Bepaling van het gehalte aan soja in vleesprodukten m.b.v. ELISA-methode

Afd. Diergeneesmiddelen

1983-04-16

RAPPORT 83.36

Pr.nr. 505.0010

Onderwerp: Bepaling van het

gehalte aan

Bijlage:

1.

soja

in vleesprodukten m.b.v.

ELISA-methode.

Verzendlijst:

direkteur, sektorhoofd

(2x),

direktie

VKA,

afd.

Dierge-neesmiddelen (4x),

afd.

Normalisatie (Humme),

Projektbeheer.

Afdeling Diergeneesmiddelen

1983-04-16

RAPPORT

83

.36

Pr.nr.

505.0010

Projekt: Ontwikkeling methoden voor het

aantonen

en bepalen van

vlees-vreemde eiwitten

Onderwerp: Bepaling van het gehalte

aan soja

in vleesprodukten

m.b

.v.

ELISA-methode

Bijlage: 1

Doel:

Onderzoek naar de bepaling van vleesvreemde

eiwitten

met behulp van

ELISA-methode.

Samenvatting:

Het

gehalte soja-eiwit

is bepaald in 3 monsters vleesprodukt

met

bekende hoeveelheid

soja en

vervolgens in 7 monsters vleesprodukt

met

onbekende

hoeveelheid

soja.

De bepaling

geschiedde met

behulp van

een

ELISA methode.

Conclusie:

Uit de

gevonden

gehalten ten opzichte van de opgegeven gehalten in de

proefmonsters lijkt de methode iets te hoge waarden te

geven.

Verantwoordelijk: drs F

.

G. Buizer

lol

edewerker/Samensteller

:

mw M.A. Visser-Heijer

\

'

v

'.' • r . I

...

_·'

.: .:·

..

, 'I ' . \ ... , r .

1.

Inleiding

Vleesp~odukten

kunnen naast de vleeseiwitten ook

v~eemde

ei

witten

b

e

vatten. Deze

vleesv~eemde

eiwitten

worden

toegevoegd als

bindmiddel

om

wate~

en

vet vast te houden.

Wannee~

een

de~gelijk p~odukt

geanalyseerd

wo~dt

moeten

de

vleesv~eem­

d

e

eiwitten

onde~scheiden

kunnen

wo~den

van de vl

ee

s

e

iwitten.

Het

~appo~t

"Quantitative Methods

fo~ Diffe~entiation

of Vegetable

and

Animal

P~oteins

in Foods'' (Document CX/VP

82/4)

dat in

1982

aan

de

Codex

Alimenta~ius

Committee on Vegetable Proteins is

aangeboden,

concludee~t

o.a. dat

e~

nog

geen algemeen

aanvaa~de

kwantitatieve

bepaling

van

plantaa~dige

eiwitten in

vleesprodokten b

es

taat.

In

antwoord

op deze

situatie,

h

eef

t

Euv~epo

(dit

is een

o~ganisatie

binnen

Eu~opa,

die

zich

bezighoudt met

plantaa~dige

eiwitten)

de

ve~­

schillende

beschikba~e

methoden

geevaluee~d.

De

2

mee

s

t veelbelovende

methoden,

een

SDS

-

PAGE methode

en een

ELISA-methode,

wo~den

getest en

ve~geleken onde~ p~aktijkomstandigheden

om

te

kijken welke

als

stan-da

a

rdmethode

geb~uikt

k

a

n \Wrden.

In dit

ve~slag wo~dt

het

~esultaat

van de ELISA-method

e

besch~even.

Deze

m

e

thode

is

gebasee~d

op het

a~tikel

in

d

e

bijlag

e

.

Standaa~dsoja,

antistof

en conjugaat

we~den doo~

de

Euvrepo

te~

beschikking gesteld.

2.

Methode van

onde~zoek

De

geb~uikte

methode is een

indi~ekte

ELISA

-

methode. De sojaeiwitten

in

vleesp~odukten

zijn

mee~

of minder

gedenatu~ee~d

en onoplosbaar

doo~

de

ve~schillende

proceshandelingen. Zij kunnen wel

oplossen in

hete

u~eumoplossing.

Na

ve~wijde~ing

van de ureum

doo~

dialyse of

ver-dunning,

waa~bij

de

eiwitten

"~enatu~e~en",

kunnen

antistoffen

gemaakt

worden

.

Een

hoeveelheid

monste~oplossing

laat men

~eage~en

met een

bekende

ove~maat

antistof,

\~aa ~na

de overmaat

antistof

bepaald

\~O~d

t

.

3.

Resultaten

Aan het

~ingonde~zoek

ging

ee~st

een

p~oef

op

monste~s

met

bekend

gehalte

soja

voo~af.

De

~esultaten

hiervan

wa~en.

monste~

B

c

8336.1

o

ave

%

0

1,94

1,51

e

v

onden

%

n

.

a.-n

.

a.-n.a.

1

,

5

-2,6

-

2,2

1 8

-3,2

-

2,1

- 2

--

2

-Vervolgens werden de monsters met onbekend

gehalte

soja onderzocht

.

monster

1

2

3

4

5

6

7

%

soja in totaal produkt

2,0

-2,2

0,03-0,02

2,3 -2,0

4,0

-4,2

3,2

-2,5

1,8 -1,4

6,2

-6,0

De

werkelijke waarden

en

de waarden, die door de

andere

deelnemers

gevonden zijn, zullen

te

zijner

tijd door Euvrepo

gepubliceerd

worden.

4. Conclusie

Uit de

gevonden

gehalten ten opzichte van de opgegeven

gehalten

in de

proefmonsters lijkt de methode wat te hoge waarden te

geven.

·.

t

~.

.'; (

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!. ' · ... · -,

'·.

-

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·

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:

1 ,

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·

·'

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.

.

.

' ) ·-\.'·I I \·• , ! I ' .. r -{ I,Î

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,,,

I • • I

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-

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.

,

.

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l•o I .· : :I dJ bn& .. -~~~ ... '· 'f .. ~·· . . •;.'. · )~~ I ) ' ~, n. ::..·:, ::,·· . ' .. ·.. ·-:· :··: .. -~·.. . . .. '·. _{' '.J. ~··· ••} ·:...i I ' , . . . , .

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-

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..

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_·

_.

_·•·' ') ' )• ::. ': i t.

,·,

·:_, -~I·' .··: ·:, t'·,, !.· _. ~ ··:·.J')·),;.'. • : I ~ • • ~; •, • : • I • • ,., ~ ~ r : ) . ·-,( ,,- ),'·, '-. .-, :·· f b,',/ ·;, _'· : ' -'1 ,. ':1

,,

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..

...

_,. . ~,

~-....

,; .-

...

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.

\ ( .

,

.~

J. St·i. rood .-IJ.• riL I'}~ I. .'2, 15 7-16~

Determination of Soya Proteins in Food Using an

Enzyme-linked Immunosorbent Assay Proeeduren

Christorher H. S. Hitchcock. Frank J. Bailey. Andrew A. Crimes;

David A. G. Dean and Paul J. Oavis

Unilt••·rr l?~lrorrh l.Ahora/urin, Colk·nrlh Hnusc, Shornhronk,. B~dford MK44 JLQ

I Munu.<rripl uain•d /9 .\fuy 19/JO)

Classica! irhmunoanalyticaltechniques arebasedon native amigens and their

precipit-ation with srccific antibodies: they are nol very approprÎl:lle for the investigation of

processed denaJured mixtures. This report describes an immunoassay based on the

more recent but well-established enzyme-linked immunosorbenl assay <ELISA)

procedure, which in this case is specific for soya proteins that have been solubilised in

urea and then ·renatured' by removing or diluting the denaturant. Levels of soya protein 100 g-1 _{total protein (nominally 100%) were determined with 34 commercial} soya products, using a standard reference antieen. Normal expected levels were

ob-served with flours (average

107

~~

)

and isolates (l08%). while

resuhs~vith

concentrales

· (82%1 and texturates 179~·~) were somewhat lower. Some specialised produels gave

liltie or no response: presumably becausc they are hydrolysed. Meat, milk, egg, wheat

· and field bean proteins displayed negligible interference. These preliminary resuhs suggest thal the ELISA procedure will pro,·ide a convenient general method for the qualitative chamcterisation and quantitative est i mation of individual proteins in food prci:lucts e,·~n after severe processing: it scems more altractive than many other met hods r~p::>nc:l. I mmunoJssay is opabic of large numbers of inexpensh·e but

sorhis-tiotcd d.:termin:11ions of srcdfi~ food components. Thc food analyst shouh.l rc~:~rd

ELISA tfor th~ determination i11 situ of specific antigensin mixtures) as a powerful

techniqu~ comrlcmentuy 10 classica! chromatographic and ~lcctrophoretic methods

'~·hich depend on serariltion befare determination.

I. I ntroduct ion

I( · Dctcrmiriation of soya prof('ins in mcat produels

Processed foods based on traditional meat produels maya lso contain foreign (non-meat) proteins

addcd as binders 10 improve texture and the relention of water or fat. or as meat ex1end~rs or

analogues. In analysing such a product. the foreigri .proteins must be distinguished from 1he m~at

rroteins: 1his presems a complex problem. cspecially when the proteins have been hea1ed during

rrocessing. In elfons to find a reliahle and COO\enient mcthod that could be used routinely. s0ya

rwtcin has been dws~n as a model f(lfcign rro1cin: this is oh en addcd as a ftour. con.:cntratc.

is0latc or texturate toa n~eat product that may then he cooked, canned or dried. l\1any dilTerent

appro<Jches ha,·e been rcponed and re,·iewed.'-~

·J./.1. Hi.wo/oJ.!y

A prcliminary microscopie examination provides general qualitative subjective analysis; in

panicu-lar. calcium oxalate crystals and plant cell components indicate soya, and staining for cell-wall

•This p3per "·as ~i,·cn in· pan at a mectin~ of thc Society of C'hcmical lndustry tFood Group) dC\'Oted 10 thc

utili~ation and identification of meat analo~ues and extenders in meat products, held at Sunon Bonin!!IOn on

~0-~t ~1~rch 1980.

00::!2-.5142;1!1.0200-0157 S02.00

f

1981 Society of Chcmical lndustry

1~1

/

.

:/

158

.

,

.,c

•.

.

H. s,,Hilçl>c:od "Dl.

polysacchllride is useful. A powcrful but·'lengthy .. technique inv.QJ.vc.s, selective protein, foliowed by quantiflcation using stereological'technique:s'.'

(

\.

~~

.

J

. /./.]. /mmiiiiOfiSSU,I'

'

L_~\

.

.

._-% .. } .

staioing for soya ...

• ~ · I ' '· · ', t I~ I

.,,

Methods based on antigen-antibody interactions suffer from the variabie respon~. of individual

animals 10 the chosen antigen; they h~ve ~n used in laboratories where the supply and standard·

isation of appropriate antiserum prék'ni"no

problems.

~

Qua.n!i.téltive

i!l)

.

llJUnQa~~y

of

pr91ein

.

s.J~

.

affected by their degree

?f

denaturation and therefor.e. i.s nol

.

i

.

~edl

for

~~e

,.

~r~~

anal.ysis of heatei:JI

pr.oducts.

J

mmunochemtcal mcthods

~re

generally

.ltmJI~

-

~

.'?:

thf

qu

.

~}llai~~e

~

tr~mn

_

g

J

of

.·

raw or:

·,:.;-:

mtldly pr.oce.ssed products.4 •

1_

_..

.·

·:

.'

.

..

i

{_

t.~~~

..

_

l('.!

:

~

....

.

~

,

_ ..

:

,l' l,&t.r· • ... .,~. ·.t• • ··~·rl ... "" . , "'~ •:,,,

1.1.3. Electrophoresis ...•. ,. ,. r .

/'t:

.

•

·

~·-

" ..

..

\;

·.J:" J. • .·, •••

•·

.

-v

tl·~· :' .·

Electrophoresis of de na tured prot

ei~

·

~

.in

~~Is

containin:i;

,

d

-

~fl!·r~eF ~

-

r

L"

fl

is

t,pp~Îar ~nd easy

~

to

,...-...

pcrform

.

6

_-

_~

The reliability of the data usually depends

O~Jhi

sqlubili~Î

-

~~n

.an'd

çiis.agg~eg~ti<:>h

·

~rl

the proteins and on the quantification by dye-bindiQ.&.:<\!Vl

se<~iming;

.th.(.l;.eSQ)Jtion of I!Q.cnP..Ie11J

mixtures from heated or canned produels is often poor. ':: .. : • •;

. · •. ·,_, .,,· f<.

.

.

:.

~~

-

...

~

:

:..

\. ·

·

.

.

I

h.·()' •jt1 \ .<

~

')

.. '4!"' '4'

1.1.4. Pep11de onulys1s 1, ·. ::•, ~ r·· ... • ". ·,., ...

ï

I ~ ·l ··.· . . -~ .• '-~

I

.

Specific cle<!vage of the denatvred protein (e.g. by tryp~in),yièl'ds·:a very ~omplex n)ixture '!f,,splul?~.

peptides which may be fractionated (e.g. in an

ordina~~!:QÏI}R

acid

a~

·

~

~

l~~

·

~

.

.

a

.

n~

a

ch~!]~t

:~

{i

.

s..,~;

:

peak selectedas a measure of the parent foreign protein.10 _{'rhe.apiJ}ysis of soya protein can-be.bastQ:}

on a pentapeptide ~er-Gin·-Gln-Aia-Arg ('SP-I', a fragment of the J IS storage protein). Although

the method is cumbersome and diffic'll\}'?.s,tandardise, it has provided useful data, panicularly from

canned products.ll-13 On the other'.hànd~ torar"fl)i'drolysa_I tes.yiël(f tra,.d_l Tkional.aminolàcid _{, :} level~.· l",

which can be more easily measured; computer-assiste~ compári~d.ns wi~h,dii}áJfom kno~·n mixtures .

leads to estimates of the ratiosof proteins present,

pr~

~i

~~_d.

.

'-h~

s

,

~ a~é ~o

~t~

9.

.

~i

milar

~

n

~m)nW

.

~q{èl

·

composition.~· u c· . ₁:.. ··:! ,,.·. 1

' ;". • H • . :·

..

--:

I. I .5. Jndirectmwlysis .,. ; , • ~ .·· , .. . . ,. .. '"' , ... . '<;·~,

• ·-· !. J ·','!.·. ~ • . •

Si nee direct prorein characterisation is of ten difficult, indire1:t mc:thods.ba·~ifon.'Jevel~'of appropriate

non-protein markers associated wi~h. $Oya are. qualitatively u.sçfvl~ Th~s.e convenient techniques may

' .J ' ' \ • •• I\ i }.o t 0, '' f ' '' l lo 1°' • •' • ,\ •o ~· ' I. • ~h

ofren be made quantitative if th~ ;iÇIU;il,s,o);a

4

ingredLt;Qt,~seÖ _\11,1~\;

1

1)')~;3\:.f?f,OQ'{<;I Ï!i·~vai)ablt;separ~:.t: .. , . . r,, .· ..

ately for direct comparison. Suiráble;markers have:·been <>hosen..for,Jheir-srabiliwa.nd.~ase. or as~ay; .,;: . . . ,, ... ,,

ideally the proponion of m·arkU'ló protein shöuld 'be'i·ndepen·dclWbf-such' ~ctors as the-biologica! :·, .... · .. ~.:.. . · , -~' · vllriety, maturity and growth

cqfi~!\'

i

.

é?n~

~f'thègó);{b.!att~

·

~~

l!

1

tyl>e

d

r

·

bs·~

'

tngtè~i~nt (flour~

texll.irèd J , · .. . , , . ••

~

.

, , . , ,,. , " ' • •• •

flour, concentrate, textured

c;,;

·

~

,

dQ'i~i~

·

o:r'

i~o~~~!!~ a~1,

;~

,

tti

r;

P.i~

;~

sf~

.

r.~~

ri9Jt

t~

.

~~

-·

~

~

~

d

.(h,eaJ~

.·

~

'

.'/.:

.

. ·,

:

.. :.

;.

, ,

,

,

:

~

·

leaching ere.). The search fora so:va·index

hii!>

:

included..in

~

9.1u61G P.R!YSJl.'C

.

J:l~r!çlç

,

s

.

.

oligc;>

.

~ac;ch~[ide.~.

,._,.,, . . , ,,,. ; ...

protein-bound sugars, free amino·a·cids. free-peprides, ph)'tate·,- saponins.·sterols,and mer·als. bot none 0 :,( , ,, •

is ideal; the sratutory addition ofan anificial marker (e.g. titanium dioxide) has a lso ix!M suggested·, . ·•.

but is not fa ,·oured.

:

:

_

\:>:

,

•

;

1 • •, l ''o '! ! ' I \ ) j f) 0 • I • ~~

1.2. Enn·me-link€'<1 immunosorbent. assa,· (ELISA) of denatur€'<1 food· protèins ·

. :. ~ .

Man)•

cl~ssical

immunoamys

:

~

~~~

-

ba

·

~~

-o~

:·

nati~

·

·

.

e

:

·

a

,

nt

·

;~~g~

·

:

~nd

.

~h~]~

.

=

.

'

p

·

r~ç

.ip

ir

.

ation

,

with

:

,

sp~è

'

ii;Ç

~.

'

~

antibodies. Much of the difficuJty

e

;

~:PeÎ

.

içnced

.

il).:th.e

ap(?lie<jtip

.

~

·

o~ir{l,.;;~

·

n~che;.Jîi~lt~chn:iquesÏ~

:.··

.• ;j: .... : ... ,

, ' I • • • • • • •0 0 ' t I•· .. , •.,( ' ' ,I 1.J

food a na lysis arises through the ne~id to qet.~rminç,J:?rqçe$~<';9 (oo,~:in~r«;Pi~(l!S:"~p.ose ~rp.my.nqc~emic.a1 , , , . . ·<• ...

properties have changed, and thmugn .• a ·rç_guir~rn~.p,r:-f~r-sup.P.Ii~s,çf:~R~r~oerfa'e.Jrand~r,?i~~9. r-:nti-. ~ • : , ) ....

bodies. Koh1_{~ has demonstrateQ; ~tte fea.sibjlity, of ~~tra~!Lns .. t;he!P[9teJps _unqe[ de~~tprin&,~PP:. i· ... , ..,}

ditions, removing·the

denarur

a

rl)~

!:ly,

~iaJxsis·a~.d

.

s

.

q

.

br\~sir&

a!'

wp.tÇj'?

.

~ (o/~I!J~er~n_,

~

tf

v

.

e

,

or.

c~

·

oked

·' . '

to 7 I °C) to a common conform~tj,on.:· aflti.t>odies wer.ç ~hen ';'ll.ise,d·?gi\inst -~~~~r; ',re')at~Jre~');>rfl,teins.

which were then used to assay s(n1i!arly 'rerJaturc,<;l.: anti8-çns ,b>.·· roeket imrpuryoeleçtr,ophpre~i.s ..

Enzymc-linked immunosorbent

as~y ~

-

~()çequre$

are;

~ell esta~ii

.

~~ed

in,cli,(ii.cai

~~ál

y

.sis

;16-I·P.Ji\e., ·'

. . .

)

)':-·.î

.

~ . ~ : . .

159

Á Soya antigen _{... Î1SI step, J}

' ···I ;, •• ,,1 ~ •

,.,-<?

Rabbit anti-soya antibody .. ._ .. -ii ·"-! '"" : \ ~~j\,~. ~<! -.~,--;, ~~Ïl':l&ll j.... j:.;!' . ·--;yj :" -·~-­

,

)

·-::

.

,

:

.

;

;

nlJr.

m

i

_,

.

,:lr

.

:

;:

n

·

t.

)

.:

·

.··.~·;·· ·.2"?.~~F J:". • ,l''·,!'Jio: 'oo!.,. -~ . :' • ~I I.·' .. I i ·• '! I : • tn.T. · . · ·'· .

.

..::·· • _,, -,~-:;- J { : • 3rd

step

1 -•: t '· ..'1:. ; ; l0

<:.,

·j -:<i I ·. I ' :

:rt

'

.

, .

4th step

~

Shecv anti-rat>bit antibodöes anti~erafen.zyme coojvgate , ... :

..

.,

.... _, ~ I I : ::,·: • I •;, o: 'tl') :~u· • 'i. i • ;~.( '( e .. · ,"'; I ~!

•

:

••, '].

-..;, I ~~ .. 0 ~ . . . :·:"'.:•!.;

• Enzyme subsirale ; :, J .f!1:J.,~tJ b~ · ', · .·:J;,' ' '•'!:'.)~~ '':"','> •)f j1'r; .. i:'\; I _;"•

--17"-;c.-:::--~7"";r-:::-:-:--:-rr.:;:-~:-::c-:-~t-~·,..----r· -·-:-7· • • !>th st~-- ... ··; • ·C .• _I !J•f i.l'•' t :! ':_,; .'·:: -~

"""".,.,."''"""·"u ~r::~i:.~c

1

ï • · ··-·':·-~-~-~>~.._,:::~ :--"' :-· · l

Low soy High soy

High colour

.J

.

t

L

~w

-

c~~r

f

. ,

'

· Speclropholometer cel! a~

1

aut:J 61h step

"_:·,;·y,·: .,"

ld2JtJY..S5ilr!

1

1?1..:u.

~

~-

··

..

1 br::

,1

:r

.:

:!> ;

i

~,;

... :

. :: .. l~•::J;."r-.1. .. ::1 ~• :;~~·,~ 4,>t,f) :lj) .. ,."•l)''!o.:, 'hj ; .. 'oj '1.." 1{) ·•

Figm I .. En:.~·m.-·!in4rd immunOHI~~~1'~ .ut~-~'·'· f.~~-~·~do;n·.: .. ~~l si~~ ·,(~~-cl.!O~ ,2·~·1}; ·~r~p~roiio~ 'ó/ ~c•nJif~!d -~·~1/s~.

thc PVC mocrot!lrauon plat~ contaoncd·~6 "·clls: cac~ v.·as scn!ll,~ttr~1nlrarfil'gt'n'Ci'cr\lltllrcd·soya··proteon!Jsolaf~) ·

undu conditions which bound thc

proiéln

i&

·

lh'c~innc'o<;-''.ill';,öf.lih"ct \\'tiiSll)yr'JSa'ssi\·o lldsoi-pri~i lrod 'stem(~Jon .. ·:

2.2.3): samples and standa'rd SO)'a ~linionsitic:ni inc.ubl!.lcdb\i.•ilh J)~CP1>SifroikP.dt.l.n ~cp.~rM~;!ll.~ss ,:;_,ls"~~;f'f!! · .... '·

(w' ion 2.2.4_): re actio~ mix1ur~ wtrs :Ha(lsfcrr~d ·'~ ~-~'l~!lisç~ •. wc11.~o?.(ql~!-'.f~ .ll;'(tÎ{')Y~~.a,yo,IJ,e,9d,,~;a•p;~~;?

1

~! ;~~~ ~ ₁ , ,

ui Jnd anul>ody-;onugcn

complex~

;

~

-

1~

s•t:

.

l'

~

V~

ç

••

.

o~}}

.-.

1

)j

c

~

~c

~~-

~

-

"

~

'}"'~~dr-_!.~~d)~~

-

~

-

~r

.

h ~

_

nz

y

~c

.

~~~~~l,n<'

.

phospha12sc) "'as addtd. foliowed Dy 1r;ocu~auon and w~hlnl:·Oul. S"lh s\~p (~!"CIIon7.i:6j:'chlymc sûóS'tr.itC""(p··' ' ·'1

·

nitrophtnol phosphalc) was addcd ail'/{ii\i:J.Îbá'(ed?Üht 'rciiCflo~'Jv.'ifs -~tOpp~i}t>J;:<ti\:~Sdlulfi'.fifdroAÎd~fJo6th'~lep·· ·.':'I~

(~~ct ion 2.2. 7): \he colour intcnsily 'V.''às"' riit.Ïsul'èd .a\' 40tr>p:/to afftr. I1130SfC{lin&nsUI'!tl(!n$--IPja :\~.CIIO(lhol,q!Jl~l5r ..

ccll. prcfcrahly usint: an autom~tic $amplins jystc;m.; .;., !c ~:::rl, ·)·. · •(; (> :r·:;,n;;1;1 -~- 1) ~ ... rr. · ,;~.r-. . ,.

•;:j ::r· r •. : .. I<. ·-·~ ~·.

'.r.

.:::r

·.

·.·

..

,

.. ·ï .J lfl. : .l 'l i •; I~ •l •• • ... L · .. ·: .'t! :i :: ,, 'L''

ftuorc

s

cen

c

e

immunoasS<~y

and

radioimmunoas

s

ay.

t9~.-~~~!~~-:;~,n~i-~?~,Y _i_~tf,ra.cli;<?rJ a[~ ,f?.ll_o~~d

by

latxllin

g.

1hough

in

1his case

.

t

.

he label

.

i

s

an

en

z

y~rie rat~er

than à fluo"rescent,

cherrïilurrÎJnès

t

ent

·'-

·

L :

or

radioac1ive marker.

Since éhe

"'

réthnique

'

1

_öbe

_·

_s

_n

_'

_ói'

_'

_(Ïepen"\J

_{ör\· pr~cî~iürliörl,l'1he}

_àsS8y

_:<t

_an

1_{be~ ··::•.o·"W ..}

_r

_l'

_!;·

_r

_.

carried

OUI

using

small

a

.

mount?bnll&plé

·

.'

s~i:h

·

'

~'rll

-

~

'

JI

·

volt.im'èslófáHil's~ra a

·

re

r

e

qVifed

:

(l'

i

ni'J~án be

l:;.

I;:

t:·

... ;;

.

:

r·

. . .,.

'sufficienl for

1000

assays or

m~~·é)Tt1'a1

~

tÄiftda~disà

'

il~rrbèddm

ti

s

~

J

iss

df á

'

-pf-btilen'i:

''

Amiseru

·

m

·

rrofh

'

'J·, .. 1· .;,_ !i-·•

one

animal can

th

erefore

bc

fulfy

·1.<

h~'i~ci'

è

ti

s

eél,

'l:ö

tn;päfHfV.

1

itl

f

sintifär

antrsera

;

'

aiid ustll'fo

"

r'

J

irll<lr-

..

.

-

~'!

·'"

:

'

". V··

hboratory

asse

s

sment and

the

";f0:~iys(~1 oYi'~ousàrtlfs1bNäl-hplts:·rh'FlinHgèns

lrt

fö'

6

êrP'r

<>

du'bis

art

.1•.). •· •• :. =~=

.-

.•

,

_.

.i' , ..

more

or

less denatvred

3;nd

inio)ûi>ië:

·

àftd~H

;

elr'

v

~

rl~ó

'

s

'

tH&

é

s\lng

-

lré'it

'

rHëntS'

;'

öu\

cärdse

'

s<11Übil·

='·'

.

·

-:·H ...

F=:

·:-.

-.. ·

.'r:

oi--ised

in hot

ure.a

solutions

under~ é~:riditi"oHF~stal:ÏIÎsHéd tó·r<êleé'rrópliófèsi's.6"Be~~iü~ àntï~dy.:.! .·'J1:: ,:, . :•)) ·•

0

antigen

intera

c

tions cannot

occ

u

.

di(tH

~

p

'

r

;

èS,e~

;ëé·

·

9

r

·

Ïhe

1

_d

_é

_rtiuuránts

1

tnè

~'

latiëY

_·

_.".

_'

_él'é

_·

_fèmové

_·

_d·

_'

_o

_y

_·

·.~(

_·

_-

_''

_.

lbO C. H. S. Hi1chrod: ~~ui.

I ' > ~ ,

1 • native

state.'~

Antibodies raiscd aga,insl

J,

h

C.Ni

:

;

re

.~

atured

;

.,

a.nt~e

n

s w~r_e

mj!d~.

:

~~c',.ba

.

s

i

s

·

C!

·

~

.

an E

LI

~A

.

procedure illustrated in Figure I. ~hich .. ~as' l_ested RY applying _it_:ip, rfiripus c.cimmércial 'sè>ya ''• '·' : ' . ·'1

produels after similar

'renatur~iion'.

_'

rtii$

·

:

r~pQf.l dc

ië

dbe~ ine

~'

rpt~lh

~d:

~n

·

d

"

e

:

~cpur

·

agin~

-'

lni

üal

·I:'· · . ':

resuhs. .. • • # • • • • ~ .. I; • • • ; • I . . ~. • . --: f : • t -:: . t .! • ~. :. i ~· ,_1) ( ",/ I ,

.,

,. 2. ExperimcnUil .I • • • • .{ 1 -' .

2.). Prcparation o( immuno-rcagents

~--~ .) , \\:.1: ; , • ' \ ''•' \,; ) · . , ·.;, · ' J"'·.·.\'···.h,,\(. •.,.'•·''· '.: ·.l

2.1.1. 1_ntibodies 10 renatured st?_ya f!f,P,'e_i(l., ___ .

~·

,_ _ · ·: ,,1; ,-;-:;.-1._1 :iv _,.,_1!_·1

·.<

•i . . ,; • . 1• J' ., . ,.

'

-

,·.i·

. . ~'

'

'

\ ...

.:.

To 250 mg of soya protein isolate ('Unisol', Unimills) in a

)9.

-

~l

,~

'f;~ç~

_

lç

W9e

~·--:~

added 6 g

ur~

•. ;

'

~

~-

'·-'_··· '_ 2 mi

!

·0"'

Tris-HCI buffer al pH-.816

8JI~

.

a

,s_mall QU;_intity

_

o(

J·

~

·

a!~~

.T~e

y

.i~

.

!

'

~·as

dissolved, by ·,.,

:J;

;

·

;-

·

·

:

:

~

.

,

,

...

,._·r

w~r~mg and the volume made--up to;,IO ml:wtt·h ;water. The t4pe.;\\{as s,.!OI'.~red é!l)d heated fpr.,p . :.:·: .:1 ,,.,; ',;,~ ~ .- . :._.,.·. : mm 10 a steam-bath. A bout. 0,2r mlj l"('lerçaptpethaJ.lOI,.,was <!M~çl,,al)9 .-!h~ liJ bi; re-heated-for a .·,, . ·

. · • \ • · ~ · · ' • I • l• · I ' . ' ·• t ' '

further 30 min. On coolinga fur:ther;0:2,ml m.erçap\~lh<!r:~<;>l was ad<;I~P. ~~9. t,h.~t~!?bution all~~·éd_

1

to: ·. r, .;,-: · ----.

stand at room temperarure for-.301min. ~ ,, . . - .. . _ . ' ; j · ' . , , . . •• , ' - •

The sol ut ion was transferred to!ll dialninac iipQ

pia

l

~~d

ag~i

.

~s

-

t

i

.

l

ip::~

R~r

_

ophsopry~i~

.-;-

~

1

1

jne

:

:

·:

:·:·

-

·:.

~

11

. ,

buffer (PPS) at room temperature. PP~ COI'J$Ïsted pf:0.9;4~,-s~p_i~tJ!1.f?Yr@pho.sp~-H~·._,O.~~~ ~?_P,tUm,. ) ₁ ,

-chloride solution adjusted 10 pH 7.2 with hydrochloric acid. Aft er I 6 h the dialysate was changed for

fresh PPS and the solution dialysed fora further 24 h. The non-diffusible material was centrifuged

and the supernatant used 10 immunise raqQ.i,t~ ... f.n _amoun) equ,iv~_l-~n~

'R

,

~~~:~s.prot~in -~~~ i_l'\~~-o­

duced in Freund's complete adjuvant (water-oil-water emulsion) as a single intramusêula-r injecïió-n'.

1

' I. ' ~ •.., ~ 1 • ' ' I ' j I; :· ' ' ., ' l"" ~-I ' 1 • ' '

After 31, 100, 108 and ll5 d_ay~.the animals received further doses' of'protein·as subCûtaheous··:,i:·, · ._,

'•

,.,·,

'

.,

, ~ .: ' 'I

injections of 5 mg protein

in

-

~ol

u

_

iion ~ithou

t

adjuvanf. Setüm' s&1mplei·'W'eie:

t

à~

è

n

äi'92

~nln

-

!ü

,r·,·.-

<" ... :.

days aft er the initia! injection. The

s

·

i:~a .,;Jé~e tes;e~

for

~nt

is'óy

antibodi~s

Gsing

thè:ÉLJSA· pro'ctdu'ré= ' ;, · · ·,_ -,_.

descri bed, . I . ~ ~ •. • 0 • i . ,·,• l'~. : • . lf~·~· f:t·, • . ; • •;'1 )• ,?/ . ! _. , 1 ,! t ,•I t, •I . ~I_ • , ' _r: •,,_,_

...

.

.

.

_{l• ! . ~ ; ;,. ~ .... , r: . . ·.: . : ...} ' " I I .;.-l _{. i ~ .... ·· . ..... ; .,.· . •} • 11ë •. ~-:.ol :.

J. 1 .. 2. En:pnr-Johrl/rd shrrp-ontirahhit l!!G

•, ') ~ (f i . .

Rabbit globulin (prepared from rabbit serum by sodium sulrhate preciritarionJ was immobili5ed '!•' , • :' , . I ~

-,i: '!· •: :~:1 l I : )

onro cyanogen bromide-activated Sepharose 48 <Pharmacia) by the procedure described by t.ht;., -•.: .... _. ...• rnanufact urers. A high-titresheep

an

,

l~

;~erl!

.

m

tp

r

.~b.b

it

_

glob1Jlin

w.

.

a

.

~ pas

.

~eçl ~own

1 he colpmn and

th

~

-

·

ini_tial unretained fraction

wa~

reje~!

ed

·

.

Tti

(pq~

-

nd

·

~

·

~Ji

,

bqd

i

ê

_

sv

.

é~~~

-~

~

~·a~d

:

~~

-

·

~a's~ip#

,

0}

..

.

'

atetic .. " '''"

ac1d down t_he column. The

.

~rpt':m-cont

,

ll,JO

IP

&

fn

'!:IJ

_P~.S

.

'!:',e

,

~~

~

I?

.

~Ql~

r

~:

~~~})'S!!d a_$

~

i~

,~~-

phO~J?

,

~~te

'J,

buffered saline <PBS), and adJuSted 10 a concentra11on of 5 mg mft. A 0.1 ml al1quot of thts'w_às ,._.

used to dissolv~ J .5 mg of Sigma type V

J

l alkaline phosphata~e <s~;~pplied as a suspension in 3.21-i · '''

amm~nium sulphate). Thc: rewhant solution was dialysed against PBS and rhen mixed with 5 1d of

a 25 ~~ glutaraldehyde solution (aqueous). The reaction wirh glutaraldehyde was continucd''for·2.5 ft' · •.: 1_{' -: ·}

-.

-·

.

-at room temperaJure ànd then stópped

by di'tuiiori

ro· l ·ml'witi'(·PBS'án&·èl(tènsive·dialysis. Finally ;,, . ,

.

z.

the product was diluted 10 10 mi in

o.ó'S~o-~

TrisGHC;J buffe'l ('pR 8.0) containi·ng -.50-mg.;mJ-..

~

-

oval- ·., ·-.:· ·- ,:.-:.·. ,.

bumin, 0.'2 mg mt-1 _{magnesium clil'oriBe:-0.2\ sodium ·äzitlë'and!o'.} ~:~-1. _{metthiölan:. ..-.. · ·I) .. . · :-..}

.

.

.

~ '

.

.

,1,1 ::,I

2.2. ELJS.-\ pror<'<lurr !Figure I)

I '•I' :·• ! ... _-·~

: • 1,;'

...

·

.'

J.J. I. Prc·parmion o_( sumplrs and ca/ihration standardç

·

,

···:

-

.

''·

A bout 50 mg of sample or the soya protein isolate used in the immun.i,slltiÇ\I_l. were placed .in a1) ,

~x~elo 1 ube. Exactly 3 g ure a,~ I IJll l M, Tr!s-.HÇI buffer .,(P

1

~. ~-~l,j!nd _9.l ml,m.$!cao!O.ethan'!l we~e

added. The urea was dissolved.-psing-added wat~~ .ar;td h«:4ting, b,ç(ore mak ing. up the :soluO.ön to

-; !'I:

5 ml with water. The tube

wa~

$

10p~

.;ed

:

~n

d

.

he;ate~

in

a

stpmi~~~~J?r

.

I h_.'

Afrr~

'

èo

.

oli~

{

3~

·,..1 ·;_ -....

of

the

suspension

v.·as rransferr~d

t

oa

-~:ml

via

I

and

di.\u.ted ~'i~h

2,97 m

i

p

h

b

.

sphàre

.burrer-salin'e- ·· :, -,

Tween 20 (PBST) to give a

J:

JO<)

.ç!p·~

_

,·i~

.

n

.

-.

~

t

q~k

.

P,s~

;·_

\~·

~

s

p

-

c~p~r

.~

c(~y dis~'?

l"in

g

.

~5

g\èidiurh

··-

~

·

'· chloride, l 0. 7 g disodium hyc;\:liogen ph<;>~pha).~<-3:9 _g ~.c;>_çliu~ d}hydrQgen: phosphate dihydráte arid · o~·

5 g sodium azide in J litre of-water, Wor~in~t_PeST solution. W.'!S prc-r'a'r:éd 'b\·-dih.iiin~t the stóck l :·

io

and adding Tweeti 20 at a ra.te of 1.5-~l-iil;e--t. .. ., -- · ·. ' -. - .- ,I ·"-·:. ·

-·:· .'·1

..

'

-

.

...

.

, ' t; 1 ~ • ,CJ (

J:-:n,~·mt··linl.t-<1 immunowrt.t•nl •''-"Y for 1111• dt·INminalion of JlrOil'in 161

• . ; .. ,~". h::t' ·~ .I f .l

2.2.2. Sc•n.l'ili.wlion of lhr ll't'lh ofmjt:rolilru/ion p/o1rs .

Eac? ~

·

.cll

in a r_lare

<D~natech

,,

~iG

.

~f.

i

ii

/~

,

ri

.

?

_

r}f

~/

~\

-

..

~

~

\i~rè'e!a•e

i

.

:

i~

s

~YS

.

~

~

~

~~

~

'

fil

,

~

~

~

·

~i

-

ttl

·

ö

:-

~

.

ml

·

~nsJIISing

solu11on. Ttm was

P~if~~re~

.

:

bY

.

fn'

~

~ng ~

!:

5

-

~l

t

-

~~

r

h

f

st~~

~r:n,.m~~·sm~

-

~~!u.tt?ry

.

t

.'

ó

'rr

{at~J~g

I 0 mg ml-t antigen isolate prepar cd as descri bed' in

~ct

ion 2. LJ'with 9'.0 mi 0.21.4 '!.ódi'clin1carborHtte'

salution and 16.0 mi 0.2M sodium hydragen carbonate. lt was \hen made up 10

100

mi with water.

The loaded plate was ~led inside a polythene bag and incubated at

37

°

C

for 24 h.

11

was slored

at

4°C

till required. l.{!r.·l· ri1·J•: _ .:..

. , ,;·}t·.Jc :nA ~t ; t .,,, 'II.L

'J , . ~ ~:J i:.-w ..

2.2.3. Seriul di lulion of sumples ond s/undords und incubolion ll'ilh u111isero l•:·'·-~" -·~

·=·

••

·

:

1··-,-,.. . -~

.

)",_J ... ·;

.

.

·

The calibration standards were prepared in separate vials from the

I :100

isolate solution'(dè'sàîbed J .• ·. : • • .,_., •• ,,~.: ", in.sect ion 2.2.1)· by se ven

serial

-~ilur

i

ons inró

P'l~S"r

:'t3-

èK

l

~IJi.iildn'bèlng rhre

à imes less'·Qo.ntentrated _., rr .. •. ' ·.>·

...

)

..

thàn its

pred

ec

e

s

~r.

The volum:es taken we re siJfflëithi''toloá·d..-fou'r'róws· of

sev~n

wellsi):>et j:ilätdJTCI" - ··,.:;

ï:.'

H -t

each via I was added an equal vohime of I: 26éfdil'uiê8!'i'àbtiirtaiilisoya -sdumï'n·~·&SIJ'r( .. iitsti-RNS'): ··: :;,, _. '

and the mixture was incubated at-'3rc for 30 ml'n:

-ri!~t

~

rhp'

Jè~

·

~'.1:r'è

á'lsO'seriàllY:diruttd b\n over a

~:y"

_. .. ". ()· (' ,, - ) I • '

smaller range. Exactly 0.2 mi

of

{he I : J

00

<lih'.Jr

i'Ó

n

~

;

:pr"e-p'iired"as

·

dé

'sc

ri~

è

ll

-'

ln se

ê

tiOh

~'

]

}

l;

·we re' .

~n.

L r·O ... ,,_ -r;:

serially diluted three times with 0.4 mi PBST giving a I: 300, I: 900 and I: 2700 range. Arn~ûal,1 _{:, :l ·:'; ; · ··:- . !i·}

( lume (0.4 mi) of

'a

nti-RNS'

~

;Ólûti

ö

n'

··

was

'i

if

e

n

1

a

a&'d'i

6-

e'áêh';

·

giving

·

á

·

ii'

-àRlple

vèlUt'ne

ro

fillnwo: ·.. -!:.- ,,.

t-·"'e wells per sample. The

v

i~i

s:~

hll

-

~cub

~

h

l

6=f8hö

1

i'n1n

''

at

:

tHè

·

s11'rn'd

-~

rTie

'

as

1

1hê

'

cörr

e

s

~

onding

:;: ,· '"

I d d J I. 1•). ~:-'r<::~: '.~Jt'};~.-,:,.:.- ,riJ,•i.f l·':'-' v ., .. · .)l ' .~, .·,- ··~L' · .

san ar sou 10ns. - · - .. · · . . . · ·· . . . "·' rr ):. ,r:-·.·r:o.·

':,J:~.J:·~:il~· .. ;_:·.v l:i~·.:qs"=t ._,;,:r~~.rd:-:·1(",' "r'T _,J ~--- .-....• ·-.. ·J·. · ' • .J I ,.;· ;:,: JC ••• ·'. : •· I ~ )

·.

'l . ( ...

..

_' :•

.

....

•I .;

.

f .; ir: "·

.

.

.

.

.

.

..

. . ·! '-';(, . f. -' . .

.

; .•;(_,, :·:!

.,.

2.2.ï. Tlw ···mÎ-1111/(1/111/IÏC llll'/1.\'/lrenii·IJ'rrljrfn·l'fopnf:~Óiouf'.:; -... nr :_)~<-- 'I .:::c • ,-. 1.-i! . . ·:--:.-~ .·_. l';r', -~

The opt i cal dc!nsitv of. each

~

~mRi

~

'

an~

-

si

a ndar'((sdiL;iÓ'n ··v!·iis'

·

(n~

cl

~ó'r'ed

::

üsi

·

nd

-ai

T~hh

i

to~

-iiluio-':• ..

~

< · · ,: ':! .. ·,, sampler a nd

prop~rt

ion pump.

a

/

A

~

w

;i

h~r~ugh ~

e

i

i

a_nd à 'tinlèaM

s

~

sc2öö) pr

~

grai'nrt!

'

<~ble

spéctroA ·· · .:---- ",

.

t•

photorr.e1er. To obtain

accur

~

te

_

\·

àl~

-~s;

.'

•b

<

~.o

~~

·

.s

)•ste

1

~

!/~

9

~~

i

~~

d-m

b

rerl~an

·

l'h

~

totäl

ca~City

>

of

1

a

''

:•'L · : .-, ::·:;,;

single well <it was for this rea!>_ém"tl{<,it.éaèJ{-ánîi-R'NS'

~·

mpln

v

as

·

rn

'

iHà

:

ff

.f

è!h

•

ió

d

d an

1

<f'pla

è

edïril~wo

\·

,-.·

.... r·-r..-·:,

we lis). At t his stage the.contè(its

~(

~

upi~

é

~i

e

d '

~

e(

~~·

w

é

·

r

/~

ob'i

~

d ~n<f

'r'

r

,i'

r\s(wéd

'to tfre;sample'h :ut>s · .. ',_ ->! ! -:-·· .... , to give a workable volume. The

~

s.;

·m~1i~g

·

r

a

te

-

~

·a·s

'

.

~

e

r

~

i

~t{

é

Mi~

:.:

,

r

~'i\~

d

-

'tn'e

;

~

fló\1;

·

·r.álê'·à\

:

o

:

6

·~'lil

.

.

•

Ji~,

.-_

._,;

-

.

·-!'-'i!·

.

-

~.

. ..

min-1• Each sample was sepa"'ratéd r?om'the

~e

'

xi ~)' a

l

r

.)

Th

'

ë

'

sp

tÜ

r

6

phÖI

'o

~ete

f\~

·

~s

-

piÓ

.f

r

il

ihm~(FIO

.

;

I .. ... :. ·,, "

I hl C. 11. S. llilchrork tl of.

. .~;•

•lij ,.,1.

measure the optica) density of c<~ch sampled solution as it was fed through I he cell. A narrow range

of wavelength (4()5--404.6 nm) was scanned during each measurcment and thé"rè·sl.dts printed out at I min intervals. •. ·: ,,; :,. . ... • ,.,, :•: ·;., ... _-.>: ') ~·-~t; .• ':.· ..

. , -:,_ .. :-·\ : .• :·: · :.·~! .:·>-~ ~~·· :, .. ·.!. ,,_:, ·. L ·~.~)·i!

2.1.8. Culcululion ofyoya conn•nfr_q,tiP,J!.·~fsumple~ . .. . , _1,· .• -~i .:~··J:C1 .;·,_::> 1, .:.-:!::.:'< .•

Using the rcsuhs from the various·~tandarcJ. re.ference samples, '-a· dupli~tc~: c;ulibrátion curve was

construcÏed for e.ach plate, ·ploning<optical density.:<.OD) agains!. log•i1) '1-/(SOY.} for-each dil u! ion

(Figure 2). Provided one or m(')re.;of the 0DNaluesJor-the.~!ari'ous ..sample ~ilution§ •Jaylwithin the

linear part of this plot, their soya .concltl;l!r'àtions could.·be··obtaincö:·directlyli•However, more

accurate resuhs we re ob ra i ned osing · teg.ression line f ormula a nd a ipfogia mmá.blè.!calcula tor.

oJ') 0 ~

...

t

..,

'"0 ö y ö 0

./

.I

.I

-··

~· ~··"..• . . . . · .: t· ·~r; );,_. · ... 1:-~o.• ~"'::'P ..

Figurt 2. ELISA ro/ibrotion ru,.·~: The calibration

standards wert prcpared from a s1andard solu1ion of

renatl!rtd slandard soya prottin (scction 2.2.1) whieh

was serially diluted (seetion 2,2.3). Thc observcd op·

ticai de ruilies ~ere th~·n ~i:if!fcfaf~}.'i_st_logooi/ISOY).

Thc \'alues of logoo 1/ISbY)'or'1.1: l·and ~ corrcs·

pond 10 concentralions·of ·JOO; ·tO,··t and ·0.1 JJf!,

rcspectivcly, of. soyp protcin mJ·I of the diluted

solution whioh! "'' \ incubated with thc 'anti·.R)'J:;'

(scction 2.2.3). Thc coneentration of so)"a prottin in

sample soluti~n;_-,c~n be read dirwly frÓ~

·

i

hls

c.1libration curve by choosing an appropriai'ê i3ilut'ion

which gi\'tS an op,leal dcnsity value ~·hléh'Jies

on

thc lincar part ~f !_he cur"e (seetion :!.2.8). ... ·.~·,:

"::

.,

. .;

....

:' \

i . :: )

...

:

T2hk I. Response of commcrci~l soya produels 10 ELISA ;,r:

T~ pc of ~~mplc 1.1: Full·jill jlour Produel I 1.2: Dt{uttrtf f/our.< Prod\'cl :! ;\ 4

s

1.3: /solrnr.• Product (I 7 f; 'J ro 11 11 1:! 1.4 Conrrntrotn Produet I~ 14 IS 16 16 Response IUni~ol= 1001

..

·

·-

J:'s:

_':p~ii1E-"i;-mlur;_{d'i!èt 17">.} :s· ' 0

• :' . ·h 18 ~ .• ' 0 ' Response !Unisol= 1001 . ,.

·-·

..

..

' I ' • V• :;) . ·-1.:~6ii8 ,·,:· 61.9 ·-"ho:~'·

..

~

·

.(: _:!()l!9 .·.: . • .,,_.. '· .. ,. . • .. SU (ti,_ 7 •·. .... 1'20.0 0::. . . ... ·p~- .2t)' ;_~ . .).:: ·;:.,~~;' 65.~

'

.

··~<!.?

..

.

6 0 ° ' ; . 106 .. ~ i . I ·. T . . ·:, 128.0 91.8 91 .4 n1.~ 1:!9.S 99.S 94.9 8S.O ss.~ 81 .4 71 .4 91.1 89.2 · ... -:. .~ , - ]] ... ~I •. ,. • • ,"I ' ' , > I l ,., , 1• • ''I :, 10~ 7.1, ·8 ·•··I .,•• '"1,' ' r O> ',:,1;.1 • .·. ,1.9~,·f J • •• .. • , ••. •• ' ·' • ! · :·.:,_;~·)..; 'h·:,.· .. · (;: . .., I .,;l.\9 . .5 n·> .. :2~ &-,j,-1 ·~'! ... h\ -:- ~ 1 •• : 12Q. I , 24 76.4 :!S 16 :!7 . -~. ·" .· •. ., 1.6: Sp..riuli.<t'd Prodm·t,•t.. Product 28 29 29

:\0

)I ):! 33 )4 ~4

-··

,. ,., ~ 67.0 88.:! 89.S 5:! .-I :!.1 :!.9 0.0 0.0 0.0 66.6 147.4 132.:! '• ;··· ..

,

I< ,· · ...

:

C

•

•

.

.

.

·

·

..

:· .. i:.: i .: -<~·1!.".··i-... .

.

. · ...

.,.

(

~I) '

<

..

' ~~., ~ , I • ~ . . l

' I

~. . : .: l

Ent)'mr·llnl..t'd lmmullO><)rl~nl A>l-'1)' fur lhr drlrrminallun of proltin 163

:\ .·'x.: ~d. ;''\üOhi: ··- .. ~ .! ~:. l(•lru · .. ! é J·•1r·:r ' ir . . -, 3. Res.\))1.$.-it (· .. ..,In ..

~

·"1

·

--

·

~en

' · · ,:. ·J" '>··r·", · ··'· ·

:--• I • ~ •.' • 'I;

•• ,'( ~ - • - -• • \... -~ • - -.·· ',1. ~ •· n .

J n this preliminary application of the immunoassay, apparent soya protein levels of various samples

were

estimated by the ELISA procedure described and expressed as the percentage of s.oya protein

• .(" ~ :• i •

compared to the tol al prolein present (N x 6.2.5). A soya prolein isolate (Unisol)\:\l.·as

ûsè:(j\

à's a· '1_{''n u :'<' ·.·· 1: ·'· "}

reference standard (100%): this protein was.ide'f'ü'ical'.1.0.Jhlq·us.cd.to.;t;aise tho•anfibodie:S. :Table 1--- , ·: ;:

records the rcsults from a ·wide range of commàrch1l"pt,'Qtlu.QU,•.Whosé.>~Gtl!äl soya protei'n lt.v.o}.s '!"Cre..·,;: ,,,;i··:· .. · , . · ··. ·•·.,..,

all J 00%. Of the si x sample types, three (·I ,:.J) .gaYetrl:S.pon_ses;us.ua·IJy. s.onie"'lb·:i~~bove\ th~ srandard·:r· ''• ... , t

•t

..

.

,,.,

.s

isolate; two (4, .5) lended 10 give lower.v.a-lues. Oth,cir:spèdctlised soya·pró!lucts (lyf}G.6) gaYOllaria_ble ,,_,.

responses, some of which were negligiblyslowJ> Wïthimeach!Of..1he~·.typês, ·corisidetabk yariatiqn in ,.,

quantitative response was observed. Some variation was caused by random error: this was illuslrated

•1 Î . ,,.,.

•' I ; -; zn•'l~ .l1 .•::''Ji H~-' ~ .~t;' I'JII,l !,.. :. •':,.~ ·: 'Pt:è ,1,.,:" '::-'I.!:

~ ~ .· " ... •J· ... L ,, • ·' . :, ,·, ,., .

Tabtr 2. fl,csponse .or $O):a. be~.n .com: . . .. . .

. pon~;,'s"ió,

lJ$

"

'>

':

.

', ·

''"_~·

...

.

~.··

'

''

'

:

·: • . . . ,I

.t

l '

0

··~

"!'; " ... : lC:'

: ,,.,·: ·,oJ • · "'""' :Rt$pon~ (· ·

r

:·_,.,

..

"

Siihi'pit'<' •rlf : · ' "J: .. '<:(tJtiisol .. 100)1 ~ ... :::. · '·

r,

·.i~. j:~ t. t.·:-~:'?':s···"::....:.:!.~~ ,,~; ._!_A .. ;: ... i,.,J~. ltC'I !'!l('".ol •J:•:,;':- ')t.~ "'~ ;1··~ J I • 1 Je;..,.

~

r S:;r:erq,lcir,! ·. ~

- ~

,j

·~

.. ,, ·4

:

·

~

.

~

~·t

.

.' ·

;

·J ·: . .'.,

~;~:.

.]S-,prolç,in•' .,; , .. _{, 0} '":Jl).! I,< , •. , . . Wh~y• .. ~.: :• :1 : • • I ,_,.,., • J.1·:~

.

.

..

IC,; SBTJ• 0.0

• lsolatcd by fractionat prccipilation

• Purifird SO)'a bcan lr)'psirt inhibitor (cataloi!U< numbcr 390·0, :BrhlslJ 'Drug Houses. Poolc). ··~:.·. ' • •• t ...

..

..

.

"':r . .-:·. J ·I #". -r;

,

,

l:l:••1.• .: .. ,,., t ·~ 't: .. ,, ..• I

by repeated determination of the same solubilised sample of a canf!~d m~t prqduc1 known 10

con-tain soya. The values obtained (7.83, 6.80, 8.08, ,6.64, 7.50, 6.13)

w

e

r~

'.

c;

ol}~i&ient

within the:limits

expected for this type of assay, and confn'med the feasibili1y of the determination of soya pr.ot~n in

a severely heated mixed product. ·"

Table 2 records the different responses from

~opurified

protein frac,t!ons obtained from soxa ,f\our

by precipitalion. This.ELISA procedure recognlsed mainly the 7S fractioó, while the IJS and whey

( ~tions had weak bul significant activity. T~b!~) gives results from;~ssays of some proleiná~~ous

lvvd ingredienrs containing no soya, and con~rms the high spedficiry of the method; in pa'rtl'ci.Jlar

the non-in1erference of meat prot eins. Cross-rea'ciivity with similar a'ffligens such as legurne storage

proteins might

.

be

expecred. but field-bean protdln gave a negligible ~~sponse.

~-T2ble ~- R csrorisc of non-soya bcan

food malcrials 10 ELISA,.,>;,",.,

Response Sample (Unisol= 100) ··----

-

-

·

-·,:;

·· - - -

-

- - -

-

·

M~al Mill: powdcr Egg powdcr Whcat protein .. Field bcan protein

0.0 3.6

:u

1.1 3.~

"

I'

,.,,,

... ,, ~;.

.

':.

' ·. ;

.

.

, >l~ !)( •':' _!.,1':'

.

.

~. \\'•,.:

..

,

.. ' \ \

•

')

:

"':·

:·

.\1 , , ' ( ; ,I.~ ' . c·· .. '· ., ;

..

··

·

.

·

.

I~ C. 11. S. Hilchc-ock tt ol.

I> I ( (', • • '•

. -~· D•scusston ?I · .~ r· . !.' · :·: :':! . \

. I •;

: •. .:" .• -·r" .. •. ;,

The reporl of an EEC S\Udy

grou~

on.

vtg~lable

proleiris in

footlslüff~

:

~xptessed

I he hope !hal

further research would improve<:'~istln~1 ;néinods of anàlysi's or.·:provide··nth.,•;;tmore accurate and

~nsitive techniques. Thc UK Foo'IH>tandiitds Co'mmitr~e.=·in re-VIe\Vi·ng:ur~corr~·emional sourees of

prolein, recögnised that their usr~·Ö\.ild_Î~a)~C"analytî~arprobTerh!f,:lO bui:Îneltladology }lad notbeen

established befare their recen1 rcp·orl o'n'fn61i produëts21 _{wlilth. reéott1m·endS'cO'nltols invalving such}

methods. Preliminary results recorded here suggest 1ha1 lhe applicalion of an ELISA procedure is

more auractive 1han many melhods reviewed elsewherc,1 _{though some of these wiJl doubtless} con-tinue 10 be useful (e.g. histology).~ Basedon appropriale renaJured an1igens,1_{~ ELISA wiJl provide a}

.

,

•• f •. : 4.1 ••

-~·

j' • 1 'I'

convenient general methad for t'he qualitative ch<Haclerisation • .and -quantit.a,live eslimalion of .• :·-: _

individual proleins in food products. Pro~ld~9 lhat suitable äfitibbdië''s·.càn bé rais·ed, rapid immuno- , ..

:r .

_

11 ~ ._ •. , \ • •

chemica! screening of large numbers or' samples can be' follo\vèd

·

by'

'

à

:q'vl!t'ltitative standardised

''

''

'

~~'

'

'•

:·

assay in appropriate cases. Considering SOY'! protei.f\_!iS a m_~?del .• ~h-~. spe~!~,c;,i_l_~

()f

th~se antibodies is

demonstraled by the datll presenie-d in Table 3, but the· qullnti!,at,i_ve re.~p_pnses wi1h different in

di-vidual soya proleins (Ta bic 2) a1i'd wltli' \he various c'ommercial: forml>.!Oft·Soya proç!ucls (Ta bie .I)

are not cons1an1. Soya prolein .~omprises t.~rçe major frac1!ons (7~. I IS _and whey) of which I he

frac1ion enriched in 7S proJein appe.ars 10

&

é'

~iósi

iln1igenic ir1er ieNarl!?árioh ;·1he responses df the

whey fract ion and J IS fraction are :30-and J 8 /~ .. respect h·ely (Ta, bi;: ~--l,"hç_cqmP,,ositio.n"of lhe_s~ya · proJein (which is son·.ewhat variable) lherefore directly affeels the observed levels of total proJein by ELISA. Th is difficulty is common

l

'

o

·

many

·

fîs

~àys

(o( fbreig.n''jJtolein:s in mè.at pr.oducls, and Jea.ds lP

th) .

~

·

..

.

similar uncenainly unless soya il)gr.ed.ients 9.f the same cqmposi1ion are available for comparison: .. - --~~

-The ELISA procedure i1self

carri~s

-

~xpe~i~-en

\~1

er

·

r~~~

-

b~i

'1hesè ·accumiil<iled

·

u'tl

cerrain

tie~ c'anhót

explain all the varia:ions

from

·

~

HX>

-

~1,;

recorded.·in .. Tablo l. 11

i~

~

evidc;Jlt

thal._tl]e solubilis.alion'; ·

rena1Ura1ion procedure does nol·álways coi'l\'e·rnhe·soya pro1ein'•in

all·lhe

.

samp

l

e~

-

10

quite.the sa.me anlil!enic form. 'i. ' ~-:: , I

·

.

·

'

• I . \

'

.

• 0 • t, .-'

J

n

~

genera I. "a lues considerably under -,

00

~-:,

·

~ou

.

ld o

~c

ui

.

w~\\

;

tht'

.

s

.

~f_~

pr

~:

llei

.

~

does no:c in1erac1 · " .

quantitalively with I he a nl ibodies.under le:SI conditi9fl$: Ihi:s could b,e

ca

~11ie

d

;by,-lhe epitapes

~i

ng

·

-

-

~ ·

'

.

~-

--.

_;

~

:.:

' · -'·, .·

..

.

-rendered una\'ailable during processing (e.g. by hydrolysis·,-:direcl:cbemical r~-!lÇi_ion or occlus.ion via.

cross-linking) or by 1here bc:ing less elf the highly·antigenic 7S-prorein-prèsenl (relative 10 the • ..

..

-.

con1rol, Unisol). Hi!!h va lues ( > 100~~~ would indicate hieh levels of 7S protein or the exposure of

.,. • • •1 ' \ "' 0 f > - • : I • •'• • \

-ex1ra antigenie siles in 1he sample. Table J shows tha1 expec1ed' nórm'al'rèsuhs \vere observed with full-fal tlour (lype J.l; average: 99·~;~1 llnd defaued-tlours.-{!ype,.l.2; av,er.age, I !_l ~·~) and isola1es I lype 1.3; averllge, J OS'!~). E"idently 11ie manufaclure of conccnt'ra1es (type I .4 ): involves a solvent

washand hea1 1r~atmen1 that if1Jerfered wi1h the assay (average, 82 %), possibly by cross-linking the

pro1ein .. Low responses were

al~o

ob;·e·r,ve.o

..?,.;_,p

-

~qme

e

~·

truq~

;

d

.

~r

:

8_d~cis

·

llype ·1.5), for they are of1en made from concen1ra1es by hol

ex~rusion;

1he liverage 'véÏiue

ls

'

'79~

~

·

.

th'ough 1his.'is anificially

high due to 1he unC:Xplained exceptiona'l'résults with products;2~ al')_d'l:Z3, Pr<;>ducts 2~-33 include whipping agen1s and acid-soluble specialised produc1s; I'J)éy'·gllve> lö,•?Cvalues.·presumably because

they are ranially hydrolysed. The presence. of added léèltt,ln:in ·product 34 may account for an

abnormally high value. The ELISA -procedure-al$~>:gav,c_.~omp>.•hat high respon~es wi1h some of the

isolates 1csted: these mt~y retlec1 different me1hods of manufaciUre. for inst<Ïnce in prc:cipilaling

condi1ions lresuhin!! in rellllively high le,·c:Js of 7S-fr;_,Cii·on9 or in dt:nlllllring processes.

Funher research is needed be(ore 1p~ differenl responses can be unders10od. bul already these

preliminary resuhs demonsirale that "the

1echn

·

i

'q

,;

~

mà'y b't·used fonl réiriä qualitative screening programme and, in addi1ion. for a.convenicn.l quaptitati~'.c;.estiiT)~tipn _of soya levels in products, especially if I he type of soya ingredienl is known or can be deduced. Moreover, I he choice.of a more sui1a bie ren at u red ani i gen may lead\o more gcne'ra'lly:éonsisfènl resuhs. The principle of this mei ho.d

can be applied 10 1he delermination of each protein separa1ely: for instance, ELISA procedures

might be developed to es1imate individual components of mcat producrs- muscle protein (Jean meal). collagen Iconneelive lissue). blood proleins e1c .. and addi1ives such as soya (legume). whea1

lcereall. Célsein I mil~). egg and colher foreign pro1eins.

Hi1her1o the 1radi1ional approach 10 the complex problems of food a na lysis is the isolation of 1he

. r ·. -~ . ,\, l. \ \' • _·. '~ -~ I •

.

...

,_ I • : .;._, .: t, • . •

,,

-' ol',

(