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Voeono-oeditsinski Zburnal, 1961, Vol. 10p pp. 65-68.

The Use of the Indirect Hemorlatinati.n Reaotion for Detection of

By: Va•or of Med. Corps V, A. SINITSY5

(Ratered January 1960)

(Tranlasted by: Edward Lachowics, Maryland, Medical-Legal Youn-

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

The preparation of a quick method for a laboratory diagnostic

of the types A and 3 botulin toxins appears to be an actual problem.

The isolation of Bao. botulinus from foodstuffs, or from substances

of a patient or cadaver, cannot be regarded as a determinant in

the diagnostics of botulism. The only authentication can be a de-

.e4tior, (f the toxin and determination of its type. P.A. CHIRTIOTA,

5.:. hA.LEDYCHAVA, F.D. *OLMATCHIN]O and other authors tried to use

for thie purpose a rxag-precipitation reaction. However, the methcd

was nut sufficiently developed and It failed to find the acceptance.

For these reasons also the reaction of the complement fixation did

not become prevalent. S.M. MIIERVIN et al. proposed a method of

deteraLmation of the phagocytic index In order to detect botulin

toxin and this test Is about to !be approved, N....

The purpose of the current work is to discuss BOIDEN'S method

of the indirect hemoglutination reaction, which we modified for

the purpose of determination of the types A and I botulin toxins

in some foodstuffs* T. RYTSAI used this principle for the first

time In 1956 In order to determine botulin toxin type A.

The data and the imestieative mothodots Botulin toxins types

A wAd I were obtained by the following methods. We poured into a

bottle of 3 liter capacity 2.5 liters of HNOTINGE1'S broth that

contained 200 no of alt~gen smaiLe. Then, WO inserted a cello-

phane sac with 200 al of the phosphate buffer (pH 6.8) in a physio-

logical solution of table selt. To covered the broth and the

phosphate buffer with a layer of vaseline oil. The entire arrangement

wa sterilised in an autoolave. Having checked the sterility, we

Inoculated the cellophane saes with 1 ml each of a 2-day culture

of Pac. botaulius type A (strain 98), or with the type B (strain

255). This we cultivated for 7 days at 370 temperature. After this

perlod, we pumped the culture liquid from the cellophane saes,

separating it from microbic t-odies and we centrifuged It at 8,000

revolutions per minute for 20 minutes. Then, the obtained botulin

teoxn was titrated on white nice weighing 10 to 12 gm. We used for

; DL a s iLlml amount of the toxin, which, after intraabdoainal

adainistratlon In a volume of 0.5 al, caused death of mice within

96 hours. One LD part of botulln toxin type A was equal to C.000015

~ ml and that of the type 3 toxin was equal to 0.0003 ml.

The serums obtained from the [HAR]OT Scientific Research

Institute of the Armed Porces were purified by the wdlafers-3"

method of the Academy of Medical Sciences LUma we used ther- as

antibotulin serums.

or nonspecifIc antigen we used the culture liquid of Bae.

histolyticus No.o, Vibrion septique 1o.59, aec. vedematiens No.

277, Pac. perfringenm type A, Sac. sporogenes, also diphtherial

and tetnic anAtoxins. We cultivated Sac. botulinue and causative

agents of the gaseou gangrene group aocordirg to the Indicated2e

method of RIGG-'BOTSTSZ. We used for nonspecific antiserum diph-

thoeria. antitoxin from TOMgE Scientific Research Institute of the

Armed Pores*.

Sheep corusoles, .1e took sheep blood Into a sterile vessel,

of ibriiated and stored it irn a refrioerator st #41 temperature.

In the 4Uy of experiment ae aashel oreythzocytea five times with

a khytailogical solution of table ealt -'p 7.2) and we Prekared

in it a 3% suspension.

Ta1nn€.L 4.dn We prepared 0.1; solution of tannic acid in a

physiological solution of table ealt (Cp 7.n) every time prior to

experiment.

Solution of normal rabbit oeram. We took rabbit blood from

the heart the day before the experiment. On the day of experiment

we Inactivated the sertu In, a 0Ator bath at 560 temperature with-

Ir P s nt. T-j order to .void nonepecfic aegglutinins, we used

adsortion b; theeo corkjfcles. go adde4 C.5 volume if a 3% sus-

pension of sheop coryuscols to one volume of a whole set-a of a

rabbit. Then, we left this at room temperature (17 to 250) for

10 mi4teos and later centrifuged, p*mpod and diluted with a physio-

logical sclution of table aIlt (9H 7.0) at 1:100.

Research maeri al. Prior to carrying out the experiment, and

in order to avoid a nocapooific agglutination, wo adsorbed the

material by means of sheep corpuscles (two drops of precipitated

erythrooytoo per I al of investigated liquid) at repoo temperature

within 10 minutes. The suspension was centrifiged; as thw liquid

above sedimentation was separated, we made from it necessary di-

O lutjons in a solution of a normal rabbit's sor-am.

-~ 3 e--

j•neleuLni of srythrocytee with %wmiio acid. We added one

voluae of a tannic acid solution to one volume of a 3% suspension

of erythraoytes. Then, we stirred the contents of the test tubes

anl we hestel them for 10 minutes in a water bath ut 370 (er-thro-

"ytess sho-Au. settle down almost fully on the butto-c 3f a teat tube

fallow.ng a proper selection of the concoentration oO tannio acid).

Atfter heating, erythrooytes were waahed oncd with u, phyaiological

solution of table malt kpH 7.2) and resuspended in a phyoialogical

solution of table salt (pil 7.0). The reausplenoin of erythruejtee

should provide a stable bomogeneo--a suepers'..na. The pretience of

inaot cougloeerates and their rapid settl.ing on the bottom of a

test tube indioate that a larger %mokint of tannin acid was 'iseo.

Similarly tAnned erythrooytes art not *,d1table for carrying otat

an experiment, because they produce ; spontaneoiks agglutination

in the course of a basic experiment. If, however, afer centri-

fugnm, loose sediment of erythrocjt-os L3 on the bottom of a test

tube, busing them w111 lead to a sharp d reaseye of the sensitivity

if reaction. ienoe, for ueing A new consignment of tanntc acid we

recommend that its optimum :oncentration be determined in advance.

one shou,• cousider as an optimum do*e esch, with which the hemo-

6latination titer to the highest In the basic experiment with

botulin toxin smd with a speciftc serun.

esnLitisation of sheep corpuscles. To one v•lljne of erythro-

cytes we added 0.5 volume of whole antibotulin serum type A, or

one volume of antibotulin ioerm tjpe B and a fourfoll amo•int (ac-

cording to the feorm) of physiological solution of table salt

lrE 6.4). To mized this carefully and then we kept it itn water bath 9

Ink

.4 a : y%

0'a0 S.4 w' .4 00 I*--C 0VC D

0 A Nr ~ ~ 4CJO4

12 4 so1%

0 o 0lp

04 0 V4+

0a l s~ m lobt al +~*0 440 -0

00 w40 1#4 0 14 * 04 4

W.4 No 44 + ,g*4,- . 4 imm si+ + +4 4 4D

P0 bb +~ 4. ++++4

IS ~ .01 1

4* 040 4D 4* 4'0

ie 0 0 3 4 4+ *

40 P.0 0 M

S r-P4 YCk sIss I * + *4+ .+ a.dL *'m lo +1 +~ + +44 + s

.0!V4b

0'f40 0 4

r4 4001414 '-4 OV-co C4

V- V-.- 0_ 9- 0 - V- W M

MO 0.f 4o .

.94 0ma4 o.4b

.44 0.00. ~ .o

0 4ft 14 0 .6.40

at 370 for 15 ainutes. Subsequently, we oentrifuged this, washed

twi10 with a physiologioal solution of table s@lt (pH 7.0) and

"srouspeaded In a physiologoal solution of a normal seram of rabbits

In a volume equal to the volume of the tanned erythrooytes.

Auranoament of the beals exoerimenlt, We poured Into test

tubeoowith inner disaeter of 8 to 10 mm, 0.5 al each of dilution

of the examined liquid, then we added 0.1 a] each of the suspension

of seneitised orythpooytoes we stirred this and placed under thermo-

statle control at 370 until the passing time of the control "aotion.

Usually, the boooadtIon of reaotiou was possible within 1.5 hours.

BIvry investigation was accompanied by the following oontrol:

1) 0.5 al of the minimum diluted investigated material + 0.1 al

of nonseenitised sheep corpusolese 2) 0.5 al of solution of normal

serm of a rabbit + 0.1 al of nonsonsitised sheep erythrooyteal

3) 0.5 al of normal serum of a rabbit + 0.1 al of nonsentioed

sheep erythrocyte@.

So used antidiphtherial antitoxic serum for the specificity

test as a control during the examinstion of various foodstuffs, an

well as in testing soil and water.

Bioloalcal test on white aLoe. Since we worked having previous

knowlod4e of the types of botulin toxins, we did not effset the

neutr•elsation roaotion. Te made biological tests In the following

way. To Sdministored to white aloe (eoighing 10 to 12 gi), intra-

abdominally, various solutions of the extract of Investigated a&-

terual in a volume of 1 al each.

Before wo passed to the basic investigations, we checked the

epoeltielty mothsd with th culture liquid of Bec. hystolitioun ie.5, o

-6-.

Tibrion septique No.59, Bae. oedematiena No.277, Bac. parfringena

No.28 type A, Bac. sporogenes, also of diphtherial and totanic

anatoxins.

The examination revealed that the idirect reaction of homo-

glutlnation is strictly specific and it gives positive results

only with a combination of antigen with a specific antiserum. More-

over, while working with antibotulin serums types A and B (series

133 and 154), we noted a reciprocal agglutination between the types

A and B. Pollowing a specific ezhaustion of these serums by botulin

toxins, we did not observe a reciprocal reaction and, at the same

time, the hemoglutination titer of serums remained as before.

Determination of botulin toxia in artificially infected salted

horrin. A brief medical-uhomioal analysis of the herringo a slight

meell of ammonium; the litmus reaction slightly alkaline; the am-

monia reaction positive; the hydrogen sulfide reaction slightly

positive and the 9.9% content of table salt.

We out the herring into pieces weighing 20 gm each and we

administered to each piece 0.5 ml of botulin toxin type A, or

I ml of type B. We ground this in a porcelain mortar and added

for each experiment 10 ml of solution of normal rabbit's serum

and left the mixture at room temperature for 1.5 hours. Subse-

quently, we transferred the paste-like mass on a double-layer

gauze-napkin and suapended It in a 5-mL centrifugal toot tube

for a 15-minute centrifuging at 4,000 revolution& per minute. We

pumped the liquid from the teoo tube and centrifuged it again for

10 minutes. Then, we prepared necessary dilutions from the liquid

found above the sediments. In order to avoid a spontaneous aggluti-

-- e 7 --

natiOn of erythrocyteM, we heated the tissue extract in water

'bath at 56° for 2 so 3 minutes until ItS coagulation sot Ing and

them wo centrifuged for 5 aInuteo at 4,000 revolutions per minute.

the 14quid obtained above the sediment was peufeotly clear. to

plepast a nonspooelfe agglutinStion, one can uoe 0.1 N solution

of hydroohlorie aold, which Is neoessar7 to add to the extract by

drops during sonstant mixing until the Coogulation bete in. A further

preparation Is cealied out analogisally to the first method after

heating.*

To used for biologlial tests the extract from a herring with-

out any preparation. As is obvious from the results of one invooti-

gaoton (see table 1), the doteotlo of botulin toxin in a herring

Is possible with the aid of the indirect hoeoglutinstion reaction.

The method of the indirect hemoglutination reaction is more sonsi-

tive than the biologloal test on white mice. While heating the

extract that io prepared from a herring, a higher homoglutination

tiser can be obtained than by the acid method.

DeterEminton of botulin toxin In artifioially infected sausage.

we used for this purpose two kinds of sausagesO * seotionod", a nd

eauosovite", i.e. bolted and thoroughly smoked.

Weighed 5 gm portions of each kind of sausage were ground In

a poroelain mo r. We added to each tooe tube 0.5 ml of botulin

toxin type A, or 1 al of type 5, mixed it and loft It at room temper-

&*ta" for I hour. Subsequently, we added to each 10 al of solution

of a normal rabbit's serum and kept this 30 minutes longer wader

the some ool|dtions. W•hen we centrifuged It, pumped off the liquid

9

above the sediment and, after adsorption by sheep corpuscles, we

prepared from this suitable dilutions, using the solution of normal

rabbit's serum for indirect hamoglutination reaotion, and the

physiologioal solution for biological test.

The results of the examination of sausage as to the presence

of botulin tozin indicated that the method of determination of

botulin toxin with the aid of the indirect hemoglutination reaction

to also more sensitive than the biological test on white zlee.

The..ai•i•ation of cagMed areen peas and flounder canned in

t t u ao revealed pertinently to the presence of botulin

toxin that the toes of indirect henoglutination reaction Is effective

in determination of botulin toxins in the ¢ans. The metkod of the

Indirect hemoglutination reaction Is specific and more sensitive

than the biological test on white mice.

Hence, the test of the Indirect hemoglutination reaction is

htghl.j specific. Due tc the reciprocal agglutination of types A

"and B toxins, It is necessary to carry out a specific exhaustion

of the antibotulin serums. The Indirect hemoglutination reaction

permits to determine and to differentiate a type of botulin toxin

(A from B) In foidstuffs with malted herring, In sausage and in

canned goods (green peas and flounder in tomato juice). This re-

action is more sensitive than biological tests on white mice; d0-

pendimg on material, the Investigation time is reduced by 3 to 5

hours, The test of the Indirect hemoglutination reaction can be

used as a quick method for checking foodstuffs that are suspected

of ontanlAlzg botulin toxins of A and B types.

-9-

ausaian word "a robirovat"

use•an-Znglish Dictionary, Prof. V.K. WULLLR, 1949, pade 20:

bprobatsiya a approbation, approval; aurobirovat - to

approbate, approve.

useian-Rugli.h Technical and Chemical Dictionary, 1,UDbILLA

IGNATIEV CALLAHA•., 1947, page 19: aprobatelya = approbation,

approval; aprobirovat a approbate, approve.

ueeian-Lnglimh I'hyuice Dictionary, IRVING EMIN, 1,163, page 19:

aprobataiya w approbation, approval, confirmation;

aprobirovannyi - approved, tested;

a=robiroyl approbate, approve, test.