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TitleBIOCHEMICAL STUDIES ON THE INCREASE AND FORMATION OF ANTIBODIES WITH CHYMOPAPAININ VITRO : I. STUDIES ON THE INCREASE OF DIPHTHERIAL ANTITOXIC TITERS FROM THE PEPSIN-DIGESTED ANTITOXIC GLOBULINS
Author(s) ITO, Tokiya
Citation Japanese Journal of Veterinary Research, 2(4): 143-156
Issue Date 1955-01-31
DOI 10.14943/jjvr.2.4.143
Doc URL http://hdl.handle.net/2115/1662
Type bulletin
File Information KJ00002372907.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
BIOCHEMICAL STUDIES ON THE INCREASE AND FORMATION OF ANTIBODIES
WITH CHYMOPAPAIN IN VITRO
I. STUDIES ON THE INCREASE OF DIPHTHERIAL ANTITOXIC TITERS FROM THE PEPSIN
DIGESTED ANTITOXIC GLOBULINS
Tokiya ITO Laboratory of Biochemist1"y, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo, .Japan
(Received for Publication, Feb. 21, 1954)
PAULING has published an interesting theory on the structure and process of formation of antibodies which was developed from available informations about immunology and physico-chemistry. The prediction was made from this theory that antibodies could be manufactured in vitro from normal serum r -globulins by use of a suitable physico-chemical method.
PAULING and CAMPBELL believed that they succeeded to manufacture antibodies in vitro by mild denaturation and renaturation of normal bovine serum r -globulins in the presence of certain dyes (methyl blue and 1, 3-dihydroxy-2, 4, 6-tri (pazophenylarsonic acid) benzene) and of antigens including the type III pneumococcal polysaccharide. The precipitates formed during these procedures were considered to contain antibodies, and those authors stated that on separating the antigens, they obtained the antibodies which react speCifically with the antigens used to manufacture them. Later, FRIEDRICH-FREKSA and LOISELEUR have published the papers studying on the synthesis of antibodies. CAMPBELL has stated that these trials have been mostly negative although a few preparations gave slight specific reactivity with the antigens used.
As was the case with the much earlier works'1) reviewed in the Jonrnal of the American Medical Association, these studies do not present the full details of the control experiments necessary for a proper evaluation of their data, so that the identity of their preparations and unique "antibodies" is far from established.
The present author(~) has carried out and communicated his works on the increase of diphtherial antitoxic titers from low potent immun9 equine serunl globulins, which were obtained at regular intervals during the progressive immunization process with diphtherial toxoid and toxin and on the formation of antitoxin from normal serum globulins using enzynla tic methods.
Jap. J. Vet. Res., Vo1. 2, No.4, 1954
144 ITO, T.
Incubating the preliminarily pepsin-digested incomplete antitoxins, which were prepared from the imlllune sera, obtained at the initial stages of immunization, with addition of suitable amounts of purified oxidized chymopapain(5) and purified concentrated diphtherial toxin under suitable conditions of pH, Eh, temperature, and the presence of suitable amounts of oxidizing agents with continuous supply of hydrogen peroxide, the increase of the antitoxic titers has been proved not only by RA)ION'S flocculation test but by protective test using guinea pigs.
Moreover, incubating the heat-weak-alkali treated equine serunl globulins, prepared frOlll normal sera preliminarily containing natural antitoxins, under the essential same conditions and treatments as the above, it has been found that more or less 5% of the starting globulins is made into large molecular incomplete antitoxins, which specifically protect guinea pigs from the challenge of large amounts of the toxin.
On the other hand, preparing the normal equine globulins, those prepared from nornlal sera containing none of natural antitoxin, these trials were entirely negative.
In this paper, the studies on the ihcrease of the antitoxic titers from the prelimin.arily pepsin-digested antitoxic globulins in vitro are described. '
MATERIALS AND METHODS
1. Preparations of the Purified Native Equine Antitoxic Eu - and ,Pseudoglobulin Solutions
Five horses were subcutaneously injected daily with diphtherial toxoid solutions (Lf 20--35 u/ml) in the initial three weeks and with toxic solutioJ;ls (Lf 30~45 u/ml) for the following 5 to D weeks. Eyery weeks 300,...,., 1 ,000 ml of sera (Lf 10--1,700 u/ml) were obtained throughout the progressive immunization. Antitoxic eu- and pseudoglobulin solutions (Lf 10,.....,3,600 u/ml; prote,in-N 13",,14 mg/ml) were prepared from every pooled sera by five-repeated fractionating precipitation with 1/3 and 1/2 saturation of ammonium sulfate, following dialysis against distilled water and concentration to the volumes equivalent to the corresponding starting serum protein concentrations.
2. Pre-digestion of Purified Nath~e Equine Antitoxic Eu - and Pseudoglobulins with Pepsin
Antitoxic eu- and pseudoglobulin solutions were made down to pH 4.4 with 10 N. He]' Adding a quantity of pepsin (Merck, 1: 3,000) equivalent to 5% of the globulins with small amounts of ascorbic acid and cysteine, they were made down to Eh 200,....,250 mY. Incubating these mixtures at 37°e, the pH, Eh, amino-N, protein-N (10% trichloracetic acid precipitable) and RAMON'S flocculation titers were
Increase and FIJrmation of Antibodies 145
determined every 12 hours. In these experimental procedures, the proteolytic activity of the enzyme deceases after 72-108 hours, and a tendency of reversing turnover appears at the hour.
:3. Incubation of the Pepsin-Digested Antitoxic Solutions with Chymopapain and Diphtherial Toxin
Catching the chance of the turnover, the mixtures were made up to pH 7.3 with NaOH. Adding a quantity of purified oxidized chymopapain(5) equivalent to 1096 of the globulins with the purified concentrated diphtherial toxic solution (L£ 1,500--2,000 u/ml: protein-N 13-14 mg/ml; prepared with repeated fractionating precipitation with 8/4 to 1/1 saturation of ammonium sulfate from the culture filtrates consisting of broth and trypsin-digested beef media inoculated with Coy.vnebacterium diphtheriae P. W. No.8 Dairen for 9 days at 34°C, and further dialysis and concentration) equivalent to 10--30 times as much as the Lf titers of the antitoxin, the mixtures were made up to Eh 450",,500 m V by the further addition of 0.6% hydrogen peroxide with small amounts of cystine, fumaric acid, CoCI~ and 1"e2 (S04\3' These mixtures were incubated at 4CtC with the continuous supply of hydrogen peroxide. During the incnbation procedures, the same determinations (except only flocculation titers) as in the pre-digestion procedures were carried out every 12 hours. The highest titers of the protein-N were found in 48--84 hours.
4. The Fractionation of Antitoxin from the Papain-Incubated Mixtures When the titers of the protein-N in the incubated mixtures reached the highest,
these were made up to G096 saturation of ammonium sulfate at pH 8.0. T'he precipitates formed were collected and electrodialyzed using PAULI'S electrodialysis apparatus, finally they were concentrated to the volumes equivalent to the corresponding starting native globulin solutions. The antitoxic titers of these final preparations were determined by RAMON'S flocculation test and by protective test using guinea pigs.
5. The Determination of Nitrogen Values of the Toxin-Antitoxin Floccules
The determinations of the N values of the pure toxin-antitoxin floceules which precipitated incubating the preparations of every four progressive procedures, viz.: 0) the original antitoxic sera, A) the purified native antitoxic globulin solutions, B) pre-digested antitoxic solutions and C) the fractionated concentrated antitoxic solutions finally prepared from the chymopapain-incubated mixtures, mixing with the equivalent titers (preliminarily titrated by RAMON'S quantitative flocculation test) of the standard toxic solutions (Lf 60 u/ml; L+ 0.16 ml; toxic N 0.0004() mg per Lf) were made by Micro-Kjeldahl method, washing the formed floccules with physiological saline three times. Pure antitoxic N were calculated out by the following formula:
Antitoxic N mg = Floccules N mg _ () 00046 mg(U) Lf .
G. The Determination of the Antigenicity of Equine Serum Globulin of Each Preparations
T""''''N>rIClQ nmrl Formation of Antibodies 147
ORIG. SERA z o
TABLE. 1 Changes of Antitoxic Titers, Pure Antitoxic N mg/Lj and
Antigenicity during the Incubation Procedures
PURIFD. GLOBULINS PRE-DIGSTD. GLOBULIN FURTHER INCUBTD.
SERUM Lf EHR,- A. N. / 1 LICH S mg/Lf No. u!m u/ml
[:: --- ------ ---. . . ~ P.N. Lf EHR,- A. N. Ant}- P. N. Lf EHR; A.N. Ant.l- Lf EHR; A.N. Ant~-~ mgl 1 u/ml LICH S mg/Lf g~m- Hrs. mg/ml u/ml LICH s mg/Lf g~m- Hrs. u/ml LICH s g/Lf g~m,~ m u/ml' clty* u/ml I Clty* / u/ml m / clty*
[
-1
-2
-3
1130J-4
\
-5
10
25
45
65
1135
-6
-7 -8
150
400
500
900
-1 10
-2 20
-5 400
-8 1100
-3 1138
[
-1 10
30
150
450
-5
-7
1
-1 15
1141 -2 30
.-6 1700
15 0.0064\ 113.8 0.0067 14.4
0.0063 13.8
100 0.0053 P 13.7
200 0.00541 8.114
.2
0.0020 13.9
0.0022 l13.8
0.0016 13.9
0.00721 f 14.0
0.0074 113.8 Ps. 0.0035 13.9
0.0016 14.2
350
25 0.0065 [ 14.4 0.0052 13.9
P8. 0.0034 14.2
0.0018 14.2
10
70
150
200
550
750
1250
2500
10
20
350
2900
15
30
550
1500
0.00721 114.4 10
0.0066 Ps. 13.9 25
0.0016 13.8 3600
20 0.0066
0.0063 2400
0.0062 2400
350 0.0038 2400
650 0.0033' 2000
00017
0.0016 2400
1.0016
0.0063
0.0066 2400
400 0.0016 2400
0.0015 2400
30 0.0063 2400
0.0038
0.0032 2400
0.0018
108 13.4 10
96 13.3 70
84 13.2 150
96 13.3 200
72 13.3 550
84 13.6 700
84 13,3 1200
72 15.5 2500
96 13.4 10
96 13.4 10
84 13.4 350
84 13.8 2800
96 13.8 15
96 13.3 30
96 13.8 550
96 13.6 1500
0.0066 2400 96 13.4 10
0.0064 72 13.5 25
0.0016 2400 72 13.3 3600
20 0.0016
0.0017
0.0017
350 0.0015
600 0.0015
0.0014
0.0014
0.0010
0.0018
0.0019
400 0.0011
00011
80
64
64
64
64
80
64
30 0.0015 64
0.0017
0.0009 120
0.0013
0.0018
0.0020
0.0010
72 70 80 0.0011
96 400 00008 16
72 950 0.0011 16
72 1300 1400 0.0011 16
72 1550 1450 0.0012 8
48 650
72 1300
84 2300
84 70
72 130
72 500
84 2500
84 95
72 180
84 2100
48 1600
72 60
84 150
72 3200
00012
0.0011 16
0.0010
0.0013
0.0013 16
350 0.0012 16
0.0009
105 0.0010 12
0.0011
0.0009 12
0.0011
0.0013 12
0.0013
0.0009 12
.... ~
~ 9' ~
r 15 0.0072\ { 14.2 15 0.0067 2400 96 13.6 15 0.0019 64 84 120
1141 -4 60 0.0034 Ps. 14.2 70 0.0033 84 13.5 70 0.0014 84 190
-8 1400 00016 13.9 3800 0.0017 72 13.3 3800 0.0011 60 3500
-1 10 15 00064\ 14.2 20 0.0086 96 13.3 20 0.0023 84 160
-2 25 0.0067 14.2 30 0.0082 96 13.5 30 0.0020 72 180
1130)-4 65 100 0.0053 Eu. 13.8 70 90 0.0063 2400 96 13.7 70 90 0.0019 64 84 500 470
-7 500 0.0022 13.7 70 0.0034 84 13.6 70 00018 72 200
-8 900 0.0016 13.6 90 00030 84 13.6 90 0.0014 84 150
r 10 00072\ \14.3 15 00090 96 13.1 15 0.0016 84 120
1135 -2 20 35 0.0085 96 13.6 35 0.0021 72 210 0.0074 J Eu. 14.2
-5 400 0.0016 13.8 200 300 0.0038 2400 108 13.2 200 300 00013 64 72 1100 1200
1138 {-I 10 25 0.0065} Eu. f 14.1 10 0.0072 108 13.4 10 0.0017 84 80
l-5 150 0.0034 1. 13.8 200 0.0038 84 13.4 200 00012 84 950
1135 f -1 10 0.0072} Ps. 114.0 10 0.0063 96 10
t-5 400 350 0.0035 l13.9 350 400 0.0016 2400 120 350
1138 { -2 30 0.0052} Ps. { 13.9 30 00038 120 30
1135{-2 20 0.0074} Eu. {14.2 35 0.0085 144 35
1138{-3 30 00052} Ps. {13.9 30 0.0032 96 13.3 30 0.0017 96 20
1135{-2 20 0.0074} Eu. { 14.2 35 0.0085 96 13.6 35 0.0021 96 20
7(. The.ae values indicate the dilution degree of the preparations stating the positive limits of precipitin reaction
with anti-equine serum globulin rabbit serum.
0.0012 16
0.0011
0.0011
0.0014
0.0013
0.0012 12
0.0012
0.0011
0.0014
0.0009
0.0009 12
0.0012
0.0010
0.0060
0.0016
0.0032
0.0076
0.0014
0.0042
~ ~ ~ ~
'G:l <'I:> ~
G:l ~ R.
~ 'i ~ G:l "'" ~. ~
<Q, ~ ~ <"10
~ c R.. ~. <'I:>
.... ~ -t
148 ITO, T.
By the determination of the dilution degree of these prepared solutions stating the positive limits of precipitin reaction with anti-equine serum globulin rabbit serum, the antigenicity of equine serum globulin of those preparations was compared with at the same concentration of protein.
RESULTS
During the incubation procedures of pre-digestion of purified native equine antitoxic en-and pseudoglobulins with pepsin, pH and Eh went downwards gradually at the beginning, and turned slightly upwards in 72-108 hours. Amino-N increased to 4-6 times within these hours. The protein-N, decreasing gradually by about 5% at the beginning, displayed a slight tendency towared a reversing turnover in 72-108 hours. There were no decrease in Lf titers during the procedures.
Incubating the pepsin-digested antitoxic solutions added with purified oxidized chymopapain, purified concentrated diphtherial toxic solution, hydrogen peroxide, cystine, fumaric acid, CoCI!! and Fe!! (504):3, pH and Eh went gradually upwards, reaching the peak 48,.....,84 hours.
With the preparations, which, after the final incubation with chymopapain and toxin equivalent to 20,.....,30 times as much as the Lf titers of the antitoxic solutions, were prepared from the mixtures by the fractionating precipitation with .60 % saturation of ammonium sulfate and following electrodialysis and concentration to the equivalent volumes of the corresponding starting native globulin solutions, it was found that the antitoxic titers have increased to 4,.....,7 times as great values as those of the starting native antitoxic globulin solutions prepared from low potent immune sera (Lf 10~ 150 u/ml; 20--200 EHRLICH'S units/ml) obtained at the initial stages of the immunization (in 1",5 weeks) (Figs. 1,......,5, 9, 10,17, 18,20, 21,23-32).
Pure antitoxic nitrogen values were found:
0) 0.003 ,...".0.009 mg/Lf B) 0.0014--0.002 "
A) 0.003 --0.009 mg/Lf C) 0.0009,.....,0.0013 "
The comparative antigenicity of equine serum. globulin of those preparations showed:
B) ]/16",,]/35* C) ]/130--1/150*
*The antigenicity of every A) was signified as ].
On the other hand, with the preparations, which were prepared by the same procedures as the above, starting from the antitoxic solutions prepared from the higher potent immune sera (Lf 400--],700 u/ml; 400-1,700 EHRLICH'S units/ml) obtained at the advanced stages of the immunization (in 6",,9 weeks), no increase of the antitoxic titers was found (Figs. 6",,8, 11, ]2, 16, 19 and 22\
Incubating the purified native antitoxic globulin solutions without p~psinpre-digestion, with oxidized chymopapain, diphtherial toxic solutions and hydrogen peroxide with other oxidizing agents under the routine procedure, no increase of the antitoxic titers was found (Figs. :33--36).
FIG. 1. Antidiphtherial Serum No. 1130-1 Pseudoglobulin
13.-tI Hl>:!;
.L'I;-A
pp6;OOO ~ po-no
Go- -0 -0 D-D D D- ff D -0--0 0--0 D
e-e-e r'''-hd,-, ~-AApil
x--x--x I'h
r111
.- -.- -_,,\ nlUla ~ l\1J~ ml
pfl41 .~~ -6-6, AA A~ 1:-~ X xX -x--.X
• /0
200 m'\" X I X-X-~X-X-X--X-)(-X 20". _ _ _ _ _ _ _ _ _______ ./ ,
~O o-oon~D0~ •• -•• Ol,-,mg--.-II-.---- .-It" .--
:: I 7'2. ~u; 101'<\ ? I 4~ .:: hrs
,\"-. P(,PS[f!
HOUR-; INlllBA Tl();';
FIG. 4. AnUdiphtheriat Serum No. 1130-.4 Pseudoglobulin
:~:;o II.
20n u.
.4-4 -<\ A-A-
L¥_A--
• • D-n-o-o O-O--Q-g1:J_-O -0-°-0/9
/' / , ,
, I : ,
v- x- T.0~X- X ~,'" / I
-~--d- -L,--6-~--fr..6-~ -' /,/
/ , , I " , , ,
, I , I
, '
x- -x -x- -x-~~~_-~~~>/
-00000000
1--.· .. ·-··,···-·· .. :?·l :>1l 1'/, 2·1 4S 72 hrs
A, Pensin
7Q'1
G:,n \l
;j:;O u
FIG. 2. Antidiphthe1"ial Serum No. 1130-2 Pseudoglobulin
I 44
r A-<X '\"
- ~~, D D--nD-n-o--~j5.-o--o--o--o-iJ Q \z..
o
v _x-~-x, -A- X
X '. X X -bo- -L:;, -A- Dc -A-A-6 f( 0.
o --X-x--x--x- X--X- -X-~
o-o-o~~~~g' ••• 11 .... __ _: • .a-_ . 'm 24 4'< ---:;;---;0;;-;;,,;
24 ~8 n
A'Pepain r:
FIG. 5. Antid?:phtherial Serum No. 1130-5 Pseudoglobulin
L-Lo. • .Dr 4- -.6-~'\-:~ b.. - /
"0"0/ p -0 -0.0- -0 0 -0 -0 _o-o-"{hO
0- // '
,:/1 /"
/1/' ///
x_.)("'-Xr"-X- _x--x--x L~--6_-.-~_-...";:_~_~-- ,," /~
/,/1,
--------- --' O-o-O;;~~o/ -X--x--x--x- -x--x
IJ--IJ--IJ-_"_IJ-_"'_~_", ...... -. .--.--24 4S 72 ms.
A" PeJlsin c
FIG. 3. Antidiphtherial Serum, No. 1130-3 Pseudoglobulin
-'0-- 6. e:,.- i">o_L:>.--6--I'r-Lo.
LJ--O--oo-o-_'5-o,o- 0--0 u-D
-6.- 4 _ X x_x-x--x-->:>. ~.,c.,.6.--L>.~ ,u
" ,x-,>.;. -,)(--x -x-x- x
I I
o D oo·oo·Q ••.•• _ •• •.•••• ii .• . 7:!..>q 2\ I" 72 hrs. 2-\
A"; Pcn.,;" «(I) _
i~' Ch'"n;"p:quln To;."in
FIG. 6. Antidiphth81'ial Serum No. 1130-6.' Pseudoglobulin
L'; ."1, -~ 4·4
g -0- 0- 0 fJ
-L', 4- 4- ~
0'00_0
-x- -X'-x--X--x--x-x
-.-.-. ~-I
\, Pepsin 48 72
x--)(- _l(- X
.-.-.--.
--*hr~.
;;. C';> ~ ~
~ CO
"" ~ ;:; ~
~ ~
~ ~ N. 0 ;:;
~ ~ ;:; "... .,.,. 0-0 ~ 0". ~ CO
~ ,;:.. ~
~ ;
FIG. 7. Antidiphtherial Serum No. 1130-7 Pseudoglobulin
A _f'!.- _Dr-L\.
'-0- __ ~_L\.-' -
. -Q._o--O_O-_O-!-~o--o--o--O--O--{] 1250 u &--0--0- -0- -0--0.. '0--0-------- -- - ._0
x-- -x-- -X: -x,- -x--x
'6- -L\.-_ 6.,- -6.,- _A- -A-.r-
--x., '1\- -x.--)(.- -x--x.--x
-.-.-.--.... -.. --. .:--.-... ---'.--. 24 ~H
A' P<,psin
72 84 24 4g (0) "' Chymopapain
TOJ(in
'/'2 hrs.
c
FIG. 10. Antidiphthe1'ial Serum No. 1135-~ Pesudoglobulin
20 u.
EY.A-_~-D.--D.
a-lX -
'0- -D-a- -0--0--0- _0-.0 -0_-0 - 0 --0 --0--0 0' 0
-x- -x- -»,~x- -x .:x: '
o_~-,:::,_..::.-":'-A--A-)i ,/
"-X'-x __ x- -x- -x--)(- ~x--x
-0-0-0-00-0.0
.. -.. -.----.-.-----1-"----.-.-----2.\ .,
A,\ Pepsin
.. ;,
7::' nil 21 4~ 72 hi'S (0) .
B" Chymopapain Toxin
FIG. 8. Antidiphtherial Serum No. 1130-8 Pse_udoglobulin
t,;~u·t 0--0-- 0.·-0 -0-0---____ :- ____ _
. ----0 A- _A--A- ..A--A--A
__ A--""-0--0- -c--o--o- -g. -0- -0- -0--0- -0--0--0
--A_ -A- _ x __ x.--X- - x-x- -x--x--X A -A- A--A
x, -x-- x--x--)<.-- x
_.--It- -.... -.--.--:--.--.-.--.-.... -. __ • 2. 48 72 24 4~ 72 84 hr.
( 0) B" Chymopapain . A" Pf'psin
Toxin .
FIG. 11. Antidiphtherial Serum No. 1135-5 Pseudoglobulin
400 ll.
3;:;0 u.
A-.L>c-~~-,o.
b.-A -
-0_-0._ n_ o- -0--0--%_-0'"-0---0- -0- -0---0
_~-X-*--K x/X""
."'O'-A..~~_~~~,'
_-0 -~--~-=--~*-"-'*:~~i- -- "" - - --0-0--00-0-0-0---- - - - - - ...
_.- .. _____ -- ----f --- -... --- --- ..... 24 48
A'Pepsin
72 RI 24 48 (0)
1:1'\ 'Chymopapain Toxin
--72 hrs
.;
10 u.
:FIG. 9. Antid'iphtherial Serum No. 1135-1 Pseudoglobulin
A_-c:<--A--A ,Ly-
lX-'"
-0--0__ lX-O--o--o--o--n--s. ,o_..o--o--a--O---D--a
'-~-~-~--A--A- x- _x_x--x--x--x- -x--x -A--A-~
.• 'X- -x- -K- - x- -x- -><:- -x--x
24
A' Pepsin
9G 2cl 4S (u)
B'\ Chymopapain Toxin
,/)J
R4 hrs.
FIG. 12. Antidiphthm'ial Serum. No. 1135-8 Pseudoglobulin
20f}O If 00-0-00.0-0- __
..cr_~~-A:~4 1:><-'" D
-0. Q. -Q. -0- -0- 0- %.0- 0-- -0-0- -0- -0- -0
A-6-4-A-A X'K'-*-K-~--~--x 6-~,
-x- -J(.. .,)(--~-x- -x--x
_ ......... .-............ .. 24
(I, '\ Pepsin
72 R I 21 4S (0)
B'\ Chymopapain T()xin
12 81 hl'~
~
~
1-1 >-l SJ>
~
FIG. 13. AnUdiphtherial Serum No. 1138-1 Pseudoglobulin
aou
,.6<
4-L:r~ -A
ft {J.. -0- n- 0- -0- -0 0-(J,P -0- 0- -0- -0
A .0-.0-rrAJ"
• .x -X ~ - "Y-'--3: .l«,X / Y
,4.-A-A..bbA-A-~/' / / / /
/ / / /
/ / / I
/ / / /
/ / - >: / /
~-~~~:..~~~ ... /
15 u.(j}() -00-000-0-~ .......... . ...-..... ..-.... .... ... 24 4H 72 '(2 M hrs
A"Pepein
FIG. 16. Antidiphtherial Serum No. 1138-1 Pseudoglobulin
_ -O-_~ --:A .-6-A- -A--0
J(iOU n.(I}- - - - - - - - - -- --<;;fA< ~
-n -0 ..Q. -0- ..Q- -0- -o-..Q -0 {J- ~,O---a.. Q b
A--A--A-A-A~·-AA~.X_X--X.--x.- -K X
-x- .}(_ ¥-K-X-¥-x X
........... -:-.. --..... 24 .j, 72 72: hrs1
A'Pepain
FIG. 14. Antidiphther·ial Serum No. 1138-3 Pseudoglobulin
11) D.
o ~~~ nU~nuaO{J~.Dc~no-.D
g~frD " I
I I
I f
x-/-:x:-~X--A x
~ ~ A. -A- A-A. -A-~/
~ -X -«- ,X-~ x- -X- .x / OQO-OQO.o-D
l<"
I
I
I (
... - . .......... --.-... . ..... ... 2'-1 48 n 7~ l"1 hra
A' Pepsin c
FIG. 17. AnUdiphthe1'ial Seru,m No. 1141-1 Pseudoglobulin
a--Cr -6
A,6'Ls:-
'~ ~' '0_0--0--0--0--00 .0--0--0--0--0
0',0'
.X ¥:>- ~-b - -b-A 4- ..A-~'
-oX- - -x- X---X-x- -x- -x-.;>(
_-x--x--x ;x:,X"
/0
/'
, "
A. .-..... '\.J -00-0-00-00' ..... -. . -.... - ,.-.. -.-.. -
~-t .t~ 7"2 96 24 -l'j i~ hrs.
A'\ Pepsin c;
FIG. 15. AnUdiphtherial Serum No. 1138-5 Pseudoglobulin
9 / \
I \
! ' / \
I b A A'&'"
p..Dcr ~
A'~ / \
~~U~fiO-~n ~o-bfrGG ~ 0-.0 / 0'0
I !
I
'C>...A-.AA-AA-A-~X1-X-XX*~X I ' X
:'50 u, tTl- - - - - - - - - - -d
.. ........ ~...... --.-
48 72 R4 96 19:5 hrs.
A'\ Pepsin W\, Chvrnooanain C
FIG. 18. Antidiphtherial Serum No. 1141-2 .Pseudoglobulin
a_LS _A---6--LSA2 -o.u-n--o--o--if "
0.0- -0- -D- -[J---o-~-O
{
I
_x--x--*-x--x.--x'x /
'A-~-A-'A-A-A I
"'X--x- x-'X' -X--X /
I
I
I
2" ,,([}-O O-oOO~_ • .a_ ••••• .. --. •. ~- -.- ' 4~ 72 ~4 hrs .
A'P<2!-'sin c;
~ C':I ~
'" ~ <:4
'" ~ ~ R.
~ "i
~ ~ , .... ~. ~
~ ~ ~ ~ 0-C R. ~.
~ ~ I-'
/ /
FIG. 19. Antidiphtherial Serum No. 1141-6 Pseudoglobulin
3600u h-o.-oo-o-o-o. _____ _
I ~:2:2 ..b-.a Lr~
"0. - 0.. -0. -u- .Q- - 0
c-.o- -o-.Q- -0- -0- -0
-A. _~......-*,*-l( -4-4. )t'-K
..A..-A-....o.
....................... ............. 48 72 24 48 72 84 hr •.
i 0)
A "p&pSin B'\ Chymopapain Tnvi ..
FIG. 22. Antidiphtherial Serum No. 1144-8 Pseudoglobulin
3800 u. O-O-OOOO-________ -q",
-A,-A--b-":>'-->4,D .L:>: :l:> LS
-0--0- -0- -0-0-0 ..0--0--0 -0- -0 '0 0--0-
"'.,)( -~ -x -~-x '>(
x' 'A--A-~-AA-A
.. -..-........... .... -.. -... 48 - 72 84 hr •.
A' Pl;'psin c
2011
FIG. 20. Antidfphtherial Serum No. 1144-1 Pseudoglobulin
at'
L!r..::.-.o.-Q-A zr--A
.0.. -nn. .c...o G -O-D '""-- -0- -0- -0- -0- -0 o--D-~
9 I
I
oK- '* ,,-1t' ~ )<~
x I ><' I
n...~Q..~o-A-4A I
I I
I
I I
I I
I I
I
I
o-oo-o-oo-~"'" -- •• -... -............. ..
24 48 72 72 84 .hr!
A' Pepsin
FIG. 23. Antidiphthm"ial Serum 'No. 1130-1 Euglobulin
o -a. A-AAA-Ar-A--/>.
-0.-0. A / -D--O--Q-D---Orr..rr-rr-c--o--<;r-o 0" 1 /
I /
I /
..x--kK-x.-X ,..x'x- / .
~ )( I
--A. A. 6-4-4-A"4 /
"'-x- -X_-.l<_x-*-x--x--x
I
I
1
0-00-0-o-ooi ..... ___ .... -11 . ....... ......... ---24 4l! 72 72 S4 hrs.
A'\ PC!)., .. ,
70u
FIG. 21. Antidiphthe'l"ia.l Seru.m No. 114/;-/; Pseudoglobulin
o / '0
tt /'
-A_=-=--u--n-fA-~-l:I. ~ I
-D- . I
-0 0- -0- -0-- 0- -c -0- -0--a: -0- -O-.Q.-o V-0 ,-
I / ! I
I I
,~_-X--X--)H<-~ ~ x->E-/
- &--D,. -6-":::"".0<.:::.--A /1
OQ-0000<:1
''X-*-x--x--x-x-x . .... -............. ~ ---.... -... --
24 48 72 72 96 hra.
A" Pepsin
FIG. 24. Antidiphtherial Serum No. 1130-2 Euglobulin
30 u
0 1
I I
A-A-4~ pA I
-t.J.. -D- -D- -0- -0- 0- {]_ g 0-.0- -a-- -0-, In- U 0-
f
X' ,J(-)(- -x. ~ ~,x- I
A_A-A-A-A-A-6-~ /
-x -x-x- -x -x-- * .x..')</ o 00--0 Oo-o--c)
f
I
.-... - .......... :----.---- ........ !?4 ~8 n 4S 7:! hrs
A,P~psin c
'"'" ~ l~
H ~ ~o
~
:'11 , AI) u
15u.
FIG. 25. Antidiphtherial Serum No. 1130-4 Englobnlin
o I-II
-0 II -0- -Ir-Q--O--Q- -0- 0 .a-Q,o-tro-~
. a- -<,>-6'~""-<>;" -"<\
L':r" /1
/ I
I
II II
.i-><.'-x.-·x-~ U'-44-4-4-~--4.4 x·J(,J
X' " , ~
" " --X--X-~'~-~"X"X''''' /' -------- --.:.- .. -f'/ 00-0-000-0-<'
~ .. -......•...•... o ~ ~ n % ~ a UMbra
(0 I A'\Pepsin H'\ ChVlnopapain
TO:"l:in
FIG. 28. AntidiphtheraiZ Serum No. 1135-1 Euglobulin
A -LT ~ -I': -A-.6 8.
'-0- LX -DUD-D--O.-o.-B':.a.D-D-fr -Q--O- -0 0
0-
q
.. x--K .X' -x- x/~x--x x--X F/
;'
'1<- 'X--X-7(- -k'X- -x.--x
. ----.. -.. -.... ~tl; 2,~ 40.; f"',1 hrs.
\" A,j'cfx;.in Cn:m'lpa)Yll!l C
FIG. 26. Antidiphtherial Serum No. 1130-7 EuglobuUn
<;:
/
&"'L:r~/-4-A L:r~ t
'0- -0- aD- -o-u- -g.-o -0- -0-, h- pO ,
I:~K·>(--){-}( -)(-~ ...
-..-, >< -.<1-_6-~-O--.«..c. ;'
,!)uffi-o-oO-Q-Oo-O
-x- -)(- x- ·X--K--X- X
...... -.. -....... : .. --.. -.. ~ 48 72 84 24 43 72 bra
A, Pepsin (0) B' Chymopapain
Toxin
FIG. 29. Antidiphtherial Serum No. 1135-2 Euglobulin
o I
I , I
" -L>-..(:".--6 ~-Ls".LY I'
5 f
-0- -0- -0- -0- -0--0- -0--0 .... ,1..0--0 -0.-07'
0--0- /
I
:~~-.x--x X ,.x:r' x-- ,
-6--.c.--D.-A-6-A--4-4 ,/
~ -x. , ~--x.~.x·X·-X;: :55 u <D-O{3.o-ou-000
, ,
.. -.--.-.. .... -•.. 11- -...... -. .-.-.
2,1 48 72 % 2,1 48 72 hrs,
A'\ P<'n"'.f", c
FIG. 27. AntidiphtheriaZ Serum 1180-8 Euglobulin
fHI u.
No.
-A--4A -L::t;:-LY-A" 0
L't;-A '
'0---0-_ -D--G--o-D--g-O __ cr--O--D~9'-o--D
, -x--x--x·-x > ·x·X"
0-0- O-o-O-oO',X->--6-", __ c.. 6--<'. __ L'-._~
"'X--)(--x- -x- -x--x-)':
L .• --.-.--.-. -.-1--"-·--. -.--. 24 I~
J\'\l'ePljn
n 84 24 48 (G)
IS ..... Chymop;.lpain Toxin
-a--. 7'2 84 hr::i.
FIG. 30. Antidiphther1~al Serum. No. 1135-5 Euglobulin
9 /~
/ / , ,
c;.c>.a~ -7f -C,
" u // - -0. -D-D--D- -0- -0--0- -0 _.0;:0-0- -0
0. 0 ,-0 "
/' I
/x·-x·-x-x .x--;),f.' x'" If
~ ,~' '"4-6--A-4-.c'.-<CI..-4- A I:'
;1
" " f'
f'
300U .• -- -. - -.- -- --- - - •••••• /
'3~0::-oM~~~-~
• ••• . ..... ~ .. ~ -.. 24 48 72 9G 108 24 48 72 hr •.
(0) -
B" Chymol;apain C Toxin
A'\ Pepsin
~ ~ ~ N ~ ~
N ;::l R.
~ ~
~ N <:-ioo
b' ;::l
,0 --., ~ ;::l No
~ o R. ~' ~
I-'
~
FIG. 31. Antidiphthm'ial Serum No. 1138-1 Euglobulin
lOu
I::s;~
.A~~ boA
«J. -0. 0 -0--0-on -0-D -0-0-0-.0- -0 0-.0--0"
~A.A.A.A.66..o.~
A"Pepsir.
X"**.)( xx-K"
9
.f
48 12 ~4 brs.
c
FIG. 34. Antidiphtherial Serum No. 1135-5 Pseudoglobulin
_--~------&- ---- -6-------6.-----_-A
-- - --0-- --- --0--- - - - -0-----.--0- - -- --{J
._- _--~ --.-- -x--- ---~----- -~- --- --x 350u
---- --- ------0
------... ------.-_._- .------24 48 72 % 120 hr8.
AB" Chymopap.in C Toxin
FIG. 32. Antidiphtherial Serum .No. 1138-5 E1.tglobulin
200 u
o f
I I
L!tcC>£i:rA -AL!tc I
.LS I £f
Q.Q-.Q-nOD-{J 0-0-00-0 n-O" I
Er " I
.. iK·J<:'~-K ~>t I
>( I I
~A-A6~ I I
I I
~et>M
/ I
.. -.-.-. ........... -. ----... 24 48 72 84 24 48 72 84 hrs
(0 ) .\ "Pepsin Il" Chymopapsin
Toxin
FIG. 35. Antidiphtherial Serum No. 1138-2 Pseudoglobulin
-- - --A-----A------A------A~ ----4 --- --0-----0------0-----·-0--- __ 0
- -- -- -x.-----:.--- - -- )(..---- -*------ X
3jl u.\Ij-- __________ - ------____ ---------4.
---~----------~----~----.. o 24 48 AB, Chymopapain
Toxin
n 96 120 hra C
FIG. 33. Antidiphtherial Serum No. 1135-1 Pseudoglobulin
~- -6.- --.c.---O---~--A- --6.---6---4 --0---0---0---0---0----0---0-_-0
_______ *------ -x-------x---- _ ~
·10 u.!n-- - - --- -------- --- - - - --- --0 _._ .. __ .. ___ • __ • __ • ___ -e- __ iI
o 24 48 AB" Chymop&p&in
Toxin
72 96 Ilrs_ C
FIG. 36. Antidiphtherial Serum~ No. 1135-2 Euglobulin
35 u.
----0-----0-----0 -----0---_-0 _____ -0 -----A-----4-----A-~--4 ____ A- ___ %:\
----*---.---------x----- -.)(- ---:)(-----X
----------------------0
I It---~---------~---~----~---~ o . 24 48 12 96 120 . III hrs. AB'I. Chymopapain
Toxin C
~ at .;:a
1-4 ~ ~O>
!"3
Increa8e and Formation of Antibodies 155
Incubating the pepsin-digested antitoxic globulin solutions without the presence of oxidized chymopapain under the routine procedure, also no increase of antitoxic titers was found (Figs. 37 and 38\
FIG. 37. Antidiphtherial Serum No. 1138-3 Pseudoglobulin
~--~--~--~-~~
o.-o--o-a--e-00-g._-a---O----O --a
--1<--~---x---x
SOU --O--Q---o---0--:------_ -0 . ..---.--.. --.. ----- --.--.. -- --.
o 24 84
A" Pepsin
72 96 2,1 48 (0 ) U'\ Toxin
72 96 hrs.
e
DIRC'URRION
FIG. 38. Antidiphtherial Serum No. 1135-3 Euglobulin
A--~- -.C>r--A--A
''(1..-- -0.---0--- "8 __ -0----0----0---0
-- A--.or __ 4- __ ~--*---x--- -- -)<:
--,x---x- .. x---)(
35u. --0---0---0--0.,--0 _________ 0
96 24 4R 72
A' Pepsin «()) B' Toxin c
The present data indicate the fact that fragmented small diphtherial antitoxins produced from the large incomplete antitoxin molecules with pepsin digestion, those produced in horses at the initial stages of the immunization with diphtherial toxoid and toxin, are converted into small complete antitoxins by incubating procedures with suitable amounts of oxidized chymopapain and toxin, under certain definite convenient conditions of pH, Eh and temperature in the presence of oxidizing agents.
It is considered that the fragments, produced from the large incomplete antitoxins with pepsin digestion, are converted into small complete antitoxins being affected by the antigenic action of the toxin and polymerizing action of the enzyme in the further incubating procedures.
Also, it is considered that the fragments, produced with pepsin digestion from the small complete antitoxins (antitoxic N 0.0016--0.0020 mg/Lf), those produced at the advanced stages of the immunization, have no other spaces to be converted into antitoxin newly in the further incubating procedures.
156 ITO, T.
It is concluded that incubating the fragmented antitoxins produced with pepsin digestion from large incomplete antitoxins produced at the initial stages of the diphtherial immunization, in order to convert them into small complete antitoxin molecules, it is essentially necessary to prepare antigenic activity of toxin, polymerizing activity of enzyme under the suitable conditions of pH, Eh, temperature with supply of hydrogen peroxide and other oxidizing agents.
SU:\DfAH.Y
Purified eu- and pseudoglobulin solutions of equine diphtherial antitoxic sera obtained at the initial stages of the immunization, were incubated with pepsin at 37°C, pH 4.4 and Eh 200--250 m V. In 72-108 hours, pH was adjusted to 7.3, and the solutions were incubated further mixed with purified oxidized chymopapain, purified concentrated diphtherial toxin being supplied hydrogen peroxide and other oxidizing agents at 40°C and Eh 450-500mV. In 4g--84 hours, when the protein-N values reached their highest, the mixtures were made up to 6096 saturation of ammonium sulfate at pH 8.0. The formed precipitates were electrodialyzed and finally concentrated to the volumes equivalent to those of the corresponding starting native globulin solutions. The antitoxic titers of these preparations were found to be 4 to 7 times as great as those of the each starting native globulins by RA:\lON'S flocculation test and by protective ~est using guinea pigs. 'fhese preparations, prepared by these procedures, are consisted of largely small but complete avid and less antigenic antitoxins.
The author wishes to thank Prof. Dr. Koji ANDO for his aid and helpful suggestions, and
Dr. K. MANAKO and Dr. R. OYAMA for their technical assistance.
REFERENCES
1) ANONYMOUS, (1942): .T. Am. Med. Assoc., 119, 1376.
2) CAMPBELL, D. H. (1948): Ann. Rev. Microbial., 2, 269.
3) FRIEDR.ICH-FREKSA, A. H. (1946): Z. Naturjorsch., 1, 44.
4) ITO, T. (1950): Med. & Riol., 17, 42 (Japanese).
5) JANSEN, E. F. & A. K. BALLS (1941): .T. Riol. Chem., 137, 459.
6) KABAT, E. A. (1943): .T. Immunot., 47, 513; KABAT, E. A. (1949): Experimental
Immunochemistry, Springfield, Illinois.
7) LOISELEUR, J. (1947): Compt. rend. Acad. Sci., 224, 687.
8) PAULING, L. (1940): .T. Am. Chem. Soc., 621 2643.
9) PAULINC. L. & D. H. CAMPBELL (1942): .T. Exp. Med., 76, 211.