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

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-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 ,

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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

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( 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

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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

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a .X I eidsT

.XI eidsT QSI Jess To do l q ms iimia .6

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vino ben immedsb ed use a has X To eeu Eev ed? sai l.ziezsmi x omqqs

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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

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as: , .s.i n ,As .31a mi has ,e.o n h .I 3 1a 8 1

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,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

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edi bus bevmesdo ed: messi ed J nemeemgs eeo i o vd ev s

.ai iuseu bedsiuois e

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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

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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

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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

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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 .

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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

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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

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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

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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

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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

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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

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.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

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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

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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

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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

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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

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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 .

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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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