antitoxin for the Flocculation Test (DIF), the plied by the Bulgarian Research Institute of Infec-
potency of which is stated to be 1800 Lf-equivalents tious and Parasitic Diseases, Sofia, Bulgaria. It has a
per ampoule. A separate DIF has been established purity of 1660 Lfimg protein nitrogen.
because the diphtheria antitoxin standard (DI) has
lost its ability to flocculate (or perhaps never had Tetanus preparations
any?). The International Standard for Tetanus Antitoxin
Recently an international collaborative study was (TE) is the second TE and was established in 1969.6T7
arranged with the purpose of elucidating whether the The preparation consists of a number of ampoules
suggested use of two toxid preparations for defining each containing the same amount of a pool of hyper-
Lf-units would be a convenient and practical way of immune horse sera purified by pepsin treatment and
doing things. The present paper describes these absorption to aluminum hydroxide gel. This prepar-
results. ation also defines the Lf-unit for tetanus toxoid (and
toxin) as it has already been described.
The calibration in IU was made relative to the first
Materials and methods international standard. The results of the collabora-
tive study has lead to the definition of a potency of
Diphtheria preparations 1400 IU/ampoule.’ The calibration by the Ramon-
The International Reference Preparation of Diph- version of the flocculation test against the Danish
theria Antitoxin for the Flocculation Test (DIF) is the Standard for Tetanus Antitoxin for Flocculation Test,
fifth DIF and was established in 1971.’ The reference a preparation which had been used widely interna-
preparation consists of a number of ampoules each tionally for many years, showed a lower potency than
containing the same amount (1800 Lf-equivalents- the above definition and led to the definition of 1000
by definition) of a pool of hyperimmune horse sera Lf-equivalents per ampoule.6
purified by pepsin treatment and ammonium sul- The International Standard for Tetanus Toxoid
phate precipitation. The first reference preparation (Plain) (TEX) is the first TEX and was established in
was established in 1935.l’ 1951.l’ The standard consists of a number of
The International Standard for Diphtheria Toxoid ampoules each containing the same amount of a plain
(Plain) (DIXP) is the second DIXP and was estab- tetanus toxoid with a purity of 1400 Lf/mg protein
lished in 1975.i1 The standard consists of a number of nitrogen. Each ampoule contains by definition 833 IU
ampoules each containing the same amount of a plain of Plain Tetanus Toxoid. Measured in Lf each
diphtheria toxoid with a purity of 1540 Lf/mg pro- ampoule contains about 300 Lf (see Results). Origi-
tein nitrogen. Each ampoule contains by definition nally, the toxoid content per ampoule was given as
200 IU of Plain Diphtheria Toxoid. Measured in 420 Lf.
Lf, each ampoule contains about 2000 Lf (see The proposed international reference reagent of tet-
Results). anus toxoid for flocculation test (TEFT) is a purified
The proposed international reference reagent of tetanus toxoid also given to WHO by the Connaught
diphtheria toxoid for flocculation test (DIFT) is a Laboratories. The purity of the preparation is 1100
purified diphtheria toxoid generously donated to Lf/mg protein nitrogen and the toxoid was supplied in
WHO by the Connaught Laboratories Limited, Can- the same medium as DIFT and was treated in the
ada. The purity of the preparation is 1270 Lf/mg same way in Copenhagen. Each freeze-dried ampoule
protein nitrogen. The toxoid was supplied in a contains approximately 1000 Lf (see Results). The
medium containing sucrose (50/o), tryptone T (oxoid; potency per ampoule in IU of Tetanus Toxoid, Plain or
1%); monosodium glutamate (l%), and in Copen- Absorbed has not been estimated.
 LF-units for diphtheria
Tetanus toxoid TA is a liquid toxoid, lot XXXIV,
from Copenhagen. It has a purity of 1840 Lf/mg
protein nitrogen.
Tetanus toxoid TC is a dilution (1 + 1.4) of TA.
Tetanus toxoid TB is a liquid toxoid supplied by the
Bulgarian Research Institute of Infectious and Para-
sitic Diseases, Sofia, Bulgaria, for use in this study. It
has a purity of 1013 Lfimg protein nitrogen.
The f/occu/ation test
This test compares a toxin or toxoid and a corres-
ponding antitoxin and estimates an equivalence ratio
between the two complementary preparations, the
‘limit of flocculation’ of Glenny and Oke11,5the ‘preci-
pit6 indicateur’ of Ramon.
The end-point is defined as that mixture of the two
components that will show visible flocculation most
quickly. It is obviously possible to arrange the series
of mixtures of the two reactants, which must be
prepared in order to find the optimal mixture, in
different ways.
The antigen concentration can be kept constant
and different amounts of antitoxin added. This is
what Ramon did and therefore this method is often
called the Ramon method. It is also known as the
beta-procedure.
The system might be turned around, keeping the
antitoxin concentration constant and adding differ-
ent amounts of antigen. This method has been called
the Dean-Webb method,13 or alpha-procedure.
Finally it is also possible to vary the concentrations
of both components simultaneously in a systematic
way. Such a method was suggested by Levine and
WymanI and has been called the Levine-Wyman
method.
A discussion of the various modifications of floccu-
lation test is given by Lyng and Weis Bentzon.15
In the collaborative study all three modifications
were used.
Other tests
Obviously a number of other in vitro tests could be
used for estimating equivalence ratios between anti-
gen and antibody or for comparing different antigens
(or antibodies) quantitatively. Two such tests have
been investigated by Ljungqvist and Lyng, namely
the radial immuno-diffusion test (Mancini test) and
the rocket immuno-electrophoresis test.i6
Collaborative study
Fourteen laboratories all over the world agreed to
take part in the study. Assay preparations were sent
to them in the beginning of 1987. It was stressed that
and tetanus toxoids 13
the primary purpose of the study was the calibration
of the two proposed flocculation toxoids DIFT and
TEFT.
Laboratories were, however, also requested to com-
pare the two proposed flocculation toxoids with some
other toxoids by means of flocculation as well as by
means of other techniques, if such were available.
The participating laboratories are listed in Appen-
dix I. In the report, laboratories are referred to by
numbers that are not related to the order in which
laboratories are found in Appendix 1.
Results and discussion
The primary purpose of this exercise was to calibrate
the two toxoid preparations DIFT and TEFT in Lf-
units for diphtheria and tetanus, respectively.
The calibration of DIFT and TEFT must, of neces-
sity, be made against the International Reference
Preparation of Diphtheria Antitoxin for the Flocu-
lation Test (DIF) and the International Standard for
Tetanus Antitoxin (TE), respectively. These two anti-
toxin preparations carry the presently valid Lf-
definitions.
The original definition of the Lf-unit (by Ramon
and Glenny and Okell-see Introduction) was the
amount of toxin or toxoid equivalent in The Ramon
uersion of the flocculation test with one unit of anti-
toxin. Since the different versions of the flocculation
test when used for such an estimate (comparing only
antigen and antibody, not anitgen with antigen via
an antibody or vice versa) will give different equiv-
alent amountsi* the calibration of the proposed tox-
oids must necessarily be based on the Ramon version.
The results of these calibrations in the partici-
pating laboratories are shown in Table I. Only one
figure is shown for each of the preparations for each
laboratory, although all laboratories repeated the
test several times. And no indication is given of the
variation between repeated estimates within individ-
ual laboratories since this variation is so small that it
is insignificant. The figures given in Table 1 are
geometric means of repeated estimates. The figures
in parentheses are the number of estimations made in
each laboratory.
Objective estimation of an end-point in the Ramon
test is not possible. In some cases the estimates
reported by a laboratory have been used and in others
an end-point has been estimated at the International
Laboratory for Biological Standards, Copenhagen.
Such estimations have then been made by means of a
graph relating volume of antitoxin to the logio-value
of the flocculation time.
14 J. Lyng

Table 1. Calibration of the two proposed interna- the different versions of the flocculation test. The
tional reference materials DIFT and TETF in 13 differences are not significant.
laboratories using the Ramon flocculation test In Table 3 a number of diphtheria and tetanus
DIFT TEFT toxoids have been titrated by the different versions of
Laboratory No. Lfiampoule Lfiampoule the flocculation test. The differences between the
versions however, have been eliminated by using
2 1080 (6) 1168 (7) DIFT and TEFT as reference toxoids, assuming a
3 900 (2) 1000 (2) potency for DIFT equal to 900 Lf/ampoule and for
4 963 (4) 952 (4) TEFT of 1000 Lf/ampoule. In cases where laborato-
5 910 (41 1000 (4) ries did not make the titrations in such a way that the
6 880 (4) 960 (4)
7 toxoids were directly compared (using the antitoxin
900 (5) 1014 (5)
8 886 (4) 1013 (5) only as a reagent) corrections have been made by
9 896 (16) 983 (15) calculation. In most laboratories DIF and TE were
10 889 (5) 926 (3) used as antitoxin reagent. Clearly it would have been
11 855 (7) 946 (9) advantageous if laboratories had used local anti-
12 886 (3) 944 (3) toxins.
13 980 (5) 1050 (41 In Table 3 mean potencies (in Lf/ampoule or ml) are
14 931 (4) 1069 (5) given for each toxoid, each laboratory and each
Geometric mean 918 1000 version of the flocculation test. Also the geometric
mean potency for each preparation has been cal-
The number of estimations are given in parentheses. culated.
Finally the ratios DA/DC and TA/TC are given. DC
is a dilution (1 + 1) made from DA. The ratio, there-
Table 1 also gives overall geometric mean antigen fore, should be 2. TC is a dilution (1 + 1.4) of TA.
contents in Lfiampoule of the two preparations. Therefore in this case the ratio should be 2.4.
Based on these results it appears reasonable to Four laboratories also compared the toxoids by
define the two Lf-units by stating, that: means of radial immunodiffusion (Mancini) tests
(1) DIFT-ampoules contain 900 Lf/ampoule of diph- and/or rocket immunoelectrophoresis tests. The
theria toxoid; and results of these comparisons are shown in Table 4.
(2) TEFT-ampoules contain 1000 Lf/ampoule of The relative potencies obtained by these methods
tetanus toxoid. are comparable to the potencies obtained by floccu-
These figures will be the basis for the results given lation although certain differences are observed. The
below. material does not allow a closer analysis of the
Table 2 compares the results obtained by means of differences.

Table 2. Comparison of results obtained by the three versions of the flocculation test: Ramon-,
Dean-Webb- and Levine-Wyman-test
DIFT TEFT
Levin* Levine-
Ramon Dean-Webb Wyman Ramon Dean-Webb Wyman
Laboratory No. test test test test test test
2 1080 (6) 1053 (13) 1168 (7) 1126 (8)
4 963 (4) 810 (4) 855 (2) 952 (4) 1072 (4) 1007 (2)
5 910 (4) 900 (4) - 1000 (4) 1000 (4) -
6 880 (4) 900 (4) 960 (4) -
11 855 (7) 926 (5) 946 (9) 102;(3) 1065(4)
12 886 (3) 859(3) 946 (3) 990 (1)
14 931 (4) - 904 (4) 1069 (5) - 1010 (4)
Geometric
mean 927 866 932 1002 1031 1051
 LF-units for diphtheria and tetanus toxoids 15

Table 3. Relative antigen contents of various diphtheria and tetanus toxoids as estimated by flocculation,
relative to DIFT and TEFT, assuming DIFT = 900 Lf/ampoule and TEFT = 1000 Lf/ampoule

Floccu- Diphtheria toxoids Tetanus toxoids

Laboratory lation
No. method DIXP DA DB DC DA/DC TEX TA TB TC TA/TC
2 D-W - 2028 179 887 2.3 - 1010 808 329 3.1
L-W - - - 1411 984 329 4.3
3 Ramon 2000 2100 170 1050 2; 2% 720 600 300 2.4
4* Ramon 2000 2260 200 1000 2.3 256 800 640 300 2.7
D-W 1852 2000 158 1000 2.0 246 767 678 321 2.4
L-W 1954 2006 164 996 2.0 222 772 682 297 2.6
5* Ramon 2000 2000 200 1000 2.0 360 780 720 330 2.4
D-W 2000 2000 182 1111 1.8 727 751 280 2.6
6* Ramon 2120 - - - 377 778 842 275 2.8
D-W 1887 2000 169 1175 1; - -
7 Ramon 2092 2146 170 1025 2.1 273 710 6% 300 2.4
L-W 2198 2225 179 1084 2.1 350 817 727 349 2.3
Ramon 2002 2110 205 1093 1.9 258 774 680 305 2.5
Ramon 2085 2000 180 - - 410 723 - 244 3.0
Ramon 1556
D-W 1932 2433 236 902 2.1 276 681 632 312 2.2
11 L-W 2061 2138 200 1029 2.1 335 808 682 307 2.6
12 Ramon 2000 2200 200 1000 2.2 280 800 720 330 2.4
D-W 2221 2107 201 1002 2.1 277 727 653 316 2.3
L-W - - 266 656 624 315 2.1
13* Ramon aGo 2250 180 1100 2; 320 825 600 360 2.3
14* Ramon 2064 2167 177 1064 2.0 242 735 581 311 2.4
L-W 2060 2130 179 1025 2.0 244 739 586 307 2.4
Geometric
mean 2002 2118 185 1028 2-03 288
787 684 309 2.55
* Theseresults were obtained by direct comparisonwith the international antitoxins DIF and TE, but have been corrected
for DIFT and TEFT end-points.

Table 4. Relative antigen contents of various diphtheria and tetanus toxoids as estimated by gel diffusion
(Mancini) test and rocket immunoelectrophoresis tests relative to DIFT and TEFT, assuming DIFT = 900
Lf/ampoule and TEFT = 1000 Lf/ampoule
DA/ TA/
Laboratory No. Method DIXP DA DB DC DC TEX TA TB TC TC
2 Mancini 800 590 400 2-o
Rocket 1910 2700 200 1200 2.7
3 Rocket 2090 2360 150 1300 1.8 170 790 600 434 .
7 Rocket 2211 2408 172 1044 2.3 278 706 696 268 iii
12 Rocket 2800 2604 230 1180 2.2 330 890 880 360 2.5

Stability
The storage temperature for the international
standard preparations kept in Copenhagen is -20°C
or lower.
The stability of DIFT and TEFT was investigated
by storing samples at various elevated temperatures
for some time. Then these samples were compared
directly with samples from the stock at -20°C.
Titrations were made by the Dean-Webb method
and both equivalent amounts of toxoid dilutions and
the so-called Kf-values (the time from the start of the
16 J. Lyng

experiment until the first flocculation is observed), 7. WHO Expert Committee on Biological Standardi-
were used for the assessment of stability. zation. Twenty-second Report. WHO Tech Rep Ser
No indication of instability was observed after sto- 1970; 444: 17.
8. WHO. Biological Substances.International Standards
rage for 191 weeks (3.7 years) at +2O”C, 96 weeks (16 and Reference Reagents 1986. Geneva: WHO.
years) at +37”C or 32 weeks (0.6 years) at +5O”C, 9. WHO. Expert Committee on Biological Standardi-
neither by means of equivalent amounts nor by Kf- zation. Twenty-fifth Report. WHO Tech Rep Ser 1973;
values. Prolongation of the flocculation time is often 530: 11.
the first indication of degradation of toxoids. It is 10. LON. Report of the Permanent Commissionon Bio-
logical Standardization. Q Bull Health Org 1936; 5:
concluded that both DIFT and TEFT are very stable 577-578, 695-701.
preparations which can probably be kept for hun- 11. WHO. Expert Committee on Biological Standardi-
dreds of years at -20°C without noticeable degra- zation. Twenty-seventh Report. WHO Tech Rep Ser
dation. 1976; 594: 15.
12. WHO. Expert Committee on Biological Standardi-
zation. Fifth Report. WHO Tech Rep Ser 1952; 65: 5.
13. Dean HR, Webb RA. The influence of optimal propor-
Conclusion tions of antigen and antibody in the serum precipi-
tation reaction. J Path Bact 1926; 29: 473-492.
It has been shown that flocculation tests using refer- 14. Levine L, Wyman L. The flocculation test and the law
ence toxoids are very reproducible and reliable but of massaction. J Immunol 1965; 94: 586591.
other methods give similar results. 15. Lyng J, Weis Bentzon M. The quantitative estimation
The two proposed reference toxoids were found to of diphtheria and tetanus toxiods 1. The flocculation
be very stable. test and the Lf-unit. J Biol Stand 1987; 15: 27-37.
16. Ljungqvist L, Lyng J. Quantitative estimation of diph-
The WHO Expert Committee on Biological Stand- theria and tetanus toxoids. 2. Single radial immuno-
ardization established:17 diffusion tests (Mancini) and rocket immuno-
electrophoresistest in comparisonwith the flocculation
- DIFT as the International Reference Reagent of test. J Biol Stand 1987; 15: 79-86.
Diphtheria Toxoid for Flocculation Test with a 17. WHO. Expert Committee on Biological Standardi-
defined content of 900 Lf-units of Diphtheria Tox- zation. Thirty-ninth Report, in press.
oid per ampoule; and
- TEFT as the International Reference Reagent of
Tetanus Toxoid for Flocculation Test with a Appendix 1
defined content of 1000 Lf-units of Tetanus Toxoid D.D.R. Dr W. Ktinzel,
per ampoule. Staatliches Institut fur Serum-und
A suggestion for how the two reference reagents Impstoffprtifung
can be used is given in Appendix 2. Wollankstrasse 15-17,
DDR-100 Berlin,
D.D.R.
References Belgium Dr M. Turneer,
1. Ehrlich P. Die Wertbemessung desDiphtherieheilser- Institut Pasteur du Brabant,
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2. Ramon G. Floculation dans un melange neutre de Belgium
toxine-antitoxine diphteriques. Compt Rend Sot Biol
1922; 86: 661-663. Hungary Dr M. M. Adam & Dr G. Nyerges,
3. Ramon G. Sur une technique de titrage in vitro du National Institute of Hygiene
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711-712. Gyali ut 2-6
4. Ramon G. A propos du titrage in vitro du serum anti- H-l 097 Budapest,
diphterique par la floculation. Compt Rend Sot Biol Hungary
1922; 86: 813-815.
5. Glenny AT, Okell CC. The titration of diphtheria toxin France Prof. Edgar Rekyveld,
and antitoxin by flocculation methods. J Path Bact Institut Pasteur Fondation,
1924; 27: 187-200. Unite des Vaccins Bacteriens,
6. Spaun J, Lyng J. Replacement of the International 3, bd Raymond Poincare
Standard for Tetanus Antitoxin and the use of the F-92380 Garches,
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 LF-units for diphtheria and tetanus toxoids 17

India Dr S. N. Saxena, Yugoslavia Dr Miroslav Beck, Sc.D., Director,

Central Research Institute, Institute of Immunology,
Kasauli (HP.) Rockefellarova 2,
India Zagreb,
Yugoslavia
Italy Prof. Clelia Collotti & Dr Giuliano
Gentili, China Dr Xu Xi-rong,
Immunological Products Unit, Chief of Division of Serum,
Isituto Superiore de SanitB, National Institute for the Control of
Viale Regina Elena 299, Pharmaceutical & Biological Fhducts,
I-001 61 Roma, Temple of Heaven,
Italy Beijing,
China
Bulgaria Assoc. Prof. G. Guencheva, MD, PhD,
Institute for State Control of Drugs,
Boul. ‘Vl. Zaimov’ 26,
Sofia,
Bulgaria Appendix 2
U.S.A. Dr Ralph Vosdingh, How to use the international flocculafion toxoids
Technical Services, The new international reference reagents defining
Connaught Laboratories Inc., the Lf-units for diphtheria and t.etanus toxoids might
Swiftwater PA 18370, be used differently.
U.S.A. Laboratories might choose to calibrate local
reference toxoids in Lf-units by comparing such a
Iran Dr S. Aleagha, Head, toxoid with the relevant international toxoid. In this
Institut D’Etat Des Serums et Vaccins case any of the three versions of the flocculation test
Razi, or any other type of test, e.g. gel diffusion tests, and
Hessarak-Karadj, any suitable antitoxin reagent can be used. In
P.O. Box 11365-1558, routine, toxoid is then compared with toxoid using an
Teheran, antitoxin as an intermediate reagent.
Iran Most laboratories will probably choose to calibrate
a suitable antitoxin in Lf-equivalent units. Then, in
Poland Prof. M. Korbecki & Dr E. Kaczuba, the routine, a toxin or toxoid can just be flocculated in
Serum and Vaccine Research this serum, estimating the flocculation equivalent. If
Laboratory, this method is used, it is important to use the same
30/34 Chocimska Street, version of the flocculation test for the calibration of
PL-OO-725 Warszawa, the local flocculation reference antitoxin as is used
Poland routinely. Otherwise an error is introduced which will
make the results incomparable to Lf-estimations
Poland Dr D. Rymkiewiez & Dr M. Gaswinska- made in other laboratories. It would appear that for
Goloboro, estimating Lf-values for toxoid preparations, the
National Institute of Hygiene, Dean-Webb version is the most logical, since here the
Chocimska Street 24, toxoid concentration is the independent variable.
PL-000-791 Warszawa, Whichever method is chosen the international
Poland preparations should only be used occasionally to
ascertain that the calibration of local reference
Canada Dr J. C. W. Weber, material is correct.
Connaught Labdratories Ltd,
1755 Steeles Avenue West,
Willowdule, Ontario, Received for publication 22 December 1988;
Canada M2R 3T4 accepted 20 May 1989.