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Enzyme-Linked Immunosorbent Assays for Determination of Plasma Aldosterone Using Highly Specific Polyclonal Antibodies a
a
a
Fidi Schwartz , Eran Hadas , Marcel Harnik & Beka Solomon
a
a
Department of Biotechnology, Faculty of Life Sciences , Tel-Aviv University , Tel-Aviv, 69978, Israel Published online: 23 Oct 2006.
To cite this article: Fidi Schwartz , Eran Hadas , Marcel Harnik & Beka Solomon (1990) Enzyme-Linked Immunosorbent Assays for Determination of Plasma Aldosterone Using Highly Specific Polyclonal Antibodies, Journal of Immunoassay, 11:2, 215-234, DOI: 10.1080/01971529008053270 To link to this article: http://dx.doi.org/10.1080/01971529008053270
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JOURNAL OF IMMUNOASSAY, 11(2), 215-234 (1990)
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ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR DETERMINATION OF PLASMA ALDOSTERONE USING HIGHLY SPECIFIC POLYCLONAL ANTIBODIES
F i d i Schwartz, Eran Hadas, Marcel Harnik and Beka Solomon* Department of Biotechnology, Faculty of L i f e S c i e n c e s , Tel-Aviv U n i v e r s i t y , Tel-Aviv 69978, Israel.
ABSTRACT Two enzyme-linked immunosorbent assays were e s t a b l i s h e d and compared f o r t h e e s t i m a t i o n of plasma a l d o s t e r o n e . I n t h e f i r s t method i m m o b i l i z e d a l d o s t e r o n e - p r o t e i n complexes on t h e ELISA p l a t e s compete with a l d o s t e r o n e t o be determined f o r t h e binding o f c e r t a i n amount o f a n t i - a l d o s t e r o n e a n t i b o d i e s . The s e n s i t i v i t y of t h i s method depends on t h e p r o t e i n carrier used t o conjugate with aldosterone. I n t h e s e c o n d method, a n t i a l d o s t e r o n e a n t i b o d i e s a d s o r b e d on ELISA p l a t e s compete f o r b i n d i n g of known amount o f t h e e n z y m e - l a b e l e d a l d o s t e r o n e and a l d o s t e r o n e t o be determined. The h i g h l y s p e c i f i c r a b b i t a n t i a l d o s t e r o n e a n t i b o d i e s were obtained by i n j e c t i o n of a l d o s t e r o n e oxime t h y r o g l o b u l i n . The d e t e c t i o n l i m i t of a l d o s t e r o n e i n both methods ranged between 2-20 pg. The proposed assays are s u i t a b l e f o r t h e d e t e r m i n a t i o n of a l d o s t e r o n e i n b i o l o g i c a l f l u i d s compared with o t h e r r e p o r t e d ELISA assays, as w e l l as with R I A . KEY WORDS:
Aldosterone, enzyme immunoassay
INTRODUCTION Aldosterone, mineralocorticoid,
the
potent naturally
occuring
i s uniquely synthesized i n t h e zona g r a n u l o s a
of t h e adrenal cortex.
*
most
I t s main p h y s i o l o g i c a l f u n c t i o n i s
To whom correspondence should be addressed
215 Copyright 0 1990 by Marcel Dekker, Inc.
216
SCHWARTZ ET AL.
r e g u l a t i n g t h e metabolism of sodium and potassium i o n s , mostly by s t i m u l a t i n g sodium r e a b s o r p t i o n i n exchange f o r p o t a s s i u m and hydrogen i o n s i n t h e d i s t a l t u b u l e of t h e kidney.
Determination
of a l d o s t e r o n e i n b i o l o g i c a l f l u i d s is important f o r t h e diagnosis of a v a r i e t y of d i s e a s e s r e l a t e d t o hypertension.
The a l d o s t e r o n e
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c o n c e n t r a t i o n i n human p l a s m a and s a l i v a a r e r e l a t i v e l y low i n comparison w i t h
o t h e r s t e r o i d hormones. The a l d o s t e r o n e l e v e l
v a r i e s between 18 between 38
-
- 79
140 pg/ml
pglml,
and a f t e r hormone s t i m u l a t i o n
(1). T h e r e f o r e , s e n s i t i v e and s p e c i f i c
assays a r e needed f o r i t s determination i n b i o l o g i c a l f l u i d s . The e a r l i e s t method f o r a l d o s t e r o n e d e t e r m i n a t i o n i n v o l v e d the double
i s o t o p e approach
( 2 ) which
radioimmunoassay p r o c e d u r e s ( 3 inconveniences
of
h a s been d e v e l o p e d .
the
above
- 6).
methods
was
f o l l o w e d by
Due t o t h e o b v i o u s an immunoenzymatic assay
T h i s method was r e c e n t l y a p p l i e d f o r t h e
determination of s e v e r a l s t e r o i d hormones been publi.shed r e c e n t l y
(7). Two a r t i c l e s have
d e s c r i b i n g t h e establishment of an enzyme
immunoassay f o r u r i n a r y and plasma aldosterone u s i n g monoclonal a n t i b o d i e s a g a i n s t a l d o s t e r o n e (8,9). I n t h e p r e s e n t study w e developed and compared two d i f f e r e n t
ELISA assays f o r plasma a l d o s t e r o n e using h i g h l y s p e c i f i c p o l y c l o n a l a n t i - a l d o s t e r o n e antibodies. The s e n s i t i v i t y
of t h e
a s s a y s w a s e v a l u a t e d a c c o r d i n g t o t h e l i m i t of d e t e c t i o n o f aldosterone,
d e f i n e d as t h e l o w e s t c o n c e n t r a t i o n d i f f e r i n g
s i g n i f i c a n t l y from t h e z e r o s t a n d a r d .
C o n c e n t r a t i o n of
a l d o s t e r o n e needed f o r 50% competitive i n h i b i t i o n of binding of a
21 7
ENZYME-LINKED IMMUNOSORBENT ENZYMES
known amount of antibodies by a certain amount of labeled aldosterone was used as another criterion for evaluation of sensitivity of the proposed assays. Both methods proposed are characterized by
simplicity, reproducibility and high
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sensitivity required for their clinical applications.
MATERIALS AND METHODS The following products were purchased from Sigma Chemical Co.: Bovine Thyroglobulin (TG) #T1001; Carboxypeptidase A (CPA) #C0386; Alkaline phosphatase (AP) #P5521; (ADH)
#A3263 and
peroxidase
Diaphorase
Alcohol dehydrogenase
( D I ) #D2381.
Horseradish
(HRP), and f3-galactosidase labeled Goat anti-rabbit
IgG, p-nitrophenyl phosphate (pNPP) and o-nitrophenyl B-Dgalactopyranoside (oNPG) were purchased from Amersham. Preparation of aldosterone-protein derivatives Aldosterone-21-acetate and aldosterone 18.21-diacetate were prepared according to Bayard et al.
(2).Aldosterone-3-
carboxymethyloxime 18,21 diacetate was prepared as described by Janoski et. al. (10). Its purity was checked by TLC on silicagel using the following solvent system: acetone/rnethanol 1:8 and to luene/acetone/methan01 1:1:1. Aldosterone-3-carboxymethoxime 18,21-diacetatewas conjugated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (11.12) to the following proteins: thyroglobulin. carboxypeptidase A
and
alkaline phosphatase.
The conjugates were denoted as Aldo-TG,
Aldo-CPA and Aldo-AP.
Determination of the number of steroid
SCHWARTZ ET A L .
218
r e s i d u e s p e r one p r o t e i n molecule was c a r r i e d o u t by s p e c t r o m e t r i c a n a l y s i s a t 240 nm o f t h e s t e r o i d - p r o t e i n c o n j u g a t e s i n PBS. compensated by t h e s u b t r a c t i o n of t h e absorption due t o t h e amount of t h e p r o t e i n p r e s e n t i n t h e complex (13). The a l d o s t e r o n e - a l k a l i n e phosphatase complex, i n a d d i t i o n as
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a c o a t i n g antigen, was used as enzymatic marker f o r determination of unknown amounts of plasma aldosterone. The enzyme a c t i v i t y of conjugated a l k a l i n e phosphatase w a s measured by both, t h e conventional assay using pNPP as s u b s t r a t e and t h e a m p l i f i e d method (14).
T h i s was done by a d d i n g 10 pl
samples c o n t a i n i n g a range of concentrations of t h e conjugate t o m i c r o t i t r e p l a t e w e l l s and assaying them a s follows: C o n v e n t i o n a l a s s a y : 80 1.11 o f 5 mmol/l pNPP i n 0.9 m o l / l d i e t h a n o l a m i n e b u f f e r , pH 9.8, c o n t a i n i n g 0.5 m m o l / l MgSO4 w a s added t o each w e l l and incubated f o r 30 nmin a t 25OC.
The enzyme
r e a c t i o n was t h e n s t o p p e d by a d d i t i o n o f 270 ~1 of 2 m o l / l N a O H and t h e i n c r e a s e i n a b s o r b a n c e a t 405 nm o v e r a r e a g e n t b l a n k recorded. Enzyme a m p l i f i e d assay: 80 pl of s u b s t r a t e c o n s i s t i n g of 0.2 mmol/l NADP i n 50 m m o l / l diethanolamine b u f f e r , pH
9.5, c o n t a i n i n g
1.0 m m o l / l MgC12 was added t o e a c h s a m p l e t o b e a s s a y e d by t h e a m p l i f i e d method and i n c u b a t e d f o r 15 min a t 25OC.
Further
phosphatase a c t i v i t y was then i n h i b i t e d and c y c l i n g commenced by t h e a d d i t i o n o f 220 u 1 o f a s o l u t i o n c o n s i s t i n g ADH,
of:
0.4 mg/ml
0.4 mg/ml diaphorase and 0.55 mmol/l p-iodonitrotetrazolium
v i o l e t ( I N T ) i n 25 mmol/l containing
4% ( v / v ) e t h a n o l .
sodium p h o s p h a t e b u f f e r ,
pH 7.2,
The c o l o u r development was stopped
219
ENZYME-LINKED IMMUNOSORBENT ENZYMES
a f t e r a f u r t h e r 15 min i n c u b a t i o n by a d d i t i o n o f 50 p l 0.4 m o l / l
H C 1 and t h e i n c r e a s e i n absorbance a t
492 nm
o v e r a reagent b l a n k
recorded u s i n g a T i t e r t e k Multiskan MC m i c r o t i t r e p l a t e reader. Preparation
of TG-aldosterone
antiserum
T h r e e r a b b i t s were immunized w i t h t h e Aldo-TG
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according t o Vaitukaitis et.
al.
(15).
conjugate
E a c h r a b b i t was
i n t r a m u s c u l a r l y i n j e c t e d with a t o t a l of 2.5 mg of t h e conjugate e m u l s i f i e d i n Freund's c o m p l e t e a d j u v a n t . After two and t h r e e months booster i n j e c t i o n s of 1.5 m g o f conjugate each i n Freund's incomplete adjuvant w e r e given.
The a n t i s e r u m w a s p a r t i a l l y
p u r i f i e d by s u c c e s s i v e p r e c i p i t a t i o n w i t h s a t u r a t e d ammonium sulfate,
d i a l y s e d and t h e amount of p r o t e i n d e t e r m i n e d by
s p e c t r o p h o t o m e t r i c measurement a t 280 nm. constant
of
t h e s e a n t i b o d i e s was
The a p p a r e n t b i n d i n g
calculated
from t h e f r e e
c o n c e n t r a t i o n of t h e a n t i b o d i e s which bind 50% of t o t a l amount of coated antigen (16).
The a n t i b o d y t i t e r s o f t h e a n t i s e r u m were
measured by an ELISA method using Aldo-CPA,
as c o a t i n g antigens.
The amount of bound a n t i b o d i e s was monitored
u s i n g a second a n t i - r a b b i t galactosidase.
Aldo- AP, CPA and TG
immunoglobulin l a b e l e d
with
p-
Enzyme a c t i v i t y of p - g a l a c t o s i d a s e was determined
according t o manufacturer.
The s p e c i f i c i t y of t h e antiserum f o r
a l d o s t e r o n e was determined by measuring t h e c r o s s - r e a c t i v i t y with v a r i o u s s t e r o i d s by radioimmunoassays ( R I A ) . Preparation
of
aldosterone sample from human plasma
F r o z e n male p l a s m a was a l l o w e d t o thaw and a
was s h a k e n w i t h 12.5 m l of d i c h l o r o m e t h a n e .
0.5 m l s a m p l e
The e x t r a c t was
e v a p o r a t e d w i t h n i t r o g e n and t h e r e s i d u e was d i s s o l v e d i n PBS
SCHWARTZ ET A L .
220
The e x t r a c t i o n was r e p e a t e d t w i c e
c o n t a i n i n g 0.1% o f g e l a t i n .
and amount of e x t r a c t e d a l d o s t e r o n e was determined by measuring t h e o p t i c a l d e n s i t y a t 240 nm.
The E240 - e x t i n c t i o n c o e f f i c i e n t
o f p u r e a l d o s t e r o n e i s 17000. p e r f o r m e d as f o l l o w s .
Recovery
A l d o s t e r o n e (5-10 pg/ml)
human p l a s m a and e x t r a c t e d i n a s i m i l a r way.
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e x p e r i m e n t s were
was
added t o
The d e t e c t i o n o f
added a l d o s t e r o n e , as w e l l as t h e a l d o s t e r o n e from human plasma, were measured and compared.
The c a l i b r a t i o n c u r v e w i t h n a t i v e
a l d o s t e r o n e and s t e r o i d - f r e e human plasma were performed )under s i m i l a r experimental conditions.
Competitive assay with immobilized antigen Aldo-AP o r Aldo-CPA o r Aldo-TG (0.1 were
- 7.3)
Pg/ml (100
pl)
added t o e a c h w e l l o f a n E L I S A p l a t e (Nunc) and i n c u b a t e d
overnight a t 4OC.
A f t e r washing t h e p l a t e w i t h PBS c o n t a i n i n g
0.05% Tween 20 n o n - s p e c i f i c s i t e s were b l o c k e d w i t h 0.1% o f gelatin.
The a l d o s t e r o n e a n t i s e r u m (2)lg/ml) was added a t a
d i l u t i o n corresponding t o t h e i n f l e c t i o n p o i n t of t h e t i t r a t i o n curve,
t o g e t h e r w i t h known amounts o f a l d o s t e r o n e ,
1-1000
p g / w e l l , o r with t h e sample t o be determined i n a t o t a l volume of
100
pl.
37OC.
The r e a c t i o n m i x t u r e s were i n c u b a t e d f o r 1 h o u r a t
The amount of bound antibody was measured by t h e a d d i t i o n
of e i t h e r p-GAL
o r HRP conjugated a n t i - r a b b i t
a c t i v i t y o f p - G A L o r H R P were d e t e r m i n e d manufacturer's i n s t r u c t i o n s .
IgG.
The enzymic
according t o t h e
The s t a n d a r d c a l i b r a t i o n c u r v e was
obtained with known amounts of aldosterone.
221
ENZYME-LINKED IMMUNOSORBENT ENZYMES Competitive assay with immobilized antibody Aldosterone-TG a n t i b o d i e s (1 )Ig/ml, 100 )11 i n
PBS) were
added t o t h e w e l l s o f ELISA p l a t e s and i n c u b a t e d a t 4OC.
After
washing and b l o c k i n g , as d e s c r i b e d a b o v e , a known amount of a l d o s t e r o n e t r e a t e d s i m i l a r t o t h e s a m p l e was i n c u b a t e d w i t h a
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c o n s t a n t amount o f Aldo-AP
c o n j u g a t e i n PBS c o n t a i n i n g 0.1%
gelatin.
A f t e r incubation f o r 1 hour i n 37OC t h e wells were washed
and
amount o f
the
AP c o r r e s p o n d i n g t o t h e amount of
bound
a l d o s t e r o n e w a s measured e i t h e r by a standard method employing pn i t r o p h e n y l phosphate as s u b s t r a t e , Johannsson e t a 1
o r by t h e a m p l i f i e d method of
( 1 4 ) u s i n g i o d o n i t r o t e t r a z o l i u m v i o l e t as
p r e v i o u s l y described.
RESULTS Aldosterone d e r i v a t i v e s were conjugated with v a r i o u s p r o t e i n
c a r r i e r s , s u c h a s CPA, AP a n d TG, i n o r d e r t o e l i c i t t h e production of h i g h l y s p e c i f i c antibodies, f o r t h e l a b e l i n g of a l d o s t e r o n e , a s w e l l as f o r a d s o r p t i o n
o f a l d o s t e r o n e on t h e
ELISA p l a t e s . Conjugation with TG y i e l d e d a product c o n t a i n i n g an average of
44 a l d o s t e r o n e (Table 1) r e s i d u e s p e r p r o t e i n molecule.
This
conjugate was i n j e c t e d i n t o r a b b i t s i n o r d e r t o e l i c i t production of a n t i b o d i e s .
The t i t e r o f a n t i b o d i e s was d e t e r m i n e d by t h e
ELISA assay u s i n g Aldo-CPA
and/or Aldo-AP as shown i n Fig. 1.
As
c a n be deduced from t h e t i t r a t i o n c u r v e , 50%b i n d i n g o c c u r s a t a d i l u t i o n of 1:9000 f o r Aldo-AP and 1:20.000 f o r Aldo-CPA (Fig. 1).
2 22
SCHWARTZ ET A L . TABLE
A
C o m p a r i s o n o f t h e s e n s i t i v i t y o f t h e d i f f e r e n t enzyme immunoassays f o r determination of plasma aldosterone.
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A.
Molecular Moles of Aldosterweightof Aldosterone con j u g a t e the one p e r carrier mol of (kD) protein
Detection Transformation l i m i t pg number(+)
*
Aldo-CPA
35
22
0.63
500 +/- 50
Aldo-TG
660
44
0.067
20 + / - 7
7
0.060
Aldo- AP
116
Concentration of Aldostero n e which i n h i b i t s 50% of binding pg/well
5000
+I- 50
70 +/- 7
2 +/ -0.2
20 + / - 2
B. Antialdosterone
*
5 +/- 0.5
50
+/- 5
Each f i g u r e i n t h e t a b l e r e p r e s e n t s t h e mean d a t a obtained from f i v e d i f f e r e n t experiments with a standard d e v i a t i o n varying between ‘+-la%.
( + ) moles of aldosterone per k i l o d a l t o n of p r o t e i n
The c a l c u l a t e d a p p a r e n t b i n d i n g c o n s t a n t o f t h e p o l y c l o n a l - ~ Fig. M 1 a n t i b o d i e s t o a l d o s t e r o n e was found t o b e ~ x I o ~(see
insert).
*
The antibody t i t e r obtained from t h e s e t i t r a t i o n curves
was r e l a t i v e l y high as compared t o d a t a reported i n l i t e r a t u r e , probably due t o t h e high content of a l d o s t e r o n e molecules bound t o
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ENZYME-LINKED IMMUNOSORBENT ENZYMES
223
ANTIBODY DILUTION (10-l)
FIGURE
1
Antibody d i l u t i o n c u r v e s obtained with d i f f e r e n t a l d o s t e r o n e - p r o t e i n c o n j u g a t e s c o a t e d on t h e ELISA p l a t e s . The c u r v e s show t h e percentage of binding of decreasing concentrations o f a n t i - a l d o s t e r o n e a n t i b o d i e s t o t h e complexed a l d o s t e r o n e ( B ) , r e l a t i v e t o t h e s a t u r a t i o n v a l u e (Bo) AP ( n o t shown), TG and CPA were u s e d f o r comparison a s c o a t i n g a n t i g e n s . The amount o f bound a n t i b o d y was f o l l o w e d by d e t e r m i n a t i o n o f enzymic a c t i v i t y o f B - g a l a c t o s i d a s e 1a b e l e d a n t i - r a b b i t immunoglobulin.
I n t h e i n s e r t of t h e Figure, t h e binding constant of a n t i - a l d o s t e r o n e t o Aldo-CPA i s measured, based on t h e concentration of f r e e antibody which binds 50% of t h e t o t a l antigen.
224
SCHWARTZ ET AL. TABLE
2
C r o s s - r e a c t i v i t y of anti-aldosterone antibodies with v a r i o u s s t e r o i d s measured by radioimmunoassy. These d a t a were o b t a i n e d from P r o f e s s o r V e c s e i , Department of Pharmacology, U n i v e r s i t y of Heidelberg. FRG.
% Cross-reactivities with a n t i - a l d o s t e r o n e
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Steroids
100
Aldosterone 5a-Dihydroaldosterone
64.4
Tetrahydroaldosterone
4.6
Corticosterone
0.4
18-Hydroxycorticosterone
0
Cortisol
0
Progesterone
0
t h e TG.
CPA, AP and TG were used f o r c o a t i n g as n e g a t i v e c o n t r o l .
E v a l u a t i o n of t h e enzyme-immuno assays proposed i n t h i s paper f o r t h e d e t e r m i n a t i o n of p l a s m a a l d o s t e r o n e and a l d o s t e r o n e i t s e l f were c h a r a c t e r i z e d regarding s p e c i f i c i t y , s e n s i t i v i t y , p r e c i s i o n and accuracy. (17).
S p e c i f i c i t y of t h e antiserum f o r a l d o s t e r o n e
was d e t e r m i n e d by m e a s u r i n g t h e c r o s s - r e a c t i v i t y w i t h v a r i o u s s t e r o i d s by R I A a s s a y and r e s u l t s a r e p r e s e n t e d i n T a b l e 2.
The
c r o s s - r e a c t i o n with c o r t i c o s t e r o n e . c o r t i s o l and progesterone were below t h e d e t e c t i o n l i m i t o f t h e a s s a y , s u g g e s t i n g t h e h i g h s p e c i f i c i t y of those a n t i b o d i e s t o a l d o s t e r o n e , confirmed a l s o by t h e h i g h b i n d i n g c o n s t a n t s o f t h e immuno complex ( - 1 0 + 9 M - 1 )
of
ENZYME-LINKED IMMUNOSORBENT ENZYMES
225
__________ Sensitivitx
a l d o s t e r o n e and i t s a n t i b o d i e s .
of these
immunoassays are e v a l u a t e d according t o c u r v e s e n s i t i v i t y which i s d e f i n e d by
d e t e c t i o n l i m i t f o r i d e n t i f i c a t i o n o f pure a n a l y t e i n
b u f f e r e d s o l u t i o n s , whereas t h e a s s a y s e n s i t i v i t y is
d e f i n e d by a
d e t e c t i o n l i m i t f o r t h e a n a l y t e i n t h e presence of a v a r i e t y of
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p o s s i b l e i n t e r f e r r i n g components, steroidals.
b o t h s t e r o i d a l s and non-
I n e x t r a c t i o n immunoassays, s o l v e n t e x t r a c t i o n may
sometimes minimize t h i s i n t e r f e r e n c e and p r o v i d e a system i n which o v e r a l l s e n s i t i v i t y and c u r v e s e n s i t i v i t y do n o t d i f f e r significantly.
I n a l l t h e experiments performed f r e e a l d o s t e r o n e
w a s t r e a t e d i n t h e same way a s a l d o s t e r o n e o f human p l a s m a t o a v o i d t h i s inconvenience.
Another parameter used i n t h i s work t o
c h a r a c t e r i z e t h e s e n s i t i v i t y of t h e assay was t h e c o n c e n t r a t i o n of a l d o s t e r o n e t h a t induced 50% c o m p e t i t i v e i n h i b i t i o n of b i n d i n g a f i x e d amount o f
antibodies
by
a known
amount
of
labeled
aldos terone. D e t e r m i n a t i o n o f p l a s m a a l d o s t e r o n e , as w e l l as t h e c a l i b r a t i o n curves of aldosterone i t s e l f , e x t r a c t e d i n a similar way t o i n two d i f f e r e n t ways.
i n buffer and/or
plasma a l d o s t e r o n e , was performed
I n t h e f i r s t method t h e c a l i b r a t i o n c u r v e s
o f a l d o s t e r o n e , as w e l l as t h e p l a s m a a l d o s t e r o n e t o b e determined, were c a r r i e d o u t by c o m p e t i t i v e a s s a y with immobilized antigens.
Aldo-CPA (18 pg/ml),
p e r mole of p r o t e i n ,
c o n t a i n i n g 22 moles a l d o s t e r o n e
a d s o r b e d on t h e ELISA p l a t e s was r e a c t e d
w i t h a m i x t u r e o f unknown amounts o f f r e e p l a s m a a l d o s t e r o n e . preincubated with a n t i - a l d o s t e r o n e a n t i b o d i e s and q u a n t i t a t e d by
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226
SCHWARTZ ET AL.
04 ' 0 '1
I
I
I
I
I
2
5
10
20
50
I
I
100 200
I
1000
ALDOSTERONE (pg/weJl)
FIGURE
2
t y p i c a l standard curve f o r the displacement of aldosterone a n t i b o d i e s from i m m o b i l i z e d a l d o s t e r o n e - p r o t e i n c o n j u g a t e s by d i f f e r e n t amounts o f a l d o s t e r o n e i n s o l u t i o n . P l a s m a a l d o s t e r o n e d i l u t i o n s , as w e l l as f r e e a l d o s t e r o n e e x t r a c t e d i n a s i m i l a r way a s plasma samples, are i n good c o r r e l a t i o n . The c o e f f i c i e n t of v a r i a t i o n f o r t h e i m p o r t a n t p o i n t s of t h e c u r v e ( d e t e c t i o n l i m i t and concentration of a l d o s t e r o n e a t 50% i n h i b i t i o n ) are g i v e n i n Table 3. The w e l l s c o a t e d w i t h t h e Aldo-AP (200 n g / m l ) (100 u l ) and t h e a l d o s t e r o n e a n t i b o d i e s d i l u t i o n used was 1:9000.
A
a d d i t i o n of
enzyme l a b e l e d a n t i - r a b b i t
IgG.
F i f t y percent
i n h i b i t i o n occurs a t r e l a t i v e l y high concentrations a l d o s t e r o n e . The d e t e c t i o n l i m i t of a l d o s t e r o n e observed i n
of this
system was 500 pg (see Table 1). The measurements of a l d o s t e r o n e concentration with another immobilized antigen, Aldo-TG a l d o s t e r o n e p e r mol p r o t e i n ) (10 yg/ml),
(44 moles
by u s i n g a n t i - r a b b i t
Ig
a n t i b o d i e s c o n j u g a t e s w i t h HRP, improved t h e s e n s i t i v i t y of t h e assay.
F i f t y percent
i n h i b i t i o n o c c u r e d a t 70 p g / w e l l
of
22 7
ENZYME-LINKED IMMUNOSORBENT ENZYMES
The s e n s i t i v i t y o b t a i n e d by t h i s a s s a y i s s u f f i c i e n t
aldosterone.
f o r t h e measurement o f a l d o s t e r o n e i n body f l u i d . results
were o b t a i n e d by c o a t i n g w i t h Aldo-AP
The b e s t
(0.2 n g / m l )
c o n t a i n i n g 7 mole of a l d o s t e r o n e / m o l p r o t e i n ( T a b l e l a , F i g 2 ) , f o l l o w e d by s e q u e n t i a l a d d i t i o n of f r e e a l d o s t e r o n e and/or plasma
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d i l u t i o n s p r e v i o u s l y incubated with p o l y c l o n a l a n t i - a l d o s t e r o n e . The r e s i d u a l amount o f a n t i b o d y which r e m a i n s a f t e r b i n d i n g o f a l d o s t e r o n e t o be determined w a s measured with commercial a n t i b o d y l a b e l e d w i t h HRP
.
second
This assay has t h e higher
s e n s i t i v i t y , a d e t e c t i o n l i m i t of 2 pg
-
and 50% c o m p e t i t i o n
between t h e free and bound a l d o s t e r o n e on t h e p l a t e occured a t 20 pg/well.
The r e s u l t s are shown i n Fig. 2 and summarized i n T a b l e
la. I n a n o t h e r t y p e o f a s s a y t h e a l d o s t e r o n e c o n c e n t r a t i o n was measured by c o a t i n g with a n t i - a l d o s t e r o n e a n t i b o d i e s , f o l l o w e d by c o m p e t i t i o n b e t w e e n a l d o s t e r o n e t o be measured and Aldo-AP conjugates
(Table l b ) .
The enzymic a c t i v i t y o f
alkaline-
p h o s p h a t a s e c o m p l e x was f o u n d t o b e 80% as compared w i t h t h e a c t i v i t y of t h e amount of experimental
conditions.
native
enzyme
By t h i s
measured
under
same
method 50% i n h i b i t i o n was
o b t a i n e d a t 50 p g / w e l l and t h e d e t e c t i o n l i m i t
of a l d o s t e r o n e was
5 Pg. I n a v a r i a n t o f t h i s method,
t h e a c t i v i t y of t h e AP was
d e t e c t e d n o t o n l y through t h e u s e of t h e s t a n d a r d s u b s t r a t e b u t by t h e u s e o f a n a m p l i f y i n g c h a i n o f enzymes, as was p r e v i o u s l y d e s c r i b e d by J o h a n n s s o n e t . a l .
( 1 4 ) ( F i g . 3).
The u s e o f t h i s
SCHWARTZ ET AL.
228
E C (u
Q)
t
E C
m 0 t
1.8
1.4 1.2 -
1.6
1-
v
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-
-
w 0
0.8
a
0.6 -
Z
m 0 0.4v) m 0.2 a 02
-
0 0.2 0.4 A P - A L D O S T E R O N E ( p g ml)
I
FIGURE
3
Determination of enzymatic a c t i v i t y of Aldo-AP complex bound t o antia l d o s t e r o n e a n t i b o d i e s measured by s t a n d a r d methods ( /i & ) and by ). Enzyme a c t i v i t y of a l k a l i n e a m p l i f i e d e n z y m a t i c method (24%rphosphatase, determined by s t a n d a r d method, w a s f o l l o w e d by i n c r e a s e i n o p t i c a l d e n s i t y a t 405nm and t h e a m p l i f i e d enzymic method by i n c r e a s e i n o p t i c a l d e n s i t y a t 462nm ( f o r d e t a i l s see experiment).
m o d i f i c a t i o n r e s u l t e d i n o v e r a l l h i g h e r OD v a l u e s , i n c r e a s i n g t h e sensitivity
_________ Precision
-
of t h e a s s a y f o r low c o n c e n t r a t i o n s of a l d o s t e r o n e . The p r e c i s i o n of t h e s t a n d a r d c u r v e o f human p l a s m a
a l d o s t e r o n e and a l d o s t e r o n e i t s e l f are a p p r e c i a t e d by i n t r a - a s s a y and i n t e r a s s a y c o e f f i c i e n t s of v a r i a t i o n and r e s u l t s summarized i n Table
3.
The s t a n d a r d d e v i a t i o n of t h e r e a d i n g of t h e mid-point
o f t h e s t a n d a r d c u r v e was + - 10%. Both d e v e l o p e d a s s a y s a r e
229
ENZYME-LINKED IMMUNOSORBENT ENZYMES TABLE
3
The r e p r o d u c i b i l i t y of t h e enzyme immunoassay f o r determination of aldosterone.
Coefficient
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Aldosterone
of
variation $
Intra-assay (n = 5)
Inter-assay (n = 5)
20
pg/well
0.32
0.04
2
pg/well
6.32
1.28
s e n s i t i v e enough f o r t h e assay of plasma a l d o s t e r o n e l e v e l and t h e r e p r o d u c i b i l i t i e s adequate €or c l i n i c a l assays. Accuracx o f t h e a s s a y was checked by t h e l i n e a r i t y o f d a t a obtained by assaying s e v e r a l d i l u t i o n s of sample c o n t a i n i n g known c o n c e n t r a t i o n s o f s t e r o i d , o r m o n i t o r i n g t h e r e c o v e r y o f known amounts of s t e r o i d added t o b i o l o g i c a l s a m p l e s . F i v e - t e n p g a l d o s t e r o n e added t o 100 u l o f p l a s m a o r b u f f e r i n c r e a s e d t h e measured a l d o s t e r o n e content by 5-10 pg +/-
(0.5-1).
Recovery of
a l d o s t e r o n e i s q u a n t a t i v e , i n d i c a t i n g t h a t i n normal c o n d i t i o n s no i n t e r f e r e n c e of o t h e r components with a n t i - a l d o s t e r o n e occured. DISCUSSION Many i n v e s t i g a t o r s
have been t u r n i n g t o enzyme immunoassay
t o r e p l a c e radioimmunoassay s i n c e i t frees them from t h e problems
SCHWARTZ ET AL.
and h a z a r d s o f h a n d l i n g r a d i o i s o t o p e s and t h e n e c e s s i t y f o r expensive counting instruments.
S e v e r a l such a s s a y s have been
developed f o r v a r i o u s s t e r o i d s and r e c e n t l y f o r a l d o s t e r o n e
(7-9).
An e v a l u a t i o n of s e n s i t i v i t y of v a r i o u s ELISA d e t e r m i n a t i o n s on plasma a l d o s t e r o n e ,
w i t h r e g a r d t o d e t e c t i o n l i m i t and i t s
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c o n c e n t r a t i o n i n b i o l o g i c a l f l u i d s t h a t i n d u c e 50% c o m p e t i t i v e b i n d i n g i n h i b i t i o n o f a f i x e d amount o f a n t i b o d i e s by a known amount of l a b e l e d a l d o s t e r o n e , as w e l l as s p e c i f i c i t y , p r e c i s i o n and a c c u r a c y of t h e a s s a y s , a r e d i s c u s s e d .
Comparison o f t h e
r e c o r d s o b t a i n e d i n e a c h ELISA u s i n g i m m o b i l i z e d a l d o s t e r o n e complexed with v a r i o u s p r o t e i n s (CPA, AP. TG) i n d i c a t e d t h a t t h e t r a n s f o r m a t i o n number ( t h e number o f a l d o s t e r o n e r e s i d u e s p e r k i l o d a l t o n of p r o t e i n ) seems t o determine t h e s e n s i t i v i t y of t h e assay. Apparently,
low d e n s i t y o f a l d o s t e r o n e o n t h e p r o t e i n
carrier y i e l d s a more s e n s i t i v e assay. can be argued as f o l l o w s :
sites €or i t s antigen. con j u g a t e
Presumably t h i s c o n c l u s i o n
Each antibody m o l e c u l e h a s two b i n d i n g When e x p o s e d t o a p r o t e i n a l d o s t e r o n e
containing
many
aldosterone residues
(high
t r a n s f o r m a t i o n number) t h e a n t i - a l d o s t e r o n e antibody would b i n d t o t h e c o n j u g a t e v i a b o t h i t s b i n d i n g s i t e s and t h e r e f o r e w i t h a h i g h e r a p p a r e n t b i n d i n g a f f i n i t y t o t h e conjugate than t o t h e f r e e d i l u t e d aldosteroned solutions.
T h i s would r e s u l t i n l o w e r
s e n s i t i v i t y measurements o f f r e e a l d o s t e r o n e .
When exposed t o a
conjugate w i t h low t r a n s f o r m a t i o n number, ( i d e a l l y one a l d o s t e r o n e molecule p e r p r o t e i n ) o n l y one o f t h e b i n d i n g sites o f t h e antibody would b i n d t o t h e conjugate and under such c o n d i t i o n s t h e
231
ENZYME-LINKED IMMUNOSORBENT ENZYMES
a f f i n i t y o f t h e a n t i b o d y f o r t h e c o n j u g a t e s h o u l d be s i m i l a r t o t h e a f f i n i t y of t h e antibody f o r t h e f r e e aldosterone, r e s u l t i n g i n higher d e t e c t i o n s e n s i t i v i t y of f r e e aldosterone. The production of a n t i - a l d o s t e r o n e antibodies using complex containing
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on immunogen
Aldo-TG
44 moles of a l d o s t e r o n e per mole of p r o t e i n as
was a good c h o i c e w i t h r e s p e c t t o t h e h i g h
s p e c i f i c i t y and a f f i n i t y of t h e antibodies. t h e ELISA p l a t e ,
Aldo-AP
However, f o r c o a t i n g
complex c o n t a i n i n g o n l y
7
moles
a l d o s t e r o n e per mole of p r o t e i n increased a t l e a s t t e n times t h e s e n s i t i v i t y of t h e assay as compared with d a t a obtained with AldoTG o r Aldo-CPA.
The e s t a b l i s h e d a s s a y s f o r p l a s m a a l d o s t e r o n e ,
using
immobilized antigen (Aldo-protein complexes) o r immobilized a n t i aldosterone antibodies, possess for RIA
high s e n s i t i v i t y , a s mentioned
( 3 - 6 ) , and p o s s e s s a l l t h e a d v a n t a g e s of ELISA a s s a y s ,
such as simple non-hazardous m a t e r i a l s and r e p r o d u c i b i l i t y .
The
normal r a n g e o f p l a s m a a l d o s t e r o n e , which v a r i e d between 18-79 pg/ml (1). a s w e l l a s t h e e l e v a t e d v a l u e s observed a f t e r hormone s t i m u l a t i o n a s s o c i a t e d with p a t h o l o g i c a l s t a t e s , can be measured
a t high accuracy by both methods. A comparison between t h e two a s s a y s proposed i n t h i s p a p e r
showed t h a t t h e y d i f f e r p r i m a r i l y i n t h e n a t u r e of t h e r e a g e n t s used, which make t h e f i r s t type of assay more convenient.
In t h i s
a s s a y ( w i t h i m m o b i l i z e d a n t i g e n ) o n l y one t y p e of a l d o s t e r o n e p r o t e i n c o n j u g a t e n e e d s t o be p r e p a r e d .
I n p r i n c i p l e , t h e same
conjugate can be used f o r both immunization of t h e r a b b i t s and f o r
232
SCHWARTZ ET AL.
running t h e assay.
The b i o l o g i c a l a c t i v i t y o f t h e c o n j u g a t e d
p r o t e i n i s o f n o i m p o r t a n c e and c o n j u g a t e s w i t h h i g h and low t r a n s f o r m a t i o n number may b e e a s i l y p r e p a r e d , and t h e r a b b i t antiserum used as a second antibody is a commercial p r e p a r a t i o n . Moreover t h e n a t u r e o f t h e second antibody may be e a s i l y changed
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and d i f f e r e n t methods f o r s i g n a l a m p l i f i c a t i o n compared e.g. u s e o f t h e a v i d i n b i o t i n system.
the
The s e c o n d t y p e o f a s s a y ( w i t h
immobilized antibody) is more d i f f i c u l t t o set up.
Two c o n j u g a t e s
are needed, one with high t r a n s f o r m a t i o n number f o r immunization, and a n o t h e r w i t h low t r a n s f o r m a t i o n number f o r t e s t i n g .
The
p r o t e i n u s e d f o r t e s t i n g h a s t o b e a n enzyme and i t s e n z y m a t i c a c t i v i t y h a s t o be r e t a i n e d a f t e r conjugation. f o r t h e c o a t i n g should be a t
The antibody used
least p a r t i a l l y purified.
A l t o g e t h e r , t h i s comparison i n d i c a t e s t h a t t h e f i r s t type of a s s a y
i s f a r e a s i e r t o s e t up and i s a l s o easier t o m a n i p u l a t e and improve.
REFERENCES
1.
Hubl, W., Aldosterone, I n : U l r i c h B.H. ( e d . ) . Methods of Enzymol. Anal. (3rd e d . ) 8. VCH: Weinheim, F e d e r a l Republic of Germany. 1985. 216-66.
2.
Bayard. F . , Kowarski, A., Weldon, V . and Migeon. C.J., Appraisal o f t h e double i s o t o p e d i l u t i o n technique f o r t h e measurement o f p l a s m a a l d o s t e r o n e . J. Lab. C l i n . Med. 1970. 75 :347-52.
3.
F a r m e r , R.W., Roup,Jr.. W.G., P e l l i z z a r i , E.D. and F a b r e , J r . . L.F. A r a p i d a l d o s t e r o n e radioimmunoassay. J . C l i n . Endocr. 1972. 34: 18-22.
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4.
Ito. T., Woo, J.. Haning, R. and Horton. R. A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of a1ternate techniques. J. Clin. Endocr. 1972. 34: 106-12.
5.
Vecsei, P., Penke, B. and Joumaah, A . Radioimmunoassay of free aldosteron and of its 18-oxe-glucoronide in human urine. Experientia. 1972. 2815: 622-24.
6.
Farmer, R.W., Brown, D.H., Howard, P.Y. and Fabre, Jr.. L.F. A radioimmunoassay for plasma aldosterone without chromatography. J. Clin. Endocrinol. Metab. 1973. 36: 460-65.
7.
Joshi. U.M. Non-isotopic immunoassays for the estimation of steroid hormones. Immunoassay Technology. 1985. Vol. 1. 151-81. Walter de Gruyter and Co. Berlin, New York.
8.
Hanquez, C., Urios, P, Desfosses, B., Samake, H., Lince, E., Rajkowski, K.M. and Cittanova, N. Enzyme linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone. 1987. Clinica. Chimica Acta 164, 71-82.
9.
Hanquez, C., Rajkowski, K. M., Desfosses, B. and Cittanova, N. A competitive microtitre plate enzyme immunoassay for plasma aldosterone using a monoclonal antibody. J. Steroid. Biochem. 1988.31, 939-45.
10
Janoski, A. H., Shulman, F.C. and Wright, G.E. Selective 3-(O-carboxymethyl) oxime formation in 3,20-diones for hapten immunospecificity. Steroids. 1974. 23, 49-59.
11
Erlanger. B.F., Borek, F., Beiser, S.M. and Lieberman. S. Steroid-protein conjugates. 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and cortisone. J. Biol. Chem. 1957. 228: 713-27.
12. Hansen. W. A simplification of Kind and King’s methods for determination of serum phosphatase. Scandinav. J. Clin. & Lab. Investigation. 1966 18: 353-56. 13. Hosada, H., Takasaki, W., Aihara. S. and Nambara, T. Enzyme labeling of steroids by N-succinimidyl ester method. Preparation of alkaline phosphatase-labeled antigen f o r use in enzyme immunoassay. Chem. Pharm. Bull. 1985. 33: 5393-98. 14. Johannsson, A., Stanley, C.J. and Self. C.H. A fast highly sensitive colorimetric enzyme immunoassay system, demonstrating benefits of enzyme amplifications in clinical chemistry. Clinica. Chimica Acta. 1985. 148: 119-24.
2 34
SCHWARTZ ET AL.
15. V a i t u k a i t i s , J . . Robbins, J . P . , Nieschlag, E. and R o s s , G.T. A method f o r producing s p e c i f i c a n t i - s e r a with s m a l l doses of
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immunogen. J. Clin. Endocrinol Metab. 1971. 31: 988-90.
16.
Pinkard, R . N . and Weir, D.M. In: Handbook of Experimental Immunology (Weir, D.M., ed.) 1978. C h a p t e r 1 6 , pp. 16.1 - 16.20. Blackwell S c i e n t i f i c , Oxford, England.
17.
Fewy. D . , Chandry, S. and James, H.T. 1984, 21:87-92.
J . S t e r o i d . Biochem.
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Enzyme-Linked Immunosorbent Assays for Determination of Plasma Aldosterone Using Highly Specific Polyclonal Antibodies a
a
a
Fidi Schwartz , Eran Hadas , Marcel Harnik & Beka Solomon
a
a
Department of Biotechnology, Faculty of Life Sciences , Tel-Aviv University , Tel-Aviv, 69978, Israel Published online: 23 Oct 2006.
To cite this article: Fidi Schwartz , Eran Hadas , Marcel Harnik & Beka Solomon (1990) Enzyme-Linked Immunosorbent Assays for Determination of Plasma Aldosterone Using Highly Specific Polyclonal Antibodies, Journal of Immunoassay, 11:2, 215-234, DOI: 10.1080/01971529008053270 To link to this article: http://dx.doi.org/10.1080/01971529008053270
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JOURNAL OF IMMUNOASSAY, 11(2), 215-234 (1990)
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ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR DETERMINATION OF PLASMA ALDOSTERONE USING HIGHLY SPECIFIC POLYCLONAL ANTIBODIES
F i d i Schwartz, Eran Hadas, Marcel Harnik and Beka Solomon* Department of Biotechnology, Faculty of L i f e S c i e n c e s , Tel-Aviv U n i v e r s i t y , Tel-Aviv 69978, Israel.
ABSTRACT Two enzyme-linked immunosorbent assays were e s t a b l i s h e d and compared f o r t h e e s t i m a t i o n of plasma a l d o s t e r o n e . I n t h e f i r s t method i m m o b i l i z e d a l d o s t e r o n e - p r o t e i n complexes on t h e ELISA p l a t e s compete with a l d o s t e r o n e t o be determined f o r t h e binding o f c e r t a i n amount o f a n t i - a l d o s t e r o n e a n t i b o d i e s . The s e n s i t i v i t y of t h i s method depends on t h e p r o t e i n carrier used t o conjugate with aldosterone. I n t h e s e c o n d method, a n t i a l d o s t e r o n e a n t i b o d i e s a d s o r b e d on ELISA p l a t e s compete f o r b i n d i n g of known amount o f t h e e n z y m e - l a b e l e d a l d o s t e r o n e and a l d o s t e r o n e t o be determined. The h i g h l y s p e c i f i c r a b b i t a n t i a l d o s t e r o n e a n t i b o d i e s were obtained by i n j e c t i o n of a l d o s t e r o n e oxime t h y r o g l o b u l i n . The d e t e c t i o n l i m i t of a l d o s t e r o n e i n both methods ranged between 2-20 pg. The proposed assays are s u i t a b l e f o r t h e d e t e r m i n a t i o n of a l d o s t e r o n e i n b i o l o g i c a l f l u i d s compared with o t h e r r e p o r t e d ELISA assays, as w e l l as with R I A . KEY WORDS:
Aldosterone, enzyme immunoassay
INTRODUCTION Aldosterone, mineralocorticoid,
the
potent naturally
occuring
i s uniquely synthesized i n t h e zona g r a n u l o s a
of t h e adrenal cortex.
*
most
I t s main p h y s i o l o g i c a l f u n c t i o n i s
To whom correspondence should be addressed
215 Copyright 0 1990 by Marcel Dekker, Inc.
216
SCHWARTZ ET AL.
r e g u l a t i n g t h e metabolism of sodium and potassium i o n s , mostly by s t i m u l a t i n g sodium r e a b s o r p t i o n i n exchange f o r p o t a s s i u m and hydrogen i o n s i n t h e d i s t a l t u b u l e of t h e kidney.
Determination
of a l d o s t e r o n e i n b i o l o g i c a l f l u i d s is important f o r t h e diagnosis of a v a r i e t y of d i s e a s e s r e l a t e d t o hypertension.
The a l d o s t e r o n e
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c o n c e n t r a t i o n i n human p l a s m a and s a l i v a a r e r e l a t i v e l y low i n comparison w i t h
o t h e r s t e r o i d hormones. The a l d o s t e r o n e l e v e l
v a r i e s between 18 between 38
-
- 79
140 pg/ml
pglml,
and a f t e r hormone s t i m u l a t i o n
(1). T h e r e f o r e , s e n s i t i v e and s p e c i f i c
assays a r e needed f o r i t s determination i n b i o l o g i c a l f l u i d s . The e a r l i e s t method f o r a l d o s t e r o n e d e t e r m i n a t i o n i n v o l v e d the double
i s o t o p e approach
( 2 ) which
radioimmunoassay p r o c e d u r e s ( 3 inconveniences
of
h a s been d e v e l o p e d .
the
above
- 6).
methods
was
f o l l o w e d by
Due t o t h e o b v i o u s an immunoenzymatic assay
T h i s method was r e c e n t l y a p p l i e d f o r t h e
determination of s e v e r a l s t e r o i d hormones been publi.shed r e c e n t l y
(7). Two a r t i c l e s have
d e s c r i b i n g t h e establishment of an enzyme
immunoassay f o r u r i n a r y and plasma aldosterone u s i n g monoclonal a n t i b o d i e s a g a i n s t a l d o s t e r o n e (8,9). I n t h e p r e s e n t study w e developed and compared two d i f f e r e n t
ELISA assays f o r plasma a l d o s t e r o n e using h i g h l y s p e c i f i c p o l y c l o n a l a n t i - a l d o s t e r o n e antibodies. The s e n s i t i v i t y
of t h e
a s s a y s w a s e v a l u a t e d a c c o r d i n g t o t h e l i m i t of d e t e c t i o n o f aldosterone,
d e f i n e d as t h e l o w e s t c o n c e n t r a t i o n d i f f e r i n g
s i g n i f i c a n t l y from t h e z e r o s t a n d a r d .
C o n c e n t r a t i o n of
a l d o s t e r o n e needed f o r 50% competitive i n h i b i t i o n of binding of a
21 7
ENZYME-LINKED IMMUNOSORBENT ENZYMES
known amount of antibodies by a certain amount of labeled aldosterone was used as another criterion for evaluation of sensitivity of the proposed assays. Both methods proposed are characterized by
simplicity, reproducibility and high
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sensitivity required for their clinical applications.
MATERIALS AND METHODS The following products were purchased from Sigma Chemical Co.: Bovine Thyroglobulin (TG) #T1001; Carboxypeptidase A (CPA) #C0386; Alkaline phosphatase (AP) #P5521; (ADH)
#A3263 and
peroxidase
Diaphorase
Alcohol dehydrogenase
( D I ) #D2381.
Horseradish
(HRP), and f3-galactosidase labeled Goat anti-rabbit
IgG, p-nitrophenyl phosphate (pNPP) and o-nitrophenyl B-Dgalactopyranoside (oNPG) were purchased from Amersham. Preparation of aldosterone-protein derivatives Aldosterone-21-acetate and aldosterone 18.21-diacetate were prepared according to Bayard et al.
(2).Aldosterone-3-
carboxymethyloxime 18,21 diacetate was prepared as described by Janoski et. al. (10). Its purity was checked by TLC on silicagel using the following solvent system: acetone/rnethanol 1:8 and to luene/acetone/methan01 1:1:1. Aldosterone-3-carboxymethoxime 18,21-diacetatewas conjugated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (11.12) to the following proteins: thyroglobulin. carboxypeptidase A
and
alkaline phosphatase.
The conjugates were denoted as Aldo-TG,
Aldo-CPA and Aldo-AP.
Determination of the number of steroid
SCHWARTZ ET A L .
218
r e s i d u e s p e r one p r o t e i n molecule was c a r r i e d o u t by s p e c t r o m e t r i c a n a l y s i s a t 240 nm o f t h e s t e r o i d - p r o t e i n c o n j u g a t e s i n PBS. compensated by t h e s u b t r a c t i o n of t h e absorption due t o t h e amount of t h e p r o t e i n p r e s e n t i n t h e complex (13). The a l d o s t e r o n e - a l k a l i n e phosphatase complex, i n a d d i t i o n as
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a c o a t i n g antigen, was used as enzymatic marker f o r determination of unknown amounts of plasma aldosterone. The enzyme a c t i v i t y of conjugated a l k a l i n e phosphatase w a s measured by both, t h e conventional assay using pNPP as s u b s t r a t e and t h e a m p l i f i e d method (14).
T h i s was done by a d d i n g 10 pl
samples c o n t a i n i n g a range of concentrations of t h e conjugate t o m i c r o t i t r e p l a t e w e l l s and assaying them a s follows: C o n v e n t i o n a l a s s a y : 80 1.11 o f 5 mmol/l pNPP i n 0.9 m o l / l d i e t h a n o l a m i n e b u f f e r , pH 9.8, c o n t a i n i n g 0.5 m m o l / l MgSO4 w a s added t o each w e l l and incubated f o r 30 nmin a t 25OC.
The enzyme
r e a c t i o n was t h e n s t o p p e d by a d d i t i o n o f 270 ~1 of 2 m o l / l N a O H and t h e i n c r e a s e i n a b s o r b a n c e a t 405 nm o v e r a r e a g e n t b l a n k recorded. Enzyme a m p l i f i e d assay: 80 pl of s u b s t r a t e c o n s i s t i n g of 0.2 mmol/l NADP i n 50 m m o l / l diethanolamine b u f f e r , pH
9.5, c o n t a i n i n g
1.0 m m o l / l MgC12 was added t o e a c h s a m p l e t o b e a s s a y e d by t h e a m p l i f i e d method and i n c u b a t e d f o r 15 min a t 25OC.
Further
phosphatase a c t i v i t y was then i n h i b i t e d and c y c l i n g commenced by t h e a d d i t i o n o f 220 u 1 o f a s o l u t i o n c o n s i s t i n g ADH,
of:
0.4 mg/ml
0.4 mg/ml diaphorase and 0.55 mmol/l p-iodonitrotetrazolium
v i o l e t ( I N T ) i n 25 mmol/l containing
4% ( v / v ) e t h a n o l .
sodium p h o s p h a t e b u f f e r ,
pH 7.2,
The c o l o u r development was stopped
219
ENZYME-LINKED IMMUNOSORBENT ENZYMES
a f t e r a f u r t h e r 15 min i n c u b a t i o n by a d d i t i o n o f 50 p l 0.4 m o l / l
H C 1 and t h e i n c r e a s e i n absorbance a t
492 nm
o v e r a reagent b l a n k
recorded u s i n g a T i t e r t e k Multiskan MC m i c r o t i t r e p l a t e reader. Preparation
of TG-aldosterone
antiserum
T h r e e r a b b i t s were immunized w i t h t h e Aldo-TG
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according t o Vaitukaitis et.
al.
(15).
conjugate
E a c h r a b b i t was
i n t r a m u s c u l a r l y i n j e c t e d with a t o t a l of 2.5 mg of t h e conjugate e m u l s i f i e d i n Freund's c o m p l e t e a d j u v a n t . After two and t h r e e months booster i n j e c t i o n s of 1.5 m g o f conjugate each i n Freund's incomplete adjuvant w e r e given.
The a n t i s e r u m w a s p a r t i a l l y
p u r i f i e d by s u c c e s s i v e p r e c i p i t a t i o n w i t h s a t u r a t e d ammonium sulfate,
d i a l y s e d and t h e amount of p r o t e i n d e t e r m i n e d by
s p e c t r o p h o t o m e t r i c measurement a t 280 nm. constant
of
t h e s e a n t i b o d i e s was
The a p p a r e n t b i n d i n g
calculated
from t h e f r e e
c o n c e n t r a t i o n of t h e a n t i b o d i e s which bind 50% of t o t a l amount of coated antigen (16).
The a n t i b o d y t i t e r s o f t h e a n t i s e r u m were
measured by an ELISA method using Aldo-CPA,
as c o a t i n g antigens.
The amount of bound a n t i b o d i e s was monitored
u s i n g a second a n t i - r a b b i t galactosidase.
Aldo- AP, CPA and TG
immunoglobulin l a b e l e d
with
p-
Enzyme a c t i v i t y of p - g a l a c t o s i d a s e was determined
according t o manufacturer.
The s p e c i f i c i t y of t h e antiserum f o r
a l d o s t e r o n e was determined by measuring t h e c r o s s - r e a c t i v i t y with v a r i o u s s t e r o i d s by radioimmunoassays ( R I A ) . Preparation
of
aldosterone sample from human plasma
F r o z e n male p l a s m a was a l l o w e d t o thaw and a
was s h a k e n w i t h 12.5 m l of d i c h l o r o m e t h a n e .
0.5 m l s a m p l e
The e x t r a c t was
e v a p o r a t e d w i t h n i t r o g e n and t h e r e s i d u e was d i s s o l v e d i n PBS
SCHWARTZ ET A L .
220
The e x t r a c t i o n was r e p e a t e d t w i c e
c o n t a i n i n g 0.1% o f g e l a t i n .
and amount of e x t r a c t e d a l d o s t e r o n e was determined by measuring t h e o p t i c a l d e n s i t y a t 240 nm.
The E240 - e x t i n c t i o n c o e f f i c i e n t
o f p u r e a l d o s t e r o n e i s 17000. p e r f o r m e d as f o l l o w s .
Recovery
A l d o s t e r o n e (5-10 pg/ml)
human p l a s m a and e x t r a c t e d i n a s i m i l a r way.
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e x p e r i m e n t s were
was
added t o
The d e t e c t i o n o f
added a l d o s t e r o n e , as w e l l as t h e a l d o s t e r o n e from human plasma, were measured and compared.
The c a l i b r a t i o n c u r v e w i t h n a t i v e
a l d o s t e r o n e and s t e r o i d - f r e e human plasma were performed )under s i m i l a r experimental conditions.
Competitive assay with immobilized antigen Aldo-AP o r Aldo-CPA o r Aldo-TG (0.1 were
- 7.3)
Pg/ml (100
pl)
added t o e a c h w e l l o f a n E L I S A p l a t e (Nunc) and i n c u b a t e d
overnight a t 4OC.
A f t e r washing t h e p l a t e w i t h PBS c o n t a i n i n g
0.05% Tween 20 n o n - s p e c i f i c s i t e s were b l o c k e d w i t h 0.1% o f gelatin.
The a l d o s t e r o n e a n t i s e r u m (2)lg/ml) was added a t a
d i l u t i o n corresponding t o t h e i n f l e c t i o n p o i n t of t h e t i t r a t i o n curve,
t o g e t h e r w i t h known amounts o f a l d o s t e r o n e ,
1-1000
p g / w e l l , o r with t h e sample t o be determined i n a t o t a l volume of
100
pl.
37OC.
The r e a c t i o n m i x t u r e s were i n c u b a t e d f o r 1 h o u r a t
The amount of bound antibody was measured by t h e a d d i t i o n
of e i t h e r p-GAL
o r HRP conjugated a n t i - r a b b i t
a c t i v i t y o f p - G A L o r H R P were d e t e r m i n e d manufacturer's i n s t r u c t i o n s .
IgG.
The enzymic
according t o t h e
The s t a n d a r d c a l i b r a t i o n c u r v e was
obtained with known amounts of aldosterone.
221
ENZYME-LINKED IMMUNOSORBENT ENZYMES Competitive assay with immobilized antibody Aldosterone-TG a n t i b o d i e s (1 )Ig/ml, 100 )11 i n
PBS) were
added t o t h e w e l l s o f ELISA p l a t e s and i n c u b a t e d a t 4OC.
After
washing and b l o c k i n g , as d e s c r i b e d a b o v e , a known amount of a l d o s t e r o n e t r e a t e d s i m i l a r t o t h e s a m p l e was i n c u b a t e d w i t h a
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c o n s t a n t amount o f Aldo-AP
c o n j u g a t e i n PBS c o n t a i n i n g 0.1%
gelatin.
A f t e r incubation f o r 1 hour i n 37OC t h e wells were washed
and
amount o f
the
AP c o r r e s p o n d i n g t o t h e amount of
bound
a l d o s t e r o n e w a s measured e i t h e r by a standard method employing pn i t r o p h e n y l phosphate as s u b s t r a t e , Johannsson e t a 1
o r by t h e a m p l i f i e d method of
( 1 4 ) u s i n g i o d o n i t r o t e t r a z o l i u m v i o l e t as
p r e v i o u s l y described.
RESULTS Aldosterone d e r i v a t i v e s were conjugated with v a r i o u s p r o t e i n
c a r r i e r s , s u c h a s CPA, AP a n d TG, i n o r d e r t o e l i c i t t h e production of h i g h l y s p e c i f i c antibodies, f o r t h e l a b e l i n g of a l d o s t e r o n e , a s w e l l as f o r a d s o r p t i o n
o f a l d o s t e r o n e on t h e
ELISA p l a t e s . Conjugation with TG y i e l d e d a product c o n t a i n i n g an average of
44 a l d o s t e r o n e (Table 1) r e s i d u e s p e r p r o t e i n molecule.
This
conjugate was i n j e c t e d i n t o r a b b i t s i n o r d e r t o e l i c i t production of a n t i b o d i e s .
The t i t e r o f a n t i b o d i e s was d e t e r m i n e d by t h e
ELISA assay u s i n g Aldo-CPA
and/or Aldo-AP as shown i n Fig. 1.
As
c a n be deduced from t h e t i t r a t i o n c u r v e , 50%b i n d i n g o c c u r s a t a d i l u t i o n of 1:9000 f o r Aldo-AP and 1:20.000 f o r Aldo-CPA (Fig. 1).
2 22
SCHWARTZ ET A L . TABLE
A
C o m p a r i s o n o f t h e s e n s i t i v i t y o f t h e d i f f e r e n t enzyme immunoassays f o r determination of plasma aldosterone.
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A.
Molecular Moles of Aldosterweightof Aldosterone con j u g a t e the one p e r carrier mol of (kD) protein
Detection Transformation l i m i t pg number(+)
*
Aldo-CPA
35
22
0.63
500 +/- 50
Aldo-TG
660
44
0.067
20 + / - 7
7
0.060
Aldo- AP
116
Concentration of Aldostero n e which i n h i b i t s 50% of binding pg/well
5000
+I- 50
70 +/- 7
2 +/ -0.2
20 + / - 2
B. Antialdosterone
*
5 +/- 0.5
50
+/- 5
Each f i g u r e i n t h e t a b l e r e p r e s e n t s t h e mean d a t a obtained from f i v e d i f f e r e n t experiments with a standard d e v i a t i o n varying between ‘+-la%.
( + ) moles of aldosterone per k i l o d a l t o n of p r o t e i n
The c a l c u l a t e d a p p a r e n t b i n d i n g c o n s t a n t o f t h e p o l y c l o n a l - ~ Fig. M 1 a n t i b o d i e s t o a l d o s t e r o n e was found t o b e ~ x I o ~(see
insert).
*
The antibody t i t e r obtained from t h e s e t i t r a t i o n curves
was r e l a t i v e l y high as compared t o d a t a reported i n l i t e r a t u r e , probably due t o t h e high content of a l d o s t e r o n e molecules bound t o
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ENZYME-LINKED IMMUNOSORBENT ENZYMES
223
ANTIBODY DILUTION (10-l)
FIGURE
1
Antibody d i l u t i o n c u r v e s obtained with d i f f e r e n t a l d o s t e r o n e - p r o t e i n c o n j u g a t e s c o a t e d on t h e ELISA p l a t e s . The c u r v e s show t h e percentage of binding of decreasing concentrations o f a n t i - a l d o s t e r o n e a n t i b o d i e s t o t h e complexed a l d o s t e r o n e ( B ) , r e l a t i v e t o t h e s a t u r a t i o n v a l u e (Bo) AP ( n o t shown), TG and CPA were u s e d f o r comparison a s c o a t i n g a n t i g e n s . The amount o f bound a n t i b o d y was f o l l o w e d by d e t e r m i n a t i o n o f enzymic a c t i v i t y o f B - g a l a c t o s i d a s e 1a b e l e d a n t i - r a b b i t immunoglobulin.
I n t h e i n s e r t of t h e Figure, t h e binding constant of a n t i - a l d o s t e r o n e t o Aldo-CPA i s measured, based on t h e concentration of f r e e antibody which binds 50% of t h e t o t a l antigen.
224
SCHWARTZ ET AL. TABLE
2
C r o s s - r e a c t i v i t y of anti-aldosterone antibodies with v a r i o u s s t e r o i d s measured by radioimmunoassy. These d a t a were o b t a i n e d from P r o f e s s o r V e c s e i , Department of Pharmacology, U n i v e r s i t y of Heidelberg. FRG.
% Cross-reactivities with a n t i - a l d o s t e r o n e
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Steroids
100
Aldosterone 5a-Dihydroaldosterone
64.4
Tetrahydroaldosterone
4.6
Corticosterone
0.4
18-Hydroxycorticosterone
0
Cortisol
0
Progesterone
0
t h e TG.
CPA, AP and TG were used f o r c o a t i n g as n e g a t i v e c o n t r o l .
E v a l u a t i o n of t h e enzyme-immuno assays proposed i n t h i s paper f o r t h e d e t e r m i n a t i o n of p l a s m a a l d o s t e r o n e and a l d o s t e r o n e i t s e l f were c h a r a c t e r i z e d regarding s p e c i f i c i t y , s e n s i t i v i t y , p r e c i s i o n and accuracy. (17).
S p e c i f i c i t y of t h e antiserum f o r a l d o s t e r o n e
was d e t e r m i n e d by m e a s u r i n g t h e c r o s s - r e a c t i v i t y w i t h v a r i o u s s t e r o i d s by R I A a s s a y and r e s u l t s a r e p r e s e n t e d i n T a b l e 2.
The
c r o s s - r e a c t i o n with c o r t i c o s t e r o n e . c o r t i s o l and progesterone were below t h e d e t e c t i o n l i m i t o f t h e a s s a y , s u g g e s t i n g t h e h i g h s p e c i f i c i t y of those a n t i b o d i e s t o a l d o s t e r o n e , confirmed a l s o by t h e h i g h b i n d i n g c o n s t a n t s o f t h e immuno complex ( - 1 0 + 9 M - 1 )
of
ENZYME-LINKED IMMUNOSORBENT ENZYMES
225
__________ Sensitivitx
a l d o s t e r o n e and i t s a n t i b o d i e s .
of these
immunoassays are e v a l u a t e d according t o c u r v e s e n s i t i v i t y which i s d e f i n e d by
d e t e c t i o n l i m i t f o r i d e n t i f i c a t i o n o f pure a n a l y t e i n
b u f f e r e d s o l u t i o n s , whereas t h e a s s a y s e n s i t i v i t y is
d e f i n e d by a
d e t e c t i o n l i m i t f o r t h e a n a l y t e i n t h e presence of a v a r i e t y of
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p o s s i b l e i n t e r f e r r i n g components, steroidals.
b o t h s t e r o i d a l s and non-
I n e x t r a c t i o n immunoassays, s o l v e n t e x t r a c t i o n may
sometimes minimize t h i s i n t e r f e r e n c e and p r o v i d e a system i n which o v e r a l l s e n s i t i v i t y and c u r v e s e n s i t i v i t y do n o t d i f f e r significantly.
I n a l l t h e experiments performed f r e e a l d o s t e r o n e
w a s t r e a t e d i n t h e same way a s a l d o s t e r o n e o f human p l a s m a t o a v o i d t h i s inconvenience.
Another parameter used i n t h i s work t o
c h a r a c t e r i z e t h e s e n s i t i v i t y of t h e assay was t h e c o n c e n t r a t i o n of a l d o s t e r o n e t h a t induced 50% c o m p e t i t i v e i n h i b i t i o n of b i n d i n g a f i x e d amount o f
antibodies
by
a known
amount
of
labeled
aldos terone. D e t e r m i n a t i o n o f p l a s m a a l d o s t e r o n e , as w e l l as t h e c a l i b r a t i o n curves of aldosterone i t s e l f , e x t r a c t e d i n a similar way t o i n two d i f f e r e n t ways.
i n buffer and/or
plasma a l d o s t e r o n e , was performed
I n t h e f i r s t method t h e c a l i b r a t i o n c u r v e s
o f a l d o s t e r o n e , as w e l l as t h e p l a s m a a l d o s t e r o n e t o b e determined, were c a r r i e d o u t by c o m p e t i t i v e a s s a y with immobilized antigens.
Aldo-CPA (18 pg/ml),
p e r mole of p r o t e i n ,
c o n t a i n i n g 22 moles a l d o s t e r o n e
a d s o r b e d on t h e ELISA p l a t e s was r e a c t e d
w i t h a m i x t u r e o f unknown amounts o f f r e e p l a s m a a l d o s t e r o n e . preincubated with a n t i - a l d o s t e r o n e a n t i b o d i e s and q u a n t i t a t e d by
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226
SCHWARTZ ET AL.
04 ' 0 '1
I
I
I
I
I
2
5
10
20
50
I
I
100 200
I
1000
ALDOSTERONE (pg/weJl)
FIGURE
2
t y p i c a l standard curve f o r the displacement of aldosterone a n t i b o d i e s from i m m o b i l i z e d a l d o s t e r o n e - p r o t e i n c o n j u g a t e s by d i f f e r e n t amounts o f a l d o s t e r o n e i n s o l u t i o n . P l a s m a a l d o s t e r o n e d i l u t i o n s , as w e l l as f r e e a l d o s t e r o n e e x t r a c t e d i n a s i m i l a r way a s plasma samples, are i n good c o r r e l a t i o n . The c o e f f i c i e n t of v a r i a t i o n f o r t h e i m p o r t a n t p o i n t s of t h e c u r v e ( d e t e c t i o n l i m i t and concentration of a l d o s t e r o n e a t 50% i n h i b i t i o n ) are g i v e n i n Table 3. The w e l l s c o a t e d w i t h t h e Aldo-AP (200 n g / m l ) (100 u l ) and t h e a l d o s t e r o n e a n t i b o d i e s d i l u t i o n used was 1:9000.
A
a d d i t i o n of
enzyme l a b e l e d a n t i - r a b b i t
IgG.
F i f t y percent
i n h i b i t i o n occurs a t r e l a t i v e l y high concentrations a l d o s t e r o n e . The d e t e c t i o n l i m i t of a l d o s t e r o n e observed i n
of this
system was 500 pg (see Table 1). The measurements of a l d o s t e r o n e concentration with another immobilized antigen, Aldo-TG a l d o s t e r o n e p e r mol p r o t e i n ) (10 yg/ml),
(44 moles
by u s i n g a n t i - r a b b i t
Ig
a n t i b o d i e s c o n j u g a t e s w i t h HRP, improved t h e s e n s i t i v i t y of t h e assay.
F i f t y percent
i n h i b i t i o n o c c u r e d a t 70 p g / w e l l
of
22 7
ENZYME-LINKED IMMUNOSORBENT ENZYMES
The s e n s i t i v i t y o b t a i n e d by t h i s a s s a y i s s u f f i c i e n t
aldosterone.
f o r t h e measurement o f a l d o s t e r o n e i n body f l u i d . results
were o b t a i n e d by c o a t i n g w i t h Aldo-AP
The b e s t
(0.2 n g / m l )
c o n t a i n i n g 7 mole of a l d o s t e r o n e / m o l p r o t e i n ( T a b l e l a , F i g 2 ) , f o l l o w e d by s e q u e n t i a l a d d i t i o n of f r e e a l d o s t e r o n e and/or plasma
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d i l u t i o n s p r e v i o u s l y incubated with p o l y c l o n a l a n t i - a l d o s t e r o n e . The r e s i d u a l amount o f a n t i b o d y which r e m a i n s a f t e r b i n d i n g o f a l d o s t e r o n e t o be determined w a s measured with commercial a n t i b o d y l a b e l e d w i t h HRP
.
second
This assay has t h e higher
s e n s i t i v i t y , a d e t e c t i o n l i m i t of 2 pg
-
and 50% c o m p e t i t i o n
between t h e free and bound a l d o s t e r o n e on t h e p l a t e occured a t 20 pg/well.
The r e s u l t s are shown i n Fig. 2 and summarized i n T a b l e
la. I n a n o t h e r t y p e o f a s s a y t h e a l d o s t e r o n e c o n c e n t r a t i o n was measured by c o a t i n g with a n t i - a l d o s t e r o n e a n t i b o d i e s , f o l l o w e d by c o m p e t i t i o n b e t w e e n a l d o s t e r o n e t o be measured and Aldo-AP conjugates
(Table l b ) .
The enzymic a c t i v i t y o f
alkaline-
p h o s p h a t a s e c o m p l e x was f o u n d t o b e 80% as compared w i t h t h e a c t i v i t y of t h e amount of experimental
conditions.
native
enzyme
By t h i s
measured
under
same
method 50% i n h i b i t i o n was
o b t a i n e d a t 50 p g / w e l l and t h e d e t e c t i o n l i m i t
of a l d o s t e r o n e was
5 Pg. I n a v a r i a n t o f t h i s method,
t h e a c t i v i t y of t h e AP was
d e t e c t e d n o t o n l y through t h e u s e of t h e s t a n d a r d s u b s t r a t e b u t by t h e u s e o f a n a m p l i f y i n g c h a i n o f enzymes, as was p r e v i o u s l y d e s c r i b e d by J o h a n n s s o n e t . a l .
( 1 4 ) ( F i g . 3).
The u s e o f t h i s
SCHWARTZ ET AL.
228
E C (u
Q)
t
E C
m 0 t
1.8
1.4 1.2 -
1.6
1-
v
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-
-
w 0
0.8
a
0.6 -
Z
m 0 0.4v) m 0.2 a 02
-
0 0.2 0.4 A P - A L D O S T E R O N E ( p g ml)
I
FIGURE
3
Determination of enzymatic a c t i v i t y of Aldo-AP complex bound t o antia l d o s t e r o n e a n t i b o d i e s measured by s t a n d a r d methods ( /i & ) and by ). Enzyme a c t i v i t y of a l k a l i n e a m p l i f i e d e n z y m a t i c method (24%rphosphatase, determined by s t a n d a r d method, w a s f o l l o w e d by i n c r e a s e i n o p t i c a l d e n s i t y a t 405nm and t h e a m p l i f i e d enzymic method by i n c r e a s e i n o p t i c a l d e n s i t y a t 462nm ( f o r d e t a i l s see experiment).
m o d i f i c a t i o n r e s u l t e d i n o v e r a l l h i g h e r OD v a l u e s , i n c r e a s i n g t h e sensitivity
_________ Precision
-
of t h e a s s a y f o r low c o n c e n t r a t i o n s of a l d o s t e r o n e . The p r e c i s i o n of t h e s t a n d a r d c u r v e o f human p l a s m a
a l d o s t e r o n e and a l d o s t e r o n e i t s e l f are a p p r e c i a t e d by i n t r a - a s s a y and i n t e r a s s a y c o e f f i c i e n t s of v a r i a t i o n and r e s u l t s summarized i n Table
3.
The s t a n d a r d d e v i a t i o n of t h e r e a d i n g of t h e mid-point
o f t h e s t a n d a r d c u r v e was + - 10%. Both d e v e l o p e d a s s a y s a r e
229
ENZYME-LINKED IMMUNOSORBENT ENZYMES TABLE
3
The r e p r o d u c i b i l i t y of t h e enzyme immunoassay f o r determination of aldosterone.
Coefficient
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Aldosterone
of
variation $
Intra-assay (n = 5)
Inter-assay (n = 5)
20
pg/well
0.32
0.04
2
pg/well
6.32
1.28
s e n s i t i v e enough f o r t h e assay of plasma a l d o s t e r o n e l e v e l and t h e r e p r o d u c i b i l i t i e s adequate €or c l i n i c a l assays. Accuracx o f t h e a s s a y was checked by t h e l i n e a r i t y o f d a t a obtained by assaying s e v e r a l d i l u t i o n s of sample c o n t a i n i n g known c o n c e n t r a t i o n s o f s t e r o i d , o r m o n i t o r i n g t h e r e c o v e r y o f known amounts of s t e r o i d added t o b i o l o g i c a l s a m p l e s . F i v e - t e n p g a l d o s t e r o n e added t o 100 u l o f p l a s m a o r b u f f e r i n c r e a s e d t h e measured a l d o s t e r o n e content by 5-10 pg +/-
(0.5-1).
Recovery of
a l d o s t e r o n e i s q u a n t a t i v e , i n d i c a t i n g t h a t i n normal c o n d i t i o n s no i n t e r f e r e n c e of o t h e r components with a n t i - a l d o s t e r o n e occured. DISCUSSION Many i n v e s t i g a t o r s
have been t u r n i n g t o enzyme immunoassay
t o r e p l a c e radioimmunoassay s i n c e i t frees them from t h e problems
SCHWARTZ ET AL.
and h a z a r d s o f h a n d l i n g r a d i o i s o t o p e s and t h e n e c e s s i t y f o r expensive counting instruments.
S e v e r a l such a s s a y s have been
developed f o r v a r i o u s s t e r o i d s and r e c e n t l y f o r a l d o s t e r o n e
(7-9).
An e v a l u a t i o n of s e n s i t i v i t y of v a r i o u s ELISA d e t e r m i n a t i o n s on plasma a l d o s t e r o n e ,
w i t h r e g a r d t o d e t e c t i o n l i m i t and i t s
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c o n c e n t r a t i o n i n b i o l o g i c a l f l u i d s t h a t i n d u c e 50% c o m p e t i t i v e b i n d i n g i n h i b i t i o n o f a f i x e d amount o f a n t i b o d i e s by a known amount of l a b e l e d a l d o s t e r o n e , as w e l l as s p e c i f i c i t y , p r e c i s i o n and a c c u r a c y of t h e a s s a y s , a r e d i s c u s s e d .
Comparison o f t h e
r e c o r d s o b t a i n e d i n e a c h ELISA u s i n g i m m o b i l i z e d a l d o s t e r o n e complexed with v a r i o u s p r o t e i n s (CPA, AP. TG) i n d i c a t e d t h a t t h e t r a n s f o r m a t i o n number ( t h e number o f a l d o s t e r o n e r e s i d u e s p e r k i l o d a l t o n of p r o t e i n ) seems t o determine t h e s e n s i t i v i t y of t h e assay. Apparently,
low d e n s i t y o f a l d o s t e r o n e o n t h e p r o t e i n
carrier y i e l d s a more s e n s i t i v e assay. can be argued as f o l l o w s :
sites €or i t s antigen. con j u g a t e
Presumably t h i s c o n c l u s i o n
Each antibody m o l e c u l e h a s two b i n d i n g When e x p o s e d t o a p r o t e i n a l d o s t e r o n e
containing
many
aldosterone residues
(high
t r a n s f o r m a t i o n number) t h e a n t i - a l d o s t e r o n e antibody would b i n d t o t h e c o n j u g a t e v i a b o t h i t s b i n d i n g s i t e s and t h e r e f o r e w i t h a h i g h e r a p p a r e n t b i n d i n g a f f i n i t y t o t h e conjugate than t o t h e f r e e d i l u t e d aldosteroned solutions.
T h i s would r e s u l t i n l o w e r
s e n s i t i v i t y measurements o f f r e e a l d o s t e r o n e .
When exposed t o a
conjugate w i t h low t r a n s f o r m a t i o n number, ( i d e a l l y one a l d o s t e r o n e molecule p e r p r o t e i n ) o n l y one o f t h e b i n d i n g sites o f t h e antibody would b i n d t o t h e conjugate and under such c o n d i t i o n s t h e
231
ENZYME-LINKED IMMUNOSORBENT ENZYMES
a f f i n i t y o f t h e a n t i b o d y f o r t h e c o n j u g a t e s h o u l d be s i m i l a r t o t h e a f f i n i t y of t h e antibody f o r t h e f r e e aldosterone, r e s u l t i n g i n higher d e t e c t i o n s e n s i t i v i t y of f r e e aldosterone. The production of a n t i - a l d o s t e r o n e antibodies using complex containing
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on immunogen
Aldo-TG
44 moles of a l d o s t e r o n e per mole of p r o t e i n as
was a good c h o i c e w i t h r e s p e c t t o t h e h i g h
s p e c i f i c i t y and a f f i n i t y of t h e antibodies. t h e ELISA p l a t e ,
Aldo-AP
However, f o r c o a t i n g
complex c o n t a i n i n g o n l y
7
moles
a l d o s t e r o n e per mole of p r o t e i n increased a t l e a s t t e n times t h e s e n s i t i v i t y of t h e assay as compared with d a t a obtained with AldoTG o r Aldo-CPA.
The e s t a b l i s h e d a s s a y s f o r p l a s m a a l d o s t e r o n e ,
using
immobilized antigen (Aldo-protein complexes) o r immobilized a n t i aldosterone antibodies, possess for RIA
high s e n s i t i v i t y , a s mentioned
( 3 - 6 ) , and p o s s e s s a l l t h e a d v a n t a g e s of ELISA a s s a y s ,
such as simple non-hazardous m a t e r i a l s and r e p r o d u c i b i l i t y .
The
normal r a n g e o f p l a s m a a l d o s t e r o n e , which v a r i e d between 18-79 pg/ml (1). a s w e l l a s t h e e l e v a t e d v a l u e s observed a f t e r hormone s t i m u l a t i o n a s s o c i a t e d with p a t h o l o g i c a l s t a t e s , can be measured
a t high accuracy by both methods. A comparison between t h e two a s s a y s proposed i n t h i s p a p e r
showed t h a t t h e y d i f f e r p r i m a r i l y i n t h e n a t u r e of t h e r e a g e n t s used, which make t h e f i r s t type of assay more convenient.
In t h i s
a s s a y ( w i t h i m m o b i l i z e d a n t i g e n ) o n l y one t y p e of a l d o s t e r o n e p r o t e i n c o n j u g a t e n e e d s t o be p r e p a r e d .
I n p r i n c i p l e , t h e same
conjugate can be used f o r both immunization of t h e r a b b i t s and f o r
232
SCHWARTZ ET AL.
running t h e assay.
The b i o l o g i c a l a c t i v i t y o f t h e c o n j u g a t e d
p r o t e i n i s o f n o i m p o r t a n c e and c o n j u g a t e s w i t h h i g h and low t r a n s f o r m a t i o n number may b e e a s i l y p r e p a r e d , and t h e r a b b i t antiserum used as a second antibody is a commercial p r e p a r a t i o n . Moreover t h e n a t u r e o f t h e second antibody may be e a s i l y changed
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and d i f f e r e n t methods f o r s i g n a l a m p l i f i c a t i o n compared e.g. u s e o f t h e a v i d i n b i o t i n system.
the
The s e c o n d t y p e o f a s s a y ( w i t h
immobilized antibody) is more d i f f i c u l t t o set up.
Two c o n j u g a t e s
are needed, one with high t r a n s f o r m a t i o n number f o r immunization, and a n o t h e r w i t h low t r a n s f o r m a t i o n number f o r t e s t i n g .
The
p r o t e i n u s e d f o r t e s t i n g h a s t o b e a n enzyme and i t s e n z y m a t i c a c t i v i t y h a s t o be r e t a i n e d a f t e r conjugation. f o r t h e c o a t i n g should be a t
The antibody used
least p a r t i a l l y purified.
A l t o g e t h e r , t h i s comparison i n d i c a t e s t h a t t h e f i r s t type of a s s a y
i s f a r e a s i e r t o s e t up and i s a l s o easier t o m a n i p u l a t e and improve.
REFERENCES
1.
Hubl, W., Aldosterone, I n : U l r i c h B.H. ( e d . ) . Methods of Enzymol. Anal. (3rd e d . ) 8. VCH: Weinheim, F e d e r a l Republic of Germany. 1985. 216-66.
2.
Bayard. F . , Kowarski, A., Weldon, V . and Migeon. C.J., Appraisal o f t h e double i s o t o p e d i l u t i o n technique f o r t h e measurement o f p l a s m a a l d o s t e r o n e . J. Lab. C l i n . Med. 1970. 75 :347-52.
3.
F a r m e r , R.W., Roup,Jr.. W.G., P e l l i z z a r i , E.D. and F a b r e , J r . . L.F. A r a p i d a l d o s t e r o n e radioimmunoassay. J . C l i n . Endocr. 1972. 34: 18-22.
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233
4.
Ito. T., Woo, J.. Haning, R. and Horton. R. A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of a1ternate techniques. J. Clin. Endocr. 1972. 34: 106-12.
5.
Vecsei, P., Penke, B. and Joumaah, A . Radioimmunoassay of free aldosteron and of its 18-oxe-glucoronide in human urine. Experientia. 1972. 2815: 622-24.
6.
Farmer, R.W., Brown, D.H., Howard, P.Y. and Fabre, Jr.. L.F. A radioimmunoassay for plasma aldosterone without chromatography. J. Clin. Endocrinol. Metab. 1973. 36: 460-65.
7.
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