BIOCHEMICAL
Vol. 62, No. 3, 1975
ENHANCERENT OF Ifil’lUNE
RESPONSE BY THE PROTEINACEOUS
S.S.S.U.
Received
and Y.1.
Prasad
Shathna
and pharasCoIo9y Labor atory, Indian Science, Bangalore-S60012, India.
November
CRYSTAL OF
THURINGIENSIS VAR THURINGIENSIS
BACILLUS
flicro biology
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inetitu
te of
30,1974
The proteinaceoua cryatal o? &cillu& Shurinaianea var thurir&enaie maa found to anhance humoral immune reeponee in rata and guinea piga immunieed with aheep red blood cella. The enhancement uaa due to the increeead lavela of both 19s and 7s antibodiea in the sera of the treated animals. A novel eyathaaie of 7s haanolytic antibodies wae observed in case of cryetal treated animals. SUflRARY 8
INTRODUCTION Nonepacific agente like
the
immunoenhancement
lipopolyaaccharide pertucreie
(S),
plexee o? synthetic bovine
cells
endotoxine
Vaccine
methylated
killed
heat
Purified
antftuaour
agent
of
zymoean
serum
like
thurinaienei
lyaed having
This
sarcoma
into
unique crystal
larvae
(10)
we have reported with high
Eordetu
walls (6)
and the
cob
poly A and U, poly C conjugated However, little
from
is known
with
regarding
componente of baoterial
Eecherichia
&,
a well known
by the proteinaceoue
thurinaiensiq
We report
crystale
hare
of
is composed
of an alkali is highly
soluble protein
toxic
for Lepidop-
with least toxicity
for mammals(11,12).
antitumour
activity
ageinet
index for this
crystal
therepeutic
Cop.vright o 19 7.5 hv Academic Press, Inc. AN rig/z t,v 1,~‘ reprotlu~tion in anv form reserved.
produces a crystalline
the medium when the eporangium ir
a molecular weight of 200,000 which
taroua insect Recently
Eacillua
which ia liberated
(9).
(4),
(3),
4.
During eporulation materiel
oervtq
to suppress immunity (8).
found
of
B
cell
of pure protein
L-aeparaginase
by a number
Entsrobacterieceaei
the enhancement of immune response ue
ebout
of BCG(1,2),
albumin (7).
has been
brought
?rOQ yeast
nucleotidae
the immunoenhancementeffect origin.
is
517
Yoehida
aecitaa
preparation
(12).
Vol. 62, No. 3, 1975
Further
the
BIOCHEMICAL
crystallization
purification,
of the biologically Crystale
alSO
against
w88
humoral
enhanced
Yoahide ascites study in
the
(15).
PlATERIALS
AND
by
antibodies
Of
this
in
H.
toinaceous
rsjection
This
Of
protected
and
the88
animal8
detection
cell-free
On
of
from
rxtract
initiated
humoral
atUdi8S
The
immunity in
observation
pr0p8rStiOll
result.8
the
Antitumour
aura against
(14).
var thurinnionai~
ua to
immune
hsva
b88n
r88pDnSe preSent8d
serotype
I
was
kindly
Parrteur, Paris. The purs d88Cribod sarlirr (13). The proprepared freshly by aiXing the aalins such that the required concen-
de Barjac of the wa8 obtained 88 auspen8ions were
crystal mSteri81 in sterile wsre contained in 0.2
trations
tumour
achieved (13).
been
among
COll8.
the
Crystal
fhurinaieneig
by Dr. preparetion
freeze-dried
has
characterization
PlETHODS
Bacillus supplied cyetal
subunit
tumor
demon8tratSd
The preliminary
recently
with
partial
and
immunity
oarcorns cells
eff8Ct
reta.
protein
long-188ting
Chsllenge
animals
the88
active
indUo
further
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ml
of
Inetitut
suspeneion.
Wistar A/Iisc rats of eith8r seXI weighing 100-120 gms wore The guinea pigs used for obtaining cofnpleaent used immUni88tiOn. w8r8 randomly bred and maintsined in our laboratory. Sheep blood collected in Ahmver*e solution, Wa8 centrifuged et 1,000 g and washed thrice with sterile physfological saline to separate red blood CellSe fresh with sheep erythrocyter at 4 C was used guin88 pig serum, pr8adSOrbSd Inbred for
a8
the
8ource
of
complement.
fl8t8 W(sP8 immUni8ed With 3 X 10" sheep red blood cells (SRBC) Different doses of the crystal intrapsritoneally (i.p) on day zero. preparations w8re adminirtored 24 hr after antigenic etimulus 88 a sing18 or divided do888~ Dntreeted immunised 8nd treated uninmunised rat8 aeruad a8 the controls. Animal8 wers sacrificed 7 days after entigenic stimulus, blood collrcted, serum separated, inactivated at 56 C for 30 lain and storrd frozen Until further use. The dOS8 of antigen usrd and the time of bleeding were earlier found to elicit maximula antibody Antibodies in the aera were quantitatively estimated by response (16). haeaegglutination and haeololyain titration8 (IT). RESULTS
with
AND The
haomagglutiflation
the
crystals
untreated observed
DISCUSSION
showed
controls to
be
(Table the
minimum
and haemolysin titers a significant A dose
I). effective
enhancement of do88
518
of
the ower
animals the
treated
imtnunised
1 rig/Kg which was sarlier against
Yoshida
aSCite
Vol.
62, No.
Table
BIOCHEMICAL
3, 1975
AND
BIOPHYSICAL
EFFECT OF PROTEINACEOUS CRYSTAL HAEflOLVSIN AND HAENAGGLUTINATION RATS IPlMJNISED WITH SRBC
1.
Ooae o? crystal*
OF
RESEARCH
&
COMMUNICATIONS
THURINGIENSIS OF ANTISERA
Haasagglutination
Hauolyein
titer**
unite/ml+’
0.1
2560
4835
1
2560
4794
2.5
5120
9641
5
2560
4217
10
2560
3092
5+
5120
0774
5+
5120
7336
320
1341
1 x IO
x
Untreated
immuniaed
Troatad unimmuniaed l
+* +
0
Rg of crystal/Kg body wright antigenic stirrulus. Average ot six eni8al~. Ona dose/day for 5 daya.
ON fROlg
TITERS
titer
100
adminieterad
i.p.
24
hra
a?t6r
Hauagglutinatlon and haeaolyain titrations wara carried out according to Caapbmll A&. (13). The reciprocal of highoat dilution showing haaragglutination uaa taken a8 hamagglutination titer. Thr haaaolyain titration8 wara portormsd with a alight roditication uheroin the final reaction volwr uaa raducmd to 1 l l. The data warm vralyaed by Uankrogh’s equation and the haamolysin titer unit,&81 obtained.
sarcoma dose
of
than
a single
such
or
&owed
the
doss
of
breakdown
out,
a8
five
10
days,
rag/Kg.
immuniaation
with
shorad The
products the
of immune response.
enhancement
for
1 s&Kg/day
waa ruled
prior
also
(12)
might
animals
not
pronounced
that
poaaibility bring
administered
SRBCdid
more
about with
shola
A divided
the
enhancement
crystals
the
immunoenhancamant
crystals
alone
haemagglutination
or
without haeno-
lyaiso Raximun
enhancement
in
haeaagglutination
519
and
haemolyain
titers
as
Vol.
62, No.
Table
3, 1975
AND
BIOPHYSICAL
ot crystal
administration
antigenic
m--w-
24
hrs
520
were
12760
+ 40
hrs
5,120
8774
+ 72
hrs
640
1930
+ 96 hrs
640
149s
320
1553
ot six
when
crystal
whereas
mice
rat
serum
individual
tound
was
administered
i.p.
along
with
tractions
SRt3C
19s (Igm), increased
is
the
to
all
the
ot 19s
immunised
with
and
and 75 (IgG)
after
hr
antigenic
response
immune
is
SRBC
presented
of
Table
in
case ot guinea
in
mediated
G200
Sephadex
depicted crystal antibodiee
enhancement
75 antibodies,
and
and
Fig.
in treated
which
haemolysin
and
known
the 19s antibodies
that
the
ettective
haemagglutination are
on
24
3,
pige
a poor re8ponseb
cryetal
by
crystal
equally
showed
levels analysed
ot
administered
immunised be
rabbits
was
were
attect
to
whether
to increased
protiles
The
animals
and
verity
crystals
the
2).
was
due
It
weight
animals.
experimental
that
body
a*
(Table
To
7059
imunised
stimulus
The
5,120 10,240
obtained
other
1544
hrs
2.5 mg ot cry&al/Kg
titer
Haeinolyain uni ts/m1**
+ 24
Untreated
*
THURINGIENSIS
---m-w
0 hr
l
8.
-UII-..------~-~~~~~ -
animal Average
OF
COMMUNICATIONS
OF ANTISERA FROB
Haomagglutination titera+*
atimulua*
---
l
RESEARCH
EFFECT OF TIRE OF ADl’lINISTRATION Of CRYSTAL ON HAEIlAGGLUTlNATION AND HAEIIOLYSIN TITERS RATS IARUNISED WITH SAX.
2.
Time atter
BIOCHEMICAL
haemagglutination
520
ot
the SRBC immunized
gal tiltration. haemolyain 1 (a) aera
and
showed
are also
ia
immunity
elution
The
titere (b).
of’ It
enhanced a88oCfated
the
is
evident
levels with
titers. are much more ettective
in
of
62,
Vol.
3, 1975
No.
Table
3.
BIOCHEMICAL
BIOPHYSICAL
0068
Haeaagglutination titer** --w--------
ot
crystal*
--w-w-----pig
2.5 1 x
FliC8
5+
4560
5200
2086
Rabbit
1892
Nil
320
1110
2.5
160
778
320
1042
5+
Nil
160
ot guinea pigs gma
in
,
Table
Averag8 OP six animals. One
dose/day
tor
haamolysfs the
nia8d 88ra
shoved
75
than 75
contirmed
by
with
O.lM
2-fiarcaptoethanol
contaminating
gal
tiltration
heemolytic
lytic with
and the
that
treating
in the
on
used
were
400,
24 bra after ths basis ot
haemagglutination The
from
the
the
antiresults
titers. results
t'ractiona
to inactivate waa
assayed
The
same
procedure
was
that
tha
IgC
88ra
treat8d
lain
RerC8ptOathanOl
521
observation
crystal
30
synth88iS
nOV81
This
from
the
to untreated
induce 8
at 37 C tar
and
immu-
crystal-treated
animals.
fraction
8r8Sti ng 0 bSerV8tiOn
contrast
in
trsated
(19).
625
An int
th8 Crystal8
(20fiE)
5ephad8x
IgPl tractions.
(18).
activity
IgG
IgM fraction on
selected
obtainsd
antibodies
was
the
administered
antibodies
?S
sntibodias
good
haemolytic
rabbits
5 days.
immunised 88Pa, indicating of
and
1.
**
that
mice
body weight The do888 w8r8
+.
is
Swiss
642
respectively.
crystal/Kg genie stimulus. seen
2420
640
1 x
The weights 20 and 1200 * mg oP
1240
1280 5+
ON FROfl
Haeaolysin titer units/ml"*
320
2.5 1 x
hare
COMMUNICATIONS
2560
Nil
inducing
RESEARCH
EFFECT OF PROTEINACEOUS CRYSTAL OF 8. THURINGIENSIS HAEMOLYSIN AND HAERAGCLUTINATION TITERS OF ANTISERA OTHER EXPERIRENTPL ANIRALS IFlPliJNISED WITH SRBC.
Animal
Guinea
AND
removed for
waa Traction
by
haemor8peated tram
Vol. 62, No. 3,1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.8 UNTREATED 0.6
6
D.4
4
0.2
2
E E 0 0
N0 t6 F’ @
0
A
CRYSTAL
Id
.x*1
I 26
20
4.. 30
FRACTION
ELUTION
PROPILES
TREATED
-
3
36 NUMBER
Of CRYSTAL TREATECJ AN0 UNTREATED SERA ON SEPHAOEX
c200 COLlJlvlN 0.5 rl
serum Prom untreated
(a) and treated (b) animals was put (2 x 50 cm) and eluted with 0.15 M phosphete-saline bufier at pH 7.4. Three ml Praotions were collected. Absorbance at 280 rime haemolyrie and haeaagglutination titers OP the individual fraetione were determined as deecribed earlier. 0 0.0. at 280 nm, A Haemolyeis, 0 Haemagglutination titer. OP
on Sephadex
the
crystal
Igfl
Praction
sera
the
column
treated
sera
had lost
the
haemolytic
was thus
retained
the
The possibility
2-4X. for
G200
that activity
haetnolytic the
haemolytic activity
contaminating
exhibited
aotivlty aPter IgA
whereas rdth
trsatmmt
might
by IgG fractions
the
be responsible oP treated
ruled out.
These
results
suggest
crystal
oP 1.
thurinaienelp
and long
lasting
regression
the
that
the
enhancement
oP immune
may be one oP the immunity
against
522
reeponsa
way by which
Yoshida
asoites
by
tumour sarcoma
BIOCHEMICAL
Vol. 62, No. 3, 1975
is like
brought
BCG, endotoxine
i8
their
1.
thurinoieneig
ita
The major
about.
toxic
known
of
aid8
ha8t
AND BIOPHYSICAL
drawback
with
the
Enterobacteriaceaq,
8ff8Ct8 prova
toxicity
for
well
itoelt
known
Bordetella
The proteinaceoue
(20).
might
RESEARCH COMMUNICATIONS
a8 an ideal
mammalian
adjuvants
mrtussip Cry8td
adjuvant
vaccine o?
in
view
of
eyetern.
ACKNOWLEOGPENTS We thank for
their
helpful
IndU8tri81 ship
Professor
di8Cu8dOn8
Research,
to one of
Dr.
T. Aamakrishnan,
Row Delhi
end to tor
the
awarding
R. Nayak
Council
ot
a Senior
and
K.S.
Scfsntitic Research
Prasad and Fellow-
u8 (S.S.S.V.P.).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Old, L.3., Benacerrat, B., Clarke, D.A., Carswell, E.A. and Stockert, E. (1961). Gamer Res., 2, 1281-131)l. Zbar, E., Bornetein, I.* Tanaka, T. and Rapp, 3.H. (1970). Science, 170, 1217-1218. Neveu, T.;-?jranellec, A. end Biozzi, G, (1964). Ann. Inst. Pasteur, 106, 771-777. Straueeer, H.R. and Bober, L.A. (1972). Cancer Res., & 2156-2159. Plunoz, 3. (1964). 3. Immunol., 90, 132-139. Culter, J.L. (1959). Federation PrOC., & 33. Braun, W. and Nakano, fl. (1967). SCi 8flC8, a, 819-821. 8rembilla, G., Parod, S., Cavanna, R., Caraceni, C.E. and Baldini, L. (1970). Cancer Rec., 30, 2666-2670. Hannay, C.L. and Fitt-J8m88, p.C.7195S). Can. 3. flicrobiol., 1, 694.710. Hannay, C.L. (1953). Nature, 172, 1004. ToChniCel Bulletin (1969). Crop Aid PrOdUCt. 08partment. Interneti onal flineral and Chemical COrpor8tiOn, Illinois, U.S.A. Prased, S.S.S.U., Lalithakumari, H. and Shethna, Y.1. (1973). Curr. Sci. s, 568-570. prasad, S.S.S.V. ad Shathna, Y.I. (1974). 8ioahim. 8iophy8, Acta, J6& 558-566. Praead, S.S.S.V. and Shethna, Y.I. Unpubliehed data. praead, S.S.S.U. and Shethne, Y.1. 1973. Proceedings of the Society of Biological Chemists (India), a, 9. Nayak, RI 1973. Ph.D. Thesis. Indian fnstitute of Science, BafIQ8lOr8, India. Campbell, D.H., Carvey, S.J., Cramer, N.E. and Susedort, D.H. (1963). method8 in ImmunoloQy , W.A. Benjamin, Inc., New York. Sterzi, I. and Rlha, I. (1965). Nature, a, 858. DeutOh, H.F. end #o&on, 3.3. (1957). Scfenco, J&S, 6OCb06Ol. Aunoz, J. (1964). Advon. Immunol. 5, 397-440.
523
Vol. 62, No. 3, 1975
ENHANCERENT OF Ifil’lUNE
RESPONSE BY THE PROTEINACEOUS
S.S.S.U.
Received
and Y.1.
Prasad
Shathna
and pharasCoIo9y Labor atory, Indian Science, Bangalore-S60012, India.
November
CRYSTAL OF
THURINGIENSIS VAR THURINGIENSIS
BACILLUS
flicro biology
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inetitu
te of
30,1974
The proteinaceoua cryatal o? &cillu& Shurinaianea var thurir&enaie maa found to anhance humoral immune reeponee in rata and guinea piga immunieed with aheep red blood cella. The enhancement uaa due to the increeead lavela of both 19s and 7s antibodiea in the sera of the treated animals. A novel eyathaaie of 7s haanolytic antibodies wae observed in case of cryetal treated animals. SUflRARY 8
INTRODUCTION Nonepacific agente like
the
immunoenhancement
lipopolyaaccharide pertucreie
(S),
plexee o? synthetic bovine
cells
endotoxine
Vaccine
methylated
killed
heat
Purified
antftuaour
agent
of
zymoean
serum
like
thurinaienei
lyaed having
This
sarcoma
into
unique crystal
larvae
(10)
we have reported with high
Eordetu
walls (6)
and the
cob
poly A and U, poly C conjugated However, little
from
is known
with
regarding
componente of baoterial
Eecherichia
&,
a well known
by the proteinaceoue
thurinaiensiq
We report
crystale
hare
of
is composed
of an alkali is highly
soluble protein
toxic
for Lepidop-
with least toxicity
for mammals(11,12).
antitumour
activity
ageinet
index for this
crystal
therepeutic
Cop.vright o 19 7.5 hv Academic Press, Inc. AN rig/z t,v 1,~‘ reprotlu~tion in anv form reserved.
produces a crystalline
the medium when the eporangium ir
a molecular weight of 200,000 which
taroua insect Recently
Eacillua
which ia liberated
(9).
(4),
(3),
4.
During eporulation materiel
oervtq
to suppress immunity (8).
found
of
B
cell
of pure protein
L-aeparaginase
by a number
Entsrobacterieceaei
the enhancement of immune response ue
ebout
of BCG(1,2),
albumin (7).
has been
brought
?rOQ yeast
nucleotidae
the immunoenhancementeffect origin.
is
517
Yoehida
aecitaa
preparation
(12).
Vol. 62, No. 3, 1975
Further
the
BIOCHEMICAL
crystallization
purification,
of the biologically Crystale
alSO
against
w88
humoral
enhanced
Yoahide ascites study in
the
(15).
PlATERIALS
AND
by
antibodies
Of
this
in
H.
toinaceous
rsjection
This
Of
protected
and
the88
animal8
detection
cell-free
On
of
from
rxtract
initiated
humoral
atUdi8S
The
immunity in
observation
pr0p8rStiOll
result.8
the
Antitumour
aura against
(14).
var thurinnionai~
ua to
immune
hsva
b88n
r88pDnSe preSent8d
serotype
I
was
kindly
Parrteur, Paris. The purs d88Cribod sarlirr (13). The proprepared freshly by aiXing the aalins such that the required concen-
de Barjac of the wa8 obtained 88 auspen8ions were
crystal mSteri81 in sterile wsre contained in 0.2
trations
tumour
achieved (13).
been
among
COll8.
the
Crystal
fhurinaieneig
by Dr. preparetion
freeze-dried
has
characterization
PlETHODS
Bacillus supplied cyetal
subunit
tumor
demon8tratSd
The preliminary
recently
with
partial
and
immunity
oarcorns cells
eff8Ct
reta.
protein
long-188ting
Chsllenge
animals
the88
active
indUo
further
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ml
of
Inetitut
suspeneion.
Wistar A/Iisc rats of eith8r seXI weighing 100-120 gms wore The guinea pigs used for obtaining cofnpleaent used immUni88tiOn. w8r8 randomly bred and maintsined in our laboratory. Sheep blood collected in Ahmver*e solution, Wa8 centrifuged et 1,000 g and washed thrice with sterile physfological saline to separate red blood CellSe fresh with sheep erythrocyter at 4 C was used guin88 pig serum, pr8adSOrbSd Inbred for
a8
the
8ource
of
complement.
fl8t8 W(sP8 immUni8ed With 3 X 10" sheep red blood cells (SRBC) Different doses of the crystal intrapsritoneally (i.p) on day zero. preparations w8re adminirtored 24 hr after antigenic etimulus 88 a sing18 or divided do888~ Dntreeted immunised 8nd treated uninmunised rat8 aeruad a8 the controls. Animal8 wers sacrificed 7 days after entigenic stimulus, blood collrcted, serum separated, inactivated at 56 C for 30 lain and storrd frozen Until further use. The dOS8 of antigen usrd and the time of bleeding were earlier found to elicit maximula antibody Antibodies in the aera were quantitatively estimated by response (16). haeaegglutination and haeololyain titration8 (IT). RESULTS
with
AND The
haomagglutiflation
the
crystals
untreated observed
DISCUSSION
showed
controls to
be
(Table the
minimum
and haemolysin titers a significant A dose
I). effective
enhancement of do88
518
of
the ower
animals the
treated
imtnunised
1 rig/Kg which was sarlier against
Yoshida
aSCite
Vol.
62, No.
Table
BIOCHEMICAL
3, 1975
AND
BIOPHYSICAL
EFFECT OF PROTEINACEOUS CRYSTAL HAEflOLVSIN AND HAENAGGLUTINATION RATS IPlMJNISED WITH SRBC
1.
Ooae o? crystal*
OF
RESEARCH
&
COMMUNICATIONS
THURINGIENSIS OF ANTISERA
Haasagglutination
Hauolyein
titer**
unite/ml+’
0.1
2560
4835
1
2560
4794
2.5
5120
9641
5
2560
4217
10
2560
3092
5+
5120
0774
5+
5120
7336
320
1341
1 x IO
x
Untreated
immuniaed
Troatad unimmuniaed l
+* +
0
Rg of crystal/Kg body wright antigenic stirrulus. Average ot six eni8al~. Ona dose/day for 5 daya.
ON fROlg
TITERS
titer
100
adminieterad
i.p.
24
hra
a?t6r
Hauagglutinatlon and haeaolyain titrations wara carried out according to Caapbmll A&. (13). The reciprocal of highoat dilution showing haaragglutination uaa taken a8 hamagglutination titer. Thr haaaolyain titration8 wara portormsd with a alight roditication uheroin the final reaction volwr uaa raducmd to 1 l l. The data warm vralyaed by Uankrogh’s equation and the haamolysin titer unit,&81 obtained.
sarcoma dose
of
than
a single
such
or
&owed
the
doss
of
breakdown
out,
a8
five
10
days,
rag/Kg.
immuniaation
with
shorad The
products the
of immune response.
enhancement
for
1 s&Kg/day
waa ruled
prior
also
(12)
might
animals
not
pronounced
that
poaaibility bring
administered
SRBCdid
more
about with
shola
A divided
the
enhancement
crystals
the
immunoenhancamant
crystals
alone
haemagglutination
or
without haeno-
lyaiso Raximun
enhancement
in
haeaagglutination
519
and
haemolyain
titers
as
Vol.
62, No.
Table
3, 1975
AND
BIOPHYSICAL
ot crystal
administration
antigenic
m--w-
24
hrs
520
were
12760
+ 40
hrs
5,120
8774
+ 72
hrs
640
1930
+ 96 hrs
640
149s
320
1553
ot six
when
crystal
whereas
mice
rat
serum
individual
tound
was
administered
i.p.
along
with
tractions
SRt3C
19s (Igm), increased
is
the
to
all
the
ot 19s
immunised
with
and
and 75 (IgG)
after
hr
antigenic
response
immune
is
SRBC
presented
of
Table
in
case ot guinea
in
mediated
G200
Sephadex
depicted crystal antibodiee
enhancement
75 antibodies,
and
and
Fig.
in treated
which
haemolysin
and
known
the 19s antibodies
that
the
ettective
haemagglutination are
on
24
3,
pige
a poor re8ponseb
cryetal
by
crystal
equally
showed
levels analysed
ot
administered
immunised be
rabbits
was
were
attect
to
whether
to increased
protiles
The
animals
and
verity
crystals
the
2).
was
due
It
weight
animals.
experimental
that
body
a*
(Table
To
7059
imunised
stimulus
The
5,120 10,240
obtained
other
1544
hrs
2.5 mg ot cry&al/Kg
titer
Haeinolyain uni ts/m1**
+ 24
Untreated
*
THURINGIENSIS
---m-w
0 hr
l
8.
-UII-..------~-~~~~~ -
animal Average
OF
COMMUNICATIONS
OF ANTISERA FROB
Haomagglutination titera+*
atimulua*
---
l
RESEARCH
EFFECT OF TIRE OF ADl’lINISTRATION Of CRYSTAL ON HAEIlAGGLUTlNATION AND HAEIIOLYSIN TITERS RATS IARUNISED WITH SAX.
2.
Time atter
BIOCHEMICAL
haemagglutination
520
ot
the SRBC immunized
gal tiltration. haemolyain 1 (a) aera
and
showed
are also
ia
immunity
elution
The
titere (b).
of’ It
enhanced a88oCfated
the
is
evident
levels with
titers. are much more ettective
in
of
62,
Vol.
3, 1975
No.
Table
3.
BIOCHEMICAL
BIOPHYSICAL
0068
Haeaagglutination titer** --w--------
ot
crystal*
--w-w-----pig
2.5 1 x
FliC8
5+
4560
5200
2086
Rabbit
1892
Nil
320
1110
2.5
160
778
320
1042
5+
Nil
160
ot guinea pigs gma
in
,
Table
Averag8 OP six animals. One
dose/day
tor
haamolysfs the
nia8d 88ra
shoved
75
than 75
contirmed
by
with
O.lM
2-fiarcaptoethanol
contaminating
gal
tiltration
heemolytic
lytic with
and the
that
treating
in the
on
used
were
400,
24 bra after ths basis ot
haemagglutination The
from
the
the
antiresults
titers. results
t'ractiona
to inactivate waa
assayed
The
same
procedure
was
that
tha
IgC
88ra
treat8d
lain
RerC8ptOathanOl
521
observation
crystal
30
synth88iS
nOV81
This
from
the
to untreated
induce 8
at 37 C tar
and
immu-
crystal-treated
animals.
fraction
8r8Sti ng 0 bSerV8tiOn
contrast
in
trsated
(19).
625
An int
th8 Crystal8
(20fiE)
5ephad8x
IgPl tractions.
(18).
activity
IgG
IgM fraction on
selected
obtainsd
antibodies
was
the
administered
antibodies
?S
sntibodias
good
haemolytic
rabbits
5 days.
immunised 88Pa, indicating of
and
1.
**
that
mice
body weight The do888 w8r8
+.
is
Swiss
642
respectively.
crystal/Kg genie stimulus. seen
2420
640
1 x
The weights 20 and 1200 * mg oP
1240
1280 5+
ON FROfl
Haeaolysin titer units/ml"*
320
2.5 1 x
hare
COMMUNICATIONS
2560
Nil
inducing
RESEARCH
EFFECT OF PROTEINACEOUS CRYSTAL OF 8. THURINGIENSIS HAEMOLYSIN AND HAERAGCLUTINATION TITERS OF ANTISERA OTHER EXPERIRENTPL ANIRALS IFlPliJNISED WITH SRBC.
Animal
Guinea
AND
removed for
waa Traction
by
haemor8peated tram
Vol. 62, No. 3,1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.8 UNTREATED 0.6
6
D.4
4
0.2
2
E E 0 0
N0 t6 F’ @
0
A
CRYSTAL
Id
.x*1
I 26
20
4.. 30
FRACTION
ELUTION
PROPILES
TREATED
-
3
36 NUMBER
Of CRYSTAL TREATECJ AN0 UNTREATED SERA ON SEPHAOEX
c200 COLlJlvlN 0.5 rl
serum Prom untreated
(a) and treated (b) animals was put (2 x 50 cm) and eluted with 0.15 M phosphete-saline bufier at pH 7.4. Three ml Praotions were collected. Absorbance at 280 rime haemolyrie and haeaagglutination titers OP the individual fraetione were determined as deecribed earlier. 0 0.0. at 280 nm, A Haemolyeis, 0 Haemagglutination titer. OP
on Sephadex
the
crystal
Igfl
Praction
sera
the
column
treated
sera
had lost
the
haemolytic
was thus
retained
the
The possibility
2-4X. for
G200
that activity
haetnolytic the
haemolytic activity
contaminating
exhibited
aotivlty aPter IgA
whereas rdth
trsatmmt
might
by IgG fractions
the
be responsible oP treated
ruled out.
These
results
suggest
crystal
oP 1.
thurinaienelp
and long
lasting
regression
the
that
the
enhancement
oP immune
may be one oP the immunity
against
522
reeponsa
way by which
Yoshida
asoites
by
tumour sarcoma
BIOCHEMICAL
Vol. 62, No. 3, 1975
is like
brought
BCG, endotoxine
i8
their
1.
thurinoieneig
ita
The major
about.
toxic
known
of
aid8
ha8t
AND BIOPHYSICAL
drawback
with
the
Enterobacteriaceaq,
8ff8Ct8 prova
toxicity
for
well
itoelt
known
Bordetella
The proteinaceoue
(20).
might
RESEARCH COMMUNICATIONS
a8 an ideal
mammalian
adjuvants
mrtussip Cry8td
adjuvant
vaccine o?
in
view
of
eyetern.
ACKNOWLEOGPENTS We thank for
their
helpful
IndU8tri81 ship
Professor
di8Cu8dOn8
Research,
to one of
Dr.
T. Aamakrishnan,
Row Delhi
end to tor
the
awarding
R. Nayak
Council
ot
a Senior
and
K.S.
Scfsntitic Research
Prasad and Fellow-
u8 (S.S.S.V.P.).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Old, L.3., Benacerrat, B., Clarke, D.A., Carswell, E.A. and Stockert, E. (1961). Gamer Res., 2, 1281-131)l. Zbar, E., Bornetein, I.* Tanaka, T. and Rapp, 3.H. (1970). Science, 170, 1217-1218. Neveu, T.;-?jranellec, A. end Biozzi, G, (1964). Ann. Inst. Pasteur, 106, 771-777. Straueeer, H.R. and Bober, L.A. (1972). Cancer Res., & 2156-2159. Plunoz, 3. (1964). 3. Immunol., 90, 132-139. Culter, J.L. (1959). Federation PrOC., & 33. Braun, W. and Nakano, fl. (1967). SCi 8flC8, a, 819-821. 8rembilla, G., Parod, S., Cavanna, R., Caraceni, C.E. and Baldini, L. (1970). Cancer Rec., 30, 2666-2670. Hannay, C.L. and Fitt-J8m88, p.C.7195S). Can. 3. flicrobiol., 1, 694.710. Hannay, C.L. (1953). Nature, 172, 1004. ToChniCel Bulletin (1969). Crop Aid PrOdUCt. 08partment. Interneti onal flineral and Chemical COrpor8tiOn, Illinois, U.S.A. Prased, S.S.S.U., Lalithakumari, H. and Shethna, Y.1. (1973). Curr. Sci. s, 568-570. prasad, S.S.S.V. ad Shathna, Y.I. (1974). 8ioahim. 8iophy8, Acta, J6& 558-566. Praead, S.S.S.V. and Shethna, Y.I. Unpubliehed data. praead, S.S.S.U. and Shethne, Y.1. 1973. Proceedings of the Society of Biological Chemists (India), a, 9. Nayak, RI 1973. Ph.D. Thesis. Indian fnstitute of Science, BafIQ8lOr8, India. Campbell, D.H., Carvey, S.J., Cramer, N.E. and Susedort, D.H. (1963). method8 in ImmunoloQy , W.A. Benjamin, Inc., New York. Sterzi, I. and Rlha, I. (1965). Nature, a, 858. DeutOh, H.F. end #o&on, 3.3. (1957). Scfenco, J&S, 6OCb06Ol. Aunoz, J. (1964). Advon. Immunol. 5, 397-440.
523
