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Zhubrn. Mikrobiol., 33, June 1962, pp. 77 - 82. J- -- - - - -- --- - - - - - - - - - - - - - - - - - - - - - - -

St'uadee on Some Factors Influencing the Reliability of Botuline

and Tetanus AnatoziA Titers According to the Flocculation Reactiol

Bys A.A. VOaROBSV and Z.A. VORONOVA

(Received September 28, 1960)

(Translated by; Edward Lachowicz, Maryland, Medical-Legal Foun-

dation, Inc., 700 Pleet Street, Baltimore, Maryland)

T he probloa of a possible determination of antigenic proper-

ties of toxins and anatoxir.a by simple, quick and economical sero-

logical methods has attracted attention of investigators for a

long time. Yet, in spite of very many reports written on the

subject, the serological methods and, particularly, the floccu-

lation reaction have not yet encountered any broad practical ac-

ceptance in development of tetaniA and botuline preparations. This

can be explained by variances occurring in the results fro& the

titration of seeujs and anatoxins in vivo and in vitro, as well

as by a lack of unified opinion pertinently to the nature and

mechanism of serological reactions.

Only a few authors who studied the flocculation phenomenon

(RAMON, GLHNY, S•IRTOVNXKOVA and GOROKHOVI•IOVA, PAPrENI•IEIMR)

assumed that the formation of visible flocculent Is conditioned

by reaction between the toxin and anatoxin. On the other hand,

many investigators (BROU•IBRENUR and REICHERT, ZINGIHER, 13LZR

and KOVACS, et al.) explained this by reciprocal action between

the exolusively bacterial procipitinogens present in a toxin and

9 beoterlal preelpitia that form concurrently tith the antitoxin

#.

- - -- - - --. --l II I -I Il I I

during Immunization processes.

Contradietoa7 opinions also exist about the question whether

the flooculation resotion is a true index of the antigenic value

with resard to tetanio and botulino anatoxins. According to con-

eluslios of RAMO#, PRIGIGINA, VOLXOVA, VOROEZV, KOLESNIKOVA and

$0OIOV, the antlgnlo properties of botuline and tetanic anatoxins

are directly proportionsl to the flocculation act-vity. BRONP•N-

SMM and UICwT, also NIGG, ]KHOTTL et al., KOERBER, ?.ICE,

PALL==, etc. do not emphasize the same conformity.

Contradiotory results obtained by various investigators in

using the flocculation reaction are explained by a lack of standard

flocoulation sorums (KHALYAPINA and iIXUOLS•AYA, SHAIN). Every re-

searcher umes arbitr•aily selected serum to effect the reaction

sad, at the same time, the selection of serums for flocculation

is made by empirical method out of dosens, even hundreds of thoere-

poutiLo oozsm%

In stuay•,4i a possible utilization of the flocculation reaction

in order to determine antigenic proportieo of botuline toxins type

A, B and Cp also tOeanio smatozins, we attempted to clarify the

reason for incogrutoes found in many Instances of flocculation

liters at snatzins and their antitoxin binding activities with

the intent on finding objective criteria for selection of floccu-

lation serums.

We determined the flocculation capacity of crude and concoent-

ate&d retaic and botalune anatoxins by using refining and con-

.eatr•t•on methods of combined dialysis and also the Odieofew-30

Ma"o4 with eut•ittano sand antibotulleS howse serums (with -iters p

__________ _________

-- 2 -

wj

from 4,000 to 16,000 AN in I ml), diluting then to 100 AR '.finti-

totanic serum), or to 1,000 AE (antibotuline serms) pez. 1 ml.

The method to resolve flocculation reaction comprised the

following schedules into a number of test tubes re poured serums

F -in inoreasing amounts, beginning with 0.05 ml and successively

with 0.1 ml interval during the preliminary experiment, then with

0.02 to 0.04 ml in the subsequent experiment (in order to obtain

more precise results); we added 0.5 to 1 ml of anatoxin to each

test tube (undiluted anatoxin ir. the preliminary experiment; di-

luted to 200 Lf-dose in subsequent test); we stirred the mixture

of serum and anatoxin and then loft it to incubate at 450. Iext,

we recorded the time of the initial flocculation appearance (Lf).

The antigen concentration (flocculation titer) was expressed in

Lf in accordance with the following equation (VARKOVICH, KHAUSTOVA,

CHIBOTA.M3VICH):T - c n'

where y is the antitoxic titer oA' serum; whore Yc is the flocculation

dose or serum, namely the volums of diluted serum in which initial

flocculation was obtained; where m is the dilution of serum; where

n is the dilution oa anatoxin.

Our findings revealed that the minimum amount of anatoxin we

could determine by the flocculation reaction was 50 to 100 Lf for

botuline and 5 to 10 Lf for totanic anatoxins.

The antitoxin binding capacity of anatoxins was determined by

titration on white aice, according to the method of TARASEVICH'S

' State Control Institute, with the use of expanded control series

-- 3--

in eiperimental dose of the toxin (MARKOVICH and VOROREV). The

"0410lt were expressed In binding units (SC) computed am a per-

ocealege flB

At the time of the seleotion of flocculation serums our at-

teation was usually centered on clearness and rapidity of the

floooesu"Sn 1A a single narrow gone. This, however, often proved

to be Imsuffioient. One should always take into account the ex-

Istenoe of absolute quantitative correlations between the an.-

toxiss' titers as revealed by Lf according to the flocculation

"raeOtiOa an by 30, according to the antitoxin's binding reaction.

If the flocculation reaction is specific, the correlation of IC/Lf

for each anatozin should be equal to one unit, provided that 1 IC

and 1 Lf are equal to 1 Al.

Out of 15 botuline and antitetanic sCr-was we selected 5 as

the fastest flooculating substances. The correlation of ZC/Lf equal

to ome unit was obtained for one serum only (type A serum, series

10.33) out of 15 snatoxin serie* used in the experiment (see Table

1).

The magnitude of 30/Lf in remaining serums varied considerably,

msils y toward exaggeration of the flocculation reactioi' s results.

UsIxg as an example entibotuline serums type A, series No.33

and 1, we tried to clarify the reasons why one serum (series No.33)

induces the obtainment of reliable flocculation resalts from all

snatoxln used In the experiment, while another serum (series No.1)

]Woduoes exaggeration of the Lf value in other section of anatoxins.

In studying a possible determination of antigenic properties

of botuilne sntoxins according to the ring-precipitation reaction

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(TORONOTA), we concoluded that reliable results can be obtained only

when antibodies to the heat-resistant bacterial precipitinogens

are absent in the serum, since they condition the group specifici-

ty of a strain. We obtained such type-specific precipitating oerums

after inoculation of rabbits with microbic cells of a strain of

other serological group (according to agglutination reaction), but

not of a strain used for the preparation of anatoxins.

We assumed that the dependability of the flocculation titers

proceeds from the factor that in this reaction and in the precipi-

tation reaction participate baoterial (group) precipitinogens -

procipitins In addition to specific antigens and antibodies (ana-

toxin - antitoxin).

The investigations proved that flocculent antibotuline and

antiTetanic serums contained In addition to the serum series No.33

also agglutinates and group precipitins (1:8 and 1:16) in higher

titers (1:2,000 and 1&5,000). As long as botuline anatoxins and

serums of the A and B types have been prepared with the use of

strains that contained usual group precipitinogens of microbic

cell, we observed a cross flocculation between the serums and ana-

toxins of these typeso). With depletion of the serum type A by

anatoxin type B and, vice versa, type B serum by the type A ana-

tozin, we were atle to obtain serums flocculent and free from group

precipitins, but they fully preserved their antitoxic properties.

The freeing of type A serun, series No.1, from ,roup precipi-

tine was accomplished In the following way: we determJned the opti-

Ser.ms of A and B types were monovalent and contained no ad-

mixtures of antitoxin of other type.

-6-

___________________

0

mum quantity of serum and anatoxin B in the cross flocc2ulation

reaction, then we mixed equivalent quantities of serum and antigen

and, after separation of precipitate, we placed the test tubes in

a refrigerator; next day we separated the sediment by centrifuging

action and we used the suprasediment fluid as the flocculent serum

type A.

The invetigstiond proved (see Table 2) that type A serum,

series No.1, freed from nonspecific antibodies, flocculated with all

anatoxins type A in a zone that agreed with their antitoxin' a

binding activity (IC/Lf* 1), while no such conformity was observed

prior to depletion. It should be added that depleted serum floccu-

lated considerably slower and, at the same time, a delicate and

thin flocculate was formed.

We also obtained good results from type-specific, precipitated

rabbit serums that contained no group precipitins; they coincided

with the titration results on animals (see Table 3).

Following a mixing of botuline anatoxins types A and B witb

purely antibacterial precipitating serums that contained no anti-

toxin, the precipitate separated immediately after addition of

anatoxin, i.e. simultaneously in all test tubes regardless of the

quantity of serum.

Hence, we assume that the flocculation phenomenon, observed

after mixing anatoxins with suitable antitoxic serums, is a com-

bined effect that is conditioned by the reaction of antitoxin and

by concurrent reactions of bacterial precipitates - precipitinogens.

Taking into account the effect of the purity of serum (presence

Q of precipitls in it) on the flocoulation "eaction's titer, we de-

-- 7-1

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cided to examine the Importance of the purity degree of anatoxin

with relation to nonspecific ballast proteins and the products of

decomposition of cells, as this affects the eagnitude of the ana-

toxzn's flocculation titer with a given serum. Pigure 1 shows a

change in the ratio of ZC/Lf, relevant to 26 series of tetanic

a a Purification degree of anatoxins (EC/mg of common nitrogen).

Figure 1 - A change in the ratio of EC/Lf for tetanic anatoxlnswith relation to their degree of purity.

anatoxins concentrated by the same method and plotted against a

degree of their purity (IC in 1 mg of common nitrogen). With the

advanced degree of anatoxin's purity from 90 to 300 ZC per 1 mg

of common nitrogen, the ratio of EC/Lf increased gradually and

approximated 1. Subsequently, the ratio remained constant and

equalled 1 for anatoxins that attained the purity degree of 300 EC

per 1 mg of common nitrogen, or higher. This rule has been recorded

in connection with botuline anatoxins of the A, B and C types.

The discussed findings indicate that, in using a serum which

oontainls bactorlal precipitins, the flocculation titers, that agree

with the results of the antitoxin's binding reaction, can be ob-

served only when onCoentrated enatoxins Are sufficiently purified.

-- 10 -

In crude botulin*e anatoxins we encountered at least 10 times

higher concentrations of group precipitinogena per I E0 than in

concentrated anatoxins per 1 IC. Thus, as a rule, we observed two

flocculation zones in crude botuline anatoxins. The actual zone

represented a smaller quantity of the flooculent seum•; it appeared

later than the false flocculation, which along with its appearance

involved a greater quantity of serum.

The investigation of the flocculent and antitoxin binding

activities of crude botuline anatoxins of the A, B and C types

/obtained from cultures of diverse growth periods (4, 7, 15 and

30 dayag revealed that the Lf magnitude of the false flocculation

zone increased 5 to 6 times with the process of the culture's

o growth, also parallel with the increase I.n the concentration of

the group precipitinogens.

Obviously, with the concentration of anatoxins, a partial

freeing of specific antigen takes place from group precipitin-

ogens and this leads to a shifting of the false zone of floccu-

lation toward the actual one; consequently, the flocculation that

started in the false zone overlaps the actual flocculation zone.

In such case, only one, the initial flocculation is registered

with the exaggerated values of Lf.

Hence, the results of the anatoxins' titration are influenced,

according to the flocculation reaction, b) the presence of bacterial

precipitins in flocculent serums and by pertinent bacterial antigens

in anatoxina.

The reactions formed between them are analogous to a specific

Q r'eaction of anatoxin, but antitoxin distorts the results of the

-- 11 --

-- - - -

latter. Consequently, in arranging the flocculation reaction one

can 004 use such serums, which are freed from bacterial proopi-

tins. According to our opinion, thnem serums can be obtained by

Inoculation of sanmals with antigen substances that contain no

bacterial group preoipititnoens, namely with preparations that are

produced from a *train of other serological group than the strain

used In the production of anatoxinz, or, as is customary, from

highly purified, but not orude, anatoxins.

Por the same reason one should use a highly purified concent-

rated anatoxin &a a staLdard antigen during titration of antitoxic

serums according to th& flocculation reaction.

Conclus ions

I-, Inasmuch as seraum used in the flocculation reaction are

obtained by inoculation of animals with antigen that has been ac-

cepted for the production of anatoxin, the results of the floccu-

lation reaction, essentially specific, may become distorted on

account of the concurrent reaction of group (bacterial) precipi-

tinogens - preolpitins.

'Z, Therapeutic, antitoxic, antibotuline serums can be used

as flocculents, if they are purified and concentrated according to

the Odiaferm--0 method, and if they are not of agglutinating strain

that has been accepted for the production of proper anatotins. The

ratio of 3C/L. to the serum in question should equal 1 even for

lest purified anatoxins.

3 The titer of botuline and totanic anatoxins conforming to

SUte :flocculation reaction from the same flocculent serum depends

up" a degree of the anatoxin's purity, namely on the antigen content

-12--

0

per I mg of common nitrogen.

Literature Cited

VOLKOVA, Z, M. Zhurn. Mikrob. 1937, vol.18, No.1, p.56. - VO-

ROBV,A.A. Purified tetanic anatoxin, sorbic by aluminum hydroxide.

Candidate's dissertation. Leningrad, 1951. - VOROXOVA,Z.A. Zhurn.

Uikrob. 1960. No.4, p.94. - KOLESNIXOVA, M.H. and SOKOLOV, S.K.

Zhurn. Wikrob. 1958, 10.3, p.92. - KBRTOVNIKOVA, V.A. and GORO-

HOVNIXOTA, A.I. Hygiene and Epidemiology. 1929. No.3-4, p.90. -

&UKOVICH, A.T. and VOROBEV, A.A. Problems in Medical Chemistry.

1955. No.2, p. 10 2 . - NKMOVICH, A.V., XHAUSTOVA, I.N. and CHZBO-

TAESVICH. Zburn. Mikrob. 1951, No.9, P.53. - FEIGINA, S.YA. Zhurn.

Niorob., Patholog. and Infect. Diseases. 1929, vol.6, No. 3-4,

p. 138. - XHALYAPIRA, X.T. and NIXOLSKAYA, V.A. Book: Problems

in infectious pathology and immunology. Moscow 1949. No.1, p. 2 2 4.

- SHAI, IBA. Books Problems in the production of serums. Moscow,

1960, p.65. - BRONPNBRIN, J., BEICHERT, P. Journ. Rxper. Ned.

1926, vol. 44, p.553. - ZISLRR, U. and KOVACS, N. Zbl. Bakter. 1,

Abt. Orig. 1926, vol.99, p.518. - GLIMY, A.T., STEVENS, M.P. Journ,

Roy. Army Ned. Corps, 1938, vol. 70, p. 308. - XKORBSR, W. Journ.

Immun. 1943, vol.46, p. 391. - NIGG, C. et al. Journ. Immun. 1947,

vol. 5 5 . p. 245. - PAPP3NHMIW, A.M. Journ. Bacter. 1942, vol. 43,

p. 273. - RICZo C.,, SMITH, L.C., PALLISTUR, I.P. et al. Canad.

Jours. I ea. (3), 1947, vol. 25, p. 1 75. - RAMONA,G. R ev. Ias.nol.

(Peais), 1956, vol.20, p. 2 8 9. - ZINGHBM, A. Proo. Soc. Zxper. Blol.

(N.Y.)# 1925 vo1. 22, p.454.

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