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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

https://eprints.lib.hokudai.ac.jp/dspace/about.en.jsp

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

~ ,;:.. ~

~ ;


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 .


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


_ -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-'

/ /


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 ..


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


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


_--~------&- ---- -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


-- - --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


~- -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\


~--~--~--~-~~

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


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.

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