Journal o[ hnnmnological Method.s-. 14tl ( Itlt12) 237- 2 ~f,

237

a_) 1992 Elsevier Science Publishers B.V. All rights lesetwetl (11122175~)./()2/$1~.1111

JIM I)6275

Superiority of sandwich EI,ISA over competitive RIA for the estimation of ANP-270, an analogue of human atrial natriuretic factor S . H . l n g w e r s e n ", P . N . J o r g c n s c n b, H. E i s k j t e r ~, N. L a n g e l a n d J o h a n s e n d, K. M a d s c n ~l a n d P. F a a r u p ~ " Plulrmac'okineti¢.~. a Peptidc Chemi.~tr?,'. '" Medi~'ml'l (.'henli.~tr~'. ('N,~i Dit i~ion. Not o Nordi.~k A / S . ,~,l~uthJel. D~'nmark. t, Moncn'hmal Antihod)' Lahorator¢. Rio~'~'it'nce. M n o ,~h,rdi~k A / S . Iiag,t uer~L Denmark. and ' Dep, rttmult of Medi~'ine trod NtT,hrology C. Ske/by Unit er~tt)' Ih~v~itul. .4arhtt~ N. lh'nmark

(Received 24 June lqgl. ,cvir,ed received 12 Scplembcr I~1~)1.iJcceptetl 2 January I~J92|

A N P - 2 7 0 is a 26 a m i n o acid a n a l o g u e of naturally occurring atrial natriuretic factor ( A N F ) which it was anticipated would be of value for the t r e a t m e n t of congestive heart failure a n d acute renal failure. T w o sensitive assays - a r a d i o i m m u n o a s s a y ( R I A ) a n d a sandwich e n z y m e linked i m m u n o s o r b c n t assay ( E L I S A ) - were d e v e l o p e d a n d validated for use in clinical investigations. T h e R I A utilized a single C t e r m i n a l m,)noclonal antibody w h e r e a s two monoclonal antibodies directed against different epitopcs were u s e d for t h e ELISA. T h e two assays were c o m p a r a b l e with respect to sensitivity a n d precision, but assay results o b t a i n e d on s a m p l e s from n o r m a l v o l u n t e e r s dosed intravenously with A N P - 2 7 0 differed widely. T h u s , in o n e v o l u n t e e r the elimination half-life was e s t i m a t e d to be 123 rain using R I A results but 6 m i n using the E L I S A results. By reversed p h a s e liquid chrom~:tographic fractionation of p l a s m a extracts followed by R I A a n d EL1SA, t h e s e discrepancies were shown ,o be d u e to f r a g m e n t s of A N P - 2 7 0 cross-reacting in the R I A b u t not in the ELISA. C o n s e q u e n t l y , the sandwich E L I S A was the m e t h o d o f choice for e s t i m a t i n g this c o m p o u n d in plasma. Key ~vords: ELISA; Radioimmunoa~,say:Atiia: natriuretie faet~r: Anah~guc

Introduction A N P - 2 7 0 is a synthetic a n a l o g u e of the n a t u rally occurring a l p h a hut.nan atrial natriuretic fac-

Correspondence to: S.I-I. Ingwersen. Pharmacokinetics. CNS Division. Now~ Nordisk A/S. DK-276(I Maaloev. Denmark. Abbretiutions: ELISA, enzyme-linkt:d immunosorbent assay; RIA. radioimmunoassay; ~hANF. alpha human atrial natriuretic factor: BSA. bovine serum albumin: BGG. bovine gamma globulin: IRMA. immunor~,diometric assay; IIPLC. high performance liquid chromatographic as.',ay.

tor ( a h A N F ) . A N P - 2 7 0 consists of the 26 amint~ acid derivative A c - A r g 4, t>-Ala '~, lie ~-', Leu ~'~, t>Ala 2'~, D.Ala 2-~, M e P h e 2~'. o.Tyr -~, Pro-~".NH, a h A N F ( 4 - 2 9 ) (sec Fig. 1). A N P - 2 7 0 was sclectcd from a series of a n a l o g u e s for clinical studies a n d in n o r m a l male v o l u n t e e r s it was shown to possess natriuretic, diuretic a n d blood pressure lowering properties (Eiskjzer et al., 1991). T h e two m e t h o d s described here for assay of A N P - 2 7 0 in h u m a n plasma wcrc developed for use in p h a r m a c o k i n e t i c studies. Both assays were based on monoclonal antibodies. "Fhc radioim-

23S purified by passage through a Milli-Q R plant (Miilipore, Milford, MA, USA).

t~c-Arq-Sor-ser-cys-Phe-p._~L~_-Gly-ArtJ-I - - l e - A ~ p - ~ r c jl

I

s

ilo

l.

. . . . . .

.

.

i

23

15

I.... I

A r q - ~ e P h e - S e r - A~ n - Cy s - ~ a 24

.

- L e u -D~/~J,_q - S e r - l.,~u - A 1 a

22

21

20

19

18

The

amino

17

0-Tyr.-Fa-_o-NI L 2a Fig.

2') 1. C h e m i c a l

residues

were

structure

numbered

28)). In ANP-270.

amino

of

ANP-270.

by analogy acid residues

with

t~hANF

acid

(ANF(I-

n o 4, 9, 12, 18. 21}. 2 2 .

26.28 and 29 weredifferent from ahANF.

munoassay (RIA) utilized a single C terminal reacting antibody whereas the enzyme-linked immunosorbent assay (ELISA) was performed by means of two monoclonal antibodies directed against different epitopes of ANP-270. Both assays were acceptable with regard to sensitivity and precision, but the ELISA was devoid of cross-reactivitics with C terminal fragments of ANP-270. The importance of this specificity difference became obvious when plasma samples obtained from healthy volunteers dosed intravenously with ANP-270 were assayed by the two methods.

Materials

and methods

Chemicals and reagents Aprotinin and ahANF as well as ANP-270, and fragments thereof were from Novt: Nordisk (Bagsvaerd, Denmark). Bovine serum albumin (BSA), phenol red, gelatin (frem cold water fish), and bovine y-globulin (BGG) were from Sigma (St. Louis, MO, USA). Freunds complete and incomplete adjuvants were from Difco (Detroit, MI, USA), polyethylene glycol 6000 and Tween 20 were from Merck (Darmstadt, Germany). Human serum albumin (~3RHA 20) was from Behringwerke (Marburg, Germany). 1,7-phenylenediamine, rabbit anti-mouse immunoglobulins, and swine anti-rabbit i,amunoglobulins were from Dako (Glostrup, Denmark), and horseradish peroxidase avidin D was purchased from Vector (Burlingame, CA, USA). Deionized water was

Synthesis of peptides and albumin conjugate ANP-270, fragments thereof, and trhANF were prepared by solid phase synthesis as described by Stewart and Young (1984). The purity of the peptides was assessed by C~8 reversed phase HPLC in two different systems and found to be > 95%. Hydrolysis and quantitative amino acid analysis indicated that the peptides were of the intended structure. A conjugate of des-acetylANP-270 and bovine serum albumin (BSA) was prepared by coupling with glutaraldehyde as described for coupling of glucagon by Reichlin (1980). The conjugate contained 6.2+0.3 mol ANP-270 per mol BSA, as determined by hydrolysis and amino acid analysis, based on the ratio of the amino acids Asx, Arg, Ala and Gly present in BSA and ANP-270, compared to Glx, His and Val only present in BSA.

Preparation of ANP-270 monoclonal antibodies Four RBF(8.12) mice (Capanna et al., 1976) were immunized once i.d. with 25 /zg ANP-270BSA conjugate in Freunds complete adjuvant followed by s.c. immunizations with 2.5 /~,g conjugate in Freunds incomplete adjuvant every 14 days. One mouse was boosted intravenously with 2.5 txg of conjugate 4 days before fusion of mouse spleen cells with FOX-NY myeloma cells as described by Taggart and Samloff (1983). Cells were cloned by limiting dilution and screened for production of specific antibodies. Positive hybridomas were recloned twice before production of antibodies commenced. Antibodies were biotinyfated as described by Hudson and Hay (1989).

Preparation of radioactice tracer [12sI] iodotyrosy128-ANP-270 was prepared by the lactoperoxidase method (Jorgensen ,~d Larsen, 1980). The reaction product was pu,!fied by gel filtration using Sephadex G-25 sup,.~rfine (Pharmacia, Uppsala, Sweden) followed by high performance liquid chromatography on a reversed phase column (Vydac Protein C4). The specific activity was estimated in RIA (Chiang, 1987) at 990 Ci/mmol corresponding to 343 i.tCi/l.tg. The purified tracer was diluted in RIA

23() buffer (see below) to a radioactive concenlration of 50110 c p m / 1 0 0 #1 before use.

Blood sampling Blood samples for drug assay and for the preparation of spiked plasma were collected in precoolcd polyethylene vials (Minisorp, Nunc, Roskilde, Denmark) containing aprotinin, 600 K I U / m l blood and EDTA, 0.78 m g / m l blood. The vials were kept on ice until ccntrifugation (2000 × g, l0 rain at 4°C). Plasma was transferred to Minisorp vials and stored at - 18°C until assayed. lnformed consent was obtained from volunteers participating in pharmacokinetic studies of ANP-270. R/A Prior to assay, ANP-270 was extracted from plasma by a solid phase technique using reversed phase Bond Elut Ct~ columns (Analytichem Int., Harbor City, CA, USA). The columns were eluted by low speed centrifugation (128 × g ) . The columns were activated by 2 ml of methanol followed by 2 ml of water. 0.5 ml plasma was applied to the columns followed by I ml of water. The columns were washed with 2 ml of water and 2 ml of a solution of 20% ( v / v ) methanol, 0.5% ( v / v ) trifluoroacetic acid in water. ANP-270 was eluted from the columns by 2 ml of 75% ( v / v ) methanol, 0.5% ( v / v ) trifluoroacetic acid in water. The eluate was evaporated to dryness in a Speedvac SVC 200 H vacuum concentrator 'Savant, Farmingdale, NY, USA). Extracts were redissolved in 0.5 ml RIA buffer containing 0.1 M sodium phosphate buffer, pH 7.4, NaCI, 0.05 M, Triton X-100, 0.1% (v/v), human serum albumin, i g / I and aprotinin, 6 × l0 s KIU/I. Standards containing 0, 5, 10, 25, 50, 100 and 200 pM ANP-270 were prepared in RIA buffer. The RIA was performed as a sequential assay: 1011 V.I standard or plasma extract and 10(I/.tl of antibody FI4 (diluted 1/800t)) were incubated for 2 h at 4°C in Minisorb polyethylene vials (Nunc, Roskilde, Denmark). 100 ,ul of tracer were added and incubation was continued for 20-24 h. Free and bound radioactivity were separated by the addition of 100 ~1 of rabbit anti-mouse im-

munoglobulins diluted 1/10 in RIA buffer followed by I ml of swine anti-rabbit immunoglobulins diluted 1/1011 in RIA buffer containing, in addition, polyethylene glycol 60110, 80 g/I. The tubes were incubated for 30-60 rain at 4°C and centrifuged (20110×g) for 10 min at 4°C. The supernatant was aspirated and the radioactivity of the precipitate (bound fraction) was counted in a LKB 1270 scintillation counter (LKB, Turku, Finland).

ELISA A sandwich ELISA was performed using 96well microtiter plates (lmrn, uplate Maxisorp, Nunc). The phosphate-buffered saline (PBS) used contained 10 mM sodium phosphate, pH 7.2 and 145 mM NaCI. The microtiter plates were coated with I00 ul capture antibody (I(1 mg/l) in PBS and incubated overnight at 4°C. The plates were blocked by incubation for I h with 200/.tl blocking buffer (PBS containing gelatin, 5 g / l ) per well. The plates were washed with washing solution (PBS containing Tween 20, 0.5 m l / l ) before assay. The ELISA buffer consisted of PBS containing (NH4),SO ~, 0.35 M, Tween 20, (1.5 ml/I, gelatin (45%), 10 ml/I, bovine y-globulin, 1 g / L and mouse y-globulin, (1.1 g / l . 25/.tl plasma and I00 /.tl detecting antibody (biotinylated F5 diluted 1/400 in ELISA buffer) were added and incubated for 2 h at room temperature with continuous shaking. Incubation was stopped by emptying and washing. 100 ~1 of avidin-peroxidase (diluted 1/10,0110 in ELISA buffer) were added, and after 1 h of incubation the plates were washed and the peroxidase enzyme reaction was started by addition of 100 ~1 substrate solution containing 1,2phenylenediaminc, 0.4 g / l in 0.1 M sodium citrate buffer, pH 5.2 containing 4.25 mM sodium perborate. The enzymatic reaction was stopped by the addition of 150 V.I sulfuric acid (0.1 M). Absorbance values were read on an automatic dual wavelength plate reader (ImmunoReader Model N J-2000, lnterMed, Roskilde, Denmark) run at 49(1 nm and 620 nm. Raw data were transferred on-line to a microcomputer (IBM P S / 2 Model 80-1111 and processed using the software ELISA AID (lnterMed).

ItI'LC J}'actionation of plasma t:~tracts In order to investigate possible interferencc from circulating fragments of ANP-270, extracts of plasma samples obtained from a healthy volunteer dosed intravenously with ANP-270 were subjectcd to H P L C fractionation prior to assay by RIA and ELISA. Phtsma was extracted as described above. Dried extracts were redissolved in I(X) # t elucnt A (see below) and injected on a Vydac C a column (4.6 × 25(1 mm). Two eluents were used for HPLC: clucnt A, acctonitrile-water ( 10 : 9(1) containing 0. 1% ( v / v ) trifluoroacctic acid; and eluent B. acctonitrile-water (90:10) containing 0.1% ( v / v ) trifluoroacctic acid. Elution was performed using a linear gradient running from 90% A at 3 rain to 30% A at 30 rain. The flow rate was 1 m l / m i n and the run time was 35 rain. T;;c elucn~ from :he column was collected in I ml .ractions, each of which were divided into two 9ortions and evaporated to dryness. The two portions of each fraction were dissolved in R I A buffer and ELISA buffer, respectively and assayed as described abovc.

Addition of hemolysate to human plas/ela In order to investigate possible interference w h e n using h c m o l y s e d samples, increasing amounts of hemolysate was a d d e d to a pool of human plasma spiked with 50 pM ANP-270. A hemolysate of human erythrocytes containing 89 g / I hemoglobin was added in amounts of 0, 5, 10, 25 and 100 ~1 to I ml portions of spiked plasma. Physioiogical saline was a d d e d to make up to a final volume of 1.1 ml. The concentrations of added hcntoglobin were O, (}.40, 0.8l, 2.4,. 4.05 and 8.09 g / l .

Results

Monoclonal antibodies 22 hybridomas were producing antibodies 4 weeks after the cell fusion. The monoclonal antibodies were characterized for immunoglobulin class and affinity for ANP-270. Aliquots of these antibodies were biotinylated and examined in a checkerboard analysis for their ability to cooperate in the sandwich ELISA. Antibody FI4 was chosen for use in R I A due to its high affinity for ANP-270 (affinity constant 1.9 × I() tt I/tool estimated by Scatchard analysis). FI4 was also chosen as the capture antibody in ELISA, whereas antibody F5 was chosen as the detecting antibody (used as biotin conjugate). The affinity constant of F5 was 3.6 × 10 "j I / m o l . Using the OuehterIony technique FI4 was classified as l g G l and F5 as IgG2a. The epitopes o f F5 and FI4 were m a p p e d using competition with ANP-270 fragments in R1A. As seen in Table I, F14 cross-reacted with C terminal fragments o f ANP-270 containing as few as six amino acid residues (as in ANP-270 (24-29)). That FI4 was a true C terminal antibody was obvious from the lack of cross-reactivity with fragment ANP-270 (4-28). No cross-reac-

TABLE I CROSS-REACq'IVITIES OF FRAGMENTS OF ANP-270 AND OF SOME NATURALLY OCCURRING PEPTIDES IN THE RADIOIMMUNOASSAY FOR ANP-270 UTILIZING TIlE MONOCLONAL ANTIBODY FI4. The er(~ss-reacliviliesare e',pre,~,~ed us Ihe ratios of molar concentra'.ions neeessar.,' to displace 50~;~ of tht: bound radioactivity relative to ANP-270. Peptidc

Cross-reactivity (,"/~)

ANF-270 (4-29) ;' ANP-2711(5-29) AN P-2711(24-29) ANP-27(I (4-28) c(hANP Iluman insulin Porcine glucagon Porcine pancreatic polypeptide Somatostatin

1()~ 81 51 < l(I- "~ < 1(~-3 < 1(1 '~ < I1)- 3

Addition of lipid to human plasma In order to investigate possible interference in lipemic samples, lipid was a d d e d in increasing amounts to a pool of human plasma spiked with 50 pM ANP-270. Intralipid (KabiVitrum, Stockholm. Sweden) containing 200 g / I lipid in amounts of 0, 5, 10, 25, 50 and 100 tzl was a d d e d to 1 ml portions of spiked plasma. Physiological ~uline was a d d e d to make up to a final volume of 1.1 ml. The concentrations of a d d e d lipid were 0, (I.9. 1.8. 4.6, 9.1 and 18.2 g / l .

" Identical to ANP-270.

< I11- 3 < l(l - 4

tions were observed with naturally occurring human atrial natriuretic peptide (~hANF) or other endogenous peptidcs unrelated to ANF. F5 cross-reacted with fragment (4-23) but not with fragment (24-29) (data not shown). Thus, I:5 was directed against an N terminal or a midmolecule cpitopc. The specificity of F5 was further investigated in the ELISA by running standard curves of fragments (5-29), (6-29) z~nd (729). As seen in Fig. 2 these fragments all showed considerable cross-reactions, the last two fragments binding with ,;yen higher affinities than the parent compound. Also from Fig. 2 it is obvious that neither ANP-27(I (4-28) nor a h A N F crossreacted in ELISA which is in accordance with the results obtained in RIA.

5O 40

20 10 0

o/°

o.o/

f

,50

260 360 4o° ANP-270 (pm01/1) Fig. 3. Standard culvt:sof RIA obtained in buffer (e) lind (if ELI.t;Aobtained in plasma((,).

Opthnization of RIA Initial experiments showed that the biqding of tracer to antibody was considerably 'reduced when plasma was present in the incubation mixture (data not shown) and for this reason plasma had to be extracted prior to assay. The extraction procedure used here for RIA was a modification of a commonly used pro ..d,tre fgr extraction of

2~/.~.----=-'-'-°

2 ~-

-,~ ~ ~.5

,

10 0.5

0~0°-

c~hANF from pl;isma (Gutkowska, 1987). We used Bond Elut Cis extraction columns which were cluted by mean:, of centrifugation. ANP-270 was eluted fr,,;va th: columns with methanol-water (75 : 25) containi ag 0.5% (v/v) trifluoroacetic acid, and this cluent was optimal for recovery of the ~nalyte. The sequcn, ial incubation procedure employed in the IHA was introduced in order to improve assay ~cnsitivity. Preincubation of antibody and sample for 2 h before the addition of tracer resulted in a steeper standard curve than that obtained with equilibrium conditions. Prolonging the first incubation beyond 2 h did not further improve the sensitivity. The triple antibody-PEG procedure for separating frcc and bound radioactivity was introduced in order to accelerate the separation. Separation was complete 30 rain after the addition of the third antibody. The non-specific binding witl~ this procedure was 2-4%. A standard curve in the concentration range 5-200 pM obtained using the optimized conditions is shown in Fig. 3.

10;

10 2

10 3

10 4

10 5

10 e

10 ~

Peptide concentration (pmolll) Fig. 2. Cross-reactionsof fragmentsof ANP-27(Iand ahANF in the ELISA procedme. Key to symbols: &, ANP-271): e. ANP-2711 fragment (4-28): II, ANP-270 fragment (5-29); .:,. ANP-270 fragment (6-29); [3 ANP-27t)fragmcnl (7-2~) and trhANF.

Optonization of ELISA The two monoclonal antibodies FI4 and F5 wcrc found to bc compatible in a sandwich assay using FI4 as capture antibody and biotinylatcd F5 as detecting antibody. Initial experiments showed plasna and buffer standard curves to differ significmtly, but plasma standard curves

242 fnom different plasma s a m p l e s differed only marginally. T h u s , one important prerequisite for a direct (non-extraction) assay was met. T h e o n e - s t e p p r o c c d u r c of s i m u l t a n e o u s incubation of samplc and detecting antibody in coated microtiler plates was chosen for convenience. 11 was shown to yield s t a n d a r d curves almost identical to a two-step incubation procedurc. T h e stability of microtitcr plates coated with c a p t u r e antibody and blockcd with buffer c o n t a i n i n g gelatin was shown to bc at least 2 weeks at 4°C. A s t a n d a r d curve obtained with p l a s m a standards in the c o n c e n t r a t i o n range 5-4011 pM is shown in Fig. 3.

100o

Sensiticity, accum(3' and precision T h e d c t c c t k m limit defined as the c o n c e n t r a tions c o r r e s p o n d i n g to t h r e e s t a n d a r d deviations of b h m k plasma was estirvatcd at 3 p M in R I A and at 2 pM in ELISA. T h e lowest s t a n d a r d a n d hence the lower limit of the assay range was 5 p M in both assays. As scen in Table !i, the R I A intra-assay precision ranged from 3.8% at 200 pM to 8.9% at 10 pM a n d the accu,acy r a n g e d from 74% to 90%. T h e E L I S A intra-assay precision r a n g e d from 2.3r,4" at 200 p M to 5.3% at 10 p M a n d accuracy ranged from 104.0% to 117.0%. T h e total bet w e e n - r u n precision r a n g e d from 8.1%, at I0 pM to 16.3c