Download radioimmunoassay of secretin in serum

AJEBAK 57 (Pi. 1) 95-105 (1979J
RADIOIMMUNOASSAY OF SECRETIN IN SERUM
by P. HO AND J, .HANSKY"
{From the Monaslt University Department of Medicine,
Prince Henry'.s Ho.spital, Melbourne, Vietoria 3000.)
(Accepted for puhjicatian Novcniher 6', 1978.)
Summary. .\ radioimnn.moassay for tbe nieaNuremeiit of inmimiDreactivr secrctin
has been developed. Tbe assay is specific and 5en.sltive so that 2-8 .fmoi/uil can
be measured. Ba.sal secretin levels iu ninn are generally iindetcctable and a
protein-ricb meal does not elicit a secretin response. However, tbe intraduodenal
histillatlon ol HCl leads to a prompt rise in circulating immnnoreaoti\'e secrt'tin
and exogenous seeretin administered intravenously is ea.sily and accurately
inea,sured.
INTRODUCTION
Seeretin. a peptrde ol 27 amino aeid. residues, wa.s the first hormone
described (Bayliss and Starling, 1902). Since then its physiolngieLd rolo and
amino acid .sequence have been elucidated and the hormone lia.s been syntliesized
(Uodansky et ah, 1966). The first description of measurement of circtilaHng
,secretiti by radiuimintinoassay was by Young et al. (1968), and a nitmber of
laboratories have described the a.ssay ot seeretin since (hat fir.st report (Boden
and Chey, 1973; Strauss, Urbaeli and Yalow 1975a, and Byi'ucs and Marjaseni,
1976). Wo describe a further radioimminioassay for .seeretin based on lal.u'lled
Research Plus syiithetie secretin, an antibody raised in a rabbit to riyiithetic
seeretin and an extraction procc^lm'e loi- the measurement ol.' itnitumort^aetive
seerotin in seruni. Thi,s a.ssay has been used iti physiological studies oE seeretin
release in man and sheep.
Antibody production
Syntbetic .seeretin (Schwarz-Mann, New Jersey, U.S.A.) was conjuf^'ated to bovine .seriim
,illnimin by earbodiijTiide (MeGuigan. 1967). Eight New Zealand white rabbits were
primarily innmniised by foot-pad injection witb 25 Mg oi the conjugated mixture with an
equal volume of complete Freuuds' adjuvant. Tbe .subsequent injecti(jn,s weie given inontbly
" AddreKs for reprint reqnest.s
96
P. HO AND J. IIAN SKY
for 3 iiionLhs and then at 12-week intervals until a (k\sirable antibody litre was
This occurred rullowing the fourth 12-\veekly injection.
()/ pepiidc
6-tyro.syl seeretin (Schwarz-Mannl. N tt-Dt'samiiiotyro.syl-;3-alanyl-secretin (Fluka, AC,
Chemische Fabrik. CM-9470 Bntilis) and synthetic secretin from Schwarz-Mann and Research
Plus Laboratories, New jeiKey. U,S,A. were labellfd by modification of tile chloraniinf:! T
tcchiiicine according to Byrues and Marjason {1976). 10 ^.g secrt'Hu made up in O'Ol M IICl
was mixed with 2 mCi Na V--' with 15 ng chloramine T di,sso]ved iu 10 |d borate buffer
pll S-3. The reaction was terminated by ,50 ng .sndimn metabisulphite afte-r 2J4 miu.
lOO 111 of "hormone free" seruni uete adtied to the reaction mixture and put im to a
uLini-Scphaclex GIO (Fhamiaeia) colmnn. size 0-4 x 5 cm. The colunm was elntctl with
0-0,5 M sodium neetate buffer pll 5 c{>ntaiiiiug 0'2'/' bovine serum alliumiri. The iir.st ml of
effluent after the void volume containing the iodinated peptide was further purified by passing
down a Sp Sephadex C25 (Pharmacia) colunm, size 0-9 x 30 cm. The colunm wa.s eluted
with ;t gradient of 0-05 M to 0-5 M sodium acetate albumbi buffer pH 5. Collections were in
2 ml aliquots. The last peak of radioactivity was used as label. The label was stored at —20°
in acid aleohol made up of 75% etlumol and 25% HCl.
Frcptiralion of liorniaiw fret' scrum
Outdated pooled serum was filtered with coarse Whatman's filter paper. 10 g of Norit A
aetivated charcoal was mixed with ,500 ml of pooled serum. The suspension was stirred for
30 min and centrifuged at 1000 g for 20 min. Supernatant was decanted and filtered through
pore size 1-2 \\, Sartorius membrane filter (Sartorius Membi'anfilter Gnibll). Honnone-Free
sermn was prepared for each snbject studied.
Exlraclion
of scniiii- .vcij?iji/f'.v
Blood was collected in tra.sylol tubes, centrifuged and sei-um-separated and stored at
—20" until extraction. I ml of serum was extracted with 2 ml of lOOS'^ ethauol and eentrifuged
at 5000 g for 40 min (Iledinjj:, 1971). Tlie supenuitant wits deeanfed and dried niider pressuie
in a vacuum desiccator. The dried extract sample was kept at —20" until assayed for secreb'n.
Raiiioinmiiuioasstiii procedure {Taiilc 1)
TAIJIJ- 1
Proiocol of secrelin R!.\ /iroccdurc.
DAY I
Standard curve:
Sl;md;ird from 2-5 lo iOO fniol
"Hurmone-iVee"' sera extract
Antibody -h 24. llnal dilution I :4(10,000
100 ,ul
200 /A
100 fil
Diluent
Unknown serum sample exlract
+ 24. Tinal dilutiou 1 ; 400,000
100 ^itl
200 ^il
100 ul
DAY 3
Secrolin iaticl l2t)0 coiints/niin
100 ftl
DAV 5
Ciiarconl dexlran suspension
Mix. centrifuge and separate
Count in y counter
500 ftl
OR
Sera .wmple:
Assays were periuiiin't! in I x 7 em puIyslyreinK;' lubes. Standards and uuknuwn .'Samples
were a.ssayed in duplieate. Honnone-fiee serum extract of e\'ery subject studied was assayed
to monitor zeiii .seeretin Striding, 0-()4 M si>diniri i^liosphale buller, pll (i-.S eontainiug
0-5!?. bovine serum albumin, 1000 Kallikrein luactivator IL'nits (KIU)/ml trasylol, 0 2 mg/uil
lUertheolate, aud 0-02 mg/iiiI rieouiyeiu sulphate wa.s used as dilueut for standards, LUitibutly
aud tracer. Fresh seeretin standards (puTe natural pOTeint; secretin, CIH, Research Laboratory,
K;ui)liiiska Institute, St()ckl)olm. Sweden) from 2,5 fmol/ml io 1000 fmol/ml were diluted out
RADIOIMMUNOASSAY OK SEGRETIK IN SERUM
97
for each assay trom a stoek seeretin standard oi 10 pmo!/nil. Dried uiikni)\vn seruui extracts
were constit:uted to half of their originul volumes with diluent. Standards and unknown senini
extracts were pre-incubated for 4S h wifh antibody No. 24 at final titre of 1:400,000 at 4°.
Label, approx. ]200 c.p.in. was then added aud incubated for a fiirther 49 h. 0-5 ml suspension
of 25% Nnrit .\ activated charcoal and 5% dextran T500 (Fliarmaeia) made up IEI piii>sph;itii
biiHer pH 6-5 wtis added to each tube at the end of U:ie ineubatiou period. Tubes were mixed
and centrifuged immediately at lOOO g for 25 miu a(. 4°. After eejitrifugation, supematants
were aspirated b>- suetion xiump and discarded. The eliarcoal peiJels were eouuted in a gajuma
counter foe eight minutes.
Ctdculaiions
Percent bound radioactivity was calculated by using an Oli\ettf piogiaimne JO! table
calculator accotding to the foimiila
% B = - " : - ^ X J 00!S
T being the radioactivity of the charcoal pellets in the absence of antibody and F the free
tractiou of radioactivity absorl.ied into the charcoal in the standard or uukuowu tubes. Pereent
bound radioaetivit>' was plotted against eoncentmtion of .standards. Tiie unknown senmi
secrelin levels were obtained liy .subtracting the "blank" secretin values I'miri the assayed
secretin eoneentratiiin of the unknown samples. The serum seerelin level \v:is expressed
as fmol/ml.
PiTYSlOLOUICAL STUDIES
Eight patient.s wore given an lntravL'noi.is injection o( CUT secretin (Research
Laboratory, Kart)lin.ska In,stitiitc, Stockholm, Sweden) and si.x of tlin.se lioots
,srcretin ( BooLs Co. Ltd., NottJngliam, iinj^land) of 1 and 3 ulinjcal nnits pei' kg
body wejglit, ]-cspecti\-(.-ly. Rlood w;i.s collected into trayylol tnlies (1000 .KIU
h-asyloi/tube) at ]5 min before, just before ajid at 5, 10, 1,5, 20, 30, 45 and
60 min after secretin injection. Serum wa.s separated and extracted as described
previons!}' and .sccretin activity measured.
Thi-ee patients had an intravenous cannula inserted and blood was collected
at L5 min intervals tor 30 min before and 120 min after a protein-rich nieal.
Sernm seeretin levels were measnred.
Three merino sheep had 2.5 ml of 0-1 M HCl instilled into the duodenum
throngh a duodenal fistula. The dnodenum was \\-ashoc] with saline to p l l 2 and
pH 5 at tlie 31,st and 3Sth min after acid instillation. Blood was coUeeted as
above for secretin measurement at 2-5 iiiiu after acid instillafion and at 5 miu
intervals over a further 1 h period.
RESULTS
Fonr ()(' the eiglit immnnised rab))ii.s de\'e]oped a usable anti!jod\- titre after
3 months. P^u-ther inoeulation raised the titre. Rabbit 24 was fonud to have (he
highest antibod\- titre and was used in all subsequent assays at a final dilution
0/1:400,000.
Ten ju,g of synthetic secretin was labelled and purified aeeording Id the
method described. The radioiodinatifin yield defined as the fractitjn of radioactivity incoi'porated into seeretin was higher with 6-tyrosyl secretin and DATA
98
P. HO AND J. HANSKY
seeretin than Schwarz-Mann and Research Plus secretin, but imtnnnoreactivity
of 6-tyrosyl seeretin and DATA seeretin was poor. The standard curve obtained
by using I^--'' DATA secretin had a low sensilnvity (Fig. 1). Labelling SehwarzMann seeretin resnlted in a lower yield than Researeli Plus seerctiii. Con.sequently, Research Plus secretin was used in all subsequent radioiodinatiotis.
40-
30o
20-
10-
25
50
75
Secre+in fmol/tube
Fiji. 1. ("ompavisons of standard curves using C.l]\ secret in stand an.I, ;tiitisi-iiim No, 24,
final i-liliifion 1:400,000. Research Plus secretin Uibel ( •
• ) . Seliwin-z-Miinn seeretin
kibe! ( O — — O ) . IXVl'A seeretiu label (X
x ).
7'he first peak ol radioactiviLy ehited in the void vokune li'oni Sp Sephadex
C25 colnmn was free iodide (Fig. 2}, The last peak of radioactivity eontaining
iodinated seeretin was used as label. 3 Imol of the label (1200 e.p.m.) was nsed
in the assay. The label self displaeement curve was superimposable on the assay
staudard eurve, showing that the imniunoreactivity ol tlie label was equal to
tliat of the unlahelled seeretin. The speeifie actiN'it\- ol: the V~'' secretin was lotiiid
to be 1S5 jLiCi/umol. The label was stable ior about 8 weeks when stored iu acid
RADTOIMMUNOASSAY OF SECRETIN IN SERUM
99
alcoliol. 0'5 ml cliarcoal-dcxtran siLsprnsion was used to separate the bound
and free fraction. 95 to QS% of tlie radioactivity wa.s absorbed to the cliarcoal in
the ab,sence ol Lintibodv,
500-
250o
O
20
Fig.
40
60
80
Fraction number
100
120
Elation diagram of seort'tiii I'-"' on Sephadex Sp C25 sizi^ ()•() x 30 em. Craclieul
A\ilh 0-05 M to 0-5 VI sotliuui acetate buffei- p l l 5, 2 ml J'raeHoris cnlleeLerl. Tlie last
peak of radioattivity wtis used as label.
\'AMT) ATI o x
Assay .'ieii.sitii'itij and reUahiliiij
Tlie lowest seeretin dctectioii limit taken as two .standard dt'\"iati()ris from
zero Jiormone Ijindiiig (Kii-khain and Tlunter, ]971) was 1 • 12 fniol/tnbe which
was equivalent to 2-8 Fmol/ml sernm. The ID 50 (dose to inhibit Inndino; by
50;^-) mean of 20 assays was 14-8 ± 0-96 finol/tube (± 2 SE) ( Fi^. 3). The infraassay variation calculated irorri lo duplicates at a level of 16-8 fmo!/ti.i!ie was
4-SS'. The inter-assay variation calcnlated from 10 assa\'s at a Icve! of 14 f'inol
and 26 fmol/tnbe wa.s &l and 8-5% respectively at 9.5!l- confidence limif'. The
working range of the assay was from zero to 30 fniol/kibe.
Recooery of secretin afier extraction
5, 10, 2.5, 50 and 100 fmoi of seci-etin were added lo 1 ml serum. The
recovery ol added secretin after the extraelion procedure was estimated to be
106 ± 5^, 94 ± 7%, 82 ± 7-6^., 76 it 6-8'4 70-5 ± 10% ( ± 2 SE) respectively
{mean of" 4 experiments}. The recovery of labelled secretin was 88 ± l-^%.
100
p. HO ANP ]. HANSKY
50-
40-
30o
20-
10-
25
50
75
Secre+in •fmol/tube
ij;. 3 . S t a n d a r d e i n ' v e iisInK l i e s e a r c l i P h i s s r c r e t i n l u b e l , a n t i s e r u i n N t i . 2 4 . (iiial ( l i l u l i n n
4t)(l.()CH], C i l J l scLTc^tiii .sl.indarci r a n g i n g f r o m 2 - 5 f m o l t o lOO i n i o l p e r t i i b f i n " h u r i i i o i i e treo" sernrn fxlract.
Spccificiiij
Glucagon and insulin (No\o Industri A/s Denmark), vasoactive intestinal
peptide (VIP), choleeystokinin ]ianereazymin (CCK-PZ), pure CCK 33 (ProL
Victor Mnti;, Karolin.ska Instifnte, Stoekholm, Sweden), mot'ilin (Dr. Yajinia,
Kyoto, Japan), gastric ijiliibitory polypeptide (Dr. ]. Brown, Vancouver, Canada),
CCK 8 (Squibb, Now Jersey)'and gash-in G171 (I.CJ., Cheshire, U.K.) were
a.s.sa)'ed for cross-reaetivity with secretin antibody 24. No significant crossreaction was detected witJi any of' tbe above peptides.
Sti-ucturally-relatcd peptide VIP and coiitaininant.s of CCK-PZ iiihihited
p2--! secretin antibod\- binding at 10,000 fmol/tube. TJie ID 50 tif VIP and GTH
CCK-PZ was 20,000' and 26\0{)0 fmol respectively compared to 14'8 fniol of
GIH secretin.
Standard cnvve.s were set up in diluent, hormotie-lree ,serum aiid hormonefree serum extracts (Fig. 4). The hormone-free serum standard curvt' was les.s
sensitive than the other two curves at low secretin concentrations, bnt they all
RADIOIMMUNOASSAT OE SECRETIN IN SERUM
101
showed sitnilar ID 50. Serial dilution eurves of high seeretin serum extracts froni
3 .subjects were set np. They were hnmd to be parallel to one another aud
siiperimposable on (he "Iiormonedree" serum extract standard cnr\'e (Fig. 5).
25
50
75
Secre+in fmol/fube
Fig. 4. Coinparisiin of sUmcUird curves in btilTer (X
x ) . with O-I ml "lii)rini>ne-lre.(
por assay tube ( O
O ) , *'"2 ml "liormone-fret'" serum extract per Uilx'- ( •
•
PnVSTdLOCTCAL STIIOIF.S
Figure 6 shows Ihe mean ± SE fo]- 8 patient,s vvJio had lui intravenous
injection ui' 1. Clinical Unit ( C U . ) per kg GIH secretin and also 6 of wliom had
2 C.U. per kg Boots secretin. Peak activity and faJI off were similar, iiidicaMng
twice the aniount of seeretin per chtiical unit in C;iH than in Boots secretin.
Three .snbjeets had a protein meal and there wa.s no ri.se in seritm secretin
from basal levels.
Figure 7 slmws tlie mean ± SE in 3 sheep \\'hich had seeretin measured
after intradnodenal instillation of 25 ml of O'l M HCl and then iwo \\'ashe.s ol'
the duodenum to pll 2 and pH 5 (3Ist and 38th min respectively). Sernm
seeretin ro.se from a basal level oi' 4-6 fmol/ml to 56'6 fmol/ml after 5 min and
retnrned to basal levels after the second wash.
P, 110 AND J. HANSKY
102
50H
40-
30o
20-
10-
25
50
75
Secre+in fmol/fube
Fig. 5. Serial dilution curves of three high coneentration.s of endogenous secretin serum
extract (A, ^ ^nifi O) !ii'e parallel and .uiperimposabie on the secvctin curve ( •
• ) wilh
0-2 ml "'honnone-free" serum extract per tube.
DISCUSSION
pjtper describes a sensitive .specific assiiy lor measureinent ol
levels. The lowest detectable level lor this assLiy when utilizing an
extraction procedm'e for serum was 2-8 fmol/ml. This is eomparable witii the
recently reported assays ol F-Iaiissen and 7'orjesen (1977) and Scliaftalitzky
De Mnckadell and Kahrenkrng (1977) who stated sensitivity to l)e 2-5 pmoI/1
and 1-3 pniol/1 rc>speetively. Despite ihe apparent sensitivity oi' this preseut
assay .system, \ve were unable to measure lasting serum secretin Ic^vels in niosl
patients, nor wore we -able to deteet any change rollo\ving a protcin-rie1i meal.
Tlie major releasing taetor of .seeretin h-oin tlie dttodcnitm i.s acid, and instillation
of aeid eauses both sceretion of liiearhonatr Ironi the ])anereas and a rise in
iminiuioreactive seeretin. Otiiers have s!un\n that only acid ix.^leases iminnuoreactive seeretin amongst a nuaibei- of' pbysiologieal stinutli tested ( Tahreiiki-tig,
Schaffalitzky De Muekadell and Hoist, 1977), aUbo\tgh some aiitliors fiave
repoited rises after glncosc (Yovtng et a/., 1968), protein meal (Chey, Ilendrieks
RADIOIMMUNOASSAY OF SECRETIN IN SERUM
0
103
20
40
Time (min)
P'ig. 6. liiimiinoreaetive secretin in ."ienim after Bool.s .secvelin (O
O) (- unit.s/kg body
weight) and Gill seci-etin ( •
• ) ( I iiiiil/!;g body weight) iiiir;i\-cni)ii,s iiiicction.
All viilnps ± SE.
and Tai, 1977) and alcohol {Straiis.s, Urbacli and Yalow. 1975b). However, the
differences from lalxjratory to lalioratory would suggest that secretin release is
variable and that tlie onh- stiiinihis to have significantly released .secretin is
intradundenal acid hi.stillation.
The extraction procedure has improved the seiistlivit)' of tliis a.ssay. Our
recovery of secretin is 94 ± 7% at the most sensitive part ol: ttie slantlard curve
and no correction factor is applied to these figures, although aihci workers
indicated tliLit a correction factor can be applied Ijecause o\ the constant In.ss in
exh'action (Schaffalitzky Dc Muckadell and Fahrenkrug, 1977; Ihins.'^en and
Torjesen, 1977). Ttiis improvement" lias permitted mea.surement ol sniall increments oF .secretin but does not make tlu's assay any mort' sensiti\^.' than other
reported assays.
An. interesting olxservation is tbat secretin obtained trom Research Plus
Laboratories. New Jersey, U.S.A., provided a better labelling material and more
stable, reproducible label than either Scliwarz-Maun .synthetic secretin, SchwarzMaun 6-tyro.syl secretin or N a;-De.sainino-lyrosyl-/i-alanyI secretin iVom Flnka.
104
P. HO AND J. HANSKY
60-
u
©
t/1
30-
-20
0
20
40
60
Time (min)
Fig. 7, Mean seruni sccretin levels in fmol/ml befurc and alter duodenal insUlhition oF
25 ml of 0-1 M HCl ID 3 sheep. ( I ) indietited aeid institlation. Onodtnnun was \v;ished with
saline lo pH 2 and p l l 5 in wasli ! and \v;tsli 2 lespcclix-elj\ .-Ml \^a!ues are mean ± SE.
This assay pro\'ide.s a reliable nieasnre of circulating ,scrmn .seeretin. The
finding that only acid in the duodenmii releases immunoreacti\'e secretin and the
seaicity ot reports of any hyper-seeretinaemic .states stiggest that this as.say has
limited application to pathopliysiology In man. However, this assay may be
useful iu mouitoring physiological events in the experimental ariimai and tor
assessing the potency of secretin used in tests of pancreatic fuuetlou.
Acknoiohdficment-.'i. Tht^ assistanee of the lNaliLinal Health iuid Mcdieal tlesearch Coinicil
of Australia is acknowledt;ed. The studies in slieep were perlonnod In Dvs. D. Ticlven and
C. Reviiolds nf the \'eterinaiv Sehool, Melbovnne V'nivevsi[\.
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M.. and
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R-ADIOIM^'IUNOASSAY OF SECRETIN IN SERUM
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