determinación de proteínas con púrpura de bromocresol.pdf

8/12/2019 Determinacin de Protenas con Prpura de Bromocresol.pdf

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C LIN IC A L C H E M IS T R Y , V o l. 24 , N o . 1 . 1 97 8 8 1

W is.; and F iso ns S c ien tific A ppa ratu s L td ., L o ugh -b oroug h , E ng lan d) pe rfo rm sim ilar ly in the ir reactio nw ith a lbu m in .

B rij-3 5 so lu tio n . D isso lve 2 5 g o f B rij-35 (p o ly -o xye thy len e lau ry l eth er ; S i g m a C hem ica l C o ., S t. L ou is ,M o .) in d istilled w a ter , w ith w arm ing , and d ilu te to 1 00

Sto ck ace tic a cid so lu tion . D ilu te 15 0 m l of g lac ia lacetic ac id AR grade ) to 1 lite r w ith d istilled w a te r.

W ork ing B C P reag en t, 40 j.tm ol o f B C P p er lite r.D isso lv e 10 g of so d ium ace tate tr ihyd ra te AR grade)in ab ou t 8 00 m l of d is tilled w a te r . A dd 1 0 m l of sto ckacetic ac id so lu tio n , 1 m l o f B rij-35 so lu tio n , and 1 m lof s tock B C P so lu tion , and d ilu te to 1 liter w ith d istilledw a ter . C heck th e p H an d if necessa ry ad just to 5 .2 0.03 w ith sto ck acetic ac id so lu tio n o r 0 .5 m ol,lite r so -d iu m h ydrox ide so lu tion . T h e reagen t is stab le fo r a tleas t a w eek a t room tem p era tu re .

So dium chlorid e so lu tion . D isso lve 9 g o f sod iumch lo ride in 1 lite r o f d istilled w ate r .

Standards . F o r th is w e used h um an a lbum in [cat. n o .B 5 158 ; D ade B io ch em ica ls , A m erican H osp ital S u pp ly( U . K . ) Ltd. , D idco t, E ng land ] specif ied as 100% a l b u m i nby m ovin g-bou nda ry e lec trop ho re sis and assay ed by them acro -K je ldah l p rocedu re . R eco nstitu te the con ten tso f the vacuum -sea led am p ou le in 3 m l o f sod iu m ch lo -r ide so lu tion as recom m ended by th e m an ufac tu re r an dd ilu te w ith sod ium ch lo ride so lu tion to g ive co ncen -tratio ns u p to 60 g /liter .

Equipment. A utoA n aly ze r I m o du le s: M k 2 S am p ler ,M kl P u m p, C o lo rim e te r (w ith 15-m m N tub u la r f lowce ll) , and R ecorde r (T ech n icon Instrum en ts C o . L td .,B asing stok e, E n g land) .

T h e con tin uou s-f low sy stem (F igu re 1 ) g iv es a sam pled ilu tion of abo u t 2 00-fo ld . T he resam pled stream isin jec ted w ith a ir to m in im ize sam ple in teractio n (asused in a m od ified B C G m etho d ; E . C . A lbu tt, pe rson alcom m unica tion ) . P a rticu lar ca re is neces sary to ach iev ea regu la r b ubb le pa tte rn , o r n o isy peaks w ill re su lt.

B C G M eth odW e used the m e thod of N ortham and W iddo w son (1 ) ,

w ith the s tan da rd s desc ribed abo ve , and m easu red ab-so rban ce a t 63 6 n m . In th is m e tho d , w h ich is w ide lyused in th e U .K ., a reagen t w ith 40 m ol o f B C G per lite rand 1 m l of a B rij-35 so lu tion (25 0 g lite r) in citra tebu ffer (50 m m o l/liter , pH 3 .8 ) is used .

E le c t ro immu n o a s s a yT he L aurell rocke t techn iq ue w as u sed . Se rum and

s t a nda rds (a s abo ve ) w ere d ilu ted 25 0-fo ld w ith sod iumch lo rid e so lu tio n and 5 sl w as ap p lied to a w e ll 2 .5 m min d iam e te r, cu t in a ge l laye r 1 .5 m m th ick . T he g elco n ta ined 15 g o f aga rose pe r lite r, m on ova len t an ti-hu m an a lbu m in an tis erum , an d ba rb ital bu ffer (50m m ol,lite r, pH 8 .6 ) . A fte r e lectroph ores is a t a f ie ldstreng th o f 10 V cm for 3 h , the p late s w ere p ressedun de r filte r p ap er lay ers , d r ied w ith a ha ir-d rye r, andsta ined w ith C o om assie B rillan t B lu e R . P eak h eig h ts

2.........Workir ig BC PAir

F ig . 1 . F lo w d ia g ra m fo r th e B C P m e th o dIns e t s ho w s a ir re -in jec tio n system

w ere m easu red , a stan da rd cu rve w as p lo tted , an d u n-kn ow n serum alb um in co ncen tra tion s w ere d ete rm ined .E q u ipm en t and reag en ts w ere ob ta ined from H oech stP ha rm aceu tica ls L td ., H o unslo w , E ng lan d .R esu l tsReact ion b e t w een B C P an d Albumin

O ptim um con ditions . T he w o rk ing B C P reag en t(withou t Brij -35) ex h ib its abso rbance m ax im a a t 4 36and 590 nm , w ith ab so rbances o f 0 .9 1 and 0 .11 , re spec -tive ly . W ith inc rea sin g pH th e abso rb ance a t 5 90 nminc rease s, bu t th a t at 4 36 nm d ec rease s. A dd ition o fB rij-35 cau ses the op pos ite effec t, a ph en om eno n a lsono ted w ith B C G (2 ) .

A dditio n o f a lbu m in to the w ork in g B C P reagen tcauses an inc rea se in ab so rbance a t 590 n m w ith a sh iftin th e peak from 5 90 to 60 3 nm . T h e d ifference spec -trum of alb um in p lu s reagen t vs. reagen t sho w s an ab-so rb an ce m ax im um at 6 03 nm .

W e ex am ined the e ffec t o f reagen t cond itions on ab-so rb an ce of the a lbum in B C P com plex a t 60 3 nm b yadd ing 0 .1 m l o f a 4 0 g l i ter so lu tion of a lbu m in to 20 m lof B C P reag en t, u sing a reag en t b lank . C ond itions o th erthan those und er exam in atio n w ere kep t a s in theo rig in al m eth od (abov e) . T he abso rb an ce inc rea sedgradua lly ove r he pH in te rva l 5 .1 to 5 .6 , fro m 0 .275 to0 .32 4 . S im ila rly , in creas ing the B C P concen tra tio n from40 to 100 .tm o l lite r caused an inc rease in ab so rbancefro m 0 .29 2 to 0 .31 3 . A dd ition o f B rij-35 so lu tion up to0 .5 m L /liter in creased the absorb an ce, b u t from 0. 5 to1 .5 m l, liter the abso rbance w as con stan t, dec lin inggradua lly a t h ig he r B rij-35 con cen tra tions.

R eagen t co nd itio ns w ere ch osen (a ) to ach iev e thebest com prom ise b etw een m ax im um ab sorbance o f th eB C P a lbum in com p lex and m in im um reag en t abso rb -an ce at 6 03 nm , and (b ) to be su ch th a t sm a ll in accu ra -c ie s in the rou tine p repa ratio n of the B C P reag en t ( i.e .,b u ffe r , B rij-35 , and B C P con cen tra tion ) w ou ld h av em in im u m effect upo n th e rep ro duc ib ility o f the m eth -od .


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82 C L IN IC A L C H E M IS T R Y , V o l. 2 4 , N o . 1 , 19 78

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A L B U M IN g /lite rF ig . 2 . S ta n d a rd cu rve s p re p a re d w ith u s e o f a lb u m in fro m va r-io us sou rce s A , pu rif ie d h um an a lb um in (D a de B ioc he m ica ls );B , free ze -d r ie d hu m a n se ru m (0 P a k I u n a s sa ye d , T ra ve n o lL a b o ra to r ie s L td .); C , purified human albumin Hoechst Phar-m a ceu tic a ls L td .); D , purif ied hu m a n a lbu m in (S ig m a C h e m ic a lC o.). In A-D , a bso rban ce w as m easure d a t 603 nm by the B C Pm e t h o d . E , p u rifie d h u m an a lbu m in (H oe c h s t P h a rm ac e u tica lsL td .); a bs o rb a nce m e a sured a t 6 36 n m b y the B C G m eth od

Standards . T h ree p urified h um an a lbu m in prep a-ra tion s w ere ob ta ined : tha t fro m D ade B io chem icalsdesc rib ed ab ov e; from H oech st P ha rm aceu tica ls L td .(sp ec if ied as > 99% a lbu m in , w ith n o d etec tab le p ro teininpu ritie s) ; and fro m S igm a C hem ica l C o . (crysta lliz eda lbum in con ta in in g 1-3 g of g lo bu lin s pe r 1 00 g). T hesea lbum in p repa ratio ns w ere d isso lv ed in so d ium ch lo rid eso lu tion to g ive a co ncen tra tion of 6 0 g lite r. W e d id no tco rrec t fo r m o istu re o r o the r im purities . W ith the B C Pm etho d , con cen tra tion an d abso rbance w ere lin earlyrela ted u p to an a lbum in co ncen tratio n of 60 g lite r(F ig u re 2 ). T he D ade a lbum in p repa ratio n gave theh ig hest ab so rbance w ith B C P (0 .45 in a 1 -cm ligh t pa tha t 603 nm ). T he H oech st an d S igm a prepa ra tion s g avelow er abso rb an ces, poss ib ly re flec ting the p re sence o fim p urities and , in pa rticu la r , th e p re sence o f m oistu re ,b ecause o n ly the D ade alb um in w as supp lied in vac -u um -sealed am p ou le s.

A freeze -d ried se ru m ( Q P ak I un as sayed ; T rav en o lL ab orato rie s L td ., T h etfo rd , N o rfo lk , E ng land) w asreco nstitu ted in 6 m l of d istilled w ate r ra the r than thereco m m end ed 10 m l, to ach iev e the h igh a lbu m in co n-cen tra tion , w h ich w as then e lec tro im m un oassayed w ithuse o f a co m m erc ia l a ssayed se rum stand ard p rov idedb y H oech st P h a rm aceu ticals L td . L in ea rity and ab-so rp tiv ity w ere sim ila r to tha t g iv en by th e D ad e a lbu -m in s tan da rd (F ig u re 2 ) .

T he lin ea r ran ge fo r the B C G m ethod w as na rrow erth an in the B C P m eth od an d the m ax im um ab sorbancew as le ss (F igu re 2 ) .

F ig . 3 . R e co rd e r tra ce o f an a lys is o f free ze -d ried h u m ans e ru m0 P ak I u nassay ed T raven o l L ab o ra to ries L td .) w as rec on stitu ted w ith d is tilledw ater to 5 7 .5 g o f a lb um in p er l ite r . T h is w as d ilu ted in so d ium c h lo rid e s o lu tio n :1 1 in 5; 2 2 in 5; 3 3 in 5 ; 4 , 4 in 5 ; 5 u nd ilu ted . C hart s pe ed , 50 .8 c m/h

S am ple in tera ctio n . T o ev alu ate th is , w e ana lyzedgrou ps o f th ree spec im ens o f low con cen tra tion , p re-ceded by th ree o f h ig h co ncen tra tion . A t 60 sam ples h ,w ith a sam ple w ash ratio n of 1 1 , se ru m con ta in in g 1 1 .5g of a lbu m in p er lite r w as inc rea sed to an ap pa ren t 12 .1g /liter w h en assayed n ex t a fte r se ru m con ta in in g 57 .5g of alb um in pe r lite r . In teractio n from lo w to h ighspecim en s w as n eg lig ib le . A pp ro ach to the con tin uou sasp ira tion p lateau w as w ith in tw o tran sm issio n lin es(i.e ., 91% of s teady sta te in te rm s of abso rb an ce un its ,97% in term s of transm ission u n its) a t a concen tratio n

of 5 7 .5 g of alb um in p er lite r (F igu re 3 ) .Prec is ion . W ith in -b a tch prec is ion w as m easu red by

rand om ly d istrib u ting 20 spec im ens o f a lbu m in con -cen tra tion 13 .0 g /lite r (s erum p oo l d ilu ted w ith sod iu mch lo ride so lu tion ) be tw een 20 o f con cen tra tion 35 .9g l ite r (u nd ilu ted se rum poo l). Fo r bo th con cen tra tionsthe C V w as 0 .4 3% . T o asse ss be tw een-ba tch prec is io nw e p repa red tw o serum po o ls w ith alb um in concen tra-tion s o f 2 3 .1 g /liter and 3 9 .6 g l ite r (by p oo lin g pa tien tsse ra w ith lo w and norm a l alb um in con cen tra tions , re -sp ectiv ely ), d iv id ed in to a liqu o ts , an d s to red at 4 #{ 176 }C .A l iquo t s o f each se ru m p oo l w ere ana lyzed , in 20 ba tch eseach , on d iffe ren t days du rin g fou r w eek s. E ach b atchin clu ded e igh t to 15 serum specim en s. W e used the sam eman ifo ld , s tock B C P so lu tion , s tock ace tic ac id so lu tion ,and B rij-35 so lu tio n th ro ugh ou t th is pe riod .

Spec if ic i ty . A lb um in w as se lectiv ely rem ov ed fro mse rum b y affin ity ch rom ato grap hy o n cy an og en b ro -m ide -ac tiva ted ag a rose (S ep ha ro se ) ge l co va len tlylin ked to B lue D ex tran 2 000 [P ha rm acia F ine C hem i-ca ls , Ph a rm ac ia (G rea t B ritain ) L td ., L ond on ] (21). T hee lua te from the g e l co lum n s w as exam ined fo r th ep re sen ce o f a lbum in by im m u no elec tro pho re sis aga inst


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To t a l A Ib u m In concn , g/l i terproteIn B C G B C Pg / lite r m e th od m e th od RID8

3 6 .0 11 .0 0 .5 0 .73 1 .0 7 .0 0 0

1 8 .0 6 .5 0 01 7 .0 5 .5 0 0

Transfer r lnconcn

% oftransferrin

assayed asa lb u m in

8. 517 .026 .533 .542 .5

12 .019.028.035 .043 .5

H u m a n S eru ma lbum in 1 2 3

H u m a ntrans-fer r in

T im ea fte r

mix ing

lO s30 s1 m m5 m m3 0 m m6 0 m m

Alb u m in -free

globulinprepn

.310

.300

.300

.301

.305

.307

.330

.321

.320

.321

.320

.321

.281

.282

.280

.280

.282

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.060

.080

.108

.150

.190

.202

.041

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.094

.095

C L IN IC A L C H E M IS T R Y , V o l. 24 , N o . 1 , 19 7 8 8 3

T a b le 1 . A p p aren t A lb u m in C o n c en tra tio n o fA lb u m in -F re e S eru m E lu a te s P re p a red b y A ffin ity

C h r o mato g r ap h yN a tu re o fs p e c I m e n

C ro h n s d is e a seDi f fuse b and on

electrophoresisN ep hro tic s yn dro meG lo me ru la r n ep hritis

a R a di al i m m un o di ff us io n .

m on ov alen t an tihu m an-alb um in se rum an d po lyvalen tan tihu m an se rum (H o echs t P ha rm aceu tica ls L td .) .C om pariso n w ith the o rig ina l se ru m show ed th at thep rec ip itin arc co rre spo nd in g to serum alb um in hadd isappeared an d a ll o the r de tec tab le p ro te ins rem a ined .E lua tes fro m the g el co lum n s of serum fro m fou r pa -tien ts w ere ana lyzed by th e B C P m ethod , th e B C Gm etho d , an d th e M anc in i rad ia l im m un od iffusionm eth od (H o e ch st P harm aceu ticals L td .) (T ab le 1 ) . T heappa ren t a lbu m in co ncen tra tion s, a ssayed b y the B C Gm etho d , w ere b e tw een 23 an d 32 % of the to ta l p ro te inconcen tra tio n o f th e elu ate . A lb um in w as no t de tec tedby e ithe r the B C P or the rad ia l im m uno d iffu sionm e tho ds, excep t in spec im en 1 , w he re alb um in w as a lsode tec ted b y im mun oe lec troph oresis .

H um an transfe rr in (H oechst P h arm aceu tica ls L td .) ,sp ec if ied as > 9 9 % p ure w ith n o d etec tab le im p urities ,w as d isso lved in sod ium ch lo ride so lu tion to g iv e con-cen tra tion s in the ran ge 5 to 4 0 g /lite r. W e de tec ted o n lyon e prec ip itin a rc by im m uno e lec troph ore sis ag ain st apo lyva len t an tis erum . E ach so lu tion w as ana lyzed bythe B C P and B C G m ethod s. T rans fe rr in had a sign ifi-can t reac tion w ith B C G (T ab le 2 ) even in the p resen ceof a lbu m in bu t show ed no reac tion w ith B C P .

C o lo r deve lopm en t o f the B C P reagen t w ith p urif iedhu m an alb um in or se rum w as in stan taneous (le ss thanlO s a fter m ix ing ) and w as stab le fo r a t lea st 1 h . N o re -ac tion w as o bse rv ed w ith the a lbu m in-free se rumprepa ra tion s o r transfe rr in so lu tion s, eith er im m ed i-a te ly o r a fte r 1 h . S om e se rum sam ples exh ib ited a slowreac tion w ith B C G w hile w ith o the r se ra and pu rif iedh um an alb um in so lu tion s co lo r d ev elo pm en t w as in -stan tan eo us. F or tran sfe rrin so lu tion s, co lo r deve lop -m en t w ith B C G con tinued ove r a p eriod of 1 h (T ab le3) .

W e estim ated th e a lb um in co ncen tra tion o f va riousan im al-based se rum spec im ens, using D ad e hu m an a l-b um in fo r ca lib ra tion . T w o freeze -d ried bov ine -b asedq ua lity -co n tro l se ra (W ellcom e R eag en ts L td ., B eck -e n h a m , E ng lan d) had appa ren t a lbum in concen tratio nso f 31 .0 and 3 6 .0 g /lite r by th e B C G m etho d , bu t o n ly12 .6 and 1 2 .9 g /liter , resp ectiv ely , by the B C P m eth od .P ure bov ine se rum alb um in (H oechst P ha rm aceu tica lsL td .) in sod iu m ch lo ride so lu tio n a t a concen tratio n o f40 g /lite r gave an ap pa ren t a lbum in con cen tra tion b ythe B C G m etho d of 4 3 .0 g /lite r, bu t o n ly 1 5 .0 g /lite r b ythe B C P m ethod . A n unassayed eq u ine se rum h ad an

T ab le 2 . R ea ctio n o f B C G a ) w ith P u re H u m anT ran s fe rrin A lo n e , an d b w ith P u re H u m an

T ra n sferrin in th e P rese n ce o f P u re H u m a nA l b u mi n

a)A p p a r e n ta lbumin

c o n c n9/l I ter

5. 0 4. 310 .0 6. 520.0 9. 530 .0 13 .540 .0 16 .5

T ran sf er r in6 .7 g /Iite r

+ pu re h u m a na lb u m in

b )Appa re nta lb u m in

co n cn .g/ l l ter

8665474541

% oft ransferr ln

assayed asa lbumIn

5230222215

T a b le 3 . R ate o f C o lo r D ev e lo p m e n t o f th eR e ac tio n o f B C G w ith A lb u m in , S e ru m , a n d

P ro te in P re pa ra tio ns a

a Absorbance a t 6 36 n m vs . rea ge nt a t in te rva ls sh ow n , tim ed fro m m ix ing20 c l o f p ro te in so lu tion w ith 4 m l of B C G reagent .

alb um in con cen tra tion o f 3 9 .0 g /lite r by th e B C Gm eth od , 23 .9 g /lite r b y the B C P m ethod .E ffe c t o f In te rfe ring S ub sta nces on the B C PMethod

B lank correc tion . S era w ere an aly zed w ith use o f aB C P reagen t from w hich bcp itse lf h ad been om itted .N o rm al se ra had a neg lig ib le abso rb an ce. Ic te ric se raw ith b iliru b in concen tratio ns up to 5 00 izm olllite r re -qu ired b lank correc tions o f b e tw een 0 .5 and 1 .5 g o fappa ren t a lbu m in pe r lite r. S im ilar co rrec tions w erereq u ired fo r g ross ly h em oly zed se ra (app rox im a te ly 1g o f appa ren t alb um in pe r lite r a t lO g o f hem o g lob in p erlite r) . G rossly lip em ic se ra requ ired the g rea tes t co r-rec tion s, 3 .5 g /lite r be ing necessa ry in one ex trem e ex-am ple w here the se rum loo ked lik e m ilk .

Salicy la te . S od iu m sa licy late , ad ded to se rum an d topu re h um an a lbum in standa rds, d id no t in te rfe re w ith


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8 4 C L IN IC A L C H E M IS T R Y , V o l. 2 4 . N o . 1 , 1 97 8

1 0 2 0 30 40 50F ig . 4 . C o m pa rison o f res u lts b y th e B C P a nd e le c tro im m u-n oa ssa y m eth od s

the B C P m eth od a t co ncen tra tion s u p to at lea st 3 00mg/l i ter .

Bil irub in . P urified b ilirub in (S igm a C hem ica l C o .,spec ified as e ssen tia lly 10 0% bilirub in ) w as added tose ru m an d to purified hu m an a lbum in standa rds up toa con cen tra tion of 5 00 u m ol/lite r. M in or in terfe ren cew as no ted a t co ncen tra tion s ex ceed in g 17 0 /L m o l/lite r .T h e app aren t a lbu m in concen tratio n o f a 4 0 g /lite r so -lu tion w as 39 .4 g /lite r an d 3 8 .3 g /liter in the p re sence of250 zm ol, and 50 0 zm ol o f b iliru b in pe r lite r , re spec -tiv e ly . S e ra w ith h igh b iliru b in concen tratio ns tend tobe u nd ere stim a ted by th e B C P m etho d . F iv e se ra w ithb iliru b in concen tratio ns be tw een 11 3 and 19 2 m o l/ lite rw ere es tim a ted b y th e B C P m ethod an d e lec tro im m u-noa s s a y . C oncen tratio ns by the B C P m etho d w ere low erby 0 .2 g to 4 .6 g /lite r (m ean d iffe rence , 2 .4 g /lite r) .H o w ev er , fo r 17 sera w ith b ilirub in con cen tra tions inthe ran ge 25 to 79 jzm ol/lite r (m ean concen tra tion , 52m ol/lite r) , ana lyzed by bo th m e tho ds, th e m ean d if-ference in a lb um in co ncen tra tio n w as on ly 0 .1 g /lite r.Compar ison of the B C P M ethod a nd B C G M e t h o dw ith E le ctro im m u no as sa y

S era w ith a lbu m in co ncen tra tion s in the range 13 to4 5 g / lite r w ere assayed b y the tw o dy e -b in d ing m e thod sand by elec tro im m u noassay . E ach ana lys is w as pe r-fo rm ed in the sam e w o rk in g d ay , to avo id sam ple de te -r io ratio n . T he sam e p urified h um an a lb um in standa rds(D ade B io ch em icals) w ere used fo r a ll th ree m eth ods .S e ra w ere se lected to p ro v ide the m ax im u m range o falb um in , b ilirub in , an d g lob u lin concen tra tion . S e rashow ing o the r abno rm alitie s (lip em ia, hem oly sis ,p re sence o f d rug s, e tc .) w ere a lso selec ted .

R esu lts b y th e B C P and e lec tro im m uno assay m e th -od s (F igu re 4 ) ag reed w ell, w ith the reg re ssion eq ua tionbe ing y(B C P ) = 0 .95 x + 1 .7 2 (in g a lb um in /lite r , n =160 , r = 0 .9 86) . R esu lts b y the B C G m etho d w ere co n-side rab ly h ighe r than by e lectro im m un oassay (F igu re

B C G

u / / / #// /

// // // ////

Albumi g/liter E IA10 3 0 4 0 5 0

F ig . 5 . C o m p ariso n o f res u lts b y th e B C G m eth od a nd e le c tro -im m uno as say m e tho ds

5 ), the reg ress ion equ atio n be ing y(B C G ) = l .Olx + 6 .77(n= 160 ,r= O .986 ).

W e fu rthe r com pared (w ith d ifferen t se ra to th oseu sed abov e) th e B C G and B C P m ethod s, w ith pu rif iedhum an a lbum in (H oech st P h arm aceu tica ls L td .) fo rstan da rd iza tio n . S im ila r re su lts w ere ob tain ed (T ab le4) .

A freeze -d ried se ru m (V a lida te ; G en era l D iag -nostic s L td .) w as stan da rd ized b y e lec tro im m uno as sayw ith u se o f pu rif ied hu m an a lbum in (H oechst P h ar-m aceu tica ls L td .) A ltho ugh th e freeze -d ried serum forcalib ra tion gave c lose r ag reem en t b etw een re su lts by theB C G an d e lec tro im m u noassay m e thod s, it w as still no ta s go od as tha t b etw een e lectro im m un oassay and B C Pm etho ds (T ab le 4 ) .Discuss ion

T he B C P m etho d p ro v ides an alte rna tive to ex istingB C G m ethod s. T he con tinuo us-flo w sy stem desc ribeda llo w s a h igh ra te o f ana lysis w ith go od prec isio n andlittle s am ple in teractio n .

In the B C G m eth od (1 ) , BCG tend s to fo rm a greenfib rilla r p rec ip ita te w ith a lbum in , an effec t th at isg reate st at th e isoe lec tr ic po in t (pH 4 .3 ) o f th e com plex9 ). P rec ip ita tion is inh ib ited , bu t n o t e lim ina ted , bythe su rfac tan t B rij-35 . N eith er p rec ip ita tion nor theassoc ia ted nega tive base line e ffec t (1 ) w ere obse rvedw ith B C P , even in th e absen ce o f B rij-3 5 , po ssib ly dueto th e h ig he r reag en t pH used in the B C P m etho d .

S eve ral com pariso ns have b een m ade b e tw een B C Gand spec ific m e tho ds (1 1 -1 5 ) . A ltho ugh all ag ree tha tB C G m ethod s ov ere stim a te se rum a lbu m in con cen-tra tion , the m ag n itud e o f the inaccu racy va rie s . T h ism ay ref lect seve ral fac to rs: reagen t co m po sition (1 0 ) ,ca lib ratio n m a te ria l, tim e a llow ed fo r co lo r deve lop -m en t, an d the so urce o f spec im ens . In o ur stud y ,ag reem en t w ith elec tro im m u noassay w as m uch be tte rin the no rm a l range w hen freeze-d ried se ru m w as used


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yxN o. sp ec i-

m e n s (n )M ea n x(9/ l i ter )

M ea n y(g/ I i ter )

S lo p eIn terceptC orre l. co e ff.

2 7 .7 2 7 .7 2 7 .9 2 9 .4 3 1 .0 3 1 .0

2 7 .9 3 4 .8 34 .8 3 5 .8 3 2 .5 3 0 .4

0 .9 5 1 .0 1 1 .0 61 .72 6 .7 7 5 .270 .9 86 0 .9 86 0 .98 9

1 .02 0 .85 0 .9 55 .8 1 6 .2 2 1 .2 10 .9 7 7 0 .9 8 8 0 .9 8 8

C L IN IC A L C H E M IS T R Y , V o l. 24 , N o . 1 , 197 8 8 5

T a b le 4 . R eg re ss io n E q u a tio n s D es crib in gC o m p ariso n s b e tw ee n B C P , B C G , a n d

E lec tro im m u n o a ssa y E IA ) M eth o d s , w ith V a rio u sA lb u m in P re p a ra tio n s U s ed fo r S tan d a rd iza tio n

P u re h u m a na lb u m in std s fro m F re eze -d rie d

D a de H o ec hs t hu m an se rumB C P B C G B C G B C G B C G B C PE IA E IA B C P B C P E IA E IA16 0 1 6 0 16 0 1 20 8 0 8 0

SD about re- 1.3 7 1.48 1.30 1.82 1.00 1.13g ress io nl in e (S i)

ra the r than purified h um an alb um in fo r ca lib ra tion(T ab le 4 ). P ossib ly in te rfe ring pro te ins in the freeze -d ried m a ter ia l pa r tly com pensa te fo r the p re sence o fthese p ro tein s in te st se ra .

T h e slow ra te o f co lo r deve lop m en t o f B C G w ith in -terfer ing p ro tein s (14) has b een used to im prove theaccu racy of B C G m eth od s by p ro m pt m easu rem en taf te r m ix in g se rum w ith reagen t (14 , 25 , 2 6). H o w ev er ,an e rro r o f 3 g of a lbu m in pe r lite r w as still p re sen t (25).P ure hu m an transfe rr in and a lbu m in-free se rumprepa ra tion s deve loped co lo r slow ly , bu t still h ad asign ifican t reac tion w ith in 1 0 s (T ab le 3 ) . C o nsequ en tly ,th is m e tho d w ou ld no t seem to be a com ple te so lu tionto th e p rob lem and p laces cons ide rab le con stra in t o nthe ana ly tica l system th at can b e u sed .

T h e princ ipa l advan tage of B C P is its spec ific ity fo ra lbum in , a s dem o nstrated b y th e lack o f reactio n w ithpure h um an trans fe rr in o r w ith the a lbum in-free se rumprepa ra tion s (T ab le 1 ), w h ich con tain ed m o st o th erse ru m p ro te in s in the ir o r ig in al concen tra tion , a s as -se sse d im mu noc hem ica lly (21). T he app aren t alb um inconcen tratio n of th ese so lu tion s estim a ted by th e B C Gm etho d w as su ffic ien tly g rea t to exp lain the d iffe ren cebe tw een re su lts by elec tro im m u noassay and the B C Gm et h o d .In the B C P m etho d , equ in e an d b ovine serum albu -m in a re no t su itab le fo r use in ca lib ra tion or qu alitycon tro l because the ir reac tiv ity is m u ch le ss than th a to f hum an alb um in . H ow ev er , n onh um an m ate ria l isc lear ly u nsu itab le fo r u se w ith spec ific im mu nochem ica lm eth ods , and its use w ith B C G m ethod s can be m is-lead ing (1 0 ) .

D y e-b ind ing m e tho ds ar e suscep tib le to in te rferenceby c ompe t ing m olecu le s . T he B C P m eth od is un affec tedby sa licy late . Pu rif ied b iliru b in h as little e ffec t b u tspec im ens con tain ing en dog en ous b iliru b in a t h igh

co ncen tra tion w ere so m e tim es und e re stim a ted , b u tra re ly by m ore th an tw o standa rd dev ia tions from thereg res sion line o f B C P aga ins t e lectro im m un oassay . Itha s been su ggested (12) tha t the B C G m etho d sim ila rlyund ere stim a tes ic ter ic se ra . C on sequen tly , the effec t o fb ilirub in is u ndesirab le bu t it is accep tab le . C lea rly ,o the r m o lecu les , pa rticu la rly d rug s an d the ir m e tabo -lite s , cou ld a lso in terfe re and th is is p robab ly ref lectedin th e degree o f sca tte r o f resu lts ab ou t th e reg re ssionl ine.

T h e h igh sp ec if icity o f B C P for h um an a lbum in , itsin stan tan eous reac tion w ith a lbu m in , and its su scep ti-b ility to in te rference by b iliru b in cou ld w ell b e m an i-fes ta t ions of the re str ictio n o f B C P bin d ing to rela tive lyfew h igh-a ff in ity b ind ing site s on se ru m a lb um in . B C Gbind s to num erou s frag m en ts o f b ov in e serum a lbu m in ,alth oug h it also has a h igh a ff in ity fo r the fragm en t tha tis th e site fo r b iliru b in b ind ing (22). H u m an serum a l-b um in is kno w n to have tw o g en e ra l c la sse s o f b ind ingsite fo r an io n ic m olecu le s o the r th an fa tty ac ids (2 3 ) .B in d ing at th e h igh -a ffin ity site is by a com b ina tion ofelec tro static and hyd rop hob ic fo rces, w h ile b ind ing a tthe low -a ffin ity site s , w h ich a re n um erous bu t va ry innum ber, is p r in c ipa lly elec tro sta tic . B C G can b in d totw o c la sse s o f b ind ing site on serum alb um in (24) bu tall tha t is req u ired fo r the spec tra l chang e is im m obili-zatio n on a catio n ic su ppo rt (9 ) such as de tergen t m i-ce lle s , ge l, o r p ro te in . W e h ypo thesize tha t the s low re -actio n of B C G w ith in te rfe r ing pro te ins w as d ue toe lec tro sta tic b ind ing to catio n ic site s , w h ich co u ld beredu ced in n um b er by in c reasing the pH o f the reagen t.Inv estiga tion o f th is h ypo thesis m igh t pe rm it fu r the rim provem en t o f th e B C P m etho d or the d esign of d yesspec ifica lly fo r a lbu m in de term in atio n . H ow ev er , theB C P m etho d a t it s presen t stage o f d ev elo pm en t o ffe rsbe tter pe rfo rm an ce in m o st re spec ts than do the B C Gm et h o d s .

B ecause o f the lim itatio ns o f ex isting B C G m etho dsit h as been su ggested (17) tha t abn orm a l sera shou ld beana lyzed by a specif ic m e thod . S uch m e tho ds a re usu -a lly slow and im p rac ticab le fo r d ea ling w ith la rg enum bers o f sp ec im en s. T he im m uno ch em ica l m eth odsrequ ire a su pp ly o f h ig h -tite r m ono va len t an tise ra, an dar e sub jec t to seve ral t e c hn ic a l p r o b l em s (15). T he B C Pm etho d , h ow eve r, sh ow s ex ce llen t ag reem en t w ithe lectro im m un oassay , ye t re tain s the sim plic ity andcapacity o f ex isting d ye -b ind in g m e tho ds, p erm ittin ginexp en sive sam e-d ay -d ay ana lysis o f m any sp eci-m en s .

R efer en ces1 . N ortham , B . E ., and W iddo w son , G . M ., D ete rm inat ion of se rumalb um in by A uto A n aly zer us ing bro m o cre so l green . A ssoc. Gu n .B io ch em ., T ech . B u ll. N o. 11 , 1 ( 19 67 ).2 . D ou m a s, B . T ., W atson , W . A ., an d B ig gs, H . G ., A lbu m in s tan -da rds and the m easure m e nt o f seru m albu m in con cen tra tio n w ithbro m o cre so l gre en . C lin . C h in s . A c t.s 31 ,87 (19 71) .3 . M c P h erso n , I . G ., an d E v era rd , D . W ., S eru m alb um in es tim ation :M o dif ica tion o f the bro m o cre so l green m eth od . C lin . C h im . A c /a 37 ,117 1972) .4 . Lo lek ha , P . H ., and C haroenpo l, W ., Im proved au tom ated m eth od


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88 C L IN IC A L C H E M IS T R Y , V o l. 2 4 , N o . 1 , 1 9 78

fo r d ete rm in in g seru m alb um in w ith b rom cre so l gre en . C lin . C hem .20 , 6 17 (1 974 ).5 . B e ng , C . G ., and L im , K . L ., A n im pro ve d a u to m a ted m e tho d forde term ination o f serum a lbumin using bromcreso l green . A m . J . C lin .P at ho . 59 , 1 4 1973 ).6 . S ch irard in , V on H ., a nd N ey , J ., E ine v ere in fach te M ic ro m ethod ezur B estimm ung von S erum A lb um in m it H ilfe vo n B rom kre so lgru n .J . C lin . C h em . C lin . B io ch em . 10 ,338 (1972) .7 . W estgard , J. 0 ., an d P oqu ette , M . A ., D ete rm ina tion o f se rum a l-b um in w ith th e S M A 1 2/6 0 by a brom cre so l g ree n dy e-b ind ingm e t h o d . C lin . C hem . 18 ,64 7 (19 72 ).8 . L eon ard , P . J., P ersaud , J ., an d M o tw ani , R ., T he es tim ation ofp lasm a album in by BCG dye b ind ing on th e T echn icon SM A 12/60ana lyz er a nd a c om par iso n w ith th e H A B A d ye bind ing tech n iq ue .Clin . C h im . A c ta 35 , 40 9 1971 ).9. B en g, C . G ., R a s a n a y a g a m , L. , L im , K . L ., an d L a u , K . S ., S o lub ilityand a bso rp t ion spec tra of c om ple xe s resu lting from in te ract ion am on gh um an a lbum in , b ro m ocreso l g reen and de te rg en ts . C lin . C him . A cta52 , 2 57 1974).ID . S pe nce r, K ., an d P ric e , C . P ., In f lue nce o f re age n t q uali ty a ndre act ion c ond itio ns o n th e de term in atio n of se rum a lbu m in by theb rom oc reso l gre en d ye b ind ing m eth od . A n n . C lin . B io ch em . 1 4 , 105(1977) .II. W e bste r, D ., B ig nell , A . H . C ., an d A ttw oo d E . C ., A n asses sm entof the su ita b il ity o f brom ocreso l gre en for the d ete rm ina tio n of seruma lbumin . C lin . C h im . A cta 53 , 101 1974) .12 . F er reira , P ., an d P ric e , C . P ., A co m parison o f bro m o cre so l g ree nand im m uno pre cip ita t ion m eth od s for the d eterm ination of serumalbumin . C lin . C h im . A c ta 55 , 25 9 1974 ).13 . L au rel l, C -B ., T he use of elec tro im m u noa ssa y for de term in ingspe cif ic pro te ins a s a sup ple m en t to aga rose gel e lectrop ho resis. J.C lin . P a t ho . 28 , S upp l. (A sso c. G un. P a t ho E .) 6 ,22(1975) .14 . G us tafsso n , J . E . C ., Im p rov ed spe cific ity of se rum album in de -

term in atio n a nd es tim ation of ac u te pha se re acta n ts by the u se o fthe bro m c reso l g reen reac tion . C lin . C hem . 22 ,61 6 (1 976 ).15 . S late r, L. , C ar ter , P . M ., a nd H obb s, J. R ., M easure m e nt of al-bum in in th e sera o f p atie n ts . A nn . C lin . B ioch em . 12 , T ech . Bu ll. 34 ,3 3 (1 97 5).16 . In tern ationa l F ed era tio n of C lin ic al C he m is try , N e w s letter N o.1 3 , M arc h 19 76 , p5 .1 7 . W eb ster , D ., A stud y of the in teract ion of bro m o cre so l g reen w ithiso lated seru m g lob ulin fra ctio ns. C lin . C h im . A cta 53 , 1 09 (19 74 ).1 8 . P inne ll, A . E ., Inv est iga tio n o f se rum albu m in d ete rm ina tion b yd ye bind ing m ethod s. M .S c . T h esis, U niversity of B irm in gha m , E n -g lan d , 197 6 , p 111 .1 9 . K rag h-H an sen , U ., an d M { 216} lle r ,J . V ., E ffec t of pH a nd ino rga nicsalts on the phe no l red /pro tein in te rac tio n . B io ch im . B io p h ys . A c /a295 , 438 1973).2 0 . C arter, P ., U ltram icro es tim ation of hum an serum a lbum in:B ind ing of th e c atio n ic dye 5 ,5-d ib rom o-o-creso l su lpho np hth alein .Micro ch em. J. 15 , 5 31 (1 970 ).2 1 . T ra v is , J. , an d P a nne ll, R ., S elect ive re m o va l o f alb um in fromp lasm a by a ffin ity c hro m atog raph y . C lin . C him . Acta 49 , 4 9(1973) .2 2 . P ete rs, T ., J r., S erum a lbu m in : R ece nt pro gre ss in the un de r-stand ing of its stru ctu re and bio sy n th esis . Clin . C h e m . 23 , 5(1977) .2 3 . S pe cto r, A . A ., F at ty acid b ind ing to p lasm a a lbu m in . J. L ip id .R es . 16 , 1 65 (19 75) .2 4 . R od ke y , F . L ., B in d in g of b rom oc reso l green by hu m a n seru malbumin . A rch . B io che m . B io p h ys . 1 08 , 5 10 (1 96 4).2 5 . W e bs ter, D ., T h e im m ediate reac tio n betw e en brom creso l g ree nand se rum as a m easure of alb um in c onten t . C lin . C h em . 23 , 66 3(1977) .26 . C o rco ran , R . M ., an d Durran , S . M ., A lbu m in de term in ation b ya m odified b rom creso l gre en m e tho d . C lin . C he m . 23 ,76 5 (1 977 ).

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