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A STUDY OF THE HEMOLYTI C ANTIBODY—ANTI GEN COMB INATION
D I SSERTATI ON
Submi t t ed t o the D irector
in Conf ormity wi th the Requirement s for the D e gre e
of D oc t or of S c i enc e in Hygiene .
Hobart W . C romwe l l
1 9 2 2
Tabl e of C ont ent s
I . Introduc t i on .
I I . The C onc ent rat i on of the Hemol yt i c
Ant ibodi e s as a Fac t or Influenc ing
the ir Ab s orpt i on by Red C e l l s . 6
I I I . The rat e of the Reac t i on be twe en the
Ant igen and Ant ib ody , in v i t ro .
IV . The Influenc e of Temperature on the
Ab s or pt i on of Ant ib ody by Red C e l l s .
V . The Re l at ion of the Gl obul in C ont ent of
the Immun e S erum t o the Ab s orpt i on of
the Ant ibodie s .
VI . The Re l at i on be twe en the Abs orpt ion of
the Ant ibodi e s and the Tit er of the S erum .34
VI I . S e l e c t ive Ab s orpt i on . 7
VI I I . Varia t i on of the Ab s orpt i on wi th the
Length of the Per i od of Immuni t y .
IX . D i s cus s ion .
X . Summary .
X I . Li t eratur e c i t ed .
X I I . Bi ography .
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A STUDY OF THE HEMOLYTI C ANTIBODY-ANTI GEN C OMB INATI ON.
I . Introduc t ion .
Ac c ording t o Ehrl i ch ( l ) , a formed ant i gen,such
a s a red c e l l or bac t e r ium , has a de f ini t e number of com
b ining groups which have an affini t y for the ant ibody .
When the ant i gen is plac ed in contac t wi th its ant ibody ,
a reac t i on ensue s , which pro gre s s e s unt i l al l the s e bonds
are s at i s fi ed or unt i l al l the ant ibodi e s have ent ered
int o the c omb ina t i on . I t fol l ows from thi s that when al l
the c omb ining affini t ie s of the ant i gen are s at i sfi ed , no
more ant ibodi e s can ent er int o the reac t i on re gardl e s s of
the number that s t i l l remain fre e in the supe rnat ant
fluid . Thi s the ory impl i e s chemical affini t i e s and a
c omb inat i on acc ording t o the re s pe c t ive val enc ie s of the
reac t ing sub s tance s .
The Vi ew of Arrhenius was o ppo s ed t o thi s . He
regarded the re ac t i on as phys i cal and expl ained the
phenomenon as a di s t ribut i on of a s olut e b e twe en two
s olvent s,the ant ibodi e s be ing the s olut e ,
and the c e l l
prot o pl asm and the surrounding fluid the two s olvent s . He
c onc luded that " the immune bodi e s are probabl y not bound
by the erythro cyt e s but onl y ab s orbed by them? , and that
" no proof has be en given of the ir chemical ac t i on ."
~no o i o medmuu edimii eb n and tmfl imei osd 1 0 [ Leo new a as
.vbodi ims end moi vi in i l ie as evad doidw equemg ga inid
, Qbo dtina ci t fi d iw Jos in o o mi beeniq ei seni imnv edd d edw
bemeiue even seibofli ims ed i I I s i tinm d o ho tl a tis a eds
i is nsdw J edi a tdi momi emefioi i i .no i isn tdmoa end o in i
on .beri ai inu 81 3 meqti ns en} l o eeii id tfii s gfl ifi idmo o end
t o aaefbas,ed moti oned end o in ? d efi ne use eeibod tins emom
insisn i equa efli mi eemfi n iemem Li l i a Jed i medmnm ed :
3 has sei i iu ii fi s I s oimedo sei iqm} vwoedd a I dT .biui l
ed: do ae toceisv sv ti oeqeem eds o i gn tbfi o ooe do idafl idno o
.eeomei adn a gu teosei
eH .aidd o i beaoqqo sew euifi edd dA To wc tv edT
ad d bem ts I QEe Ems t ( S) {so fagnq as no ti oaem odd behasgeq
owi neewied eiu io a e do mo tiwdimi e tb s an monemoaedq
{Leo and has Qs i xties ed: ga ted aetbodi ifis add .eineyiqa
eH .eimev £ca owi ed! btmtl ga tbnnOd e add ban mes iqodomq
finn ed ion vidsdoaq ems eeibcd enmmmt edi"J edi bebu ionoo
dad: on e .?mefi i Yd bedmoeda g imo J ud BSSVOOTfi JVfi e edd md
Borde t 's idea al though not the s ame as
that of Arrhenius , was s imi l ar in that he at t empt ed t o
ex pl ain the phenomenon ac cording t o phys ical l aws . He,
however , held that the ant ibody or ” s ens i t i z ing sub s t anc e "
is ads orbed in much the s ame way that a fi l t e r paper
t ake s up a dye. He s tat e s that " the uni on of the ant i
body wi th the ant igen de pends on what is cal l ed mol e cul ar
adhe s i on or c ontac t aff ini t y , in other words ,should
b e c l as s ed in the ca t e gory of ads orpt i on phenomena ."
I t has l ong be en known that a red c e l l or bac
terium wi l l c omb ine wi th much more than the amount of
ant ibody ne c e s s ary for l ys i s . Arrhenius found that
the amount of ant ibody t aken up increas e s w i th the
c onc ent rat i on ac c ording t o a defini t e phys ical l aw ,
which he expre s s ed by the equat i on ,
B = K Cn,
in which E re pre s ent s the amount of ant ibody ab s orbed
by the c e l l s,C the c onc ent rat i on remaining in the
supe rnatant l iquid aft er ab s orpt i on , and K and n are
c ons t ant s . He found the va lue of n t o be and from
the equat i on and the cal cul at ed value of n , he int er
prat ed the reac t i on as a di s t r ibut ion of the ant ibodi e s
es emr e edi J on flnuod l is sebi e'debdod
oi beiqmci in ed J adJ mi asiiml e eb w ,euinedwd A i o ind:
, eH .ews I I soievdq o i nnibqosos sensuo usdq an: nfsiqx e
”eonn iadus an ia l J ianee
"d o wbodiims edc 38n d b l ed ,
wevewod
aeqaq mei iii a dear vaw ensa , admdoum mi b udmoabu at
-23ns end l o nc tmu odd" Jedi eeda ie eh .egb s qv saint
sa l voeiom betise ai Jsdw ad abqeqem h eating and di iw mbo d
b l o ods , abd ow weflio o i,gi indfiis mosinee 1 6 noi eedhs
”.smemoaedq mo tdqmoebs i s vi oaeiso and Hi beaaa io an
bnuoi an imoq A .eievi moi was aeeoen vbedidms'
di tw'
eeesemoni qu nefln i zbod tine in income edd
[sci agdq ei in ii eb a ct anibwo o os mo tisqiaaano o
.no& 5ru pe end wqbeseeiqx e an do tdw
and at ax inismem no i iemJneonoo add 0 ,ee c add vd
- 1 eiai ed .m is es I nv beiaiuoiso efi a has s o icsupe edi
aetbodi ins‘
sdd is moidwd id ieib s as mo tioned edc ‘beismq
be twe en the red c e l l pro t opl asm and the surrounding
l iquid as s ol vent s , and that two of the ant ibody mol e~
cul e s fre e in the s erum form thre e of the c omb ined
mol e cul e s .
Manwaring’s work (4-8 ) on thi s s ame phenomenon
fai l ed t o c onfirm the c onclus i ons of Arrhenius . He
found tha t the ab s orpt ion did no t fol l ow any s impl e
phys i c o-chemi cal l aw , and that K and n were no t c ons tant s .
Manwaring obt ained what he cal l s a " ne gative ab s orpt i on" ,
i .e., the t i t er of the s erum di lut i ons , when great c on
centrat i ons of ant ib ody uni t s were us ed,was oft en great er
aft er c ontac t wi th the c orpus c l e s than before . He c on~
el uded ,therefore , that qual i t at ive change s take pl ac e
in the amboc e pt or due t o its c ont ac t wi th the cel l s , and
" that any dire c t quant i tat ive c ompari s on be twe en it and
the unt reat ed s erum give s errone ous re sul t s ."
From the s e and other experiment s , he c ame t o
the c onc lus ion that there is a ”third c omponent " in the
s erum be s ide s the ant ibody and c ompl ement , which vari e s
in quant i ty in different animal s . Thi s third c omponent
may be " ant i l yt i c"
, or " aux i l yt i c"
, but neve r has inde
pendent hemol yt i c powe rs , al though it may b e ab s orbed
by the red c e l l s . In addi t i on , he ment i oned s eve ral
o ther fac t ors influ enc ing the ant i gen-ant ibody comb i
gn tbnuo1 mue ed: has mes iqodomq [ I eo 5 6 1 ad: neewied
« e l em vbodtins ed: do ow: Jedi has , einev £oa as bJUp I I
bemtdmo o ed: to sedd i mmoi mumee en: mi s emi s e ine
.ee[u oe£om
aomemonedq emse aid: no ( e-e) flmow s’gm imewnsm
eH .euined1 1 A To eno reuiono o ed i matinee o i be l ts?
eiqmte vme wo f toi J oe b ib noi iqd oede ed: J ed: bm moi
.sias ienoo J on emew n has H Jedi has , we £ I so l nedo~o o l avdq
,
"no tiqi oeds ev l iegeu
"e eI I eo ed dedw benieido gn iqswnsM
~HOO $39 1 3 nedw .ad oi iui tb mu wee ed: To 1 9 31 3 ed:, .e.1
d edeed g aei l o sew ,beeu s t ew s i tuu vbodi ims 30 sao iiewineo
a n oe eH .emoi ed mad: es I oeuqx o o ed: di iw J ceinoo media
eoq efie i eeqnsdo ev i is J i I evp Send te'x o
‘ied edd , fiebu [o
bee , ei ieo ed: di tw foeimoo eii 03 sub d oiqeoodms edJ at
bus 31 neew ied d oetmsqmo o ev ti si timsup J oei l b was Jedi ”
”.eiiu eeq auceuomd e aevtg mamas beised inu ed:
03 ems o ed , aimemideqx e medi c baa eeedi m a
edi mi"J meuoqmoc bards“ s at emed: Jedd notau I Od oo ed:
seix sv doidw Q inemeiqmo o bus vbodi ins ed: aefi ieed muses
Jdenoqmoo bqidi et .aI emide inei eii ib at vi i iaaup mt
- em and waves Jud d o ,
"ei ii Jme
”ed vsm
bedx oeds ed vsm i t dauodi ts .emewoq o tiviomed inebneq
I si evee bemoi inem ed ,ao ti ibbs HI .e I I eo bed ed : vd
- 1 dmoo vbodi ins~aeg t$us ed: an i omeu iint emo i oei medi c
- 4
nat i on , among whi ch are the reac t i on and s pec ifi c gravi t y
of the medium and the amoun t of inorganic s al t s .
Amat o in a re c ent s tudy of o ps oni c s ens i
ti z at i on of bac t eri a , has c ome t o the c onc lus ion that
the uni on of the o ps onins wi th the bac t e ria i s governed
by the s ame l aw that governs the uni on of hemol ys ins wi th
the ant igen . He found the s ame equa t ion appl ic abl e t o
the o ps onins which Arrhenius a ppl i ed t o the hemol ys ins
and c onc luded tha t the reac t ion is probabl y a distri
but ion of the o psanins b e twe en two s ol vent s in which
they have different mol e cul ar we i ght s .
C oul t er ( 9 and 1 0) has donefiz
ome very defini t eW
work on the influence of theAmedium on the ab s orpt ion of
hemol yt ic s ens i t i z er by red c e l l s , and on the di s s oc i at i on
of the c ombinat ion . He found pH t o be the opt imum
H- ion c onc entra t i on for the ab s orpt i on . However , he al s o
found very l i t t l e differenc e in ab s o r pt i on be twe en the
value s, pH and pH in the s al t- fre e medium ,
and a much w ide r range in medi um c ontaining s al t .
Kahn in a very rec ent work on the ab s or pt i on
of hemo l yt i c s ens it i z er , has made s ome s tudi e s on the
ra t e of the reac t i on a t the vari ous t emperature s . He
finds that the reac t i on i s compl et ed very qui ckl y ,in
every cas e at the end of fift e en minut e s . He al s o f inds
- 5 _
" tha t the extrac t i on is grea t er at 37°
than a t room
t emperature , whi ch in turn ‘ i s grea t e r than a t ice—box
t emperature ."
I t is s e en then tha t the ant igen- ant ibody com
b inat i on is influenc ed by t ime , t emperature , the reac t i on
of the medium , and the amount of inorgani c s al t s . In
addi t i on , there i s al s o c ons iderabl e var iati on due tz o
uncont rol l ed fac t ors , whi ch vary wi th the different l o t s
of s erum us ed tho s e variat i ons whi ch caus ed the
differenc e s of o pini on b e twe en Arrhenius and Manwaring .
In the fac e of al l tha t has b e en done on the
ab s orpt ion of hemo l yt i c ant ibodi e s by red c e l l s, it
would s e em almo s t ho pe l e s s t o t ry t o add anything new,
e i ther in fac t or the ory . However , it is thought that
the re sul t s of the ex periment s rec orded in thi s paper
are s i gnifi cant in sugge s t ing an expl anat ion of the
differenc e s ob t ained by Arrhenius and Manwaring , wi thout
the ne c e s s i t y of re s ort ing t o the sub-divided " third
component " ,o r to any qual i tat ive change s in the ambo
c e pt or due t o i ts c ontac t wi th the red c e l l s .
In al l the di s cus s i ons the t erms ," ambo c e pt or
"
,
" ant ibody" ,
" s ens i t i z er"
, and"immune body” , wi l l be
us ed int e rchangeabl y .
moom is asdi°TE J a veisei n ei u c1 308 1 3x e add J edJ
”
x od~eet is send esteemg si‘ nmui n } fi c tdw .edwdwwegre8
ef
zrfiw teqmei
—mo o {bodi ifi o—ueef +fi s and J edi nefi i uses at J ?
n ! .eJ I ee c innamon ! l o imuome end bme.eu ihem add i n
ad: be aune doidw eno iie twev eeofi d a been und ee do
.3n rmewmeM fine eu l nefi v neewded nc tmiqo do aeofiewei l ib
efli no en0b se e d set Jed i Tie 30 seal eni .n I
n! term tskt
dndi ddauoni mi 3 ?,mevewofl .vwosfi i 1 0
meqsq sin} n} Lebanese aimemimeqx e end i s ei iueem end
end 2 0 not iennq e as gn iieeggma mi dnnoii inn te ed s
J uoni iw ,3miwewnem bus euinefimv n gd benis ido eeon ed ei i ib
bi rds“
bebtvi bm dua end 03 antid oeed To wi tsseoeu anJ
.aiI eo D8116fi d fi d fw i onimu o si t on sub d eiqeo
.”do tqooodme
“eds sno ieauoein and i ts n I
ed I i iw ,
”wbod saumn i
"has ,
"vbod tdns
"
- 6
Ex pe rimental .
I I . The C onc ent rat i on of the Hemol yt i c Ant ibodi e s a s a
Fac t or Influenc ing the i r Ab s orpt i on by Red C e l l s .
The s e ex periment s were carri ed out in order t o
de t e rmine how many t ime s the amoun t of s ens i t i z e r
ne c e s s ary for hemol ys i s the red c e l l W1 1 1 ab s orb, and
whe ther or no t there is a defini t e s atura t i on po int ,as
mi ght b e as sumed from Ehrl i ch 's the ory,above whi ch the
red c e l l s wi l l ab s orb no more , re gardl e s s of the amount
s t i l l remaining fre e in the s erum .
The t e chni c was a s fol l ows : Rabbi t s and guinea
pigs were immuni z ed t o the red c e l l s of the she e p , by
i ntrape ri t oneal inje c t ions of the guinea- pi gs and intra
venous injec t ions of the rabbi t s . The s erum s o obtained
was inac t i va t ed a t 5 6°C. for 30minut e s and then care
ful l y t l trated agains t fre sh she e p c e l l s that had b e en
col l e c t ed in one per c ent . s odium c i t rat e and washed four
t ime s in l arge volume s of normal s al t s olut ion . The uni t
of c e l l s was arbi t rari l y cho s en as cc . of a
sus pens i on (measured in t e rms of whol e bl ood ) , and the
uni t of c ompl ement as c c . of a di lut i on of fre sh
Al l dilut i ons and sus pens i ons were made in
normal s al t s o lut i on . A sus pens i on of ce l l s means
one part of c e l l s plus thre e part s of s al t s olut i on .
d ex tj rsnee l o Javons ed: samid imam sod en imasisb
bus,J moeds l i ce [Leo bed edd siegfomsd d o ? x t seeecen
er , id ieq mo idsmudse edimtieb s 5 1 ed ema J on d o wed isdw
edd dc tdw ev ods ( Ymoefi i s'doi ifi dfl ma x i bonu s es ed idqim
income end 1 0 eeetbwsgea Qad o rn-
on J acads i f iw a I Leo bed
mumes ad d at sea? gd l niemed [lids
yr?
“w eds at “‘
to eEI ee bs‘
l mm oo‘
bee -grinned R‘THW efl iq
- s win i bus ag iq—senjug end i s ano ti oetmr I seno j iaeqsmdfl i
bea tsddo o s amuse odT .ai iddsa adj i s sec ti es gui QUOK®V
ne e d bad jsdi ei feo g esda deemi den tsgs bc3s 1 5 1 3 Q I I ui
"
sci bedesw has edst i fc mu ibo e .J weo d eq ems mi beicei ioc
a s\ i a i s .os as mesons g itmsmi idds sew arise 30
and bus , ( bo o id eicdw l o ammo ? mt bemusasm ) nei snsqau s
deed ? i s no i dfi tzb 8\ f b To .00 £ .O an snemc iqmoo l e' dimn
-8
Prot o co l 2 . Type for Ab s orpt i on of Hemol yt i c Uni t s
by She e p Red C e l l s .
number per c c .
1 CO.
a Al l the s e t s were made in dupl icat e .
es Se t s 1 1 and 1 2 are c ont rol s .
we re arranged , each c onta ining two uni t s of she e p c e l l s,
and s ens i t i z er in varying amount s . Enough s al t s olut ion
Was added tc 'each tube
,bef ore the sensiti z er
,to make the
final volume two cub i c c ent ime t e rs . The tube s we re
incubat ed in a wat e r bath for 50m inut e s at 37°
c. ,aft er
whi ch the red c e l l s were removed by c ent rifugal i zat ion
and the superna tant flui d t i t rat ed as in Prot oc o l 1 ,t o
de t e rmine the’
number of uni t s of s ens i t i z e r l o s t, i .s . ,
the number removed by the red c e l l s . The us e of al l the
mat erial s c onc erned in the reac t i on in only twi c e the
amount ordinari l y us ed for t i t rat i on overcame the
ne c e s s i t y for the us e of an undue amount of immune s erum
and s t i l l afforded suffi c i ent supernat ant fluid for the
sub s equent t i t rat i on . The me thod is shown in Protoc o l 2 .
Tab l es I and I I show the ab s or pt i on from immune
rabbi t and guinea- pig s erum in c onc ent rat ions ranging
from 5 t o 2 , 000, uni t s pe r cub ic c ent ime t e r . On the
fir s t l ine of e ach t ab l e are shown the c onc ent rat ion s
wi th whi ch the c e l l s were t reat ed , and on the fol l owing
l ine s are shown the amount s of ant ibody taken up by the
ce l l s from each c onc ent rat i on in the t e s t s made with the
different l o t s of s erum . S ome of the re sul t s are al s o
shown gra phi ca l l y in Fi gs . 1 , 2 , and 5 , in which are
, e [I eo qeeds is eaten ow: sh ia ts inoa dos e .begnsams ad ew
no isuioa dies dguoaa .ainuoms gn tzasv mi aesi i ieaee has
ed: eism oitx esi iienee eds st oi ed
eedut dose OJ babes sew
st ew BedUJ edT .sve3emt3neo o l duo ow: smufov Inu it
ve il s is seduh tm 08 t o ? died messw s at bedsducn i
ao iJ si sgui tvdneo vd bevomea emsw‘
si ieo has an: doidw
03 , I I ceodcaq at as bedsud ti btn ii ddsdsnveqns ed: bus
, .e. 1 ,J so I vex idissee to sJ inu to vacuum ed: en tmqsdeb
odd [ I s To esu ed? .ai £eo D Q T ed: zd bevomea d edmun ed:
ed: eoiw i al so m t no tiosam ed : ni , bequsoneo aisimeism
ed : smacaevo no i ismdid not been z ii a sn tbao Janene
muves enummt l o Janene eubnu as i s see ed: 1 03 vi iessoed
ed: 1 01 b tu l i Jas ismweqms ins istiiue bebvci i s l i tes has
.8 1 000301 ? mi anode at bodi es edT .no tisai ii J aecpeedus
onuwm t new? no tdqwoeds ed: weds I I has I aeI dsT
gm tqmsd ene tdsvdneonco at muses aiq~seniug has 31 005 1
and “0 .medemtJneo sides aeq adieu 03 8 mom?
anoi isu iaeonoo ed: nwode 87 8 sided dos e 1 0 sai l tea t?
an two i ioi eds no has ,bedssad emew si ieo ed: del dw ddiw
ed: vd qu fl efls i {D ecl ine to sinuoms ed: needs ems sen tI
ed: di l w ebsm s te ed ed ? a t d oii sadneemoo dose moat s ites
05 1 3 ems ai is sem ed: l o emoa .nuwes to aJ o I Ju smeii l b
01 8 dsidw a t ,8 bus .3 , 1 .asiq mi viiso idqsqg nwode
Tabl e I . Ab s orpt ion of Ant ibodi e s from Rabb i t I e
S erum by She e p Erythrocyt e s .
2 2 0 49 9 2 1 7 5 540
3 2 0 47 86 1 60 500 5 60
Uni t s
47 87 1 50
37 5
37 5
300
300
450
350
5 00
5 00
5 00
700
5 40
4 montt i t e r 1 000.
6 70
750
1 2 00 Ti t e r
87 5
as in t e s t 9 , 8 daysl at e r . Ti t er ,
pl o t t ed the l ogari thms of the amount s ab s orb ed agains t
the l ogari thms of the c onc entrat ions remaining in the
l iquid aft er the ab s orpt i on is c ompl e t ed . The pl o t,as
i s s e en , t ends t o approach a s t rai ght l ine .
A s tudy of Tabl e s I and I I brings out s everal
important fac t s . Even from c omparat ive l y l ow c onc ent ra
t i ons of ant ibody uni t s al l are not ab s orbed ,whi l e
, if
the c onc entra t i on in the l iquid is suff ic ient l y high ,
mas s ive quant i t i e s are t aken up . The onl y ins tanc e s in
which the reac t i on s e ems t o b e compl e t e are when very
l ow c onc ent rat i ons are us ed , and even here , the as sumpt i on
that al l the s ens i t i z e r i s ab s orb ed do e s no t s e em
jus t ifi ed , be caus e amount s much l e s s than one uni t per
cubic c ent ime t er c ould no t be de t e c t ed by the me thod of
t i t rat i on us ed in the s e experiment s . I t is evident ,
therefore,that the numbe r of amboce pt or un i t s t ak en up
by the she e p red c e l l s vari e s wi th the c onc ent rat i on of
tho s e uni t s in the surrounding medium ; the more c on
centrated the un i t s are , the more are ab s orbed .
Ano ther fac t t o b e not ed is that the abs orpt i on
i s nearl y always hi gher from the rabb i t s erum than from
the guinea- pig s erum , and that the ab s orpt i on vari e s
wi th the s erum of indiv idual s of the s ame s pe c i e s . Thi s
wi l l b e referred t o l at e r .
isnisgs bed1 oada s inuous ed: to smdi id sgo l ed: beido iq
add a t sn in tsmea an01 3s 1 3n00n00 edi l o smdi tmsgo l add
as ,ioiq edT .be ielqm00‘ et ne tiqmoeds edi ve i l s b tsp l i
.en i i idsfsqia s doseq s 03 shes : ,aeee si
I saeves inc aga tmd I I the I es I dsT i o zbui s A
a s x i d sonoo o vievidsasqmoo moat nevH .ei osi issgmeqml
i t ,o I tdw ,bedmoeds ion 01 s {is earns vbodiins l o snot:
,dgid z iineio tiius a t binp l i add at no tiswineonoo adj
n l seonsdsn i vino edT .qv H8X83 ems seii timsup ev teesm
( may nedw 9 1 8 eieiqmoo ed 03 smese no tioseq edi doidw
mc tdqn uass ed: .s1 en neve has ,be en ems eno tismineouoo o
mes s J on seob bedvosds at wesii ienes edi I ts Jedi
aeq J inn eno med: ssei doum sinuoms sansoed ,beii tisnt
l o bodiem odd vd befioeieb 01 J on biuoo weiemi ined o l duo
,J nebtve at 31 .s3nemid sqx e s eeds at been no tismi td
qu h ens: adimu voiqecodms i o madman ed: Jedi ,eao l eaedd
To noi isd iueod oo ed i di iweeims v sI I eo bee qesde ed i vd
-n00 saom ed: amutbem qn tbnnomdu e ed i mi s i l au eeodi
.bedvoads ems st em- ed: ,ed s si inu ed: beisu ineo
an i iqx osds and Jed i a: beach ed 03 Josi asdiomA
moat nsdi meme s i tddsm edi mom? wednl d szsw fs vit s en s t
seimsv noi iq1 0eds ed: isdi has ,m01 sa g l q-sen tug ed:
B idT .eel oeqe emss and l o sisub tv ibu l 1 0 mumse edi d i iw
.meis I 03 bsmwel eq ed I fiw
Ex pl anat i on of the Figures
Fig . l . Logari thmic pl o t of the dat a of Tabl e I I I .
The amoun t of s ens i t i z e r ab s orbed is pl o t t ed agains t the
c onc entrat i on remaining in the supernatant l iquid .
Ti t er of s erum , 1 /4 ,ooo. K n
Fig . 2 . Curv e s A and B , l o gari thmi c pl o t s of
the data of t e s t s 5 and 4 of Tabl e I I . The dat a for
the s e plot s are al s o shown in Tab l e VI . For Curve A ,
K n e For Curve B , K = l .97 , h
Fi g . 3 Logari thmi c pl ot s , A and B , re pre s ent ing
the data of Tabl e s IV and V . The s era were obtained
from rabbi t 6 9 on the l oth and 24th days re s pec t ivel y .
For Curve A,K n For Curve B ,
K n
Fig . 4 . Logari thmi c pl ot of the data of t e s t 5
Tabl e IX . K n
Fig . 5 . S imi l ar pl o t of t e s t 1 2 , Tabl e IX .
K 5 5 00, n 005 1 0
Where the pl o t dev iat e s from the s t rai ght
l ine the value s of K and n can be de t e rmined onl y
approx imat e l y .
asmug ifi add 30 so tdsns iqx fi
. I I I eI dsT l o sisb ed : 30 J o iq ormd jziagoa . 1 .g 1 1
ed: Jeniega bsdio iq a t bedmo sds mes td temea l o demons 9 LT
.biup i £ desisamequa ed : s t gnidismea ao idsmdmeomo o
.a.o a ,ez .es x .ooo.i\ x .mumes l o meJ iT
to aJ o Iq oimddimsgo i .B has A seVdUO .S .3 1%
1 03 sisb edT .1 1 sidsT so 9 has a sieei t o stab as:
.A evmuo 1 02 . IV eidsT mi an ode o s I s ems aioiq seed ?
.I 8.0 z a X ,E svmuo t o? a .8V.O n f X
ga zinesemqem .H has A .sio iq o im ddimsgod .8 .3iq
bests ido ed ew sas s edT .V has VI seI dsT To a isb add
.v£ev ti oeqaem 82 85 dat a has djoi eds no 96 J iddsm mo st
,8 ev 0 t o? n .Q .85 £ t X .A svano d o?
.b .0 2 mt8? .08 H
, 6 J ae: To e h ed: l o 301 ; o tm di fmaaod .s
a .X I eidsT
.XI eidsT QSI Jess To do l q ms iimia .6
.I a.O .O.8E H
J dg isuds edi moml eel s tveb J oLq adj emedw
vino ben immedsb ed use a has X To eeu Eev ed? sai l.ziezsmi x omqqs
a so i edi nedw vino , wevewod ,eumi ab iod no i isupe s t
e aimeeemqem , sai nati edi mi swede as .i o iq o imfi i iqs
efli mom? seis iveb i t medimsi edi has ;eu fi idg tsmi a
temp s ed: semooed eidsetiqqs d i emom edi , ad i £ insismi a
tnei smo o edi To eeuisv ed : mi mo tisimsv ad: i s eeusoed
edT .smoj ismineonoe idemeit ib ed: deew ied ,m bus H
viieoi i osmq weds A8 has i .sgiK vi beimesemqed aisei
Iqqs ed use so iisspe ed : owed o s t eio iq emii idgismis
gmev ed sqmoo ai iseed beis iu o iso has [ninemiqeqx e edT
doidw ,V I has I I I eeidsT at awoda ei as .vidsmovsi
L-e cb edT .z iev ii oeqsem .AS has I .eg iK o i bdoqsedmo o
[s imem tmeqx e To ai tmi i add m idi iw ,eeaso i scm mi .e t
bimo da .n has 3 , eias ieso o ed i To seu isv edT .1 omde
moi 8L.SS ei ,X
,
”imei ssoo d o tiqmesds
"edT .beioa
vmev at do x dw ,3.QSI K
,VI eideT 1 0? has , I I I e tdsT
medsid doum vmev o s is at d o tiqmosds edT .1 edg id doum
a To ss isv edT .V I eideT a t eao i ismid eOd oo [ I s mom?
Tabl e I I I . Experimental and C a l cu l at ed Re su l t s from
Te s t 6 , of Tabl e I .
Tab l e IV .
Rabb i t 6 9 , Ob t ained on the l oth Day .
K a
2
1 3 2 4
1 2 4
6 60 4081 6 50 1 9 5 06000 8000
the number Of un i t s ab s orbed by the c e l l s,
the c onc ent rat i on remaining aft er ab s or pt i on .
wcmi ai ivsefl beis iuo iso bus I sdmemimeqx a .I I I eideT
. I eidsT To ,6 iseT
nevi ei iueefl beisruoieo bus I s i demi x sqx a .VI eI dsT
To mvmea edi no ebsM si ne? uo t mosdA
.ys0 diOI and no ben is ido .96 i iddsfi
0008
s 8,aiiso ed: vd bedmoeds s i l mu is medmnn edi
H(3
d o i iqmo e fs mail s gu zmismem mo i ieuimeomo o efi i
Tabl e V .
~Re sul t s of Ab s orpt ion Te s t s on the Se rum
Rabbi t 6 9, Obt ained on the 2 4th Day .
0 ( ob s . ) C ( cal c . ) K n
5 5
2 01 70 1 70
309
Curve 4, Fig. 2 .
l ower thanthe value , whi ch was found by Arrhenius .
In Fi g l,n and in Fi g . 5A , n i .s ., the
the curve having the s t e e per gradi ent has al s o the hi gher
value for n . Thus , it is s e en that the cons t ant s vary
wi th the different l o t s of s erum us ed , the value of K,
de t e rmining the po s i t ion of the curve , and of n,the
gradi ent .
In Fig . 2 , there are shown two pl ot s , A and B ,
re pre s ent ing the two t e s t s shown in Tabl e VI . Curve A
deviat e s considerabl y from the s trai ght l ine s o the
value s,K an d n are ne c e s sari l y cal
cu l ated onl y approximat e l y from the average s o f s everal
de t erminations made a t int erval s al ong the curve . There
is al s o in the t abl e qui t e a di s cre panc y be twe en the
ob s erved and ca l cu l at ed re sul t s . The t abl e for curve B,
whi ch approximat e s the s t rai ght l ine very c l o s e l y , show s
a very c l o s e agre ement be twe en the ob s erved and the
cal cul at ed re sul t s .
{sh -q
.su inedmmA vd bnuoi asw do tdw .asiay edi asd i mewdi
as: , .s.i n ,As .31a mi has ,e.o n h .I 3 1a 8 1
medg id ed: oats sad inetbswg meqeeie ed: gd ivsfi evvuo eds
vmsv eins ismoo ed: Jedi se e s e! i f ,sudT .n t o? su isv
,X l o eu isv ed: ,beeu muses i s 8301 imemel i ib ed i di iw
ed:,n To bms , evmuo edi io uo idisoq ed: gu is lmwei eb
, 6 has A .aio I q owi swe de ems awed : , 9 .gifl a I
A ev1 s0 .I V sI dsT s t nwode siaei dwi edi gu i iuesemqsx
ad: 08 eel ! idgismia ad j mom? { I deveh‘efl oo seisiveb
- I eo viimseeeoen ems ,8V.0 m n has X , eeuisv
I emevea To seget evs edi mom? vieismix omqqs vino seisiuo
emedT .evmuo ed: suois e tsvmedni is seem smo t ismtnx eieb
odd fleewied weasqei oeib s ei iup aided eds mi 08 1 3 at
,8 evmuo moi s idei edT .ai I ueea beisfuoiso has bevmeadc
swede .visso ie vmev eai I ide ismi s edi seismi x omqqa doidw
edi bus bevmesdo ed: messi ed J nemeemgs eeo i o vd ev s
.ai iuseu bedsiuois e
bus neg i iflA adj neew ied no idosefl and i s sdefl ed? . I I I
.owdiv a t , 2bodl daA
d s bns nesiins ed: l o mo tmu edi Jedi mess biuow 31
at eucems iaedemi wi tsox i osx q bus ,biqsd wl emesi x e at
a t woods o ti os isflqsns edd yd bedsa ienomeb at s t .ov 1 v
beioetsi a t neg tjns eds uedw so l , aism1 x s evtsisnesWeqwfi
.wI eis ibemmi ssueoo medi c floods ed i ,msemia~boo fd ed: OJ fl I
edi o in i beioetn t nedw , tooo oomuemq have? eed (SI ) Lima
beis n i iu tqas ed 07 t l snjws saumn i as t o msewde~fiocid
s I I eo ed: ,sinemiwsqx e deidqwosds eueivewq eds ml
J us tcti iue beweb teno o sew doidw Qas i s-{rm 08 moi mwd ee
o : sense u I .mu tmd t£ tupe dos es 03 neiaoeeq ed: as? emi J
ds tids i se oJ waseeeoen asw 31 .1 evewod , s td3 toafino o
,dguone sew bewo i is eml i and i on 1 0 med iedw wiesin ii eb
odd ani oneui l at duodi iw beimsv ed biuoo i t dosm wed bus
.sJ I U89 1
x esi J isaee has s so qeede , J nem tweqx e sidd n I
i s abord eq gmiwasv so ? J os ino o ml n ismex o: beo I s eqsw
sd i a! as .moi issi ti wd beieei qu asis i insane ed: bus
.ex uod ow: asw emi t i seauo f edT .ednsmrweqx e eso tvemq
add a t stun tm eno an hei s t! at do tdw , emi i i ssisofie edT
Tabl e VI I
tabl e , c ould no t be k e pt absolu t e l y c onstant . The c or
puscl es were added to the serum di lu t i ons , shak en up
thoroughl y , and immediat e l y c ent rifuged at hi gh speed .
The t ime during whi ch the c e l l s were in fre e c ontac t wi th
the serum was c ertainl y not more than two minut e s in any
case . The resul t s are given in Tabl e VI I .
I t is evident , from this tabl e , that equi l ibrium
i s reached very qui ck l y . I t is prac t i cal l y c ompl e t e
w i thin five minut e s , ard ent ire l y so in eve ry case at the
end of fift een minut es . A sl ight di ssoc iat i on , at the
end of two hours , i s indi cat ed in the absorpt ion from
1 00uni ts ,but thi s i s no t very pronounc ed and do es no t
show at a l l in the hi gher c onc ent rat i ons . The data
given here are few,but the exper iment serve s its purpos e
as a c ont rol t o the o ther expe riment s , and shows that the
t ime al l owed for the absorpt i on may be vari ed wi thin a
wide range w i thout mat e rial l y influenc ing the resul t s .
IV . The Influenc e of Tempe rature on Absorpt i on of
Ant ibodi e s by Red C e l l s .
A t emperature of 37°C. i s empl oyed in most se ro
l ogi cal reac t i ons . The c omb inat i on of the ant i gen and
ant ibody , however , take s plac e at much hi gher and much
l ower t emperature s . D ifferenc e s of o pini on oc cur among
the different inve st i gat ors , as t o the best t emperature
for the comb inat i on . Ne i l ( 1 3 ) and Kolmer ( 1 5 ) advise
the u se of room t emperature as the be st for the sens i
t i z ati on of c e l l s , whi l e Hint on ( 1 4 ) re commends 37 l l
The se o pinions were advanc ed as the re sul t of rather
ext ensive work on the s tandardi zat i on of the Wassermann
Reac t i on . Kahn ( 1 1 ) stat es that ” the extrac t ion is
great e r at 37°
than at room t emperature , whi ch in turn
i s great er than at ice-box t emperature ."
In the experiment s here re c orded,the influenc e
of the variat i on of the t empera ture on the absorpt ion
of hemol yt ic ant ibodi es has be en observed . The ab
sorpt i on t est s were carri ed out as in the fore go ing
experiments ,using the rabb i t ant i—she e p hemol yt i c
syst em . Dupl i cat e t e st s were made for each t emperature
and c onc entrat i on . The ant ibody dilut ions we re made
first , and the c e l l s added aft er both had be en brought
t o the required t em pera ture . I t is needl e ss t o
Tabl e VI I I . C ont inued .
Absor ption from 1 09 Un i t s of Sensiti ser from
from
F 1 67 1 85 1 70 1 55from
g 1 9 9 soo 900 1 99
H 1 2 0 1 60 1 6 5 1 5 5S e f a
I so 1 20 1 00 1 00
.beuni J noO .I I LV eI dsT
mow? fi esidtemea do edisfl OO I moml n oi iqsoadA
. I 03 8 ( 3 1 98 efi i
59 as as a moidqmoédA5 9 as. as s
a
6 6 ca
06 or 05 I ax es
mow? 1 es i i tsnea To si l aU 008 meal moq wosdA . ( G )
I o i H ,se adj
say that the t e s t s a t a l l different t emperature s wi th
one l o t of serum were made on the same day wi th the
same l o t of ce l l s and c ompl ement . The resul t s are given
in Tabl e VI I I .
In the t ests wi th the sera A , B , C , and D . the
absorpt ion was great er at 37°than at the l ower t empe r~
ature s . That i s , the absorpt ion at or > 1 5°
>
In the t ests wi th the serum of rabbi t B , the abso rpt i on
was great er at 40°
than at any other t emperature .
The serum from rabb i t 487 , however,when t est ed
gave a different re sul t . S even t e st s were made on th is
one l o t of serum at different t ime s , using the various
c oncentrations l i s t ed in the tabl e . In every t e st,the
t emperature of 1 5°t o 2 5
°proved t o be the opt imum ,
l ower absorpt i ons b e ing obta ined at and and
above . The re lat ive amount s of absor pt ion for thi s serum
may be ex pressed thus : or > 37°
I t i s
t rue that the differenc es in ab sorpt ion be twe en 1 5°
and
37°
are not great,but they c ons ist ent l y appear in al l
the c onc entrat i ons t e st ed .
The re sul ts ob tained wi th this s erum sugge s t ed
that the opt imum t emperature for the absorpt i on mi ght b e
a variabl e , whi ch i s different for the diffe rent sera .
Therefore,o the r l ot s of serum , which had be en k e pt
st ored wi th phenol,were t e s t ed t o see if any would fal l
into the same c lass wi th that of rabb i t 487 . The se are
l i st ed in the tabl e as se ra E t o I , inc lu sive . The
l ast two,H and I , were from guinea- pi gs and the re st
we re from rabbi t s . Tab l e VI I I , (0) shows the absorpt i on
from 1 00uni t s of sensi t i z e r from each of the se sera at
the different t emperature s . In Tabl e VI I I , ( D ) , i s
shown the absor pt ion from 2 00uni t s . Of the se four s era ,
F and I proved t o b e in the same c lass as tha t of
rabb it 487 , i .s ., about room t emperature i s the o pt imum
for absorpt i on .
Those invest igat ors , therefore , who c laim that
e i ther room t empe rature or 370i s the be st for the
s ensi t i zat ion of the c e l l s , were doubt l ess ri ght in
their observat i ons , but any general i zat ion that a l l sera
reac t in the same way i s obvi ousl y inc orrec t . The b est
t emperature for the sensi t i zat i on of the c el l s seems ,
rather ,t o b e a variabl e , its ab solut e value de pending
on the part icu l ar l o t of se rum used in the t e s t . No
ex planat i on of this variabi l i t y is here advanc ed .
Tabl e IX . Absorpt i on from Immune Sera wi th Quant i tat ive
D e t e rminat i ons of the S erum Prot e ins .
Uni t sab sorb ed in
Te st AnimalRabbi t
l2
3 4
4 4
l7 481
481
9
481
481
1 4
l
1 8 486
1 9 Guinea
p ig2 02 1
1 93 80001 50 2 0001 50 2 5001 93 400001 75 1 00009 1 1 60 1 5001 00 800
6001 02 5001 2 0 500
140 5 00emaciat ed , di ed so on aft er .1 80 41 0 80009 1 1 60 1 5 001 5 0 1 000
8001 02 5001 20 6001 34 370 600 .2 5
emac iat ed , di ed soon aft e r .45 2 000
40 1 50033 2 000
evi isd tiasuQ di tw swea enumml movi no tiqwosdA .X1 e ideT
.en iedomq mflqea ed: 30 eno iisnimaeiea
"
.T~
I A”
5 131'
s"
q iT 005 008 001 ca 09 add mi .05 “teq
si knfl
at EB dToeds
J aeT
beib .beis £osme
0008 08I006 1 06 1 I Q
0001008006008
DYE FE!be ,beis iosme
69
serum proteins were made on each l ot of serum before
the absor pt i on t est s were done . The pro t e in determin
et i ons were made ac cording t o the microref ractometric
me thod of Robertson ( 1 9 ) wi th an Abb e refrac t ome t e r .
Serum , inac t ivat ed at 5 6°
C. , was always us ed fre sh t o
avoid the possibi l i ty of bac terial c ontaminat i on , and
spe c ial at t ent ion was given t o the c l eaning of the
gl ass-ware .
The resul t s of this work are given in Tabl e IX . 4
The re is no evidenc e , from these , that the amount s of
the serum prote ins pre sent have any re lat i on t o the
amount of sensi t ize r absorbed by the c e l l s . Rabbi ts
481 and 486 were bled and t e st ed at int erval s for 1 1 6
days aft er the be ginning of the immunizat i on . The
gl obu l ins and albumins fluc tuat ed considerabl y during
that t ime , but the fluc tuat i ons se em no t t o bear any
evident re lat i on t o the absorpt i on . At the t ime of the
l ast bl e eding both animal s were emaciat ed and the gl obul ins
of bo th sera we re very hi gh . They were about the same
in amount as at the se c ond bl e eding , but the absorpt i on
i s much more c ompl e t e .
The fi gure s given in Tabl es IX t o X I I do nota l l re present the ac tual experimental re sul t s . In manycase s the c oncent ra t ions t e st ed did nét c o inc ide withthose given in the tabl es ,
80 the fi gures had t o beint erpo lat ed from the l ogari thmic chart s , for the purpose of tabul at i on .
smo l ec muqes To 30! does no ebsm si ew ed ieiowq mwwea
~n tmmedeb n teiovq edT .enob emew adeed nef iqi osds edi
o imiemo i oswl evovoim ed: 03 sn l bvo oos ebsm emew suo tds
.meiemoJ osmi ea edda as di iw ( G I ) no siwedofi i o bodiem
o i deem? been avs s esw is bessv tioan l .numea
bus .noi isn tms dmoo I s imei osd in vi i i idiaseq ed: sieve
edi 30 ga inse to add e: sev ka sew no tdneids I s ieeqs
.evew~sss ig
a .XI etdsT m t nevig ems flvow midi To ei iueea edT
i o eiasoms ed: Jedi .saadd nevi .eenebive 6h si eqefiT
ed: 03 udi ieiea vns evsd ineseaq emieiowq mu1 ee ed:
si fddsfl .si£eo edi vd b edi cada aesidismee to Sas oms
BI I we? s fsvaedd i is beieed bus herd evew 085 has £83
esT .no tis x inummi ed: i n aninniged ed: meiis swab
amid st vI devebisnoo bedsfi douii snimud l s has en l l udo ig
vns weed o i i on mesa anoi isuiou I i ed: J ud .emi i Jedi
end io amid edd JA .no iigvosds ed: 03 d o i J s I ew inabive
sni iudo l g edi has beisicsme emew alanine died gn tbee I d deaf
emsa ea: i uods emew vedT .dg is vi ev ewew area died 30
uo iiqmoads ed: ind ,gs ibeeid emcees edd is as JHUOM E mt
.eiq moo swam down at
ion ob I I X oi X I sel dsT mi nev ta sei ugii edT s
vusm ml .eJ I usev I sdmemiveqx e Isu i ca ed i J meseaqea {Lsd i l w eb ion to o J on bib beisei sssidsaiueonoo and 35 9 5 0
ed 03 bed esaus ti end os .ceI dsd edi at nev tg eeodi~vug add i oi .eiqsdc oimddimsgoi ed i mow? bedsioqmeini
.no i is [nds $ i o eacq
VI . The Re l at i on be twe en the Absorpt ion of the
Ant ibodi es and the T i t er of the S erum .
I t has be en no t ed that the c onstant s ,K and n
,
vary in the absorpt i on from the se ra of diffe rent ani
mal s . I t has al so be en observed , inc idental l y , that
oft en the serum of the hi gher t i t er showed the hi gher
ab sor pt i on . In t es ts 2 and 3,of Tabl e I , the serum was
t est ed fre sh and then t es t ed again aft er four months '
st orage ,during whi ch t im e the t i t er dro pped from
uni ts t o uni t s pe r c c . The lat e r t est gave a
somewhat l ower absorpt ion than when the serum was firs t
drawn . Another serum was t e st ed wi th a t i t er of
uni t s per c c . , and gave a very high absorpt i on ( test 9,
Tabl e I ) . The rabb i t was k e pt and its serum drawn again
8 days lat e r ( t e st 1 0, Tabl e I ) . The t i ter had dnopped
t o uni t s per c c . , and from al l c onc entrat i ons
a mark ed l owering of the absorpt ion was observed . I t
was thought ; therefore , that there mi ght be a rel at i on
be twe en the tit er of the serum and the number of ant i
body uni ts the red c e l l s would tak e u p .
A number of fre sh sera of bo th high and l ow
t i t ers were t es t ed . Some of the animal s having serum
of hi gh t i t er were t e st ed subsequent l y , aft er the t i t e r
had dro pped . The resul t s are given in Tabl e X . In
dd To aoiiqfi osdA add neewded no tds iefl efiT . IV
.mu 1 ea en: l o maJ iT an: bus ssibodidnA
,n has X ‘
adnsdanoo end jami beion ne ed sad J I
~id s duemel i tb l o sass and mom? no isqmosds and mi vasv
J sdd .vI I s3nebioni .bswseado used o ais sad $1 .aiaa
medg id and bswods aeJ l J mang l d and l o mnwea an: medi c
saw n umas ad : .I aids ? i s (8 has 9 eiasJ n I .moi $qmoads
‘addnom «no ? medi a niaga bsi aed med: has seen? bei aei
mom? beqqoab mai l s and amid do ihw‘
gn tqub ,agamo ia
s evsg Jaed weds! sfiT .os seq afi nu oi adin u
Jam il ssw ami ss and nedw “and no tiqqosds d swoi indwemsa
l o mei i f s nJ iw bedsed saw moves wsHJ onA .nwswb
,9 seed ) noq aoads dg td vmav s svag bus .:oo seq adl fiu
beqqoqb bed meats eflT . ( I s idaT ,or deed ) we I aveb 8
ano i isaJ nsod oo I I s mom? has deg adiau oi
J I .bsvaeado ssw noiiqmoads end is an taewoi bsiqsm e
mo tds iem s ed J figim amend 33nd , evol eqsdd , $dguod3 saw
w i ins l o qadmus and has mnmas adJ i s medi i adv neewded
.qm efisd biuow a I I eb hem and sj l nu vbed
o has dais di ed l o sass nasal i o msdmum A
amass gn tvsd al anins add to emoa'
.bedaed ewes awai t:
1 6 31 3 oni'
qssisi
,vl ineupesdua s seJ st ew fl ai x i figid l o
n I .X e tdsT ml nev ig 81 8 aJ I uaem edT .bsqqomb bad
many case s,the absor pt i on se ems t o vary wi th the t i t e r
of the serum , but this is not c onstant , and the diffe renc e s
in t i t e rs above uni ts per c c . se em t o have no
def ini t e re l at i on t o the absor pt ion . Normal rabb it
serum , in spi t e of it s l ow t i t er , give s c ompl e t e absorpt ion
from the undi lut ed serum . (Tests 6 and M ost of the
other l ow- t i t ered sera were obtained from rabbi t s tha t
had formerl y had hi gh t it ers , and the absorpt ion from
these w as uniforml y l ow . Te st s 1 7 t o 2 1 , however,we re
made wi th the sera of rabbi t s 7 2 and 80, drawn dur ing
the first few days of immun i zat i on . The y a l l show
uniforml y high absorpt i on , al though the t it ers vary
from 1 00 t o uni t s per c c .
These re sul t s se em t o indi cat e , therefore , that
al though in many case s , the hi gher the t i t e r the hi gher
the absorpt ion , it is no t the t i t e r of the se rum , pe r se ,
which causes the variat ionu Rather , i t would indicat e
tha t the ext ent of absorpt ion i s de pendent on the t ime
the animal is immune , the higher absorpt i on be ing
obtained earl y in the peri od of immuni t y . This variat i on
may be caused by some inh ib i t ing substanc e whi ch i s
pre sent in the serum in larger quant i t ies lat e r in the
immuni t y peri od .
1 9 31 : ed: d i iw wi sv 03 sneea mo £$q eda ed: ,®easo imam
aeefi emei i ib end has .J nedamoo ion a t a tdi J ud , Mmqea ed: 1 0
on evsd oi mesa .oc meq 331 mm evods ax ed i i a t
diddsm Lsmaefl .mo idqmo ade ed: 03 wo tieiem edimii eb
noi iqaoeds edeiqmoo eevig .dei i i we! si t To ei tqe at .muaee
end To i aow has 8 moqi
flan: s i tddsm mom? ben fsido smew siee bemei i i -wdi.meddo
mow? mo l dqmo eds adj has , sve31 3' dqtd bed. yidsmmoi bad
st ew ,mevewod .I S o i VI siesT .o z imaoi ieu sew seed:
an tmub fi wsmb ,09 has 8? ai tddsi i o sass edi d i iw sham
wcde Lie medT .ao i is simfi m mi To swab we? iaqii l ed i
vmev ems i i i edd dauofi die .mo i iq1 03ds ng im mimmoi igm
.cc ass a i imu 000,8L o i 001 mom?
, dsd$ t e'm
'
ie'xedi Q eise rbm ? on mass ai iueem esedT
d odgid edi sed ii add medgid ed i ‘ eesso Ynsm at dauod i fs
, ee aeq ,mumee ed: 30 i ei fd edJ J on 's ! 31,mo tiqaoeda add
eis oibni biuow i i ,fl eflJ afl fimo ii siwev efi i c eases do ldw
emid and no inebaeqeb at no iiqmoede i o iuei x e edi dad:
gmied no isqmoads menaid» en3 , ewummi a} famine and
so iieiasv arfiT .qi iaummi i o Loid ag add a t g l are benis ido
s t screw eoms i adue qmi i ididni eqoa yd beeuso ed yam
efl i HI meds i seii timaup t eams ! a t muses efi i a ? J aeesmq
.boiaeq wi tnummi
VI I . S e l e c t ive Absorpt i on .
In the pre c eding se c t i on , it was indi cat ed that
the variat i on in absorpt i on mayi be due t o some disturb ing
e l ement in the serum , the l owe ring of the absorpt i on
de pending on the amount of thi s substanc e pre sent . I t
was no t possibl e t o show any influ enc e of the fluc tuat i ons
of the serum prot e ins ,so evident l y the di sturbanc e i s
not due t o the serum pro t e ins as such . The probl em
now i s t o de t ermine the nature of thi s el ement , i f
po ssibl e , and i ts mode of ac t i on ,i .e. , whe ther i t is
tak en up by the c e l l , or ac t s mere l y by virtu e of its
pre senc e in the serum .
I t has b e en no t ed before that , when the l ogari th
mic pl o ts of the absorpt i on are made , some of the curve s
re pre sent straight l ines whil e o thers do not . Curve A ,
Fi g . 3,for whi ch K approache s the st rai ght
l ine very c l ose l y . Curve B , in the same figure , repre
sent s a t est made on the serum of the same rabb it ,drawn
6 days lat e r . The t i t e r had dro pped from uni t s
t o uni t s per c c . wi th a dro p in the absorpt i on
c onstant , K The pl ot , as i s se en ,deviat e s
c onsiderabl y from the s trai ght l ine , the deviat i on be ing
mos t pronounc ed in the hi gher conc ent rat ions . Fi gs . 4
Jeni beino ’bnt new i t .mo tdoea gn ibeoeaq efi i at
ga l dmu iaib emoe 03 sub ed ivsm no tiqmoads mi ao i inimsveds
no i iqmoade edi To gn i i ewo i edi .mUd es edi m t dmemsi e
31 .J neeemq somedadua aids i o imuoms edi no gn ibmeqeb
mci isui ovfi adj l o eofi euiimi Que wens o i eidreaoq ion saw
at eonsdqude tb edi vl inebive ca .amredoaq mumea efi i To
meidcmq edT .dcue as emieiomq m az e s edi o i su b ion
31 , J aeme£ e aid: 1 0 ewudsm edi enimwe ieb o i at won
at ii medi edw ‘.e . t .moti ca i o ebom air has ‘
eI diaaoq
s i t i o eudqiv vd viewem 9 303 ac , i ieo end vd qu ne xni
.mn1 ee ed: m1 eonseemd
- di £msgoi add medw ,J sfi 3 emoied be don weed sad J I
eevmu o ed: i o emoe e sm ed s nofiqwosds edi l o eio iq el m
,A evdUO . J om ob ameddo eI tdw semi ! ddgismde imeaemqem
idg isuie ed; asdeeomqqs X doidw moi Q8 .g ifi
—edg ew .sangti emse ed: a t {a ev 1 u0 .visso ie vmev eat!
nwsmb , $ iddam emna efi i i o muwee ad j mo ebsm $36 3 8 eJ uee
ei tmu mp mi beqqoab bad mai l : edT .me is £ evsb 8
noi iqmo sde ed : a t qomb s di iw .oo meq ei iuu o i
aeis tveb ,neee 8 1 as .io iq edT .EV.OS a Xt J fi s ‘i eao o
gated noi isiveb edd ,emi i J fig I smi s ed : mom? vidsmeb iamoo
b .ag tfl .snoi dsd dneonoe medg id end at beenuonomq Jaom
Fi gure s
Logari thm i c p data
Tabl e n
S imilar pl o t of t est
S imi l ar pl o t of t est
K n
Fi g . 9 5 S imi l ar pl o t of 7, Tabl e
n
1 6 , Tabl e 1 x
8 Tabl e IX
J se i is i o iq ws iimi ‘
1 8 teed mei iml
and 5 re pre sent com parat ive l y hi gh value s for K,but in
nei ther is it so high as in curve A ,Fig . 3. The se pl o t s
,
al so , deviat e from the st raight l ine in the hi gher con
cen trat ions of ant ibody uni t s . Fi gs . 6 t o 9 a l l repre
sent very l ow value s for K , and in the se i s se en a dro p
in the curve in the int ermediat e c onc entrat i ons .
This dev iat i on of the curve from the st rai ght
l ine,
ac c ording to a physi cal int erpre tat i on , means
that there i s a se l e c t ive absorpt i on of the ant ibodi e s ,
the amount of se l ec t i on diffe ring wi th the c onc entrat i on .
S e l ec t ive absorpt i on may , al thbugh do e s no t ne c e ssari l y ,
denot e the presenc e of two or more substanc e s in the
serum capabl e of be ing absorb ed .
I t was t entat ive l y assumed , therefore , that
that the variat i ons in the absorpt i on are caused by the
presenc e of some inhibi t ing substanc e in the serum ; and
that this substance exert s i ts influenc e by virtue of
i ts own absor pt ion by the c e l l . The fol l owing "mu l t i pl e
abdorpt ion" exper iments were carri ed out t o su pport or
di sprove this hypothe si s .
She e p c e l l s , one uni t , were al l owed t o tak e up
a c ertain number of uni ts of a sensi t i z e r from a serum
that,from previ ous t est s ,
had be en found t o have a
comparat ive l y hi gh ab sorpt i on c onstant . ( The valu e of
-oa;
at Jud (X d o ? seU I sv data vievi ismaqmo c ineeemdew 6 bus
,aJ o Iq eaedT .3rfl ,
A evau o mi aardg id be i t at medi ten
-noo medard edi at emi t ifig iamde and meal eieiveb ,oa1 a
n eqqed [ I s 9 03 e .831 2 .ai tu n vbod l ima To efi o idamdmee
qomb s mesa at seen: a t has QX t el sesiav wo i vmev imea
.ad o i iamifi eomo o edaibemfi eimi edi ml ek o ed: mi
J fig tami a ed : moai evmuo sud i o mo tiaiveb at
enaem ,moi is ieqq1 ein i I aoi avflq s 03 gmibmo ocs , eni i
.ee thodiina an: i s ao i iqaoada ev tdoeiea a at eaedi Jedi
.noi ismineono o edi fi i iw animei i ib mo i ieeLea i s J auoma ed:
Qvim seeooen J on eeob flavofi dia .vam» no t$qmoeds ev ti oeiea
add a t aeons iadua emom mo owi i o eoneeemq em: edomeb
.bedmoada amiad i o eidaqeo mamas
mead , emoi emed3 ,bemuaaa vI svi iainei aaw 31
m
edi vd beeus o ems no idqmomds adJ at anoi iaiwar ed J isdd
bus {mu wee ed i ml eons iadue gu i i idifini emo a i o ecfl eaemq
l o eudmiv vd ecueu iini si t eivex e ecnaiadu a aids dad:
eiqisium"
gn iwoi l oi edT . riee edi yd mo l iqi oeds nwo ai l
d o id oqqua o i duo beimmao emew admamtweqx e”moi iqmobds
.eieeddoqzd mid i evomqetb
qu eflei'
ci bewo i ia emew,$ tm u eno , al iao qaada
mumee a mom? mesii tsaee a To si tuu io medmum aiaamee a
e evad o i bnuoi n e ed bad , ai as i auorvewq mom? ‘dedi
l o eu isv edT) .3msieaco ao tiqwoads dgid vievi iamaqmoo
K was no t de t e rmined exac t l y in the se ex pe riment s ,but
onl y e st imat ed t o be hi gh or l ow in pro port i on t o the
amount of absorpt ion from the c onc ent rat i ons t est ed . )
Ano ther simi l ar l o t of c e l l s was al l owed t o tak e up
sens i t i z er from a serum having a l ower absorpt ion c on
s tant . Bo th l o t s of c e l l s were then se parat ed from the
superna tant l iquid by c entr ifuging at l ow spe ed , and
each t reat ed wi th an equal number of uni ts of sensi t i z er
from one serum . The amount of absorpt i on was de t e rmined
as in the pre ceding experiment s .
I f this inhib i t ing el ement is real l y tak en up
by the ce l l s , those whi ch had re ce ived a c ertain numbe r
of uni t s of sensi t i z e r from a serum w i th a l ow absorpt i on
c onstant shoul d be more resistant t o further sen sit i
z ation than c e l l s which had re c e ived an equal number of
uni t s from a serum wi th a higher absorpt ion c onstant .
The resul t s of the experiment s described be l ow agre e
W I th the expe c ta t i ons .
Experiment 1
( a ) . First Absorpt i on . S e t ( 1 ) a, One uni t of
she e p c e l l s was trea t ed wi th 50 uni ts of fresh guine a
pi g immune se rum , t i t e r Ti t rat ion of the super~
Al l se ts were made in dupl i cat e .
ind ,ainemtqeqx e sasai a t g idos x e bamimmedeb Jam saw X
ed: 03 mo i imoqomq a t wo i 1 0 data ed cd beasmiiae vino
( .bedee3 ano l iamdneonoo ed s meai ‘
noi iqmoeda l a iuuoma
qu e ia i o i bawo i ia saw ai l eo hav ici aa tl mia medieflA
-moo no l iqmoads d ewo i a gaivsfi mumea a mom? mes l al aaee
ed : mom? beiamaqes nefl i ewew al ien me 8301 d ioa .$maiw
bus ,bseqs o is gfi tgu l iainec vd .b twp i i inasafl fi equa
mesi tiauea i s earn “ 30 aedmum fauna as di tw bei semi4dose
benrmmeieb saw no t aoade is dfi uoma edT .mumee sac mom?
.a$asmfi eqx e gnibecemq eds a t as
qu neiai vi l aex al Jnawaie gnid tdiflni. atdi TI
medmun a l s iwec a bev l eoem bad do idw eaead ,eI I ec ea; ad
no tdqaoeda c s fli tw mumea a mum? meai i ianee i s adiuu To
~ i i iamee med ian? o i dnada ieew 9 1 0m ad bimede iaaienoo
io medmum Taup e ms hev tesea bad do tdw ei ieo madi no l iaa
.dnsiamoe mo iiqmoads mefig id a diiw mumea a mom? s i tnu
eemga wo ied bediwoseb eiaemiqeqx e add 30 ei ruaax asT
.afl o tiai oeqx e edi di iw
I irrenri'xeqx a
To 31 m m emO ( I ) 388 - .fi o i iqmoadA Jami? . ( s )
—aenin3 daeai 30 adimu 06 di iw bedaemi aaw aI I eo qeefi a
~wequa ed: i o ao i iami tT .06 £\I 1 9 31 3 ,muwea emummi al g
.e$aoi £qub a! sham emew asaa I la
na tant l iqu id showed on l y 5 uni t s absorbed . The value
of K , therefore , is very l ow . S e t One uni t of
she e p c e l l s , treat ed wi th 50 uni t s of gu inea- pi g immune
serum , t i t er absorb ed 2 0uni t s of sens i t i z er .
The value for K is , therefore , much hi gher than for the
serum of Set al though st i l l rather l ow .
(b ) . Se c ond Absorpt ion . Each l o t of c el l s
was then treat ed wi th 50 un i t s of the se c ond serum ,
t it e r By t i trat i on of the supernatant l iquid ,
l ot 1 was found t o have tak en up 5 more uni t s , whi l e
l ot 2 absorbed 1 0more . The serum wi th the l ower
absorpt i on c onstant was , therefore , more effe c t ive in
prevent ing fur ther sens it i zat i on of the c e l l s than the
serum wi th the hi gher absorpt i on c onstant .
Ex periment'
z
( a ) . First Absorpt i on . S e t One uni t of
c e l l s,tr eat ed wi th 1 00uni ts sens i t i z e r from rabbi t
serum ,t i ter ab sorb ed 5 0uni ts . The value for
K i s ,therefore
,c omparat ive l y l ow . S e t One uni t
of c e l l s ,treat ed wi th 1 00 uni t s sensi t i z e r from rabb i t
serum , t i t er t k up 90uni t s . The value for
K then i s c omparat ive l y high .
( b ) . S e c ond Absorpt ion . Each l ot of c e l l s ,
aft er se parat i on from the supernatant l iquid , was
eu isv eflT .bedi oads ai inu 8 vino boweds btfi p if ffl e33fi
1 0 Stan eflO J ee .o Qx ev at , ei oi oqed3 ,K
'
i o
emumml grq~seuiua l o si tnu 03 fldiw fieisei i ,s I I eo qeeda
.1 es tdienes i o adinu as bedi o éds ,oooa\ 1 1 9 31 : ,mu1 se
ed i t o? nsdi aedg id dou n ,ei oi ei edi , ai X 1 03 en flev edT
.wo i z edisn I ri ia figuodd l s , ( I ) 36 6 To mUd ea
ei ieo l o 301 floss ~.noi iqmosdA bnooea .( d )
, su 1 ee bno oea ad: i s sal ad 08 ddiw beisewi mend sew
,bl upi £ ins isnuequ s add 30 mo idsai i i 78 .0006\ I Teal :
ertdw , t nu 9 1 0m 5 qu HG flEJ sved o i bafi oi sew i 301
TGWOI adj d i iw mus es edT .eqom O I bedTOEdfi S 301
m ! evi i oei ie STON , ei o ? ei sd3 ,asw ins i anoo moi iqi oede
and dad: s I I eo end To no i is s ti tsnes tenet s } an ionevetq
.3ms i anoo ao l dqi oeds fl edg l fi edi di l w muuee
“ S'
J aem ii eqx fi
l o J im” ag o , ( I ) 39 8 —.no i iqi oedA Jai l ? . ( s )
i iddei mow? Tesi disnea si l nu 001 di tw beisei i ,s I fso
1 03 eu l sv edT .si l nu 08 Dedwoeds ,OOSI \ I z eal : ,msi ea
J inn emO , (S) J ea .o vievidsi sqmo o ,ei ci e1 edi , s l H
d dB T mowi t esl i tenea si imw OO I di iw bei sei i t si l eo i o
i oi eu tsv edT .si zmfi 09 gm £033 .000d\ 1 wai ts ,mfi i ee
.dg tfi vievi iei eqmoo s t d ads X
,a I I eo i o J o I dosfl ~ .ao l iqqo adA bno oea . ( d )
sew , biup tl ins isfi i equ a ed: noni'
uo tis x sqes uai l a
c e l l s , treat ed w i th 200uni t s of sensi t i z e r from rabb i t
serum , t i t e r absorbed 80 uni t s . The absorpt ion
c onstant i s very l ow . S e t One uni t of c e l l s,t rea t ed
wi th 1 2 0 uni t s sensi t i z er from rabb i t serum ,t i t e r
t ook up 1 2 0. The absorpt i on constant ,therefore
, must
b e very high .
( b ) . Se c ond Absor pt i on . Each l o t of c e l l s, aft e r
se parat i on from the supernatant l iquid , we re t reat ed
wi th 1 2 0un it s from the se c ond rabbi t serum , t i t er
Lot 1 absorbed 40uni t s more , whi l e l o t 2 t ook up 1 1 0
more . In thi s case , al so , the absorpt ion from the se rum
having the l owe r value fo r K i s much more effec t ive
in mak ing the c e l l s resi s tant t o fur ther sensi t i zat i on
than ab sorpt ion from the serum wi th the hi gh value for K .
In every case , in the forego ing experiment s ,
the ant ibodi es tak en up by the c e l l s from the serum
wi th the l ower absor pt i on c onstant had much more in
fluenc e in inhibi t ing further sensiti zat i on of the c e l l s
than a l arger number of ant ibodi es tak en u p from the
serum wi th the hi gher absorpt i on c ons tant . I t i s
obv i ous ,therefore , that the inhib i t ing e l ement ,
what
ever i ts nature , can be taken up by the c e l l s , and
and probabl y in pro port i on t o its c omb ining aff ini t y ,
inhib i t s the absorpt i on of the ant ib odi es .
i tddew mnmi TGStJ I Efi GB To ai tnu 008 fi iiw seisemi , eI I eo
no tiqi oads edT .si tam 08 bedi oads ,OOO®\ I i si i i ,mumea
beisei i , eI ieo l o i kuu unO t (S) J ed .wo l vi ev at imaisnoo
,~ooa\ r me i ti ,mUd se i iddsi most d esi i iefl es ei imu oer di iv
i sum Q evo‘
i s'xedi , ins ieno o no tiqi oads edT .OSI qu fioo i
.dg id vi ev ed
meiis , aiieo l o i o I dosfi - .no tiqmosdA bao oea . ( d )
beisei i ewes ,bisp ti ins tsnweqve out moo ? moiisaeqes
TeJ I J ,mui se i iddsi bosses and mom} si l nu 09 1 di tw
Oi l qu floo d 8 def ei tdw ,ei om si rum
‘
oe bedsoads I i od
mui se edi mom? no tiqmosde edi (ee l s ,
eas o aid: n I .ei om
evi i oeii s ei om have at H moi es I sv aewo i eni anivad
no i is x ti tsnes median? 03 iss ieiaem sLI eo sfi i autism at
H moi eu I sv dg td edi d iiw meme s ed: mo i ? mo i iqqoads soda
0
, sinem f1 eqx e an togei oi add at ,
esso vmeve ml
meme s ed i semi s I I eo ed i vd qu nefls i eeibod tine eds
~mi ewom doom bad insi snoo mo tiqi cads mewo i ed: d i l w
s I I eo ed i i o mo tissi i ianes i onimwi an i iid l dmi mi semenil
edi moi i qu aefis i eel bod l ins i o medmun mogwai s msdi
a t 31 .ins isno o no idqwo ads d edg td odd fi i iw mumes
- dsdw,iasna£e gn ti i i ifin i ed i isdi .eioiex edi ,euo ivdo
bms , eiieo ed : vd qu fl 9 fl£ 3 ed use , e1 u isn ai l i eve
,vi ta fi is gn inidmoo adj o i mo tdi q iq a t vl dsdomq baa
.eeibodi ins edi i o mo i iqwosde edi ei ididfl l
VI I I . Var ia t i on of the Absorpt i on wi th the Length
of the Per i od of Immuni t y .
In the fore go ing sec t ion , it was found that
the di sturbing e l ement , whi ch inhib i t s the absorpt ion
of the ant ibodi e s , is ac tual l y taken up by the c e l l s
al ong wi th the ant ibodi e s . In Se c t i on VI , it was indi e
os t ed that the amount of thi s sub stanc e probabl y varied
during the t ime tha t the anima l is immune . In this
S e c t ion , it is purposed t o show how the absorpt ion
vari es during the immuni t y of the animal , whi ch varia
t i on se ems t o give some indi cat ion as t o the nature of
the disturb ing fac t or .
A ~carefu l re c ord of rabb i t s 481 and 486 was
ke pt whi l e t est ing for the re lat i on of the serum pro
t e ins t o absorpt i on . The animal s were given three
in j e c t i ons w i thin the first nine days and no morel during
the 1 1 6 days they we re under observat i on . Tabl e X I
shows the fluc tuat ions in the absorpt i on from the ir
sera ,al so the t i t e rs and the number of days aft er the
f irst in j e c t i on when the serum was drawn . In Fi gs . 1 0
and 1 1 are pl o t t ed the observat ions .
The first t e s t s, made on the l eth day ,
showed a
hi gh t i ter and a c ompara t ive l y hi gh absorpt i on . The
s e c ond t e st,on the 2 4th day ,
showed a great dro p in
Tabl e X I Absorpt i on Te st s on the Sera of Rabbi t s 481
and 486 , Drawn at nt erva l s Dur ing a Peri od
of Immunit y of 1 36 Days .
from serum of
41 0 6 70 80001 60
1 000
The se rabbi t s re ce ived thre e infle c t i ons ofwashed shee p c orpusc l e s wi thin the first nine days .
Each in j e c t i on c on si st ed of 3 cc . of the c e l l s , mea sured
in t e rms of who l e bl ood .
{as ai l idsfl i s smea end so ai ssT l noq x oedA . I R eidsT
bc tmeq s sfl i i ufl a tsv x edu l is aWs .5 8& has
si ims c di dms
bedwosdm si imfi
00001
0008 OTB
l o suo i i oegni ssmdi bevieoes siiddhm se en? s
. aqsb .su ifl dam ti sud n iddiw as! too qssde bedsswbeauesem ,
e I !eo edi 1 0 .os 8 l o bets 0 do ii esimi dosfl
.boo
Ex planat i on of the Fi gures
Fi g . 1 0. Pl o t s of the absor pt ion from the serum
of rabb i t 481 against the t ime of immuni t y . S e e Tabl e XI .
Curve A re pre sent s the ab sorpt i on from 2 00 uni t s , B
from 1 00uni t s , and Curve 0 re pre sent s the square roo t
of the t i t e r .
Fig . 1 1 . Simi lgr pl ot for rabbi t 486
,Tabl e XI .
Curve 0 re pre sent s the absorpt ion from 50uni t s , and
D re pre sent s the squane root of the t i t e r .
Fig . 1 2 . S imi lar pl o t for rabb it 6 9 , Tabl e X I I .
Curve A re present s the t absorptidh'
from 500!unit s , and
B from 2 00uni t s . The t i t e rs of the sera On the vari ons
days i s indi cat ed at the to p of the chart .
Fig . 1 3. Pl o t simi lar t o Fig . 1 1 , for the goat .
S e e Tabl e X I I .
semug iq ed: To do zi sdsq m
muses and moat no iiqd osds saw 30 aiciq .01 .31 6
s I dsT sea .2 J 1 flwmmi l o eml i eds J amisgs I 8$ i iddsd i s
6 .edin n 008 momi fisiiqmoeds and admeae%Q eu A ak 0
3001 ex sup s edd ainesedqem 0 evmuQ has .ai iuu OOI mcvi !
. I X eI dsT .ass J iddsq TOT i c ig ds i imia .I i .3ifl
bus .sdinm 00mom? no tiqwoade and s inesedqea Oevqno
.I I X s l dsT .9 6 J iddsd moi do ig ms iimia .81 .giq
has ,ad tnq Q d mom} no tdqd osds end sineaemqsd A evmuo
auoidsv and n o sass add to awai ts sdT .si inm 003 and ? a
.idsdc end is q add is beisoibmi ei avsb
.J scg ed: moi Q I I .g ifl o: as i imis J oiq .8£ .sih
.nc iiqmo ads edJ n ? qomb gn tbmoqsemmoo s has d ei i i ed:
vd ev i sqqoab meats edi .qo i b I s tdin i add ghiwo iiofi
no n ed i mow? has .vsh bash edi 3006 s I rish vi tf ubsmg
h i no r u s v weedo it chi I i dnm s J enoc vivisi biefi
d o e I fi Fsacq i cu adew efi cJ JSVd esfi o d endvufl .tsh dibi f
To e as e edi n I .sism tns and To diasb edJ to J auo oos
b l ed so ,vi rsubsqg beqqovb d ei i i end emedw .1 8& i tddsfi
gn t s J ou I i dgnodi is .no i &qmoads and,dflsdhnmo J aomis
ed: bd swod bus .insi edo o vista? bl ed oais ,isdwemos
J oai iaib s bewoda .no idsvmeado To bo ivsq ed: i o bus
muse s end meal mo i iqmoeds eds do ancicsudcs ii edT .eaim
beiman s gu iw ode ( bSOfi UOUOTq e x am ex ew as» Siddsd 1 0
eeiv heaven ,e J oe B d i tw .qoqb I s ti fin i ed: media salt
gn igbut , d evewod ‘eidsdoqq vrdg id a t 31 .bme ad: is
sa id ed: Jedi‘wo ied bediaceeb sn o iisvweedo medic most
need i t bad .1 efi3id beumi id o o évsd bison bme add is
.sdae3 t eddmu l e x am 03 sidiaseq
-g £ a bemeee ai tddss owi ssedi mom? si iuaed edT
— 1 oa .beimsin ST9 W adedio To medmum s as .insoii in
miedi o s be tb .@8 .J iddsm .eso dqecx e I ia , v£eisnui
cs is saw isog A .emed bebmcoed ion at gao i aid
agifl has I I X e I dsT .bewo i £oi vmo ieid si t baa bex imummt
aisn iqs saedi no snoi jsv 1 eedo edi weds SI has 8!
bed ts ido eaod i di tw I I ew vmev sedge si fuaem edT
w i th the other two rabb i t s . Even though the t i t er i s
not hi gh at first , the absor pt i on i s hi gh ,wi th a great
dro p wi thin the sec ond or third we ek of the peri od of
immun i t y . Fol low ing thi s ,the absorpti on hol ds about
c onstant for a t ime , aft er whi ch i t shows a more or
l e ss gradual ris e .
The his to ry of the se animal s seem? hi ghl y si g
nif icant in su ggest ing an t ex p l anat i on as t o the nature
of the di sturbing fac t or in the absorpt i on . In nearl y
every case , the sudden ini t ial dro p in the absorpt i on
i s ac c ompanied by a simi lar dro p in ti ter . In the
case of the goat , thi s i s not ev ident , but nine"
days
had e lapsed be twe en the f irst and se c ond t e st s , during
whi ch the t i t er may have gone h igher and dro pped back
t o
I t is c onc e ivabl e that , as the amboc e pt or c ont ent
of the animal ’s bl ood- s tream dro ps , the dest ruc t i on of
the ant ibody oc curs Ln progre ssive stages , the first
s tage be ing mere l y the l oss of the power t o sensi t i z e
the c e l l s t o the ac t i on of c ompl ement . The ab i l i t y t o
c ombine w i th the c e l l would st i l l be re tained for a
t ime . The se would be , in e ffe c t , somewhat anal o gous t o
the "agg l ut ino ids
" of Ehrl i ch , whi ch were work ed out so
thoroughl y by Eisenb erg and Volk In t i t rat ing
the serum ,then
,the se de t e ri orat ion product s of the
3 ed: dguodi d eva .aJ tdds d owi med ia'sdi di tw
( i tw gdaid ai mo i rqmoads edd ,
J amii J a ,ng £fi ion
taq add 1 0 isew fluid : TO emcees ed: aI dJ i w gei b
b led no i iqmo ads ed:,at gml wo iiofi .Yi i 1 uwmi
cm s ewods i t do idw mai l s ,smti a d o} ins i amoo
mes tq I ssbsqg eas t
Esta amass a l smid s ea sdd l o Qi odeifi cd&
odd oi as ao iismsiqx e rms 3mtdeeggss. n l J as o il l n
I .mo £ dq1 oeds a dd a t d oaosi sa tdi udsib ed: 1 0
ml . 1 sii i a t q b «s l imia a 2d bs rqsamoqos at
Him J ud ,daebive i on at sidd 1 i803 eflfi to e as e
eJ aei bnocea bus jad il efl i mesw i ed bsaqsie bad
.ocos\ £ cd
iqeoodms sdi ss ‘dsdi erds viecaco at 31
fd i aeb sad,sqomb ws s1 3a~bosid a
'
I sml as ad: i s
edd , ssgsi a sv l asemgomq a t amused vbodi ius ed:
as o i x cwoq ed: 30 aecl sad wred sm gated easda
da edT .imems£qmo o l o ac tios efli 03 a I I eo add
bea tsisa ed I i ti a biuow fi l es sad di iw eul dmo o
gd isvtomed eno has ,bei oe}eb ed J on biuuw aq é ocdmw
s w e tboc’
tifis arm oa‘mo l dibbs at. m isa
‘ fieo bruaw m m
enofiT .sdoubomq mel dsmo tiedsfi ad ? is Jmuoms e l daxasv
h eatin g add t o ? vi tn ii is miefl i fienfibflem ( I i ia fidtfiw
3 sense 03 has: , aimsmrmeqx e fi ctdqx o ads sfi d m l gbiuow
edT .ee tbod tdms edd i o mo tiqd oads add 30 gfl iwewol
at aci iqmoadh ed i i o gmimewci issisemg‘
edi isfl J 303 1
add i o uoiicuaiasb b tqai deem add diiw imsbtcmios
- ds s ed : ind: evi iseggue gidi ovdi tsev at sekfiodrims
vsw eonsdi u is tb s td ? seeuso do xdw muses sdi a} eons ia
adj i o no tisumdseb add i s aibuboqq add as behiisb ed
D i scuss i on .
I t has be en found that , in the absorpt ion of
hemol yt i c immune bodi e s by red cel l s , immense quant i t i e s ,
as much. as ‘2 000uni t s or more , ma y be taken up by one
uni t of c e l l s,provided the ir conc ent ra t i on in the
surrounding medium i s suffi c i ent l y hi gh . On the o ther
hand , a l l the ant ibodi es are oft en no t tak en up even
from c oncen t rati ons as l ow as 50uni t s per c c " This is
c ertainl y not in acc ordanc e with the theory of chemi cal
val enc ies . I t has be en found , more ove r , that this absorp
t i on fo l l ows , more or‘ l ess st rict l y , the physical l aw
of a solut e be twe en two immi sc ibl e sol ven t s . The
equat i on ,B a K O
n
,whi ch re presents this l aw ,
c ould ,
how ever , wi th equal accuracy ,be appl i ed to simple
adsor pt i on phenomena ,such as the tak ing up- of ac et i c
ac id from a benz ol so l utLgn by si l i ca gel , Arrhenius
assumed that a l l absorpt ion t est s wi th the hemo l yt ic
syst em would fol l ow this l aw wi thin the l imi t s of ex
conf irmed in thi s paper , that many ,.in fac t , most of
the abso rpt i on t est s do not fol l ow th i s l aw stric t l y ,
as is shown by the devia t i on of the l ogari thmi c curves
ed: ml meidnmdueomo o fi l ed: befi ivomq ,ei £eo l o J inn
qedio adj no .daid gidmeio ti iue er unifi es gaibmuoaama
nave qu measd J on medi e et a aetbodi ine eds fie ibmsd
e ! siflT . oo seq eaten 08 as wo i as emo tis seoqoe mefi i
I so imedo mo yaoeds ed: di l w eensbmocen a t J on { I n l eJ d oo
cede sidd Judd .meveei cm ,a e7 need sad i t .eekefi eiev
weI Lsoiegdq ed J ( y idcl x i s eeeI TO eqom ,aweI I o% note
o idud tudel b add 03 eei iqqe do l fi w , au tfi efl1 1 A 3d besoqomq
edT .eJmev ice eidieeimmi ow: neewjed eJ u I cs s 30
.biuoo ‘wsi sin: seneeedqefl detdw X 8
,mo tieup e
eiqmie o i beiiqqs ed ,qosmuoos [eup e dctw, aevewod
oiieoe l o qu guided edd as done ( anemonedq mo t i eaba
em tmedd .I eg s o ti te 1 d coi iu ios l oaned a mom& bi os
otdviomed ed i di iw si sej not iqi oeds { I s Judd bemuses
~ x c l o edimri ed: nidi iw ws i sidi wo l iol biuow meiaza
has ,3a tqswmeM I d swede sew i t dud Qae'rze [s ineml x oq
do deem , J oe l a t ,yuen Jedi gaeqsq sin: mi head l ines
,z I J oiqiewa x aid: wo fioi J on ob aimed neq qoede edd
indi cat e s that the o pt imum t emperature for the ant igen
ant ibody c omb inat i on i s a variabl e , the absolut e value
of whi ch de pends on the part i cular l o t of serum used
in the test .
An endeavor was made t o de t ermine the cause of
the extreme variat i ons ob tained in the absorpt i on from
the different l ot s of serum . A large number of t e st s
were made on the different sera , at the same t ime mak ing
quanti tat ive de t e rminat i ons of the gl obu l ins and albumins .
The quant i tat ive fluc tuat ions of the serum pro t e ins were
not shown t o b ear any dire c t re lat ion t o the variat i ons
in the absor pt ion . They may , howeve r , ex er t some more
indire c t inf lu enc e whi ch was no t de tec t ed by this me thod .
Al though in many case s , the serum wi th the l ower
t i t e r gave the l owe r absorpt i on , this was no t c onstant ,
and wi th t i t ers of uni t s per c c . or hi gher the
differenc es did no t se em t o b ear any rel at ion t o the
amoun t of sensi t i z e r tak en u p by the c e l l s . Normal sera
and sera that wer e drawn earl y in the peri od of immuni t y
gave uniforml y hi gh absorpt ion , re gardl ess of the t i t e r .
This indi cat es that the l ower absorpt i on c ome s about as
a resu l t of some change in the serum aft er the animal
i s immuni z ed .
Thi s l owered abs orpt i on i s evident l y due t o
-seg idns ed: 1 03 emu dsx eqme t muml mqo odd Jedi eedsoibni
euEev eduiosds end ,e I ds tqsv s at no tdsn tdmhe tbedi dns
been nud es 30 301 asinei ii sq and no abeeqeb doidh is
.3333 sfi h at
to asses edd en l muedeb ed eb em esw ao vsebne '
ha
mow? noidqvosds end at beais ido emoi dsfasv ememdx e efi i
3339 3 To «adage GQTEI A .muves iq awe: issaeti tb add
gniflsm emi i emes ed: is , sase suedei l ib end so sham ovsw
.am tmsd£s has sni iudoig ed J to shel den l mdedeb evi isdidnsup
eaew sn ieiovq amass and To emoi ieu J oUI i evi isdiinsup -edT
eno ideivsv ed: 03 meidsiev J oev l b was weed o i swede dds
seem emos Jasx o ,vevewod ,vsm vedT .mo¢iqwoeds add a t
.boddem aid: vd beioeieb don sew do tdw echeu l in t deev l bnt
vewo i ed: di tw muvee edd , eeeeo vnsm n } figuofldih
,insiemoo don sew aid: ,no l dqvoeis aewei edd evsa
edd dedsifl d o .00 seq adiau GOD ‘ S to avai l : ddl w'
bds
eds oi doi is iea was used 03 mess dos bib eeeasi ei l ib
sass [emaow .ei £eo ed : vd qu defied vex idianes i s infl ows
vdimummt i o bo iaeq edd at t imes uweab emav Jedi ates baa
.1 ei i i ed: 30 seeibvsgeq Q fi ctiqi oeds dgid vimwoi infi eves
as duods eemoc no i iqvoeds vowel and dad: eeds oibdi at
famine ed: t ai l s m uses ed: a t egnsdc emoe to diuedm'
s
.Besihdmmi at
ca sub vidmebive at no tidaoede bedewo i eI dT
i _6 o
"agg l utinoids
" of Ehrl ich . Thi s hypo the s i s would ac
c ount , not onl y , for the great differenc e s in the
ab s or pt i on from the different sera ; but a l s o for the
3. Wi th the majori t y of s era the ex trac t i on
of s ens i t i z er by the c e l l s was found t o be gre at e s t
at a t empe rature of about Some s era , howeve r ,
gave more compl e t e ab s orpt i on at 1 5° t o 2 5
°than at
any o ther t emperature .
4 . N ei ther the quant i tat ive fluc tuat i ons of the
s erum pro t e ins nor the t i t er of the s erum was found t o
have any c ons tant re l at i on t o the variat i ons in the
ab s or pt i on .
5 . C e l l s , s ens i t i z ed with a s erum which gave
a l ow ab s orpt ion , were more re s i s t ant t o further s ens i
t i z at ion than c el l s , s ens i t i z ed wi th a much l arger
number of uni t s from a s erum which gave a hi gh ab s or pt i on .
Thi s indi cat e s that the sub s t anc e in the serum , that
inhib i t s the ab s orpt i on , i s real l y ab s orbed by the ce l l s
al ong wi th the ant ibodi e s .
6 . In every cas e t e s t ed , the ex t rac t i on of the
ant ibodi e s was grea t e s t from s erum drawn earl y in the
peri od of immuni t y , re gardl e s s of the t i t er . The
gre at e s t dro p in the ab s orpt i on was found t o b e cow»
inc ident wi th the mo s t ra pid dro p in the t i t e r .
7 . The ac cumul at ed evidenc e is forc ibl y sug
gest ive that the great variat i ons in ab s or pt i on are
Li t e rature c i t ed
Ehrl i ch,Paul ,
" S tudi e s in Immuni t y "
,1 9 1 0,
t rans la t ion by Charl e s B ol duan .
Arrhenius ,S. ,
” Immunochemi s t ry" , M acM i l l mi
1 907 , pp . 1 44- 1 6 6 ; 2 5 7 - 2 5 9 .
Borde t ,J . ,
" S tudi e s in Immuni t y" , trans l at ion
by Frederi ck P . Gay,
First Ed . 1 909 ,p . 440.
Manwaring , W.H. , A Quant i t at ive S tudy of Ant i
Manwaring , W.H.,The Third S erum C omponent ,
J our . Inf . D is. , 1 906 , g, 647 .
Manwaring ,W.H. ,
Anal yt i cal M e thods of S erum
Pathol ogy,
J our . B i ol . Chem . , 1 905 - 6, 1 , 2 1 5 .
Manwaring , W.H. , On the So- cal l ed Phys i cal
Chemi s try of the Hemol yt i c Sys t em,
J our . Inf . D i s .,1 907
, g, 2 1 9 .
Manwaring,W.H.A Fac t or s in Hemol ys i s ,
J our . Inf . D is . , 1 908 , 5 ,5 5 .
C oul t e r ,C.B . ,
The I s o- e l e c tri c Point of Red
B l o od C e l l s , and its Re l at i on t o Agglut inat ion ,
J our . Gen . Phys i ol . , 1 9 2 0- 2 1 , Q ,5 09 .
Robert s on ,T.E. ,
A M iororefraotometric Me thod of
D e t e r mining the Pe rc entage s of Gl obul in and
Albumin in very smal l Quant i t i e s of Bl ood
Se rum , Jour . B i ol . Chem . , 1 9 1 5 , 22 , 2 5 3.
Hunt oon , F.M ., Ant ibody S tudie s , I , I I , and I I I
Jour . Immun . ,1 9 2 1 , g, 1 7 7- 1 9 9 .
Ei senbeng and Volk,Zei tschr . f , Hyg ., 1 902 , fig.
Amato , A . , Ri c erche S per imental e sul l a Fago c i t o s i,
S perimenta l e ,Fl orenc e , 1 9 1 8 , 2 1 , 45 9 .
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