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Clinica Chimica Acta, 83 (1978) 25-31 @ Elsevier/North-Holland Biomedical Press

CGA 8993

RADIOIMMUNOASSAY FOR SARALASIN ANTI-ANGIOTENSIN II SERA

ANNE MARIE KAPPELGAARD, J@RN GIESE *

BY MEANS OF

HANS IBSEN, META DAMKJAER

NIELSEN

and

Department of Clinical Physiology, Glostrup Hospital, Nordre Ringvej 29-67, DK-2600 Glostrup, Copenhagen (Denmark) (Received

July 14th, 1977)

A radioimmunoassay for the angiotensin II-inhibitor saralasin has been developed. The assay is based upon an exploitation of the cross-reactivity of antisera raised against angiotensin II, with radioiodinated saralasin as the tracer peptide. Performance data for the assay and results obtained in clinical infusion studies are presented.

Introduction In earlier publications we have emphasized the point that the term specificity, as commonly employed in the radioimmunoassay field, is an operational term. With anti-angiotensin sera it was shown that the cross-reactivity of antibodies can be exploited for radioimmunological quantification of cross-reacting angiotensin peptides [ 1,2]. We now present evidence to show that it is perfectly possible to utilize antisera raised against angiotensin II in radioimmunoassay for an angiotensin IIinhibitor (Saralasin: 1-sarcosine-8-alanine angiotensin II). Patients and normal subjects The saralasin assay to be described has been used for plasma concentration measurements in 8 patients with recently diagnosed essential hypertension and in 2 normal subjects.

* To whom correspondence

should be addressed.

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Materials and methods Reagents

Saralasin was kindly provided by Eaton Laboratories, Norwich Pharmacal Company, N.Y., U.S.A. Isoleucine-5-angiotensin I and II was obtained from Schwarz-Mann, Orangeburg, N.Y., U.S.A. Na12”I free of carrier and reducing agents (Hoechst, Frankfurt, G.F.R.) was used for iodination of saralasin or angiotensin II according to a procedure described in an earlier communication [ 31. Suitable samples of anti-angiotensin II sera were selected from our stock [ 11 by preliminary trials. A total of 15 different antisera were tested. Six out of 7 antisera with a high titer towards ‘2SI-angiotensin II proved useful for saralasin measurements. Eight low-titer antisera were found to be unsuitable for this purpose. Radioimmunoassay

procedure

(standard

curves)

The equilibration system contained 20 ~11tracer peptide solution (400 cps), 50 ~1 diluted antiserum, and 1000 ~1 standard solution of unlabelled peptide (isoleucine-5angiotensin I or II or saralasin, dissolved in 0.15 M sodium phosphate buffer, pH 7.4, containing 10 mM disodium-EDTA, 5 mg/l phenylmercurie acetate and 0.1% human albumin). Equilibration was carried out for 20 h at 0°C. Separation of antibody-bound from free tracer peptide was performed by gel centrifugation with Sephadex G-25 as previously described [ 41. Saralasin assay

5-ml venous blood samples were collected in tubes containing 5 mg disodium EDTA. The plasma was separated by centrifugation and radioimmunoassay was performed immediately or after storage at -18°C. For assay, plasma was diluted with phosphate buffer (see above) to a final concentration of 0.4-10% plasma. 1000 ~1 diluted plasma were equilibrated with 20 ~1 radioiodinated saralasin solution and 50 ~1 diluted antiserum for 20 h at 0°C. Separation procedure as described above. Saralasin in fusions

Saralasin was dissolved in isotonic glucose solution and infused intravenously by means of an infusion pump. Blood pressure was measured frequently. Infusion rates varied from 0.54 to 10.8 nmol/kg body weight/min (0.5-10 pg/kg/ min). At the end of each lO-15-min infusion period, venous blood samples were collected for saralasin assay. Results Assessment

of cross-reactivity

in a conventional

radioimmunoassay

In a system based upon the reaction of radioiodinated angiotensin anti-angiotensin II serum, unlabelled angiotensin II was, as expected, efficient displacer than angiotensin I or saralasin (Fig. 1).

system

II with an a far more

27 K 17 NNN-ANGIOUNSfN 1RACLR

I

01

10

‘251-ANG10TtNSlN

10

DJ

f 97000 P

100

$tl

pephde/~O70//

Fig. 1. Anti-angiotensin II serum (low concentration) with ’ z SIangiotensin II as tracer peptide. Abscissa (logarithmic scale): concentration of unlabelled peptide in incubation mixture. Ordinate: percentage of tracer peptide bound to antibody. Displacement curves obtained after addition of unlabelled angIotensIn II (0). unlabelled angiotensin I (a). or unlabelled saralasin (0) to the equilibration system.

Assessment of cross-reactivity in a radioimmunoassay system designed for quantitation of saralasin The same anti-angiotensin II serum was used, but in a higher concentration, and. 12sI-saralasin was utilized as the tracer peptide. In this system, a tracer quantity of radioiodinated saralasin was readily bound to the antiserum. With increasing concentrations of unlabelled saralasin in the equilibration mixture, a useful displacement curve was developed (Fig. 2). Radioiodinated saralasin can also be displaced from the antibody binding sites by addition of either unlabelled angiotensin II or angiotensin I. However, the molar concentrations of these two peptides required in order to lower the binding percentage from an initial value of about 70% to a value of 50% are roughly 5 and 30 times higher, respectively, than in the case of unlabelled saralasin. The affinity constants for anti-angiotensin II serum K 17, as calculated from Scatchard plots, are given in Table I. Blank values of the saralasin assay: The possible influence of plasma componen ts 16 different plasma samples from untreated patients were diluted with phosphate buffer to a final plasma concentration of 10%. No signifiCant blank value was discernible in any of these samples. Similarly, there was no significant difference between standard curves developed with saralasin dissolved in pure phosphate buffer and curves obtained with saralasin dissolved in buffer-diluted plasma (0.1, 1, 10 and 50% plasma concentration).

28 K

17 (ANTI

TRACER:

-ANGlOltiVSIN

I7 J 1 2700

‘251-SARALASIN

w---e SAR&lASfN 60-

(I-81

o--o

ANGIOTENSfN

II(

-

ANGIOTENSIN

iTI f -101

I -8J

40-

30-

a-

I

iu

I&p

Fig. 2. Anti-an~ote~ II semm (high concentration) and symbols as in Fig. 1.

mof

pepfide/?O7o/uf

with *2SI-saraIasin as tracer peptide. Coordinates

Linearity of response in determinations of plasma saralasin concentration

Radioimmunoassay carried out directly on post-infusion plasma samples in dilutions ranging from 2.5% to 93% plasma showed a perfect straight line relationship between the volume of original plasma included in the equilibration mixture and the quantity of saralasin subsequently detected. Data from three separate experiments are shown in Fig. 3. Recovery and precision in radioimmunoassay for plasma saralasin

The recovery of unlabelled saralasin added to pooled blank plasma at 13 different concentration levels (range 5-430 pmol/ml) was 106% + 14 (S.D.). The within-assay coefficient of variation was 13%. The day-to-day coefficient of variation was 14%. No significant change of saralasin concentration was detected in plasma samples across a 6-month period of storage at -18°C. Clinical infusion studies

This saralasin assay has been utilized for plasma concentration measurements in 8 patients with recently diagnosed essential hypertension and in 2 normal TABLE I AFFINITY CONSTANTS (l/mol) FOR ANTI-ANGIOTENSIN Antiserum dilution

Tracer peptide

UnlabeIkd peptide Angiotensin II

1 : 97 000 2700

1:

1* 51-Angiotensin II 1 2s I-Saralasin

II SERUM K 17

1.7 x 1010

2.9 x 108

Angiotensin I

9.3 x 107

6.1 X 107

Sarakin 1.1 x107

1.2 x 109

29

VOLUME INCLUDED

OF IN

PO~~-INFUS~~N EQUILIt9ffAIION

PLASMA MIXTURF

Fig. 3. Graph showing the relationship between volume of poet-infusion plump included in adiohnmunoaesey equilibration mixture (abecieea) and mewed quentity of aeraladn (ordinate). Individual symbols (0. X. 0) are used for each of the three different pleama eamplee wed in the experiment.

subjects. Saralasin was infused intravenously at rates of 0.54, 2.7, 5.4 and 10.8 nmol/kg body weight/min. The highest infusion rate was left out in 5 out of a total of 13 infusion studies. The concentrations of saralasin measured in venous plasma samples collected during these studies are depicted in Fig. 4. The correlation between the infusion rates and the average steady-state plasma saralasin concentrations, as measured after infusion periods of 10-15 min, is highly significant (r = 0.99, p < 0.01). The range of plasma saralasin concentrations is approximately 20-400 pmol/ml.

PLASMA

SARALASIN

(n

PLASMA

moi/mll

SARALASIN -500

‘Yng/mll

0.5-

. -400

8 .

OI-

. .

0.3 -

-300

. 3

-200

2

0.2 -

.

!

3.

Ol-

-100

f 108 0.54 2.7 n 5.4

o-

mol/kg/mm-0

SARALASIN

100 DOSE

pg/kg/mm

5.0 25

f 05 T

10

~~

;0

Plasma concentmtions studies. Fig.

4.

;5

4;

INFUSION

TIME

(mm)

of semkein (ordinate). ar measured et different times during ~lh&pl infusion

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Discussion At present angiotensin II-inhibitors are being used to an ever increasing extent in pathophysiological studies on the possible importance of the reninangiotensin system. In addition, the clinical use of saralasin for diagnostic purposes is rapidly gaining momentum. It is, of course, very important to obtain pharmacokinetic data in the course of such investigations. This need has been met by the development of a radioimmunoassay for saralasin, based upon antibodies raised by immunization with a saralasin-protein conjugate [ 51. In view of our previous experience [1,2], it seemed a logical development to explore the possibility of utilizing antibodies raised against angiotensin II, together with radioiodinated saralasin, as the components of a radioimmunoassay system for quantification of saralasin. The feasibility of this approach would seem amply documented by our observations, as reported in this paper. Anti-angiotensin II sera should probably be available to most investigators working in this field, and the avoidance of a more or less tedious new immunization procedure represents a considerable advantage. The concept that the cross-reactivity of antibodies raised against angiotensin II can be exploited in a meaningful way for quantitation of the structural analog saralasin requires certain comments and interpretations. We suggest that the addition of radioiodinated saralasin to an antiserum raised against angiotensin II leads to a binding of the tracer peptide to certain antibodies which are present in low concentration within the antibody population. Thus, according to the concept introduced in our earlier articles, the addition of ‘251-saralasin to the antiserum occasions a selection of cross-reacting antibodies, which are presumably characterized by their avidity for saralasin. A displacement experiment can only provide information about the nature of the antibodies selected by the tracer peptide, and it is only natural that unlabelled saralasin should be the more efficient displacer in such a system. The molar concentrations of endogenous angiotensin II and exogenous saralasin likely to be encountered in clinical plasma samples should be considered. The range of plasma saralasin concentrations measured in our clinical infusion studies was 20-400 pmol/ml. These figures are comparable to those reported by Pettinger et al. [ 51. For comparison, the plasma concentrations of angiotensin II commonly encountered in clinical practice range from about 3 to 400 fmol/ml. Thus, in molar terms, the plasma concentrations of the endogenous octapeptide angiotensin II are extremely low, 50 to 130 000 times lower than the plasma concentrations of the angiotensin II analog obtained during infusion studies. It will be understood that a 20% cross-reactivity, as demonstrated in Fig. 2, for angiotensin II is of no importance whatsoever for the results obtained in radioimmunoassays for saralasin performed according to the principles outlined here. Similarly, the 3% cross-reactivity with angiotensin I does not affect the measurements. This plasma saralasin assay does not incorporate an extraction procedure. Therefore, in the general evaluation of the assay, due attention was paid to the exclusion of interference from plasma in the incubation mixture. With our antisera, we ‘were unable to detect any interference whatsoever caused by plasma

31

components. However, it would seem a wise precaution to examine this particular point for each individual antiserum prior to its use. We conclude that reliable measurements of plasma concentrations of saralasin can be obtained by exploiting the cross-reactivity of anti-angiotensin II sera. Thus, if such antisera are available, there should be no need to raise specific antibodies by immunization with saralasin for the purpose of quantitating plasma concentrations of this synthetic angiotensin II analog. References 1 Giese. J., Damkjaer Nielsen, M. and JOrgensen. M. (1971) Nature New Biol. 229.189-190 2 Giese. J. and Damkjaer Nielsen, M. (1972) in Radionuclides in Nephrology (Blaufox, M.D. and FunckBrentano. J.-L., eds.). pp. 105-113. Grune and Stratton, New York and London 3 Damkjaer Nielsen, M., Jdrgensen. M. and Giese, J. (1971) Acta Endocrinol. (Kbh.) 67.104-116 4 Giese. J., Jdrgensen. M.. Damkjaer Nielsen, M.. Lund, J.O. and Munck. 0. (1970) Stand. J. Clin. Lab. Invest. 26.355-367 5 Pettinger. W.A., Keeton, K. and Tanaka, K. (1975) Clin. Pharmacol. Ther. 17. 146-158