novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for...

Journal of Clinical Laboratory Analysis 5324-330 (1 991)

Novel and Sensitive Noncompetitive Enzyme lmmunoassay (Hetero-Two-Site Enzyme Immunoassay) for a-Human Atrial

Natriuretic Peptide in Plasma Seiichi Hashida, Naoko Yamamoto, and Eiji Ishikawa

Department of Biochemistv, Medical College of Miyazaki, Miyazaki, Japan

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for a-human atrial natriuretic peptide (a-hANP) in plasma, which uses only one monoclonal IgG for the ring structure of a-hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N-hydroxysuccinimidobiotin to biotinylate a-hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of a-hANP to trap biotinylated a-hANP The polystyrene ball was washed to eliminate unreacted N-hydroxysuccinimidobiotin and other biotinylated substances, and biotinylated a-hANP was eluted from the polystyrene ball with HCI. The eluate was neutralized and incubated with horseradish peroxidase-la-

beled antibody IgG for the ring structure of a-hANP and subsequently with two streptavidin-coated polystyrene balls. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. The detection limit of a-hANP was 20 amol, and the assay range of plasma a-hANP was 0.8-1,200 nglL using 100 pI of plasma filtrates corresponding to 75 pl of plasma. Plasma levels of hANP in healthy subjects were 9.8-21.5 ng/L. These values were significantly lower than those measured by a two-site enzyme immunoassay probably due to the presence of a-hANPs lacking some N-terminal amino acids, which were as reactive as a-hANP [l-281 in the two-site enzyme immunoassay but less reactive in the hetero- two-site enzyme immunoassay.

Key words: peroxidase, sandwich enzyme immunoassay. competitive radioimmunoassay

INTRODUCTION

On the basis of pioneering studies on atrial granules by de Bold and his coworkers, a 28-amino acid polypeptide with potent diuretic and natriuretic activity was isolated from the rat atrium, and its amino acid sequence was determined (1). Later, three distinct polypeptides called a-, p-, and y-human atrial natriuretic polypeptides (a-hANP, p-hANP, and y-hANP) were identified in the human atrium (2,3). a-hANP is a 28-amino acid polypeptide with an intramolecular disulfide linkage. P-hANP is an antiparallel dimer of a-hANP with intermolecular disulfide bridges. y-hANP is a 126-amino acid polypeptide carrying the a-hANP sequence at the C-terminus. The molecular form of plasma hANP is almost exclusively a- hANP (43).

a-hANP has been measured with high sensitivity by two- site (sandwich) enzyme immunoassay (6). A polystyrene ball coated with antibody IgG for N-terminal half of the ring structure of a-hANP was incubated with a-hANP and subsequently with enzyme-labeled antibody fragment Fab’ for the C- terminus of a-hANP. The detection limit of a-hANP was 10 amol, which was 100-fold lower than that by competitive radioimmunoassay. The assay range of plasma a-hANP was 0.6-600

0 1991 Wiley-Liss, Inc.

ng/L using 50 ~1 of plasma. Plasma levels of a-hANP in healthy subjects were measured without processes of extrac- tion and concentration, which are required when measured by competitive radioimmunoassay. However, a disadvantage of this method is that two antibodies, which can simultaneously bind to a-hANP molecules, are required.

This paper describes a novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for a-hANP, which uses only one antibody for the ring structure of a-hANP.

MATERIALS AND METHODS

N-Ethylmaleimide-Treated Bovine Serum Albumin

Bovine serum albumin (100 mg, fraction V, Armour Phar- maceutical Co., Kankakee, IL) in 2 ml of 0.1 mol/L sodium phosphate buffer, pH 7.0, was incubated with 0.2 ml of 0.1 mol/L N-ethylmaleimide in 0.1 moVL sodium phosphate buffer

Received May 6, 1991; accepted May 10, 1991

Address reprint requests to E. Ishikawa, Department of Biochemistry, Med- ical College of Miyazaki, Miyazaki 889-16, Japan.

Enzyme lmmunoassay of cx-hANP 325

ther incubated with 100 pl of 1 mol/L Tris-HC1 buffer (pH 7.0), 50 pl of 0.1 mol/L EDTA (pH 6.0), and 120 pl of 1 mol/L hydroxylamine (pH 7.0) at 30°C for 5 min. The reaction mixture was subjected to gel filtration on a column (1 .O x 45 cm) of Sephadex (3-25 (Pharmacia LKB Biotechnology AB) using 0.1 mol/L sodium phosphate buffer (pH 6.0) containing 5 mmol/L EDTA. Fractions containing mercaptoacetyl anti-a-hANP IgGl were pooled and concentrated by centrifugation in a microconcentrator (CENTRICON-30, Amicon Di- vision W.R. Grace & Co., Beverly, MA). The average number of thiol groups introduced per IgG I molecule was 3.2 ( 1 1 ).

Maleimide-peroxidase

Maleimide groups were introduced into horseradish peroxidase molecules (Grade I, RZ = 3.0, Boehringer Mannheim GmbH) using N-succinimidyl-6-maleimidohexanoate (Do- jindo Laboratories, Kumamoto, Japan) (13). The amount of peroxidase was calculated from the absorbance at 403 nm by taking the extinction coefficient and molecular weight to be 2.275 g-l .L-cm-' and 40,000, respectively (1 1).

(pH 6.0) at 30°C for 30 min. The reaction mixture was dialyzed against 0.1 mol/L sodium phosphate buffer (pH 7.0) at 4°C overnight. The amount of bovine serum albumin was calculated from the absorbance at 280 nm by taking the extinction coefficient to be 0.63 g-' .L.cm-l (7).

Buffers

The regularly used buffers were 10 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1 moUL NaCl (buffer A), and 10 mmol/L sodium phosphate buffer (pH 7.0) containing 0.3 mol/L NaCl, 1 g/L N-ethyl-maleimide-treated bovine serum albumin, 0 .2 mmol/L L-cystine, and 1 mmol/L ethylenediaminetetraacetate (EDTA) (buffer B).

Peptides

[7-281, and P-hANP were obtained from Peptide Institute, Inc. (Osaka, Japan). a-hANP [6-281, synthesized by a con- ventional solution method (8), was generously supplied from Dr. K. Kondo (Chemistry Research Laboratories, Takeda Chemical Industries, Osaka, Japan).

a-hANP [ 1-28], a-hANP [4-281, a-hANP [5-281, au-hANP

Anti-a-hANP lgGl

Ascites containing a monoclonal IgGl, which recognized the ring structure of a-hANP [ 1-28], was generously supplied from Dr. K. Kondo. a-hANP [ 1-28] was conjugated to bovine thyroglobulin (Sigma Chemical Company, St. Louis, MO) by the carbodiimide method (9). Female BALB/c mice were immunized with 250 kg of the conjugate by subcutaneous injection at 3 week intervals. Each mouse was intravenously injected with 200 pg of the conjugate 4 days before cell fusion. Cell fusions were performed using mouse myeloma cell P3X63Ag8-Ul (10). The monoclonal IgGl was prepared from ascites using protein-A-Sepharose CL-4B (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) according to the instruction of Pharmacia.

IgG and F(ab'),

IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of DEAE-cellulose, and F(ab')2 was prepared by digestion of IgG with pepsin (11). The amount of IgG and F(ab')* was calculated from the absorbance at 280 nm (1 1).

Anti-a-hANP IgGl-Peroxidase Conjugate

Mercaptoacetyl anti-a-hANP lgGl

Anti-a-hANP IgGl (1 mg) in 0.82 ml of 0.1 mol/L sodium phosphate buffer (pH 7.0) was incubated with 80 pl of 2 mmollL N-succinimidyl-S-acetylthioacetate (Boehringer Mannheim GmbH, Mannheim, FRG) in N,N-dimethylfoma- mide at 30°C for 30 min (12). The reaction mixture was fur-

Anti-a-hANP IgG,-peroxidase conjugate

Mercaptoacetyl anti-a-hANP IgGl (0.6 mg, 4 nmol) in 0 .3 ml of 0.1 mol/L sodium phosphate buffer (pH 6.0) containing 5 mmol/L EDTA was incubated with maleimide-peroxidase (0.4 mg, 10 nmol) in 30 pl of 0.1 mol/L sodium phosphate buffer (pH 6.0) at 4°C for 20 h. After incubation, the reaction mixture was subjected to gel filtration on a column (1.5 x 45 cm) of Ultrogel AcA 34 (IBF biotechnics, Villeneuve- la-Garenne, France) using 0. 1 mol/L sodium phosphate buffer (pH 6.5). Absorbances at 280 nm and 403 nm of each fraction were measured. Fractions containing the conjugate were stored at 4°C in the presence of 0.1 % bovine serum albumin (fraction V, Armour Pharmaceutical Co.) and 0.005% thimerosal. The average number of peroxidase molecules conjugated per IgG, molecule was 2.0 (1 1) .

Acid-Treatment of Anti-a-hANP lgGl Anti-a-hANP IgGl (1 mg) in 76 p1 of 0.1 mol/L sodium

phosphate buffer (pH 7.0) was incubated with 1 .5 ml of 0.1 mollL glycine-HCI buffer (pH 2.5) at room temperature for 10 min. pH of the incubation mixture was adjusted to 7.5 with 2 mol/L Tris-HC1 buffer (pH 8.0).

Biotinyl Nonspecific Rabbit IgG Biotinyl nonspecific rabbit IgG was prepared by the reac-

tion of maleimide-nonspecific rabbit IgG with N-biotinyl- 2-mercaptoethylamine (14).

Nonspecific Mouse IgG-Sepharose 4B Nonspecific mouse IgG (2 mg) was coupled to CNBr-

activated Sepharose 4B (0.2 g) (Pharmacia LKB Biotechnol- ogy AB) according to the instructions of the manufacturer.

326 Hashida et al.

Affinity-Purification of Rabbit (Anti-Mouse IgG) IgG

Rabbit (anti-mouse IgG) IgG (Medical & Biological Lab- oratories, Nagoya, Japan) was affinity-purified by elution from a column of nonspecific mouse IgG-Sepharose 4B at pH 2.5 (15).

Protein-Coated Polystyrene Balls

Polystyrene balls (3.2 mm in diameter, Immunochemical Co., Okayama, Japan) were coated with acid-treated anti- a-hANP IgGl (0.1 g/L), biotinyl nonspecific rabbit IgG (0.1 g/L), and affinity-purified (anti-mouse IgG) IgG (0.1 g/L) by physical adsorption ( 16). Streptavidin-coated polystyrene balls were prepared by incubation of biotinyl nonspecific rabbit IgG-coated polystyrene balls with streptavidin (0.1 g/L) (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, MD) at 30°C for 4 h. The protein-coated polystyrene balls were stored in buffer A containing 1 g/L N-ethylmaleimide-treated bovine serum albumin and 1 g/L NaN3 at 4°C.

Alpha-hANP Standards and Plasma Samples and Filtrates

a-hANP [l-281 (0.59 mg, Peptide Institute, Inc.) was dissolved in the mixture of 1.1 ml of 0.1 mol/L HC1 and 1.1 ml of acetonitrile, and diluted with 0.1 mol/L sodium phosphate buffer (pH 7.0) containing 0.1 mol/L NaCl, 1 g/L N-ethylmaleimide-treated bovine serum albumin, and 1 mmol/L EDTA. The amount of a-hANP [ 1-28] was calculated by taking the molecular weight as 3,081 (2).

Blood was withdrawn into chilled glass tubes containing EDTA (Venoject VT-070NA EDTA-2Na, Terumo Corp., Tokyo, Japan) and centrifuged at 500xg for 10 min to separate plasma. The final concentration of EDTA in blood was 1.5 g/L. Plasma was obtained from 10 healthy subjects, aged 23-41 years with ad libitum water intake and normal activity approximately 4 h after breakfast.

Plasma (0.3 ml) was mixed with 0.1 ml of 0.4 mol/L sodium phosphate buffer (pH 7.0) containing 1,000 kallikrein inactivator units (KIU)/ml aprotinin (Sigma Chemical Co.), and filtrated by centrifugation in a microconcentrator with polysaccharide membrane (CENTRICON- 100, Amicon Divi- sion, W.R. & Co.) at 4°C at 5,OOOxg for 30 min. The filtrates (0.12 ml) were mixed with 1/99 volume of 10 g/L N-ethylmaleimide-treated bovine serum albumin.

Biotinylation

A 100 p1 aliquot of the diluted a-hANP [ 1-28] or the plasma filtrates was incubated with 5 pl of 63 mmol/L N-hydroxysuccinimido-biotin (Zymed Laboratories, Inc., San Francisco, CA) in dimethylsulfoxide at 4°C for 1 h. The reaction mixture was further incubated with 10 pl of 2 moVL glycine-NaOH (pH 8.0) at 4°C for 1 h, followed by addition of 35 pl of buffer

A containing 1 g/L N-ethyl-maleimide-treated bovine serum albumin, 0.2 mmoVL L-cystine, and 1 mmol/L EDTA.

Hetero-Two-Site Enzyme lmmunoassay

The biotinylated mixture (1 50 pl) was incubated with an acid-treated anti-a-hANP IgGl -coated polystyrene ball at 4°C for overnight. After incubation, the polystyrene ball was washed twice by addition and aspiration of 2 ml of buffer A and was incubated with the mixture of 75 p1 of buffer B and 15 p1 of 1 mol/L HCl at 4°C for 1 h. After removal of the polystyrene ball, the remaining solution was neutralized by addition of the mixture of 15 p1 of 1 moVL sodium phosphate buffer, pH 7.0, and 15 p1 of 1 mol/L NaOH. The neutralized mixture was incubated with anti-a-hANP IgGl -peroxidase conjugate (100 fmol) and nonspecific rabbit F(ab'), (0.5 mg) in 30 p1 of buffer B at 4°C overnight. Subsequently, two streptavidin-coated polystyrene balls were added, and the incubation was continued at 4°C for 5 h. After removal of the reaction mixture, the polystyrene balls were washed twice as described above, and peroxidase activity bound to the polystyrene balls was assayed at 30°C for 60 min using 3-(4-hydroxy- pheny1)propionic acid as hydrogen donor (1 7). Fluorescence intensity was measured relative to 0.2 mg/L quinine in 50 mmol/L H2S04 using 320 nm for excitation and 405 nm for emission with a Shimadzu spectrofluorophotometor (RF-5 10, Shimadzu Corporation, Kyoto, Japan).

Expression of the Detection Limit of a-hANP [l-281 The detection limit of a-hANP [ 1-28] was taken as the min-

imal amount of a-hANP [ 1-28] which gave a bound peroxidase activity significantly in excess of that nonspecifically bound in the absence of a-hANP [ 1-28] (background). The existence of a significant difference from the background was confirmed by the t-test (P<O.OOl, n = 5 ) .

Avidin-Peroxidase Conjugate

Mercaptoacetyl avidin

Avidin (2 mg, Avidin D, Vector Laboratories, Inc., Bur- lingame, CA) was dissolved in l ml of 0.1 mol/L sodium phosphate buffer (pH 7.0) and incubated with 100 p1 of 10 mmol/L S-acetylmercaptosuccinic anhydride (Nacalai Tesque, Inc., Kyoto, Japan) in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was further incubated with 50 pl of 1 mol/L Tris-HC1 buffer (pH 7.0), 30 p1 of 0.1 mol/L EDTA (pH 6.0), and 60 p1 of 1 mol/L hydroxylamine (pH 7.0), at 30°C for 5 min. The amount of avidin was calculated from the absorbance at 280 nm by taking the extinction coefficient and molecular weight to be 1.4 g - '.L*crn- and 68,000, respectively (18). The reaction mixture was subjected to gel filtration on a column (1 .O X 45 cm) of Sephadex (3-25 (Pharmacia LKB Biotechnology AB) using 0.1 mol/L sodium phosphate buffer (pH 6.0), containing 5 mmol/L EDTA. The

Enzyme lmmunoassay of a-hANP 327

peroxidase. Bound peroxidase activity was assayed by color- imetry using o-phenylenediamine as hydrogen donor.

average number of thiol groups introduced per avidin molecule was 2.7 (11).

Avidin-peroxidase conjugate

Mercaptoacetyl avidin (1.7 mg, 25 nmol) in 0.4 ml of 0.1 mol/L sodium phosphate buffer (pH 6.0), containing 5 mmoVL EDTA was incubated with maleimide-peroxidase (1 .O mg, 25 nmol) in 0.1 mi of 0.1 mol/L sodium phosphate buffer (pH 6.0), at 4°C for 20 h. After incubation, the reaction mixture was subjected to gel filtration on a column (1.5 X 45 cm) of Ultrogel AcA 44 (IBF biotechnics) using 0.1 mol/L sodium phosphate buffer (pH 6.5). Absorbances at 280 nm and 403 nm of each fraction were measured. Fractions containing the conjugate were stored at 4°C in the presence of 0.1 % bovine serum albumin (fraction V, Amour Pharmaceutical Co.) and 0.005% thimerosal. The average number of peroxidase molecules conjugated per avidin molecule was l .5, which was calculated from the absorbances at 280 nm and 403 nm (1 1,18).

Biotinyl a-hANP fl-281

a-hANP [l-281 (0.12 mg) in 0.37 ml of N,N-dimethylformamide was incubated with 30 p1 of 1.6 mmol/L N- hydroxysuccinimidobiotin (Zymed Laboratories, Inc.) in N,N-dimethylformamide at 4°C for 1 h and subsequently with 2 p1 of 2 mol/L glycine-NaOH (pH 8.0), at 4°C for 1 h.

Competitive Enzyme lmmunoassay

Anti-a-hANP IgGl was diluted with buffer B to a concentration of 2.5 nmol/L. Biotinyl a-hANP [ 1-28] was diluted with buffer B to a concentration of 75 pmol/L. Avidin- peroxidase conjugate was diluted with buffer B to a concentration to3.3 nmol/L. a-hANP [ 1-28] was diluted with buffer B. The diluted a-hANP [ 1-28] (40 p1) and the diluted biotinyl a-hANP [ 1-28] (40 pl) were incubated with the diluted anti- a-hANP IgGl (40 1.1) at 4°C for 5 h. Subsequently, the diluted avidin-peroxidase conjugate (30 pl) was added, and the incubation was continued at 4°C for 3 h. After incubation, three polystyrene balls coated with affinity-purified rabbit (anti- mouse IgG) IgG were added to the reaction mixture, and the incubation was continued at 4°C overnight. After removal of the reaction mixture, the polystyrene balls were washed twice by addition and aspiration of 2 ml of buffer A. Perox- idase activity bound to the polystyrene balls was assayed as described above.

Two-Site Enzyme lmmunoassay

Two-site enzyme immunoassay for a-hANP was carried out as described previously (19). Immunoplates coated with monoclonal anti-a-hANP F(ab’)z were incubated with a- hANP standards or plasma (50 p1) and, after washing, with affinity-purified rabbit anti-a-hANP [6-281 Fab‘-horseradish

Measurement of Amino Groups

A 10 p1 aliquot of samples was mixed with 2.0 ml of 0.2 mol/L sodium borate buffer (pH 8.0) and reacted with 0.8 ml of 0.01% fluorescamine (Sigma Chemical Company) in acetone. The fluorescence intensity of the reaction mixture was measured using 390 nm for excitation and 475 nm for emission (20). L-Leucine was used as standard.

RESULTS AND DISCUSSION

a-hANP was biotinylated with N-hydroxysuccinimidobiotin and trapped onto a polystyrene ball coated with monoclonal IgGl for the ring structure of a-hANP. The polystyrene ball was washed to eliminate unreacted N-hydroxysuccinimidobiotin and other biotinylated substances. Subsequently, biotinylated a-hANP was eluted from the polystyrene ball with HCl, and was measured by hetero-two-site enzyme immunoassay using peroxidase-labeled monoclonal IgGl for the ring structure of a-hANP and streptavidin-coated polystyrene balls (Fig. 1).

Immobilized Biotinylated anti-a-hANP lgGl a-hANP

pH 1.0

0. Peroxidase-labeled anti-a-hANP lgGl

Immobilized streptavidin

Fig. 1. Hetero-two-site enzyme irnrnunoassay for a-hANP [ 1-28]

328 Hashida et al.

t -I

1 .o

0.8

0.6

0.4

0.2

0 I

c 10 100 1000 ._ 0 0.01 0.1 1

a-hANP (1 -28 ) ( fmol/ tube )

Fig. 2. Dose-response curves of a-hANP [ 1-28] by hetero-two-site enzyme immunoassay (circles) and by competitive enzyme immunoassay (triangles). Open and closed circles indicate bound peroxidase activity (specifically bound

peroxidase activity plus nonspecifically bound peroxidase activity) and specifically bound peroxidase activity, respectively. Vertical bars indicate standard deviations of 3-5 determinations.

Detection Limit of a-hANP [l-281

The detection limit of a-hANP [ 1-28] was 60 fg (20 amol)/tube. This was 50-fold lower than that by a competitive enzyme immunoassay using the same antiserum as used in this study (Fig. 2), 4-fold lower than that by a colorimetric two-site enzyme immunoassay (19), and 2-fold higher than that by a fluorometric two-site enzyme immunoassay (6).

Specificity

The specificity was examined using hANP-related peptides (Fig. 3). The dose-response curves of a-hANP [4-281 and a-hANP [5-281 were almost identical to that of a-hANP [I-281, indicating that there was no significant change in the reactivity of a-hANP by deletion of up to four N-terminal amino acids. However, the reactivity significantly decreased

E 1,000 a-hANP (1-28) a-hANP (4-28) a-hANP (5-28)

a-hANP (1-28) a-hANP (4-28) a-hANP (5-28)

1,000 :

P-hANP

a-hANP (7-28) 100

I I I

100 1,000 10,000

hANP ( amohbe )

Fig. 3. Dose-response curves of a-hANP 11-28] and related peptides by hetero-two-site enzyme immunoassay.

Enzyme lmmunoassay of a-hANP 329

TABLE 1. Plasma Level of hANP in Healthy Subjects

Plasma level of hANP (ngiL)

By hetero-two-site By two-site Plasma enzyme enzyme No. Age Sex immunoassay immunoassay

1 33 M 21.5 50.0 2 27 M 15.9 35.0 3 23 M 15.2 32.0 4 33 M 13.6 26.5 5 41 M 10.8 17.0 6 30 M 9.8 19.0 7 32 M 13.9 15.0 8 38 M 16.7 26.5 9 25 F 14.5 34.0 10 25 F 15.2 42.0 Mean k SD 29.7 ? 11.2 14.7 ? 3.2

by deletion of five and six N-terminal amino acids. The cross- reactions with a-hANP [6-281 and a-hANP [7-281 on amolar basis were 35% and 11%, respectively. The cross-reaction with P-hANP was 22% on a molar basis.

It remains to be investigated whether a-hANP lacking 5 and 6 N-terminal amino acids are as reactive as a-hANP [ 1-28] in hetero-two-site enzyme immunoassay using antibodies for epitopes (e.g., C-terminus) other than the epitope recognized by the monoclonal IgG, used in the present study.

Recovery of a-hANP [l-281 Added to Plasma

As the volume of plasma subjected to biotinylation in- creased, the recovery of a-hANP [l-281 added to plasma decreased. This was due to the presence of amino groups at high concentrations in plasma (approximately 60 mmol/L), while the concentration of N-hydroxysuccinimidobiotin added to the reaction mixture for biotinylation was 3 mmol/L. The maximal volume of plasma that could be used with a satisfactory recovery was only 5-10 pl.

In order to overcome this difficulty, plasma was slightly diluted and filtrated by centrifugation in a microconcentrator with polysaccharide membrane to separate peptides from plasma proteins. When plasma was diluted 1.3-fold, the concentrations of amino groups in plasma filtrates of healthy subjects aged 28-37 years at 9:OO A.M. before breakfast (n = 4) and approximately 4 h after breakfast (n = 8) were both 1.7-2.1 mmol/L. When 100 pl of plasma filtrates corresponding to 75 p1 of plasma was subjected to biotinylation, the recover- ies of a-hANP [ 1-28] (30 ng/L) added to four plasma samples containing 3.6-30 ng/L of hANP were 80- 104%.

Assay Variation

The assay variation was examined at three different plasma levels over the range of 5.5-160 ng/L for within-assay and at three different plasma levels over the range of 3.6-196 ng/L for between-assay. The volume of plasma filtrates used was 100 pl corresponding to 75 pl plasma, and the number of determinations at each level was 10 for both within-assay and between-assay. The variation coefficients of within-assay and between-assay were 4.4-7.0% and 2.4-10.5%, respectively.

Plasma Level of hANP

The assay range of a-hANP [ 1-28] in plasma using 100 p1 of plasma filtrates corresponding to 75 pl of plasma was 0.8- 1,200 ng/L, since 100 p1 of plasma filtrates corresponding to 75 pl of plasma could be used with satisfactory recov- eries of a-hANP added to plasma as described above. As a result, it was possible to measure plasma hANP levels in healthy subjects without concentration processes, which were required for competitive radioimmunoassay (21,22). The concentrations of hANP in plasma of 10 healthy subjects aged 23-41 years with ad libitum water intake and normal activity

approximately 4 h after breakfast were 14.7 2 3.2 (SD) ng/L (range, 9.8-21.5 ng/L), whichwerelowerthanthose(29.7 2 11.2 ng/L; range, 15.0-50.0 ng/L) determined by a two-site enzyme immunoassay (19) (Table 1). This might have been due to the presence of a-hANP [6-281 and/or a-hANP [7-281 in plasma, which were as reactive as a-hANP [ 1-28] in the two-site enzyme immunoassay but less reactive than a-hANP [ 1-28] in the hetero-two-site enzyme immunoassay.

Since plasma hANP level has been reported to be elevated in cardiovascular, pulmonary, and renal diseases (2 1 -24), the hetero-two-site enzyme immunoassay may aid diagnosis of these diseases.

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novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for...

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