competitive enzyme-linked immunoassay with use

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CLIN .CHEM . 22/8,1372- 1377 1976 )

1372 CLINICALCHEMISTRY,Vol.22,No.8,1976

C om petitive E nzym e-L in ked Im m un oassay w ith U se

of Soluble Enzym e/Antibody Im m une C om plexes for

L ab elin g. I. M easu rem en t o f H um an C ho rio go nad otro pin

D onald E. Y orde,’ E dw ard A . S asse,”3 T eh Vu W an g,’ R obert 0 . Hussa,2 a nd J oh n C . G a ra nc ls ’

W e d es crib e th e prin ciple of a n ew enzyme- immunoassay,

com petitive enzym e-linked im munoassay CELIA , fo rq u an tita tiv e m e a su re m e nt o f s o lu b le a n tig e ns a n d h a pte n s.Intheass ay,bindingofan tibody to an tigen -immunosorben t

is compet it ive ly inhibited by the free antigen to be m ea-

sured. The am ount of firs t antibody bound to the im mu-n os or be nt is m e as ur ed b y a n e n zy m at ic te c hn iq u e inwhich

a h ete ro lo go us b rid gin g a ntib od y a nd a s olu ble a ntib od y

e nz ym e im m un e com plex are app lied in sequence. The

solub le com plex w e used w as rabbit antiperoxidase/horseradishperoxidase.Peroxidase activitysinversely

proportionalo the concentrationinthe originalample of

thesubstanceto be assayed.T h e e n zy m e -lin k ed r ea g en ts

are potentia lly w ide ly applicable to any substance to bem ea su re d. T o d em o ns tra te th e fe as ib ility o f CELIA, w e re-

port a preliminarystudy of itsapplicationto the mea-surem ent of hum an chioriogonadotrop in in serum andu rin e. T he a ss ay d es crib ed fo r this h orm on e h as a w ork in grange of 1 to 50 m t. units per m illiliter of sam ple. T hetec hn iqu e o bvia te s the d isa dva nta ge s a sso ciate d w ithm eas ure me nt a nd h an dlin g o f ra dio is oto pe s in ra dio im -m u no as sa ys a nd th e o nly m a jo r in stru m en ta tio n re qu ire d

isa centrifugeand a conventionalspectrophotometer.

A d d It Io n al K e yp h ra s es : in te r-m et hod com pa ris o n {149}a-

dioimmunoassay {149}CELIA

The principles underly ing measurement of m inute

quantities of substances in b iological flu ids by com -

pe ti ti ve p rot ei n -b ind ing a ss ay s a nd radioimmunoassays

are w ell estab lish ed . C om petitive p rotein-binding assays

and radioimmunoassay procedures require that the

competing substance (ligand, hapten, or antigen) be

labeled w ith a radioiso tope, to achieve the desired

specificity and sensitivity. The use of radioiso topes

makes these assays very sensitive, but it a lso has some

inconveniences. For example, in many instances the

labeling process must be frequently repeated because

of the lab ility o f the labeled compound or the radioiso-

tope itself. The radioactive label may cause destruction

o f t he a nt ig en molecule, or t he labeling p ro cess its elf

may alter the origi nalantig enm olecu le. In addition,he

methods require expensive and soph isticated coun tin g

equipm ent, radioiso tope licensing, and special protec-

tiv e m e as ur es for safe handling of the radioactive re-

agents.

D epartm ents of Pathology’ and G ynecology & Obstetrics ,2 The

M edical College of W isconsin, 8700 W . W isconsin A ve., M ilw aukee,

W i s. 5 32 26 .

‘ A ddress correspondence to this author.

Received M ar. 29 , 1976; accepted M ay 25, 1976.

An alternative label that does not suffer these dis-

advantages would be an improvement. Such is the case

for methods in which an enzyme is used as the label.

Enzymatic activity can be measured in most cases w ith

conventional spectrophotom eters available in virtually

all clinical laboratories. In addition, enzyme activity

measurements can afford comparab le sensitivity be-

cause of the inheren t amplification nature of enzyme-

catalyzed reactions. Several successful enzym e im mu-

noassays or enzyme-linked methodologies have been

reported that overcome the disadvantages of radioac-

tiv ity labeling w hile m ain tain ing the sensitivity andspecif icity desired of radioim munoassay techniques.

Enzyme-conjugated antibodies have been used by

several workers to localize antigens and antibodies in

t issue sections 1), and in immunoelect rophores is an d

immunodi f fus ion 2 , 3 . O ne of the earliest reported

techn iques in which an enzym e label w as used to mea-

s ur e s olu ble ant igen in biological flu id in a comp etitiv e

immunoassay was described in 1971 by Engvall and

Perlmann. This first paper described the technique of

enzym e-linked im munosorbent assay for the m easure-

ment of rabbit IgG (4).4 In th is technique, antigen la-

beled w ith enzym e and an immunosorbent were used.A lkaline phosphatase (EC 3.1.3 .1) was conjugated to the

antigen (IgG ) by the use of glutaraldehyde according to

Avrameas 2). The immunosorbent w as prepared by

con ju gatio n of th e globulinfra ctio n o f sh ee p a nti-ra bb it

IgG serum to B rC N-activated m icrocry stalline cellu lose.

The bind ing of enzyme-labeled antigen to antibody was

competitively inhib ited by the unlabeled antigen to be

assayed. Subsequent papers on this technique have

reported the measurement of other antigens and vari-

ations in the im munosorbent utilized 5-9). Avrameas

and Guilbert have also described an enzyme-immu-

noassay, s im ila r in principle to the Engva ll-Perlm ann

te ch niq ue , fo r q ua ntita tio n of humora l antigens 1 0, 11).The antigen to be assayed competitively inhibits the

bind ing of peroxidase/antigen conjugate to antibody.

They reported both direct and indirect methods, which

are som ew hat an alog ous to sing le- an d do ub le-an tib od y

Nonstandard abbrev iations used : CELIA, competitive enzyme-

linked immunoassay; IgG , immunoglobulin G ; HCG , human cho-

riogonadotropin ; EM IT” is a registered trade mark of the Syva Corp.,

P alo A lto, C alif. 94304; PAP, soluble antibody/antigen com plex of

rabbit an tiperoxidase/horseradish peroxidase; in t. un it, the in ter-

national unit of choniogonadotropin as defined by the W HO Expert

C om mittee on B iological S tandard ization (1964) 2 2 ; GARIgG , goatanti-rabbit im munoglobulin G .



0

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1374 CLIN ICAL CHEM ISTRY, Vol. 22,No.8,1976

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k,.. .-C”--- 4OO.

F ig . 1 . P r o c ed u ra l s te p s used inCELIA

1. CompetI t ion b etw ee n s olid -p ha se I -I CG {149})nd s olu ble H CG (o.) for a lim ite d

a m ou nt o f r ab bi t a nt iH C G ( >R -) . 2. A d di tio n o f e xc es s goatn ti -r a bb it i m m u-n og lo b uii n G ( >0 -) f or s at tr at io n o f t h e ant i -HCG bound to the sepha rose /HCG.3. Addition of ex cess an ti - pe roxi d ase /p e rox i dase -< {176} - . 4. Release ofperoxidase a ctiv ity b y u re a. 5. Colorimetrlc quan ti ta t lon o f t he r el ea se d p e r-

o xi da se in t he supernate

concentration of solubleH CG inthe orig inal sam ple as

prescribed by com petitive protein bind ing princip les.

F igure 1 sum marizes these steps.

M a te ria ls and M e thods

P re lim in ary P ro ced ure s

P re pa ra ti on o f a nt i- pe ro xid as e/p er ox id as e. Horse-

ra dis h p ero xid as e (Sigma VI, lot No. 44C-9570; S igma

Chem ical Co., S t. Louis, M o. 63178) w as dissolved in

physiological sa line and em ulsified w ith com plete

Freund adjuvant (D ifco Labs, D etroit, M ich. 48232) and

injected subcutaneously into New Zealand white rab-

bits. F ive injection sites w ere used, w ith a total dose of

2 .5 mg per an imal. An identical second injection was

adm inistered after two weeks and the rabbits were bled

at irregular intervals from four to 12 w eeks after the first

injection. The sera were pooled and stored at -70 {176}C .

The so luble immune complexes of peroxidase and

anti-peroxidase were prepared as described by Stern-

berger 19). The soluble complexes are stab le for at least

a year, stored at -70 {176}C .

Purification of HCG for coupling . Com mercial H CG

(A .P .L . brand; Ayerst Laboratories, Inc., New York, N .

Y . 10017) was fractionated by ion-exchange chroma-

tography on diethylaminoethyl-Bio-Gel A (100-200

mesh; B io-Rad Laboratories, R ichmond, Calif. 94804)

as described by Bahl 20). The eluted fraction corre-

sponding to HCG was further purified by gel filtration

21 ) on Sephadex G-150 (Pharm acia, Inc., P iscataway,

N . J. 08854). The Sephadex-purified HCG fraction was

collected, dialyzed exhaustively against d istilled w ater,

a nd ly o ph il iz ed .

Coupling to Sepharose 4B . The purified HCG (60

m g was coupled to Sepharose 4B (Pharmacia) by the

method o f March et al. 21). The agarose beads (20 m l

p ac ke d g el )were activated at 24 {176}Cith 1 g o f cyanogen

b ro mi de i n 0 .5 m l o f a c et on it ri le .h e HCG /S ep ha ro se

was stored at 4 {176}Cn 10 0 ml of sodium phosphate-

buffered saline (10 r nm o l/lite r p ho sp ha te , 0.14 mol/liter

NaC l, pH 7.4) containing 0.1 g of NaN3 per liter as

preservative. Recovery experim ents indicated that

about 1.5 of the added horm one was coupled to the

Sepharose 4B , to g ive about 45 ig (or 225 mt. units) of

H CG per m illiliter of packed gel, or 0.9 ig (4.5 int. units)

per 100 M l of ge l suspension as used in the assay. T his

material is stable for at least si x months when stored at

4 {176}C.

H C G R a dlo im m u no as sa y

A d ou ble -a ntib od y ra dio im m un oa ss ay was used, for

comparison purposes, as described by Midgley 22).Rabbi t an ti-H CG antiserum fin aldilut ion 1/18 0 000,

lo t No . 1 54 7; O rth o Laboratories,Raritan, N . J. 08869)

was the first antibody, goat anti-rabbity-globulin (lot

No. 1257; ICN Pharmaceuticals, Inc., C leveland , Ohio

44128) the second.T he Second In ternational R eference

Preparation for HCG, used as a standard, was kind ly

supplied by the W orld H ealth O rganization 23). Highly

purified HCG (CR-115) for iodination was provided by

the N atio nal Institu te of C hild Health and Human

Development and the National Institute of A rthritis,

M etabolic and D igestive D isease, N IH . Na’251 for

radio-iodination was purchased from New England

Nuclear Corp ., Boston , M ass. 02118 .

Competi t ive E nzym e-Linked Im munoassay (C ELIA )

HCG s to ck s ta nd ar d was prepared from A .P.L . brand

H CG contro l No. 1V SB) after e xh au stiv e d ia ly sis

against phosphate-buffered saline. The concentration

of HCG was determ ined by the rad ioimmunoassay

procedure, w ith the International Reference Prepara-

tion of HCG as a standard . W orking standards of 1 to

50 m t. units per m illiliter w ere prepared by diluting the

stock standard w ith phosphate-buffered saline con-

taining 10 g of bovine serum album in (A rm our Phar-

maceutical Co., Chicago , Ill. 60650; Fraction V : BSA)

p er lite r.

HCG/Sepharose, 100 (containing about 0.9 g of

HCG ), w as added to 1.0 m l of solub le HCG solu tion

(standard or sample), fo llowed by the addition of 100

M lof rabbit an ti-HCG (lot No. 1515; ICN Pharmaceu-ticals, Inc.) at a dilution of 1/1000. The m ixture was

incubated for 2 h at room temperature w ith constant

stirring (glass-coated magnetic stirring bar). The im -

munosorbent beads were washed three t imes each w ith

about 10 m l of phosphate-buffered saline by suspension,

centrifugation (1100 X g, 5 mm) and asp iration of the

supernate down to a constant vo lum e of 0.5 m l. A fter the

fm al wash, the beads were suspended in 1 .0 ml of a 1/400

dilutionof goat anti-rabbitimmunoglobulin G (lot No.

2072; ICN Pharmaceuticals, Inc.). A fter a 20-mm in-

cubation at room temperature, the HCG /Sepharose was

washed as described above. The residual bead suspen-

sion w as then treated with 0.5 m l of a 1 /50 dilution of the

anti-peroxidase/peroxidase preparation for a sim ilar

20-mm incubation and was washed four tim es. The

dilutions of goat anti-rabbit immunoglobulin G and

anti-peroxidase/perox idase were m ade w ith the phos-

p hate-buffered saline/album in diluent. A fter aspiration

of th e la st w ash , 2.0 m l of 6 molt liter urea was added to

the tube, and the m ixture was incubated for 10 m in w ith



0 200 W 0O 00000 3O0

A

(400nm>

OA,tioo of AntI-HCG

Fi g.2.Effect ofdif feren tiluti onsf a n tiH C G i n t he a b se n ce o fany f ree HCG(zero s tandardcond it ions ) on enzyme ac ti vi ty re -le a se d f ro m t he H C G - Se p ha ro s eThe limit ing di lut ion o f t he 1 00 z l o f t he a ntlH CG w as fo un d to b e 1 /1 00 0 w he n1 00 f zl o f H C G /S e ph ar os e, 1 m l of 1/400 goatnt i-rabbit immun ogiobulin6,an d 0. 5 m l o f 1 /1 00 a nt i- pe ro xid as e/ pe ro xi da se w e re u se d

U I S er um /m i S es pl e

A(400ii i”)

Fig.3 . A r ep re s en ta ti ve s ta n da rd c u rv e f or H C G ( { 14 9 })S e ve ra l d il ut io n s o f serum from a choriocarcinoma patient are shown (A )

5 020 50

H CG l U/ mI Samp’e

CLINICA LCHE MISTRY ,Vol . 22,No.8,1976 1375

constant stirring . A fter centrifugation, 0.4 m l of the

supernate was transferred to a te st tu be c on ta in in g 1. 6

m l o f 1 0 mm olllite r phosphate b uffer (pH 6.0 ), fo llo wed

by 2 .0 m l o f p ero xid ase substrate (30 m g/liter H 2O 2 and

80m g/liter o-dianisidine) as described by Avram eas and

Guilbert 11), for co lor development. A fter exactly 10

mm at room temperature, the reaction was stopped with

0. 1 m l of 5 mol/liter HC1 and the absorbance was m ea-

sured at 400 nm on a Spectronic 100 spectrophotom eter

(Bausch & Lomb, Inc., Rochester, N . Y . 14625).

The series of reactions that occur, shown di-

agram atically in Figure 1, are as follow s:

- wash

Sepharose/HCG + AntiHCG + HCG (sample) -

GARIgG , wash

Sepharose/HCG/Ant iHCGPAP, wash

Sepharose/HCG/Ant iHCG/GARIgG -

ureaSepharosefHCG/Ant iHCG/GARIgGfPAP -

peroxidase in supernate

T he i ni ti al nc ub at io n t i me f or im munosorbent, free

an tig en, a nd a nti bod y was set at 2 h, to limitappro-

priately the total tim e required for the procedure. No

rate studies w ere conducted to indicate that the reaction

reached a steady-state in th is tim e. Further stud ies are

necessary to optimize th is portion of the procedure. The

concentration of antibody to be used for the assay was

determ ined by testing serial d ilu tions of antiH CG under

zero standard conditions, the object being to find the

lim iting concentration that w ill bind all the available

sites on the HCG /Sepharose. Results of this test are

given in Figure 2. The 1/1000 dilution of our antiHCGpreparation was considered optim al, and was used in

subsequent competitive-binding assays w ith this same

volume of HCG/Sepharose (100 M l). A n apparent anti-

b od y e xc es s r ea ctio n was observed at d ilu tions of less

than 1/500, for reasons unexplained. The amounts of

goat anti-rabbit im munoglobulin G and anti-peroxi-

dase/perox idase should be in excess for the experi-

m e n ta l c o nd it io n s used; both were tested in this system

fo r optimizat ion. The h ig h es t d ilu tio n s po ssible w ere

ch ose n th at w ou ld give the maximum final absorbance

values under z ero s ta nd ard condit ions and w ith the

p ro po se d in cu ba tio n tim es. Longer incubation tim es

with th e proposed amounts of goat anti-rabbit im mu-noglobulin G and anti-peroxidase/peroxidase did not

increase the final absorbance readings. W e have not

done further stud ies of shorter incubation tim es; al-

though it m ay be possible to shorten these tim e s w ith ou t

losing sensitiv ity, the am ounts of goat anti-rabbit im -

munoglobul in G and anti-peroxidase/peroxidase may

have to be inc reased.

Figure 3 shows th e standard curve for H C G . As th e

am ount of added soluble H CG was increased, th e in -

tensity of the color reaction decreased progressively.

The working range of the assay was from 1 to 50 m t.

units/m l of sample. The dialyzed com mercial HCG used

as a standard was equivalent to the In tern atio na l R ef-

e re nc e P re pa ra tio n of HCG by rad io immunoassay.

Conversely, w hen the reference material w as a ssa ye d

by CELIA, th e expected v alu es w ere o bta in ed . T he serum

from a patient w ith c ho rio ca rc in om a w as d ilu te d to test

f or pa ral le li sm. As shown in Figure 3 , cu rve s fo r sta n-

dard an d choriocarcinoma serum coincide. The

with in-run coeffic ient of variation of th e m ethod, as

determ ined by 24 replicate analyses of a 4 mt. unit /ml

s tandard, w as 9.6% . A s a pre lim inary evaluation of the

c lin ica l applicability of the CELIA method , HCG wasm easured in five sera an d five urine samples and the

results compared to results obtained by radioimmu-

noassay (F igure 4).

Discussion

There have now been several enzym e immunoassay

m e th od s p ub lis he d, varying somewhat in principle,hat

successfu lly com pete w ith radioim munoassay w ithout

th e disadvantages of radioiso tope labeling. H ow ever,

each o f t he se procedures stillhave certain inherent

limitations.For example, al l of the previous such



320

1376 CLINICALCHEMISTRY,Vol.22,No.8,1976

28 0

RI A

( lU/mi)

240

20 0

60

20

80

40

40 80 20 60 200 240 280 320

C E LI A ( (L i/ m i)

Fig. 4. A c om p a ris on o f s e ve ra l s e ru m A an d urine ( {149})CGv alu es o bta in ed b y im m u no en zy m e a ss ay CELIA and by ra-dioimmunoassayTh e least-squares regession line has a slope of 0.90 and an i n te rcept o f -l in t .units/mi. The cor re la tion coe f fic ien t fo rhese p oin ts is 0 .9 89 w ith P < 0.0005.

Th e s ta n da rd e rr or o f t he e st ima te i s 1 1 m t . u n It s/ m i

methods m entioned above requ ire a specific enzym e-

coupled reagent for each ind iv idual substance to be

m easured. Prior to the assay , these m ethods require thatthe first antigen, antibody, or hapten associated w ith

th e competi t ive o r i mm un o- re ac ti on m us t b e c ov al en tl y

coupled t o t he enzyme label.CELIA, however, has the

distinct advantage ofnot requiringprior couplingof the

e nz ym e l ab el .S in ce t he goat anti-rabbitimmunoglob-

ulin G willrecognize any rabbit antibody an d bridge

between the first antibody and the rabbit antibody in

the ant i-perox idase/perox idase, CELIA offers the ad-

v an ta ge o f a llo win g th e us e of goat anti-rabbit im mu-

noglobulin G and anti-peroxidase/peroxidase as re-

agents fo r d iffe re nt a ntig en a ss ay s. T he e nz ym e- lin kin g

reagents ar e there fore the sam e, regard less of the spe-

c ific s ub sta nc e b ein g a ss ay ed . For example, in the HCGassay presented here, rabbit antisera to HC G is used as

th e first antibody followed by goat anti-rabbit-IgG

followed by ra bb it a nti-p erox id ase/p ero xid ase com ple x.

Because m any d ifferent rabbit antibod ies as well as th e

goat anti-rabbit im m unoglobu lin G and anti-peroxi-

d as e/p ero xid ase a re c om m ercia lly a va ila ble , the method

can be read ily app lied to the m easurem ent o f m any

o th er s ub st an ce s. C EL IA o bv ia te s the more difficult

covalentcouplingsofantigenor antibody to enzyme and

the subsequent purificationteps required by the other

m et ho ds . T he E MI T m et ho do lo gy i sp ar ti cu la rl ycum-

bersome in this respect, because not on ly does the en-

zyme used h ave to b e co uple d w ith the hapten, but the

coupling has to critica lly position a num ber of haptens

on the enzym e in order that the activ ity o f the enzym e

w il lb e i nh ib it edw he n t he h ap te n/ en zy me c on ju ga te i s

b ou nd t o a nt ib od y.

Som e o f the ear lie rmethods are more susceptibleto

e n do g en o us in te rf er en ce s or inh ibitors than is the CELIA

m ethod, in which the enzym e labe l is not in troduced

untilafterthe so lu ble sa mp le c om po ne nts are removed

by washing, thereby completely eliminating th e p ossi-

bility o f any sam ple interferences. In the EMIT method

one has to b e c on ce rn ed a bo ut t he p re se nc e o f e n do ge -

nous enzym es sim ila r to the enzym e used as labe l, be -

cause the entire assay is done in the presence of sample.

T he l ys oz ym e (E C 3 .2 .1 .1 7) la be l used in some of th e

EMIT urine assayscannot be used in serum sam ples for

this reason, an d, alth ou gh rare,some patients’urine

contains lysozyme. EMIT istheoreticallyusceptibleto

enzyme inhibitorsand other interferents that m ight be

present in the sample. In most o f th e e nz ym e -lin ke d

im m uno so rb en t a ssa y te ch niq ue s a nd th e noncompet-

itive sandwich methods, th e possibility of endogenous

e nz ym e in te rfe re nc e, e nz ym e in hib itio n, o r o th er s am ple

interference is m in im iz ed , b ec au se s ep ara tio n of the

sam ple supernate from solid-phase-bound enzym e

usually involves a washing step. However, because the

enzyme label does come in contact w ith the sp ecim en,

it would theoretically be possible for an irreversible

inhibitor to in terfere w ith the test. O ther techniques

10-14) do no t in vo lv e a w ash step and so are potentially

susceptible to specim en interferences and inh ib itors.

CELIA is poten tially applicable to the m easurem ent

o f b ot h low - and high-m olecular-weight haptens. W e

have success fu ll y applied the principle of CELIA to the

m ea su re me nt o f te sto ste ro ne , a nd th is top ic w ill be thesubject of a fu tu re p ub lic atio n. The coupling of the

hapten to a so li d-phase mate rial m ay becom e som ew hat

more difficultechnically,nd one has to b e c on ce rn ed

tha t antibody recogn ition sites a re n ot l os ti n t h e c ou -

pling process. How ever, th is does not have to be a

prob lem if the same functional group on the hapten that

is u sed fo r conjugat ion to the c ar rie r p ro te in to produce

immunogenic i ty i s a ls o used in th e l inking process to the

immunosorbent . To our know ledge, no EMIT methodshave been reported for measurement of pro te ins. It

seems probab le that it m ay be difficult in the EMIT

system for the enzym e in a prote in /enzym e conjugate

to b e suffic ien tly inh ib ite d on binding to the ant ibody.It is conce ivab le that antibody b ind ing sites cou ld be far

enough away from the enzym atic site that inh ib ition on

antibody b ind ing m ay not occur. Enzym e-linked im -

m unosorbent assay and the other s im ila r techn iques

seem to be more su ited for th e m easurem ent o f pro tein

antigens ra ther than low -m olecular-w eight m olecu les.

O ne reason for not be ing as su itab le for haptens is the

sam e reason that a llows the EMIT m ethod to work:

antibody bind ing to hapten/enzym e may inh ib it the

e nz ym a tic a ctiv ity .

Theoretica lly , CELIA offers the potentia l for greater

sensit ivity than the other enzyme imm unomethods,

because h igher ra tios of enzym e to th e first immuno-

sorbent/antigen/antibody com plex m ay be form ed. In

the second step of CELIA, more than one molecule of

m ultivalent goat anti-rabb it im m unoglobu lin G may

b in d to th e S ep ha ro se /a ntig en /a ntib od y com plex, and

in the third step m ore than one com plex of anti-perox-

idase/peroxidase m ay bind per m olecu le of goat anti-

rabb it im munoglobu lin G , and fm ally th ere c an b e m ore

than one peroxidase m olecule per each com plex of

anti-peroxidase/peroxidase.t isthereforeconceivable

that ratiosof 5/ 1 or greater o f enzym e to first antigen/

antibody com plex m ay be form ed, thus am plify ing the


http://slidepdf.com/reader/full/competitive-enzyme-linked-immunoassay-with-use 6/6CLIN ICAL CHEM ISTRY , Vol. 22, No. 8, 1976 1377

p ro ce du re s en sitiv ity . This multiple binding has been

described by Sternberger et al. 18 , 19) and has been

p ro po se d a s the reason for the observed increased sen-

sit ivi tyof th e “ un la be le d antibody enzym e m ethod”

when anti-peroxidase/peroxidaseisused rather than

i mm un of lu or es ce nc e a nd o th er p er ox id as e m e th od s f or

loca lizin gtiss ueantigen . In addition, the efficiency of

covalentlylinking enzyme to immunoglobulin is quite

low yieldson the order of 4 ) for glutaraldehyde-

conjugating procedures 19). In the HCG assay de-

scribed, sensitivity could be easily increased by in-

creasing the enzym atic reaction tim e and by preincu-

bating th e sam ple w ith anti-H CG before in troducing

th e HCG /Sepharose as in a sequential competitive

protein-bindinganalysis.

The noncompetitive sandwich technique described

by M ai ol in i a nd M as se ye ff 15) and the other non-

co mp etitive m eth od s a ll s uffe r th e disa dva ntag e of de-

pending on the purity of the antibody. If ant ibody is no t

pure, something other than th e antigen to be assayed

w ill b e m e as ur ed . CELIA does not re qu ire p ure a ntib od y,

and in fact comm ercial an tisera are quite accep table.

The authors of the sandw ich technique also discoveredthat rheum atoid factor gives false-positive reactions by

bridging betw een the two antibodies w ithout the pres-

ence of antigen. T he s an dw ic h technique also w ill not

work for haptens that are univalen t.

A variation of CELIA ca n be used to titrate serial

dilutions of serum antibodies w ithout competition by

putting the antigen on the solid phase. For exam ple,

titers of antibodies in autoimmune diseases such as

antinuclear antibodies or of antibacterial antibodies can

be determ ined . The resulting antigen/an tibody com -

plexes can be m easured by the described enzym e-link-

ing process and subsequent activity m easurem ent.

CELIA therefore is a new method of enzym e im mu-noassay for the measurement of soluble haptens and

antigens. It offers a sim pler m ethodology than previous

m ethods w ith respect to preparation of reagents and is

po tentially universal in its ap plication.

This investigation was in part supported by grant C A 14232,

aw arded by the Nd, NIH , DHEW .

References

1. Avrameas, S., Im munoenzym e techniques: Enzym es as markers

for the localization of an tigens and antibodies. mt. R ev . C yt ol . 27,349

1970 .

2. Avrameas , S., C ou pling of enzymes to p ro te in s w it h g lu ta ra ld eh yd e.U se of the conjugates for the detection of antigens and antibod ies.

Immunochemistry 6,4 3 (1 96 9).

3. Stanislawski, M ., Immunologie . L ’em plo i d e I a peroxydasecommem arqueur dans la quantification im munochim ique d’antigenes et

d’anticorps. C . R. Acad. Sci. 271, 1 45 2 1 97 0 .

4. Engvall, E ., and Perhnann, P ., Enzym e-linked im munosorbent

assay (EM sA ). Quantitative assay o f immunoglobu l in G . Immun o-

chemistry 8,8 71 (1 97 1).

5. Engvall, E ., Jonsson, K ., and Perlm ann, P., Enzym e-linked im -m unosorbent assay. I I. Quan ti ta ti v e assay of protein antigen, im -

m unoglobulm G , by m eans of enzym e-labelled antigen an d anti-

body-coated tubes. B io ch im . B io ph ys. A cta .2 51 , 4 27 ( 19 71 ).

6. Engvall, E ., an d Perlmann , P ., Enzyme-linked immunosorben t

assay, E LISA . III. Quantitation of specific antibodies by enzym e-

lab eled an ti-im m uno glo bulin in an tig en-coated tu bes. J. Immunol .1 09 , 1 29 1 97 2 .

7. H offm an , D . R ., Estimation of serum IgE by an enzyme-l inked

immunosorbent a ssa y (E LIsA ). J. A llergy C lin . Im munol. 51 , 3 03

1973 .

8. V oller, A ., D rap er, C ., B idw ell, D . E ., an d B artlett, A., Microplateenzyme-linked imm unosorbent assay fo r C ha ga s’ d is ea se . Lancet i,

4 26 ( 19 75 ).

9. B elanger, L ., Sylvestre , C ., and Dufour, D ., Enzym e-linked im -

m unoassay for aipha-fetoprotein by competitive and s an dwi ch pro-

cedures. C lin . C him . A cta 4 8, 1 5 (1 97 3).

10 . Avrameas, S., and Guilbert, B ., Im munologie. D osage enzy-

moim munologique de proteines a l’aide d’im munoadsorbants et

d’antigenes m arques aux enzym es. C . R . Acad. Sci. 273, 2705

(1971).

11. Avram eas, S., and Guilbert, B ., Enzym e-im munoassay for the

m eas urem en t o f an tig en s u sin g p ero xid as e c on ju ga te s. Biochimie 54 ,8 3 7 ( 19 7 2) .

12 . Van Weem an, B . K ., and Schuurs, A . H . W . M ., Im munoassay

u sin g an tige n-e nz ym e c on ju gate s. FE BS Lett. 1 5, 2 32 (1 97 1) .

13 . Mas se yeff, R ., M aio lin i, R ., an d Bouron, Y ., A method of enzyme

immunoassay . Biomedicine 1 9, 3 14 1 97 3 .

14 . M aiolin i, R , Ferrua, B ., and M asseyeff, R , Enzymoimmunoassay

o f h um an alpha-fetoprotein. J. Im munol. M ethods 6 , 3 55 (1975).

15 . M aiolin i, R . and M asseyeff, R ., A sandw ich m ethod of enzy-

moimmunoassay. I . Application to rat and h uman a lp h a- fe to p ro te in .

J. Im munol. M ethods 8, 223 1975 .

16. Rubenstein , K . E ., Schneider, R . S., an d U llm an, E . F., “H om o-

g en eou s” en zym e im m un oassay . A new im m uno chem ical techn iq ue.B iochem . B iophys. R es. C om mun. 4 7, 24 6 ( 19 72 ).

17 . M ason, T . E ., Phifer, R . F., Spicer, S. S ., e t al., A n im munoglob-

ul in -enzyme b ridg e m etho d for localizing t is s ue a n ti g en . J. Histochem.

Cytochem. 17 , 5 6 3 ( 19 6 9) .

18 . Sternberger, L.A., Hardy, P . H ., Jr., Cuculis, J. J., and M eyer, H .

G ., T he unlabeled antibody m eth od of im mu no ch em is tr y. P re pa ration

and properties of soluble antigen-antibody complex horseradish

p ero xid ase -a ntih ors era dis h p ero xid as e) a nd its u se in id en tifica tio n

of sp i roche tes . J . H i st och em . Cytochem. 18,315 1970 .

19. Sternberger, L . A ., Immunocytochemistry, Prentice-Hall,

Princeton, N . J., 1974, p 129.

20 . Bahl , 0. P ., Hum an chorionic gonadotropin : Iso lation and

p hy sio ch em ic al p ro pe rtie s. J. B io l. C hem . 2 24, 5 67 1 96 9 .

21 . M arch, 5. C ., Parikh, I., an d C uatre ca sas , P ., A s im p lified methodfo r cy ano gen b rom ide activ atio n o f ag aro se fo r affin ity ch ro matog -

raphy. A na l. B io ch em . 6 0, 1 49 1 97 4 .

22. M idgley , A . R ., Radio immunoassay: A m ethod for hum an cho-

rion ic gonadotrop in and hum an l ut ei ni zi ng h o rmon e. Endocrinology

7 9, 10 19 66 .

23 . Bangham , D . R ., an d G rab , B ., T he s eco nd in te rn atio na l standard

f or c h or io n ic g o na d ot ro p in . B ull. W .H .O . 3 1, 1 11 (1964).

competitive enzyme-linked immunoassay with use

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