f,,, ', \ i ' i,:i/ :

Reprint f iom

.. RAD I OIM]VTUNOAS SA\-AND RELATED PROCEDURES

IN } ,TEDICINE I98 ] ' '

J u n e , V i e n n a

r s B N 9 2 - 0 - 0 1 0 4 8 2 - 7

INTERNATIONAL ATOMIC ENERGY AGENCYVIENNA. I982

IAEA-SM-2 59167

A NEW SOLID-PHASE SYSTEMFOR IMMUNOASSAYS*

H. SCHMIDT, O. von STETTEN, G. KELLERMANN,H. PATZELT, W. NAEGELEFraunhofer-Institut für Sii icatforschuns.Würzburg, andByk-Mal l inckrodt Chemische Produkte GmbH.Dietzenbach,Federal Repubi ic of Germany

Absrracr

A NEW SOLID.PHASE SYSTE\I FOR I} lMUNOASSAYS.The development of anew sol id-phase separat ion s-vsrem based on s i lane polymers rs

descnbed. A T3 radio immunoassay (RIA) was opt imized using coated tubes wi th polymer

coati.ngs containing hydrophrlic zurface aldehyde gloups for antibody coupling and a T+ RIA

deveioped on the basis of zurface anilino goup containing particles using a suspension method.

Both RIAs of fervery good performances and shorv the var iable usabüiry of the new separat ion

system.

1. In t rod .uc t ion

Immunoassays are wid.ely used, in ned.icine and. have become anind. ispensable too l for mod.ern d iagnos is . The nethod. is usec asa rout ine test in many c l in ica l laborator ies . Cormerc ia l tes tk i ts for the d .e terminat ion o f more than 1OO b iochenica l compo-nents are avai lable tod.ay. The rapid. d,e.relopment is d.enonstrated,by the inc :+as ing number o f d .e terminat ions: in the FeCera l Re-pubi ic o f C- . :nany 1979 a to ta l number o f 1? n i l l ion d .e termina-t ions was t r , : formed. . The nurabers for 1985 are est imated. as h ighas 39 mi l l ion. Assays for new analy tes (hormones, enz1rnes, v i ta-m ines , d - rugs , v i r us , bac te r i a , r es id .ues i n f ood . and o the rs ) a rebeing deveJ-oped., and on the other hand.,nethod.ological improve-nents are worked out in order to make present analys is more pre-c ise and/or more economic. Thus, much work is devoted. to mecha-n iz ing or automat ing analys is or to use other than rad. ioact ivemarkers l ike enz1rnes or f luorescent d .yes. A cons i .d .erab le par t o fthese assays is based. on so l id phase tec i rn iques as separa 'u ing

+ Supported by Bundesminister für Forschung und Technologie, Federal Republ ic of

Germanv.

l l l

t t 2 SCHMIDT et a l .

n r ^ n p r l 1 1 r a - a n r l i n ' r , ' n - r e a s e s t h e s t a b i l - i t y o f t h e i r n n o b i ' i : e a' J j . v U u u l g - 1 @ I s 4 r r 4 l * ' J

äntibod.y is be1:t,er tha:r i ,hat of antibod.y in solut ion. For tnis

r cAqön - f i r r t he r r eseaJch xo rk i n t he f i e ' o o f So l i d phase sy -I v + J v r f t

f q : v r - '

s lens is be ing carr i -eä cu* , in rnany laborator ies .

T ] : e ccup l i ng (es lec : z . * r7 ccva l en ! coup l i ng ) o f an t i boc ies t 9 - . , ,

p ro ie r sc i l a ph t= "s ge : t e ra - i 7 l equ i res seve ra l r eac t i - on s - -e lS l i . , '

ö " i i r ts : a ; ion c f s i - r ic3- ,3s ; , i -uh su i - ,ab le s l lanes anC. ccupi : -ng

an- , i -cody onto thenr ,€ .3 . Se!a- r3 t icn roed ' ia are obta ineo tha- ' per . -

fc r : ' , " -u i i in R-- { [ : : . : : -= pr : : lc ip ie , .howe' rer , is l i xn i - 'eo :o

porcus g l ass a : rC o iher ' . r=- i " .=ed. s i l i ceous subst ra+r 'es ' Th 'e : la i l

. r ' . i o e - . j - r e n t - t r i e D I ' e S 3 : : i . : - - U C ] ' ' u h e : e i o f e W a S t O d ' e ' f e l C l a q e : e : -

9 L ü - e J - , v

a- i : r arcr ica ' r le rä : .a ; l :se s , , -s ;e : i base i . or ' : rc i ' i - l iec s : i : : :e pc- ; i -" r - ' 1 t ' r R IA ' Fo r P rac - - i ca lners L: l and to prove i--s re: ' ior:rance :.n

reasons, a cCä. . ,ed tube S: . -S:en ' . / -aS Ci losen. C- ' 'her geCxle- l ] . i C ior :s

ha.re been i i : .c1ud.ei in -, : : : stud-;r l -ater on'

n ^ ^ . , " 1 + ^Z . n c S r l - l - l , D

.1 Ia . I De.relocment of # ' . r r . a m ? R T A s

V U V U L Ju v 9 v e *

Frorn [ i ] i - . l ras k;: .o'"-n ' , ,hat ' using the process of hyi 'rclyza'ui-on

and. po lyconctensa ' , ion 9 l rcnon"r ic = i l . ' , ' "= , so l id- ma+'er"a lS con-

ta i n i ng o rgano f ' nc t i o r : " r g ; ; ; ; ; - " cco rC ing ' t o equa t i on ( t ) and ( z )

may be PrePared ' , +

x s i ( o R ) 4 + v

( r )

x s i ( o ä ) U + Y

x s i ( o l { ) U + Y

R ä s i ( o n ) , + z Y ( R " ) r r s i ( o R ) 3 + ( ) + : < + 2 v + l ' ) H z o H - ( r )

( n ) ( r r i )

Räs i (on) , + z Y(R" ) r rs l (oH) , + ( \ x+2v+ lz )p 'o i l, ,+

R ä s i ( o u ) , + z Y ( R " ) r r s i ( o l i ) 3 n '

$, ' * ss#9 .n ro

( z )

-.-------..-,I

> r - uI

t l

I }jT;i1R = -crHr; R ' - -cH3, -Ph; R" - -c lz- ; Y = -NHz, '@l lär ; -cä0,

- O i + : x . 1 f . n . = 1 - 1 O O t n = j - A' . t J t r v v t J /

a:rd. thai these polyzlers rnay be used for coating of any nechani-

cai ly stable maleria' l . The polyraer surfaces are able to bind'

ant, ibod.ies covalentlY.

I A E A . S M - 2 5 9 1 6 i I i 3

Easen on t hese resu ]+ -s , t he f o i l ov : : g i nves t i ga t i ons i l e r?a ineC a i -uhe de. re ' loonent o f maier ia is su i tab le for nechanizeCr . . A - - i r r n - - r n - . ] U f e S . T n e f g f O r e - a T ? r ' a r " 1 c t . , : i - i . r c A q q p r " r W a S C h O S e nv e v * l & v J . v r ! t O L J - v L ! - u 4 v ; / 9 q a J a l

a s a n o C e r .

G ' l ass i - ubes we re used , s i nce t hey se s tab ie aga ins t t es l e ra -t i : . re t rea inent and. n ight la 'uer be use i as a cuvet te for ccror i -n e ' - r j c n e 3 s u r e n e n t s . i i - n e c e s s a r / .

Fcr the r re l i rn inarT eype: inents " ' : i : :c and an i l - ino groups ' " rere

chosen as acr- i .re f tnc+,icnal- grcr:.ps f: : counl- ing. rr ' I i t ,h the a:t i iog : "up S r ' : : a : ' r : a l oeh i roe was t aken as : : : : i i ng agen r , ' ; n i r e ; r - - . h- , ,he an i ' -nc grcup an-" iboo. ies i t rer '? bc: : : f . r ia Ciazc bo i :Cs.

F i : s t c i a r i r t he i n i l uence o f l r e S - ,ä r - - i ng con r :onen ts i r t heh - r i r i l - r =a * - i on an r i nondence l i on reac * - - : : : accc r i i ns t , o t he ecua -. - J * J L J

- , ic : rs ( t ) anC (a ) on +,he : r : re : ' t ies c l * ,he ccncensed na i er ; a l ,espe : i a l - 7 w i t h rega rC t , o * "he cca t : : : : e : i pe r i ' " e r i , was s tu i : ed . .The resur : shovs +-hat , i i und-ers to i -c : :c re t r ic ano 'unts o f a-a- -era re useC i n t he hyo rcL i yzaL ion p rccess , o l - i gcne : i c p r cduc t , s ' s i l - lbe fornec. vhich are soluble in orgar:: sol. /el+,s l- ike etha::cl- orace+-one. Af ter the in t rcd,uct ion o f a prcper anount o f a d l r ' : * 'edso l -u*r ion c f these o i igome: 's in* ,o the i rs id .e o f the tubes a nomo-genecus d is t r ibut , ion o f the so lu t io i l c - rer the inner wai r o i tne- a r - ^ + . ' ' ^ s a e h i o - r e d b w : . n f . r i j n ; - - i e t u b e s i n a h c r i z c n ! 3 ]g 4 . a : : : U t l , v = W - : . J 3 v - r 5 - / ! g e J i U U J , L T ^ I l ! l - - ( :

ncs i - - i on i n a vay t ha t t he cen - t r i f ugz i f o r ce exceeos t he g :av l -ty . On heat ing the so lvent is e . ranora ied. and the coa ' t - - ing re-maineC as a th in f i ln o f soxxe Um th ic i -ness. The whoie proceCurewas cornpl-ete1y automated..

F rg .1 shows a schema t i c ö rav ing o f + ,he coa i i ng mach ine . Thernach ine i s con t ro l l ed . by two m ic rop rcc3sso rs . One ccn t ro l s t hewho le coa r i ng p roceCure i nc l uo ing pcs i t i on ing t he t ubes on t nero+ -a t i ng cy l i nd .e rs , i n t r oduc ing t he sc lu t i on and t r anspo r t i ngthe tubes through the heat ing channe ' . The second, one con ' r , rc l -sthe homogenei ty o f the f i l -n by opc icat mearrs . A l ight sourcec m i 1 - . c l i c r h t t o e n h n i . n e e l l r . ' l r . ' n i . , h ö o c 1v v * r 1 w ! r J - L ' , - c : : . I f € s t h e t r a n s n i s s i - o n o f* ' ^ a " l " - ' ^ + * l - ' - 1 1 1 6 ' n t h e f i " l r n ? l 1 r \ . \ h ^ ^ ^ r r e c ' n e n t q n e r . t r t b e a r gU L ! = r - : - 5 . ^ f U i J . a J u E i ^ i U r f s J - : : ! I . J ) I J W \ J j ! = = . J u I g i l g t f t / J i J g : t r L l

carr ied. out us ing a sca: rn ing procedr : - : 'e . f t is poss ib le to d-ef inet . h o m e q q : r r i n n n n - i n t q < n i - h r t t h e ) r a m n - o n a ' i i r r n f o q n h n n a f . i n c r r . : nl i v u r

f v r r r e r r r v . . . v ö v - r L 4 v J v r e v q v 4 . r -

be cont rc l led. . The apparatus is ab ie to prod.uce 101000 coa+,edf r r h o a n o r Ä a r rJ . Y - ^ * * J .

Ani ro g :oup conta in ing coat ings were pro i .uced. by cohyd. ro iyze-t ion and cocondensa 'u ion of te t ranetho: rys i lane ( f ) , d inethy ld . i -

/ - - \ / - - - \€-u l ioX/Sl - lane I I I / , anO. Y-an lnoDropl r l t r le tnox i rs l - l -ane ( f I I ) accor-d . i n g t o t h e , e q p S t i o n s ( t ) a n d ( Z ) , u s i n g H C I a s c a t a l y s t a s d . e -scr ibed. in L : ,b ] . The exper iments shor- that the t i rc ,e o f cond,en-sat ion a f fec ts s t rongly the hard.ness of the f i l ! . Cond.ensa ' , ion

l l 4 SCHMIDT et al.

i 1 r - e ^ ^ F ^ . ö c - i t - t ö - ^ n r i . ' n r r . a n : , : - 1 I- - : : - - J ; - i - = : v v r v v + v r r ' ö

r i : : o p : ' c c e s s o r i o r q u a l i * u y c c n t : ' c l^ , , ^ l i + - ' ^ ^ F r - . 1q ü 4 r a u , r

c o a t e d . + - ; b e s t c c k

t u b e s ' J x d e r ' ' - r e a ' " : e : : l^ ^ - t r ö r r a Fu v - . , s J v a

rIl - l

r-:tL-J

FIC. 1. Scherne of rhe coar ing dDparatus.

Tt iLE I . ScLrenat ic tes t proced,ure for the T3 RIA

C.

\[]

E LJ-J

- ^ ^ + i - . - ^ ^ 1 . . + - ' ^ -e J a v I - r i ! J v I q - ^ J r r

{ ^ - . i -1 ! J I ^ r 5 ! \ ! r !

- i - : o - - . ' 6 6 6 6 - - ' 5 -

I l l , c L J - v ^ l . i v L v - - r

t u b e s t o c k+ : r ' r . a c l r n ' l - r

r a + e i - ' n - . " l i - ; P : ' 3i J U o u f i r 6 u J

h p a t i n c n b r . n c l

stand,ard. 1OC UL pat ient s* lp les 1OO UL

+ +1 2 5

l O O O u L ' - ' I - T 3

s o l u t i o nI

Y

incuba.bion: 2 hor:rs a+- 3ToC

IY

t ' I t t q . n ' r n o

+of the rad. ioact iv i ty

+Cet errninat i on

+B/B for const ruct ion

of the stand.ard. curte

for d.eternrination of

serun l concent ra t i .on

the sia^nd.ard. curve

yl bo

the

from

tAEA-SM-2s9167 r l 5

t ines o f more t i : .an three hours ( - lOoC, e t -hanol as so l . rent ) le"dto c i - y f i } ns ' *h i ch cou l -d no t be useC . W i th h i gh amoun ts o f t e -i . r e - o - , h o x = r : - i l a n e b r i t - . i e f i l m s c : q c k i n g d r r r i n c t h p h e q t i n o n r o -U a G - J ! - V i l J Q ! ; + - i - U ! : v v : v M i J u : ; u ; i : l - 5 u | 4 ! 1 1 6 v t l v t l u * v 4 ^ r 5 P r v

cecrure üere ob-"ai.ned.. The nr:mber of surface amino groups wasf i 3 i r - - i npÄ h ' . . us i ng a co r o r j _ne t r i c me thod [S - i . Denend - i nc ^n t hc\ i = J = - - . - : - : ' J f , v 1 B J - i l i s u v - ! - l i ; r L w r - u u g ! t r v u

l ) ) . v ! J L - r s r r a S v l r v r r e

t ' r - , e n - e ra r , i r - r . ^ i - - . ^on 10 and l+O a :n inO s t ^ouns ne t - 1OO l -2 Were- - , _ i r E \ , - U J a u r r l : t U € i r l C = : I l \ . , / d , I I u + V 4 J U J - I ] ( J

ö - v q _ v J

Y L -

a-r la '7 :ec. . No s ign i t icant d" i i fe rence wi 'bh regard. to an i ibody

c c u l l ; r g c c u " l , i b e o b s e r v e d .

A : : : l c g ro r - i x ccn * .a i n i ng - , . . l bes we :e t r ea teC w i t h a 2 . j i ( - u . , / - r . )

so l - : - - . : : i o1 g 'u- ,ar i ia loehyoe ( \ hcurs ; roon: r tenpe: 'a* -ure) a-n i w i - , ,hT j 3 : r : r s e r " - u : t ( 1 : h o u r s l r c c n * . e r : o e : 3 i r . r r e ; C i l u t i o n i : 1 J C C C O ;u : i : e l i e ; Zea la : : d rabb , - t s ) . The rec r : i uc i b i t l t , y o i t he f o * i ow : r :E

/ ^ t * ^ - n - ^ 1 . - i' L ' - 1 1 - : _ \ j c l " | = < ) ' ; L ) = t ' J - , r ; e ! = l ) . t i I = t o - u ä _ ! o l n o t n g c 3 ! a c l l : / ;

D = e i : c : i n i na t i cn ; CV = coe i f i - c i en t o i va r i a t i on ) was no -u accep 'u -ab le . Th i s m ig i : ; be due t o i r : ' en rod .uc i -b l e c ross - I i nk i ng reac t i cnsA - ' i , i c i . . r : j e - T h F - c i ^ r o r . z o ' n n r : n Ä

t h g a n t i b C C i e S - ; l : h j : -U l ö - r - ö ! * - . j - 4 * U C : ^ J u s . I l r s : = - \ J I s t w s U U u l s

azo bcncs to the sur face. The s i lane. , r i t ,h an an i l i -no grcur r ras

s7 r r . : e : i : ed ' ac : : r d . i : r g - "o t ; : e i o l i ow ing schene ( : ) ,

( :.c ) ̂ s: ( c,t, ) ,rr-^ +

.). Tvrtc, -licl- - \ J i

+

(no ),si (cn, )rnä-co*@no, di+;;

(no )rsi (cH, )rNä-cc@:,rH,

R = - C ^ H -/ 1 /

The s'.ructure of the comlound. was proveC with IR and mass spectro-scocy . Hyo ro l yza t i on and condensa t i on reac t i ons l ed t o p rod .uc t sw i t h ve ry pco r so lub i l i t y i n mos t o rgan i c so l ven - " s , even i f t hereact ion t ine vas very shor- t . Wi th a d , ipheny l s i lane ins tead of

a d- i .ne+-h l r l s i - lane (equat ion ( t ) , I I ) products vere obta ined, v i r ichi rer3 r lpr - : <o i . r ; | " i s in aCetOne, e . /en i f the COnd-enSat iOn reaC+, iOnr _ .

. : r v r

was carr led out v i th the s to ich ion€* , r ica l arnounts o f water . Thus,- ' + - ' \ ' 1 e t o n rene1 .o n rnd ' r n f . q nn t Sens i t i ve tO a i f . The- L U w d . J l J \ r ö > - i - L ; - v v ! r ! ^ w q r ' - f . J L \ J L a f U U - I I L . / u

^ ^ ^ +u \ r c r ! - r E l J l . U \ - g L I - L ^ I g L v ( . g u U s P s l l - r J r r l l g U v J

s c r i b e c a b o v e .

The an i l ino coated. tubes were t reated v i th Nal {02/HC1 at OoCand then incubaied. r.r i th t3 antiserum. Table I shows thre optirnizeCRTA r , f^ccr l r r rs and f ig .2 the s tand.ard curve. Genera l ly the fo l l -ow-i ng assa -v eha rac te r i s t i c s l r e re ach ieved . | 27 < Bo /T < 36%; D = 6> -75 i ' ;

CY < 10%; uo take : \O / ' < Bo /T < 5A I ' ; CV < 3 .5 i ' ; r ecove ry = 93 - ' l lO / ' ;

r e n g e : 2 5 - 8 O o v g / a t .

l l 6 SCHI|{IDT et al.

nc 200

s tondord ing i 100 mL

FIG.: . T3 RI-1 baset l on üt i i i r to group contain ing coatet l tubes: ant iserunt J i lut ior t "

] : 1 5 0 r ) 0 0 ; d m o u n t o ' t . s t d n d a r i : l 0 0 I J L . b u J . f e r : 0 . 0 8 m o l l L b u r b i t a l , z n d 0 . 0 3 r n , l i ' L

so t l [ t tmsa l [ c1 ' l a te . pH 5 .5 : i ncuba t ion cond i t i o r t s : 2 h ' 37 -C '

FIG.3. Standard curve for Tt Rl .1, using h7'dropt t i l ic a ldehyde group contain ing coated

tubes . An tßerum d i l u t i on : I ; 150 000 , 300 pL an t i se rum; bu f fe r : c i t ra te , pH 2 '5 :

incubat ion condi t ions for coot ing: 20 min (r t - t tat ing) at room temperatureI BolT = 357o;

D = 70%; 507o [n te rcep t = 120 ng l100 mL: NSB (non .spec i f i c b ind ing )= 0 .17o .

IIIIIr

ocDö

r0

ü rdo ?c0 (m 8m

IAEA_SM-2 59167

TABL: I I - Accuracy a ld recovery d .araT3 RfA on hydrophi l ic a loehyce group

ccn ia in i ng t ubes ( va lues i n ng /1OO nL )

l t 7

Ccn* " r c l se ra

ex!ec-- eC .ral-ue

value found.

a\,,'

/ r ^ \L ? D ^ A - r a F - ' ( n = | t l )

3 j ipea : : , : va lue

) , l ,+ +r ] 1U U

I t \t t )

- - a< a r I

J / v

7 ^ \ ^ lI \J\J

nor:nal-

1 2 O + ? q_ J l

t r ) , + r QI I T : I U

a n lI l \ - -| . v l J

l , n = 2 0 )\ r - - v l

found.

I+ZB6

- - I

J J I

636

h r r n o r

, a ^2 i \ r ^ t l

t - v v

2 L O r 1 9

^. tc/

r . - 6 )

hrrnn

' l o + 1 nt /

9 A +

ot n

q 7. ö rr a\1ra ts-,./ w L '

9 6 . oo 8 Ä/ v . v

^ - a

> i , I^ ! a

9 u . ba Q > Q . V

r-- r ;l

-t 'lI ' l

-r ) ,

fn ord-er to avo id the chemica l react ion s teps due to the an ino

and an i r i no g rcup ccn ta in i ng su r faces , d - i r ec t l y couo l i ng su r faceseon ta - i r i nc a ' l r i oh ' rÄo c r r . r r r r ' r q r ' ^ r ^ ^ ^ - - ^ ' l ̂ - . 1d . . S i l aneS COn ta in i nS* - J c j : I J ( . t g ö r U L , t l J > w C . . L C \ r s / = : L r - U C u . L J I - I G I I S - L v r ^ v e r r r r ! r b

a ldehyoe g roups we re syn+ .hes i zed . acco rd ing t o (h ) ,

(cHao )rsi ( cn, ),sti + cHr=g11-azor ) , \

( curo ) as: . ( ct i , ) ,s (cH, ) , - ,

Ant ibod. ies were bound- to the resu l t ing hydrophi l ic sur facesat pä 2-3 in c i t ra 'ue buf fer ' . Us ing the ro t ,a t ing pr inc ip le i t wasposs i -b le to red,uce the är to l ln tu o f ant ibody to 300 UL (a i lu t ion

1 1 -

a a a \1 : 1 ) C 0 0 0 ) p e r r u o e . ' r ' n e r n n o o l r r z e o a n t i b o o i e s v e r e s r a o r r l z e . rby a t reatnent wi* ,h a O.1% (o l /v ) so l -u t , ion o f bov ine serurn a lbu-m ine .

F . ' - ? ^ L ^ - - c o f r r n r ' n a " l . t a n f l . a r d . C u r v e O f a T 3 R I A u S i n g t } : e s eL L = . ) D r l \ J v { J q u J - v r L q Ä r

hyd roph i l i c t ubes . Assay cha rac te r i s i i c s a re g i ven i n t ab l -e I I .

o//"

.rl

I 1 8 SCHMIDT et a l .

FIG.1. Ta tptake wi th amino, ani l ino and aiCeh;, 'de group contain ing par: ic ies '

s-e 50

omrno grougs

n ( ' n i A

t 2 5 n 2 c / ' 0s tondord {Pg l l00 nL) .+

FIG.5. Standard curve of the Ta assay with aldehyde groups'

rAEA-SM- :59167

_aF__-__:_-

r 2

srondqrd {14/CI! ":L) _

FI ( ; .ö - S tanc ia r r j cur t 'e oJ the T t rssa \ .w i t l t . tn r l ino groups ld is . :o coup l i t ry

:_cDo 1l

CD

I

I t 9

- F ^ r r h a a n i6 r

v u ! u v l a v g f l t

t es tos+ ,e roneassays ver3

*--\

< n r \

in pre l in inary exper imenrs hyd. rcoh i l ic a ld .ehydei r r t u ' : e s r ü e r q a n n l - i e r ] t n f i r - r . h o r a . : q e : r r r ' n n ' l r r Ä . ' - -r w s _ ! a _ v ! ! { L { u u ! * . _ - - _ - r J - I I U J - U Q I I I g

and es t r i o l , w i t h reasonab le resu l t s . so f a r t hesenot oor i rn iz ea.

2 .2 . Pe r+ r i . c l es as se ra ra t i on ned . i un

Therefore, we prepa-r 'ed the s i lane condensates in forn o f par -t i - c l es w i t h a d i ane te r o f abou t 1 un . spec ia l - r eac t i on cond i -t i o n s j - n ( t ) a n d ( Z ) y i e l d p r o p e r . s i z e s . f n c a s e o f a n i n o , a : r i -l ino a : rd a ldehyd.e group conta in ing par t ic l -es i t vas Doss ib le tcd 'e f ine the i r s ize wi t t r var ia t ion o f the cata lys t con-cen ' , ra i ion(nc: - ) . Thus srov ly prec ip i ta t ing granula tes were obta ined. and.app l i ed i n f c : ' : o f suspens :ons . A r t , i bo i . y coa t i ng vas pe r fo rne iv i+ -h essen t i a l l y t he same techn iques as w i t h t ubes . G ranu la tes^ n - + o " - " - - Ä i i f e f . e n t f \ : n C t i o . " ' . 1 c r - , r r r - r c r ,L \ J^ rvd " - r . r . r - :18 n - : i l e ren t l unc t iu r -d "_ i - E : uu rJs were usec as so l_ id , phases e p a - r ' a t i o n s y s t e n i n a T \ R r A . F i g . h s h o w s t h e T h u o t a k e .

FiS.5 anC 6 show the stand.ard. c l l r r /es of the oo-, , imized Ti+ as-a e r r l r > n - * L ^ 1 , 1>,r.J. f :uru r, i le aJnl-no groups no usable siarld,ard_ curlre could. beobta: :ed, . Due to the s teeper . s lope of the s ta : rd .ard curve we ob-ta ine l*bet - ter resu l - ' i ,s w i th par . t ic les conta in ing an i l ino grours .Tab le I I I g i ves t he de ta i l _s .

n0 SCHMIDT e t a l .

' l ' i \ i l r I i , r ' / ' r c = t 1 r a \ t ? t t ' r a \ m I \ A F - ' l n I : c5 r u G - L - ^ . ! - S J J G J s = ! G l M I l } J ( Ä v ! v ! - y

n / r : t l J / \D / - \ , o t D t . r , n \ i ) l , I C C ' l ( ; .o \ l - 4 u i

c J . i )

Anil- i-no tt?

l 1 - i o l - r r i - 4 1- ^ : { !

q 2

5 - 8

REFERENCES

I i ] RATTCLIFFE, J .G . , Separa t ion techn iques sa tu ra r i cn ana lys i s , B r . med . Bu l l . 30 I( i 9 7 4 ) 3 2 .

[ : ] WEETALL, H.H., lmmunochemice] composi tes and ant igen or a-nt ibody pur i f icatron

the rew i th , US. Pa t . 3 652 76 i , Sep t . 1969 .

t3] SCHII IDT, H. , v . STETTEN, O., Stabi l is ier te b iochenische Präparate, Verfahren zu

ih re r Hers te l l ung und ih re Ver ' , vendung , DBP 2 7 - (8 507 , Dec . 1977 .

[4] SCHII ITT, H.H., WALKER, J.E. , Coupl ing capaciq. , . of sol id phase sequencing sdpports,

F e d . E u r . B r o c h e m . S o c . L e t t . S l ( 1 9 7 7 ) 4 0 3 .

[5 ] SCHr \ { IDT , H . , KA ISER, A . , Un te rsuchungen zu rH l ' d ro l yse von K iese lsäureder l va ten ,

G l a s t e c h n . B e r . 5 4 ( 1 9 8 1 ) 3 3 8 .

DISCUSSION

In response to a quest ion, lv l r . Schmidt ind icated that the s tud ies wi th cot t ings

conta in ing amino groups had shorvn l i t t le d i f ference in Bs/Tvalues betw'een tubes

t reated wi th g lu tard ia ldehyde and tubes not so t reated. The immobi l iza t ion o f Ab

in the la t ter cou ld invo lve e i ther hydrophobic in teract ion bet rveen the coat ing and

comesponding s t ructures in the Ab or ion ic or d ipo le-d ipo le in teract ion between

the amino groups and corresponding s t ructures in the Ab. Comparable resu l ts had

been obta incd in s tud ies invo lv ing the immobi l iza t ion o f enzymes on suppor ts

fn f ' : : '+ -her ' - ,€s i ls -Je lounc - -h : - - o- ,h : : q :cnet i ' i c forns. - : r -c us ing coa te i ba i l s c r coa ted . ca : i - J -a : - I s , n :7 be usee i c : 3L ) - \ * n t V U U .

T n e t ' F n r r - r + ' ^ ^ F S - ! ' ' l - - ^ t - + ; s c r . r . : - - - ; ^ - - ! ' - - - - r /L l l e f : S r : : . S O I I n e S L U : : . - S : l O V r u . l : : . - a I I i ' f : * S 3 : - l

. l - r h a s e s - r c - - a n h p c h e e n ' l o r v p l o n e r i - , h p i . l : e o s j , - . - r n r e n r r e i a l ä

g i - res h i l : : i y : ' e r ro fuc : ' : r e assa , r r ?su : * , s .

IAEA-SM.2 59167 t 2 l

conta in ing tmino -groups, a l thougi r the s iorver decay of enzyme act iv i ty in g lu ter -

d ia idehyde- t reeted es compared wi th unt rer ted tubes suggested that less s tab ie

b ind ing occurred in the la t ter .

Mr . Schmid t desc r i bed expe r imen ts (no t men t i oned i n h i s pape r ) i n wh i ch

Ab-coated tubes had been regenerated af ter use by t reatment wi th a 50% rv t vo l .

methanol i 'water mix ture for l5 min. Suc i r regenerat ion, which removed bounc i

Ag but not Ab. cou lc l be repeated up to l0 t imes wi thout loss o f Ab-b ind ing

capac i t .u* . The pr imar" - a im of t i rese exper iments had been to invest igate ihe

poss ib le use of coat ings on capi l la r ies in automat ic anai lzser svsterns opc i t t tng

on t lorv pr inc ip les. None the less. the i r impl icat ions wi th regrrd to prcspec ls for

t i re commerc ia l product ion o f coated tubes were recognzed in d iscussron.